University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA INSTITUTE FOR ENVIRONMENT AND SANITATION STUDIES OCCURRENCE OF ANTIBIOTIC RESISTANT BACTERIA IN THE LEGON SEWAGE TREATMENT PLANT AND THE RECEIVING ONYASIA STREAM; IMPLICATIONS FOR WASTEWATER REUSE BY LADY ASANTEWAH BOAMAH ADOMAKO (10167241) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE AWARD OF M.Phil IN ENVIRONMENTAL SCIENCE DEGREE JULY, 2019 ` University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Lady Asantewah Boamah Adomako hereby declare that this thesis was carried out entirely by me from research undertaken under the supervision of Dr. DzidzoYirenya- Tawiah and Dr. Daniel Nukpezah of the Institute for Environment and Sanitation Studies, University of Ghana. This thesis has never been presented, either in part or in whole for a degree in this university or any other institution. All cited works and assistance have been duly acknowledged Sign………………………… Sign…………………………… Lady Asantewah Boamah Adomako Dr. DzidzoYirenya-Tawiah (Student) (Principal Supervisor) Date…………………………… Date…………………………… Sign…………………………… Dr. Daniel Nukpezah (Co Supervisor) Date…………………………… ii University of Ghana http://ugspace.ug.edu.gh ABSTRACT Although wastewater treatment plants efficiently reduce the loads of pathogens in wastewater, the conventional treatment processes do not show significant removal of antibiotics, antibiotic resistant bacteria and antibiotic resistant genes, resulting in the introduction of these contaminants into the environment. This has human health implications for wastewater reuse and the use of effluent receiving water bodies as irrigation sources. Humans may be exposed to resistant bacteria and render treatment options for infections caused by resistant pathogens ineffective. In order to assess the occurrence of antibiotic resistance bacteria from the Legon Sewage Treatment Plant effluent and the receiving Onyasia stream, antibiotic resistance profiles of Aeromonas hydrophila, Pseudomonas aeruginosa and Escherichia coli isolated from wastewater and the Onyasia stream were evaluated for resistance to selected antibiotics. Wastewater and surface water samples were collected once per month in triplicate over a six-month period from two sampling sites in the Legon Sewage Treatment Plant (influent and effluent) as well as three sites from the Onyasia stream with reference to the treatment plant discharge point (upstream, outfall and downstream). Aeromonas hydrophila, Pseudomonas aeruginosa and Escherichia coli isolates were tested for resistance to Gentamicin, Amoxicillin clavulanate, Tetracycline, Ciprofloxacin, Cefuroxime, Aztreonam, Imipenem and Ceftazidime using the Kirby-Bauer disc diffusion method. E. coli, Aeromonas hydrophila and Pseudomonas aeruginosa isolates showed high resistance patterns to most tested antibiotics. Escherichia coli and Aeromonas hydrophila isolates were most resistant to Amoxicillin clavulanate (57% and 68% respectively), Cefuroxime (52% and 43% respectively), and Tetracycline (49% and 31% respectively). In contrast, they were susceptible to Imipenem (91% and 78% respectively) Gentamicin (83% and 91%) Aztreonam (74% and 73% iii University of Ghana http://ugspace.ug.edu.gh respectively), and Ciprofloxacin (71% and 78%). Pseudomonas aeruginosa isolates showed high resistance to Aztreonam (37%) and Ciprofloxacin (33%). Pseudomonas aeruginosa isolates sensitivity to Imipenem (96%), Gentamicin (22%) and Ceftazidime (89%) was high. Resistant rates were high in effluent, outfall and downstream isolates, Significant differences (p < 0.05) were observed between upstream and downstream sampling sites relative to the wastewater treatment plant discharge point (outfall) and the rate of Escherichia coli and Aeromonas hydrophila isolate resistance to Amoxicillin, Cefuroxime and Tetracycline and Pseudomonas aeruginosa isolate resistance to Aztreonam and Ciprofloxacin. There were also levels of multi-drug resistant isolates in downstream sampling site compared to upstream sampling site. Results show that the discharge of treated wastewater effluent into water bodies are potentially significant contributors to the dissemination and persistence of antibiotic resistance in the receiving watershed. These findings also have human health implications for effluent wastewater reuse and the use of the stream as a source of irrigation water. iv University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to my dear husband Mr. Ofori Boamah Adomako, and children Michelle and Caleb Boamah Adomako. v University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I want to express my gratitude to God Almighty for His guidance and strength accorded me throughout my study. I would like to express my profound gratitude to my supervisors Dr. Dzidzo Tawiah- Yirenya and Dr. Daniel Nukpezah both of the Institute for Environment and Sanitation Studies (University of Ghana) for their encouragement, suggestions and constructive criticisms. My appreciation also goes to Dr. Yemoh Ted Annang of the Institute for Environment and Sanitation Studies (University of Ghana) for his suggestions and corrections. I am very grateful. I express my profound gratitude to Dr Mike Osei Atweneboana the Director of CSIR- WRI, Hawa Ahmed, Mr Mark Akrong, Regina Banu, Serlorm Borbor and Mr Bello all of the Environmental Biology and Health division for their immense support and encouragement. I would also like to say thank you to Mr. Lorenzo of Noguchi Memorial Institute and Dr. Josiah Owusu Danquah of Cleveland State Univerisity for their assistance and guidance. I am also grateful to Mr Emmanuel Nartey for his assistance during sampling. Finally, I would like to thank my husband Mr. Ofori Boamah Adomako for his immense support throughout my education. He is one of a kind and His support and encouragement has been phenomenal. vi University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ........................................................................................................... ii ABSTRACT .................................................................................................................. iii DEDICATION ............................................................................................................... v ACKNOWLEDGEMENTS .......................................................................................... vi LIST OF FIGURES ...................................................................................................... xi LIST OF TABLES ...................................................................................................... xiii LIST OF PLATES ...................................................................................................... xiv LIST OF ABBREVIATIONS ..................................................................................... xvi CHAPTER ONE ............................................................................................................ 1 INTRODUCTION ......................................................................................................... 1 1.1 Background ..................................................................................................... 1 1.2 Problem statement ........................................................................................... 6 1.3 Justification ..................................................................................................... 7 1.4 General Objective ............................................................................................ 9 1.5 Specific Objectives .......................................................................................... 9 CHAPTER TWO ......................................................................................................... 10 LITERATURE REVIEW ............................................................................................ 10 2.0 Introduction ................................................................................................... 10 2.1 Antibiotic resistance ...................................................................................... 11 2.2 Emergence of antibiotic resistance ................................................................ 11 2.3 Mechanisms by which bacteria acquire resistance ........................................ 12 2.4 Antibiotic resistance in the environment ....................................................... 14 2.5 Antibiotic resistance and wastewater treatment plants ................................. 17 2.6 Wastewater reuse........................................................................................... 18 2.7 Human health and ecological impacts of discharge of antibiotic resistant bacteria from wastewater treatment plants ............................................................... 20 2.8 Tools for detecting antibiotic resistant bacteria in waste water and surface water 22 2.9 Current Knowledge on antibiotic resistant bacteria and antibiotic resistance genes in wastewater treatment plants and surface waters. ....................................... 24 CHAPTER THREE ..................................................................................................... 28 MATERIALS AND METHODS ................................................................................. 28 3.1 The Study Area................................................................................................ 28 3.1.1 Description of study site ........................................................................... 30 3.2 Study design .................................................................................................. 32 vii University of Ghana http://ugspace.ug.edu.gh 3.3 Field data collection ...................................................................................... 34 3.3.1 Wastewater collection from treatment plant .......................................... 35 3.3.2 Surface water collection from Onyasia stream ........................................... 35 3.4 Laboratory analysis ......................................................................................... 35 3.5 Quality assurance .......................................................................................... 41 3.6 Data analysis ................................................................................................. 42 CHAPTER FOUR ........................................................................................................ 44 RESULTS .................................................................................................................... 44 4.1 Occurrence of total coliforms, E. coli, A. hydrophila, P. aeruginosa in treatment plant .......................................................................................................... 44 4.2 Occurence of E. coli, A. hydrophila, P. aeruginosa in Onyasia Stream .......... 46 4.3.1 Treatment plant ............................................................................................ 47 4.3.2 Onyasia Stream .......................................................................................... 53 4.4 Field observations ............................................................................................ 55 4.5 Determination of antibiotic resistant E. coli isolates....................................... 60 4.5.1 Occurrence of all antibiotic resistant E. coli isolates .................................. 60 4.5.2 Occurrence of antibiotic resistant E. coli isolates across sampling sites ... 61 4.5.3 Multi-drug resistance in E. coli isolates .................................................... 64 4.6 Determination of antibiotic resistant A. hydrophila isolates .............................. 65 4.6.1 Occurrence of all antibiotic resistant A. hydrophila isolates ...................... 65 4.6.2 Occurrence of antibiotic of A. hydrophila isolates across sampling sites .. 66 4.6.3 Multi-drug Resistance in A. hydrophila isolates ........................................ 69 4.7 Determination of antibiotic resistant P. aeruginosa isolates........................... 70 4.7.1 Occurrence of antibiotic resistant of all P. aeruginosa isolates ................. 70 4.7.2 Occurrence of antibiotic resistance P. aeruginosa isolates across sampling sites ....................................................................................................................... 71 4.7.3 Multi-drug resistance in P. aeruginosa isolates ......................................... 74 DISCUSSION .............................................................................................................. 78 5.0 Introduction ................................................................................................... 78 5.1 Occurrence of E. coli, A. hydrophila and P. aeruginosa ................................ 79 5.2 Antibiotic resistance in E. coli isolates ........................................................... 80 5.3 Antibiotic resistance in A. hydrophila ............................................................. 83 5.4 Antibiotic resistance in P. aeruginosa isolates ............................................... 86 5.5 Suitability of treatment plant effluent for reuse in agriculture ...................... 90 5.6 Implications for guidelines and standards for compliance in effluent quality discharge into the environment and agriculture. ...................................................... 92 viii University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX ............................................................................................................ 94 CONCLUSIONS AND RECOMMENDATIONS ...................................................... 94 6.1 CONCLUSIONS ........................................................................................... 94 6.2 RECOMMENDATIONS .............................................................................. 96 REFERENCES ............................................................................................................ 99 APPENDICES ........................................................................................................... 111 APPENDIX I .......................................................................................................... 111 AEROMONAS ISOLATION MEDIUM COMPOSITION .................................... 111 APPENDIX II ........................................................................................................ 113 E. COLI ISOLATION MEDIUM COMPOSITION .............................................. 113 APPENDIX III ....................................................................................................... 115 PSEUDOMONAS ISOLATION MEDIUM COMPOSITION ............................... 115 APPENDIX IV ....................................................................................................... 116 PLATES SHOWING SAMPLE COLLECTION AND ANALYSIS .................... 116 PLATES SHOWING BACTERIAL CULTURE PLATES ................................... 117 APPENDIX VI ....................................................................................................... 119 SOME BIOCHEMICAL TESTS CONDUCTED ON ISOLATES ....................... 119 APPENDIX VII...................................................................................................... 121 WATER SAMPLING SITES AND COORDINATES .......................................... 121 APPENDIX VIII .................................................................................................... 122 BACTERIAL COUNTS, ANTIBIOTIC SENSITIVITY PATTERNS AND BIOCHEMICAL IDENTIFICATION ................................................................... 122 Negative control (blanks) analysed alongside water and wastewater samples .. 122 Levels of E. coli, A. hydrophila and P. aeruginosa in wastewater and surface water at the sampling sites. ................................................................................. 123 Levels of total coliform in wastewater ............................................................... 125 A. hydrophila breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2018) .... 126 E. coli breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2017) .... 126 P. aeruginosa breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2017) .... 127 E. coli antibiotic sensitivity quality control analysis .......................................... 127 P. aeruginosa antibiotic sensitivity quality control analysis .............................. 128 Susceptibility patterns of Aeromonas hydrophila isolates against the selected antibiotics............................................................................................................ 129 ix University of Ghana http://ugspace.ug.edu.gh Biochemical and physical confirmatory tests for E. coli isolates ....................... 145 Biochemical and physical confirmatory tests for Aeromonas hydrophila ......... 150 Biochemical and physical confirmatory tests for P. aeruginosa isolates ........... 156 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 2.1: Mechanisms of resistance and Antibiotic targets 14 Figure 2.2: A diagram depicting how human activities and the environmental compartment intersect in terms of spread of antibiotics, antibiotic resistant bacteria and genes 16 Figure 2.3: Methods used in assessing antibiotic resistance bacteria and antibiotic resistance genes present in water and wastewater 24 Figure 3.1: A map of study area 29 Figure 3.2: Schematic representation of the Legon Sewage treatment plant 31 Figure 3.3: A map showing sampling sites 33 Figure 4.1: A line chart showing levels of resistant E. coli in treatment plant 47 Figure 4.2: A line chart showing levels of P. aeruginosa in treatment plant 48 Figure 4.3: A line chart showing levels of A. hydrophila in treatment plant 49 Figure 4.4: A line chart showing levels of total coliforms in treatment plant 50 Figure 4.5: A line chart showing levels of faecal coliforms in treatment plant 53 Figure 4.6: A line chart showing levels of E. coli in Onyasia Stream 52 Figure 4.7: A line chart showing levels of in A. hydrophila Onyasia Stream 53 Figure 4.8: A line chart showing levels of P. aeruginosa in Onyasia Stream 54 Figure 4.9: A bar chart showing percentage occurrence of resistance E. coli isolates in treatment plant 62 Figure 4.10: A bar chart showing percentage occurrence of resistance E. coli isolates in the Onyasia Stream 62 Figure 4.11: A bar chart showing percentage occurrence of resistance of A. hydrophila isolates in treatment plant 67 Figure 4.12: A bar chart showing percentage occurrence of resistance A. hydrophila xi University of Ghana http://ugspace.ug.edu.gh isolates in the Onyasia Stream 67 Figure 4.13: A bar chart showing percentage occurrence of resistance all isolated P. aeruginosa. 70 Figure 4.14: A bar chart showing percentage occurrence of resistance P. aeruginosa isolates in treatment plant 71 Figure 4.15: A bar chart showing percentage occurrence of resistance P. aeruginosa isolates in the Onyasia Stream 72 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 2.1: Studies quantifying antibiotic resistance bacteria in urban wastewater treatment and receiving rivers 27 Table 4.1: Levels of P. aeruginosa, E. coli, and A. hydrophila in influent and effluent wastewater 45 Table 4.2: Level of total coliform in influent and effluent wastewater 46 Table 4.3: Levels of P. aeruginosa, E. coli, and A. hydrophila in the Onyasia stream 47 Table 4.4 Antibiotic susceptibility patterns of all E. coli isolates 61 Table 4.5: Multi-drug resistance patterns of E. coli isolates by in the treatment plant 64 Table 4.6: Multi-drug resistance patterns of E. coli isolates by in the treatment Plant 65 Table 4.7 Antibiotic susceptibility patterns of all A. hydrophila isolates 66 Table 4.8: Multi-drug resistance patterns of A. hydrophila isolates by sampling site in the treatment plant 69 Table 4.9: Multi-drug resistance patterns of A. hydrophila isolates by sampling site in the Onyasia stream 70 Table 4.10: Multi-drug resistance patterns of P. aeruginosa isolates by sampling site 75 Table 4.11: Multi-drug resistance patterns of P. aeruginosa isolates by sampling site 76 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF PLATES Plate 1: A photograph of lettuce farm located at downstream sampling site 56 Plate 2 A photograph of vegetable farm abstracting water for irrigation 57 Plate 3: A photograph of a farmer abstracting water for irrigation of vegetables 57 Plate 4: A photograph of wastewater connections from a household into the Onyasia stream 59 Plate 5: A photograph of wastewater connections from entering the Onyasia stream 60 Plate 6: A photograph of sample collection 116 Plate 7: A photograph of antibiotic sensitivity testing using Kirby Bauer Disc diffusion method 116 Plate 8: A photograph of culture plates showing P. aeruginosa (left) and E. coli colonies (Right). 117 Plate 9: A photograph of culture plates showing A. hydrophila colonies 117 Plate 10: A photograph showing antibiotic sensitivity tests of A. hydrophila. Note the zones of inhibition 118 Plate 11: A photograph a setup of oxidase positive (Left) and citrate positive (Right) 119 xiv University of Ghana http://ugspace.ug.edu.gh Plate 12: A photograph a setup of glucose fermentation (Left) and sulphide indole motility positive (Right). 120 xv University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS ARB Antibiotic Resistant Bacteria ARG Antibiotic Resistance Genes CFU Colony Forming Unit HPC Heterotrophic Plate Count WWTP Wastewater Treatment Plant WHO World Health Organization GN Gentamicin CXM Cefuroxime ATM Aztreonam IMP Imipenem AMC Amoxicillin clavulanate CIP Ciprofloxacin CAZ Ceftazidime TET Tetracycline xvi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 Background Antibiotics are one of the breakthrough discoveries in human history. Antibiotics are substances that are able to prevent the growth of or kill other organisms (WHO, 2014). They are broadly used for improving human and animal health by preventing and treating infections caused by bacterial pathogens. In animal husbandry and aquaculture, antibiotics are used extensively as growth promoters. (Roca et al., 2015; Kummerer, 2009; Baquero et al., 2008). Since their discovery antibiotics has saved many human lives, but the broadened range of antibiotic use for both medicinal and non-medicinal purposes has caused a surge in their use over the years (Bouki et al, 2013). The consequence of this includes the development and spread of antibiotic resistant bacteria (ARB) (Levy and Marshall 2004) due to the abuse and evolution through mutation. Antibiotic resistance is defined as the capacity of bacteria to withstand the effects of antibiotics previously used in their treatment (WHO, 2014). The pervasive usage of antibiotics in humans and animals results in the development of resistant bacteria, which is a major concern as antibiotic resistance results in failure in the treatment of infections that were previously treated with these antibiotics. Antibiotic resistance is one of the major health threats confronting humans (WHO, 2000). The O’Neill, 2014 Review on Antimicrobial Resistance, estimates that the 700,000 annual deaths caused by drug-resistant pathogen infections if left unchecked, will increase to 10 million by 2050 (O’Neill Commission, 2014). Resistant bacteria that cause life-threatening infections are becoming increasingly prevalent, and this limits treatment options. The WHO Global Action Plan on 1 University of Ghana http://ugspace.ug.edu.gh Antimicrobial Resistance which aims at reducing the rate at which antibiotic resistant bacteria evolve and spread (WHO, 2015). This action plan emphasizes the need to quantify environmental concentrations and total loads of antimicrobial resistant bacteria, antimicrobial resistant genes and antibiotic residues originating from humans and animals in the environment in order to provide needed evidence for decision- making and reduce exposure and human health risks (Wuijts et al., 2017). A strategic goal of the action plan is to increase surveillance and research into antimicrobial resistance. Addressing the rising antimicrobial resistance threat requires a “One Health” approach. “One Health” approach involves the collaborative efforts of multiple sectors working together locally, nationally and globally to tackle antibiotic resistance and other health issues. The goal of “One Health” is to achieve optimal health through acknowledging that humans, animals, plants and the environment are interconnected (Centers for Disease Control,2018). The initiative recognizes that, the effects of antibiotic resistance on humans, animals as well as the environment must be addressed simultaneously. This is because antibiotics, which are used in treating various infections in animals, may be the same used for human infections. Antibiotic resistance bacteria originating from animals, humans and the environment may spread within each other as well, locally and globally. In addition, resistant pathogens found in clinical environments have obtained their resistant genes from environmental reservoirs (Wright, 2010). Antibiotics enter the environment via different pathways including wastewater effluent discharge, surface runoff and leaching from agricultural lands applied with manure (Zhang et al 2009). The environment serves as potential reservoirs to antibiotic 2 University of Ghana http://ugspace.ug.edu.gh resistance bacteria (Kummerer 2009; Zhang et al., 2009; WHO, 2014). Hotspots for antibiotic resistance are within environmental compartments where environmental bacteria mix and exchange genetic material with potential pathogenic bacteria. These hotspots include sewage, wastewater treatment plants (WWTPs), animal farms and aquaculture facilities (Kummerer, 2009; Rizzo et al, 2013, Wellington et al., 2013,). Wastewater treatment plants (WWTPs) take and sewage from households, hospitals and other sources, which may contain antibiotics and antibiotic resistant bacteria. Hospital effluents, particularly, are very dangerous because they contain large amounts of residues of drugs and infectious pathogens; as such, they are an important source of antibiotics and multiple drug-resistant bacteria (Lorezo et al., 2018). During treatment of infections microbiota present in the human gut become exposed to high concentrations of antibiotics and which places selective pressure on microorganisms present and may stimulate the development of resistance phenotypes in the gut, which are then released into sewage through human excreta. A large percentage of most antibiotics administered for the prevention or treatment of microbial infections in humans and animals are excreted as an active substance, and are consequently released through sewage into the environment (Rizzo et al., 2013). WWTPs are key points for the emergence and proliferation of antibiotic resistance in environmental settings (Rizzo et al., 2013). Antibiotics, antibiotic resistant genes and antibiotic resistant bacteria have been found in wastewater treatment plants (Zhang et al., 2009; Rizzo et al., 2013). The treatment of wastewater includes different processes, which include chemical, physical, biological, mechanical, and physicochemical that may affect the outcome of antibiotics, antibiotic resistant bacteria and genes. 3 University of Ghana http://ugspace.ug.edu.gh Effluent may be discharged into the environment, and as such, they play a major role in antibiotic resistance development and proliferation (Rizzo et al., 2013). Urban wastewater treatment by biological treatment process creates a conducive environment that enables the development and spread of antibiotic resistant bacteria (Rizzo et al, 2013). This is as a result of the continuous mixing of antibiotics at low concentrations and bacteria present which leads to selective pressure resulting in the development of resistant bacteria. In most cases, although the concentrations of antibiotics may be low and not lethal to bacteria it is sufficient to select for resistance (Gullberg et al, 2011). Wastewater treatment plants may discharge their effluent into receiving environments such as rivers, lakes, or oceans after treatment, and therefore has implications for wastewater reuse and the use of contaminated water bodies as irrigation water sources. Water bodies containing antibiotic resistant bacteria may overflow during heavy rains and results in the contamination of ground water sources. Unfortunately, there are inadequate global and local regulations or guidelines for the permitted or desirable maximum limits for contaminants like antibiotics, antibiotic resistant bacteria and antibiotic resistance genes that can be discharged into the environment or reused (McConnell, 2016). Antibiotic resistant bacteria and genes which are still present after treatment end up in the environment where antibiotic resistance genes can spread among bacteria and can also be obtained by dangerous pathogens in the environment. The reuse of wastewater is being encouraged in agriculture, aquaculture and other non- critical uses in order to manage aquatic resources to meet the United Nations Sustainable Development GOALS (SDG) goal 6 of ensuring the availability and sustainable management of water and sanitation for all. Nevertheless, there is a 4 University of Ghana http://ugspace.ug.edu.gh potential risk to humans from consumption of wastewater irrigated produce as well as contact through recreational activities. As result of exposure, humans may acquire infections caused by antibiotic resistant pathogens thereby rendering treatment of infections ineffective. As such, antibiotic resistant bacteria and other contaminants in wastewater has to be monitored to protect human health if wastewater reuse is being encouraged. This research seeks to investigate presence of antibiotic resistant Pseudomonas aeruginosa P. aeruginosa) Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) isolates in the Legon Sewage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods. This will contribute to knowledge on antibiotic resistant levels in the wastewater treatment plant and the Onyasia Stream. The study will also provide evidence for the need for further treatment of wastewater to remove antibiotic resistance for the use of wastewater effluent in agriculture and other purposes. The study will also highlight the need to include antibiotic resistant bacteria in standards and guidelines for wastewater discharge and reuse. The information from this study will also inform decision making regarding the reuse of wastewater from the Legon Sewage Treatment Plant for agriculture, aquaculture and other non-critical uses. This study thus seeks to investigate the presence of antibiotic resistant P. aeruginosa, E. coli and A. hydrophila in the Legon Sewage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods and the environmental health implications of the treatment plant discharge. 5 University of Ghana http://ugspace.ug.edu.gh 1.2 Problem statement Inadequate sanitation and lack of access to safe and clean water threatens human health. Due to water scarcity, there has been recent efforts at considering the promotion of recycling and reuse of wastewater to reduce pressure on fresh water. Recycled wastewater is being recommended for irrigation, car washing and other non-critical uses. However, effluents discharged by wastewater treatment plants into the environment may contain antibiotics, antibiotic resistant bacteria and antibiotics, and may end up in water and soil (Aslan, 2018). Wastewater and agricultural runoff are considered as sources of antibiotic resistance in the environment (Li et al., 2010). Water contaminated with antibiotic resistant bacteria can affect aquatic biodiversity (Aslan, 2018) and human health adversely (Hong et al., 2018). Antibiotic resistance bacteria could come into contact with drinking water resources, the food chain through irrigation as well as contact from recreational use rendering treatment options ineffective (Blaustein et al., 2016; Wang et al., 2014; Frey et al., 2015; Zhang et al., 2015). As such antibiotic resistance is a major health concern worldwide, and World Health Organization has recognized antibiotic resistant bacteria as an emerging water pollutant of concern (WHO, 2014). In order to improve and expand sewerage and sanitation system for disposal of wastewater in Accra that meets environmental standards, the Legon Sewage Treatment Plant was constructed as part of the Accra Sewage Improvement Project (ASIP). The treatment plant is located in Legon, Accra and operates on the concept of waste stabilization ponds. Effluents from the plant is discharged into the Onyasia Stream, a nearby stream and part of the Odaw catchment. The Onyasia stream, which empties into the Odaw River, serves as a source of irrigation water to some vegetables farms along the stream in Dzorwulu, Airport and other parts of Accra (Drechsel and Keraita, 2014). It is also a water source for car washing in these communities. It is not known if it also serves other purposes in times of water shortages in the area. 6 University of Ghana http://ugspace.ug.edu.gh Monitoring of antibiotic resistance in clinically relevant bacteria in treated effluent leaving the treatment plant and the receiving stream is essential to afford knowledge on the type of resistant bacteria being released into the environment. This information is necessary for the identification of public health risks to inform decision-making regarding improvement of removal rates of antibiotic resistant bacteria and genes to minimize exposure to humans. 1.3 Justification Wastewater treatment plants reduce the concentrations of pathogens in wastewater, but the treatment processes are not able to substantially remove all antibiotic resistant genes and bacteria (WHO, 2014). Treatment of wastewater by use of stabilization ponds in particular have been shown to enhance the transfer of resistant genes among bacteria when compared to other treatment methods like chemical and mechanical treatment (Neudorf et al., 2017). Wastewater treatment plants are therefore salient hotspots and risk environments for the emergence and distribution of antibiotic resistance (Kummerer, 2009; Rizzo et al., 2013). Surface waters are often the recipients of treated wastewater from treatment plants. These water bodies, which are also discharge points often, serve as irrigation water sources, posing a health risk to humans. In a period of water scarcity, wastewater reuse is encouraged in agriculture, aquaculture and other non-critical uses in order to manage aquatic resources to meet the SDG 6 to ensure the availability and sustainable management of water and sanitation for all. The United Nations Educational Scientific and Cultural Organization (UNESCO) recommends treated wastewater reuse for irrigation in order to reduce 7 University of Ghana http://ugspace.ug.edu.gh pressure on freshwater resources. This is because the use of water for agriculture makes up 70% of global water withdrawals. There is a potential risk to humans from consumption of wastewater irrigated produce as well as contact through recreational activities. As result of exposure, humans may acquire infections caused by antibiotic resistant pathogens thereby rendering treatment of such infections ineffective. As such, antibiotic resistant bacteria and other contaminants in wastewater has to be monitored to protect human health. Antibiotic resistance bacteria and genes are now considered as environmental pollutants and as such, severe measures need to be taken to prevent their further spread (WHO, 2014). Better understanding of antibiotic resistance reservoirs is needed to fight the resistance threat in the clinic and the environment. There is limited knowledge on the antibiotic resistance occurrence as well as resistance types in the wastewater and wastewater affected environments. In the era of increasing antibiotic resistance and the potential reuse of wastewater, it is necessary to examine the contribution of wastewater treatment plants to resistance in the environment. Mapping antibiotic resistance in wastewater environments in Ghana is essential in order to increase knowledge about the insights into extensive occurrence and range of antimicrobial resistance in non-clinical environments. This will also provide guidance for decision making regarding additional treatment of sewage needed to reduce the risk of development and spread of antibiotic resistance. This thesis reports on a study designed to determine the presence of antibiotic resistant enterobacteria like P. aeruginosa, E. coli and A. hydrophila in the Legon Sewage 8 University of Ghana http://ugspace.ug.edu.gh Treatment Plant and the receiving Onyasia stream, using bacterial resistance data from culture-dependent methods. The aim is to determine the whether these resistant and potentially pathogenic microbes would likely compromise the quality of the effluent from the treatment plant. Further to this, the study also discussed the likely impact of the quality of the effluent on reuse in agriculture and other uses, as well as the general environmental health of the stream and its users. 1.4 General Objective The primary objective of this study is to determine the presence of antibiotic resistant P. aeruginosa, E. coli and A. hydrophila in the Legon Sewage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods, and assess the environmental health implications of the treatment plant discharge on wastewater and stream use. 1.5 Specific Objectives 1. Assess the level of occurrence of E. coli, P. aeruginosa and A. hydrophila in wastewater from the Legon Sewage Treatment Plant and the Onyasia stream. 2. To assess the occurrence of antibiotic resistant E. coli, P. aeruginosa and A. hydrophila isolated from the Legon Sewage Treatment Plant effluent. 3. To assess the occurrence of antibiotic resistant E. coli, P. aeruginosa and A. hydrophila isolated from sampling sites in the Onyasia stream relative to the wastewater treatment plant in order to determine the impact of the treatment plant effluent discharge on antibiotic resistance profiles of surface water bacteria. 4. To evaluate the safety of wastewater effluent for reuse options. 9 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.0 Introduction Antibiotics discovery is probably one of the greatest inventions of all times, as without them humankind would have been wiped out by diseases. Antibiotics are compounds that cause microbial cell death or inhibit cell growth due to specific interactions with bacterial targets. Antibiotics are essential in the treatment of infections in humans and animals. Antibiotic resistance occurs when bacteria are altered in such a way which makes medications that will otherwise kill or inhibit their growth ineffective in curing infections caused by the microorganisms. (Nordgård, et al., 2017). The overuse of antibiotics has resulted in the selection of antibiotic resistant microorganisms. Resistance of microbes to antibiotics is a serious human and wildlife health threat in terrestrial or aquatic environments. The WHO, has asserted that humans may be entering a post-antibiotic era when simple bacterial infections that were previously treatable are no longer responding to antibiotic treatment resulting in death (O’Neill Commission, 2014). The 2014 O’Neill report commissioned by the UK government estimated that globally by 2050, antimicrobial resistant infections may become the leading cause of death (O’Neill Commission, 2014). Although global increased levels of antibiotic resistance has been linked to the misuse of antibiotics in medical and agricultural practices, the contribution of the natural environment to the development and spread of resistance has not received a lot of attention until now. Human activities may contaminate the environment with antibiotic resistant bacteria and antibiotics which results in the acceleration of the development 10 University of Ghana http://ugspace.ug.edu.gh and spread of resistance. Sources of antibiotic and antibiotic contamination in the environment include animal and human waste, and pharmaceutical manufacturing waste. 2.1 Antibiotic resistance Antibiotic resistance may be intrinsic or acquired. Natural resistance bacteria have a natural ability for Deoxyribonucleic Acid (DNA) recombination and genetic mutation can occur in all bacterial isolates. However, intrinsic resistance occurs when the target of drug action does not exist or is inaccessible. For example, gram-negative bacteria have intrinsic resistance to some antibiotics, including erythromycin, because of the outer membrane being impermeable (Alkhaleefah, 2015). The genetic mechanisms underpinning acquired bacterial resistance development are gene transfer and mutation (Alkhaleefah, 2015). Acquired resistance results from selective pressure exerted by antibiotic agents present in the environment (Osińska et al., 2017). 2.2 Emergence of antibiotic resistance There is a significant correlation between antibiotic consumption and antibiotic resistance (Karkman, 2015). Although the phenomenon of antimicrobial resistance was existent prior to mass production and clinical use of antibiotics, human activities have contributed massively in the development of environmental reservoirs of antibiotic resistance. 11 University of Ghana http://ugspace.ug.edu.gh Large amounts of antibiotics manufactured since the 1940’s have been used and released into the environment. A small proportion of antibiotics in clinical use is contributed by organisms that produce antibiotics naturally (Helt, 2012), with the bulk of commercial production accounting for the vast amount of antibiotics currently used. These antibiotics have spread widely providing continuous selective pressure on bacteria resulting in resistant strains in the environment (Davies and Davies, 2010). Clinically relevant pathogens resistant to penicillin were recorded not long after it was discovered. The first Tetracycline resistant Shigella strain was isolated five years later after the antibiotic was discovered in 1948. Horizontal transfer of genes has contributed to the evolution and transmission of resistant genes (Davies & Davies, 2010). Superbugs are commensal and pathogenic bacteria that have acquired multiple resistance genes (Karkman, 2015). An example is Methicillin Resistant Staphylococcus aureus (MRSA). Some strains have in addition acquired increased virulence and enhanced transmissibility (Karkman, 2015). Due to massive antibiotic use, many pathogens associated with epidemics of human disease have acquired multiple resistance determinants. Multiple antibiotic resistant bacteria are causing serious problems in healthcare system worldwide. The World Health Organization (WHO) has raised concern about antibiotic resistance amidst fears the human race is heading back to pre-antibiotic era if serious action is not taken. (World Health Organization, 2014). 2.3 Mechanisms by which bacteria acquire resistance Through evolution, some bacteria have developed various mechanisms to resist the effects of antibiotic compounds. Antibiotic resistance genes (ARGs) genetically code for resistant traits. Resistance can be acquired through mutation and gene transfer 12 University of Ghana http://ugspace.ug.edu.gh (vertical and horizontal). Horizontal gene transfer occurs between microorganisms by means of use of mobile genetic elements including plasmids, integrons and transposons. These genetic elements contain genes that are transferred between bacteria (Marti et al., 2014). Resistant genes in mobile genetic elements control specific mechanisms, which fight an antibiotics ability to harm bacteria. Horizontal gene transfer mechanisms through which bacteria take foreign DNA are transformation, transposition, transduction or conjugation (Dantas and Sommer, 2014). Transformation occurs when bacteria obtain free DNA from the environment, and transduction occurs when a bacteriophage (a virus that infects bacteria) carries DNA from one bacterial cell to another. Conjugation involves physical contact between a donor and a recipient cell via a conjugation pilus, through which genetic material is transferred, therefore involving live bacteria (McConnell, 2016). Mobile genetic elements have a major role as vectors for accumulation and spread of antibiotic resistance genes among bacteria. This is due to their ability to encode conjugative transfer of genes, which enable the movement of genes between bacteria of different species. Resistance genes located on plasmids and other genetic elements can carry one or more genes. This enables various mechanisms of resistance to multiple antibiotics and other compounds like heavy metals. The capacity of microorganisms to share genetic material through horizontal genes transfer implies that genes that occur in environment can potentially transfer resistant genes from animal to human pathogens within bacterial communities (Nordgård et al., 2017). The environment is therefore a large reservoir of potential resistance genes and this can be referred to as environmental resistome (Wright, 2010). Resistome is a term used to describe the occurrence and transference of antimicrobial resistance genes between and 13 University of Ghana http://ugspace.ug.edu.gh within different environments (Alkhaleefah, 2015). Figure 2.1 shows some mechanisms of action of antibiotic classes and the resistance mechanisms that exist to counteract them. Figure 2.1: A diagram showing mechanisms of resistance and antibiotic targets (Adapted from Wright, 2010) 2.4 Antibiotic resistance in the environment Antimicrobial resistance hotspots occur in clinical settings as well as environmental settings that have been impacted by anthropogenic activities. Antibiotic resistant 14 University of Ghana http://ugspace.ug.edu.gh bacteria and genes and antibiotics have been found in different environmental niches including wastewater treatment plants, sediment, surface water, seawater and drinking water (Xi et al., 2009; Rizzo et al., 2013; Berglund et al., 2015; Berglund et al.,2014; Zhang et al., 2015; Zhang et al., 2016; Rizzo et al., 2013). Antibiotics and resistant genes may persist in the environment, after the use antibiotic has ended. Although resistant microorganisms can be found naturally in all environments, their occurrence has increased over the years, a phenomenon attributed to the increased usage of antibiotics in addition to exposure to other substances, which promote resistance for example, some heavy metals, biocides and disinfectants. (Martinez, 2009; Berglund et al., 2015). The environment has different functions in the emergence and spread of antibiotic resistance. Firstly, the environment serves as a receiving receptacle of resistant bacteria from animals and humans that have ingested antibiotics. These bacteria may encounter other bacteria already present in the environment enabling the exchange of resistant genes between species. The environment also facilitates antibiotic resistant bacteria and resistance genes spread through air, groundwater and surface water (WHO, 2014). In addition, the environment serves a cache for natural resistance genes. Lastly, humans and animals are exposed to antibiotic resistance directly and indirectly through the environment (WHO 2014). Figure 2 depicts human activities and the environmental compartment interact in terms of spread of antibiotics, resistant bacteria and genes. Hotspots are environmental compartments where environmental bacteria mix and exchange genetic material with potential pathogenic bacteria. These hotspots include sewage, wastewater treatment plants, animal farms and aquaculture (Kummerer, 2009; Wellington et al., 2013). 15 University of Ghana http://ugspace.ug.edu.gh Municipal wastewaters are particularly an important environmental source for antibiotics, antibiotic resistant bacteria and genes (Rizzo et al., 2013). They also provide a conducive environment for the transfer of resistance genes between bacteria in the course of the treatment process or after discharge into the aquatic environment. Figure 2.2: A diagram depicting how human activities and the environmental compartment intersect in terms of spread of antibiotics, antibiotic resistant bacteria and genes. (Adapted from Dantas and Sommers 2014). 16 University of Ghana http://ugspace.ug.edu.gh 2.5 Antibiotic resistance and wastewater treatment plants Wastewater treatment plants are key antibiotic resistant bacteria sources (Kümmerer, 2009; Figueira et al., 2011a; Lupo et al., 2012 Rizzo et al., 2013). Antibiotics, antibiotic resistant genes and bacteria have been found in wastewater and activated sludge (Dröge et al., 2000; Auerbach et al., 2007; Zhang et al., 2009). Antibiotics consumed by humans are released into municipal treatment plants partly metabolized or unused (Bouki et al, 2013). These antibiotics present in wastewater, even at low concentrations, apply selective pressure on bacteria present, which results in emergence of antibiotic resistant bacteria (Kummerer 2009). Most wastewater treatment processes have primary, secondary, and tertiary treatment stages. Physical operations to remove solids in the primary treatment process, where the use of bar screens to remove large particles like sand, gravel and other heavy particles are found. Biological and chemical processes are used in the secondary treatment to remove organic matter. In tertiary treatment, other processes are used to take out other components such as nutrients and toxic materials, which are not removed during the secondary treatment. In the course of wastewater treatment distribution of bacterial populations are altered. Due to the constant mixing of antibiotics at sub lethal concentrations with bacteria, the treatment process provides favorable conditions for resistance development and spread (Rizzo et al., 2013). Although there is a remarkable reduction in the quantity of bacteria in treated wastewater, wastewater treatment process may not remove all antibiotics present and may contain higher amounts of resistant bacterial populations when compared with the 17 University of Ghana http://ugspace.ug.edu.gh corresponding proportions contained in surface water (Huang et al., 2012). In addition, antibiotics present in final effluents even after treatment may persists and exert selective pressure on bacteria present (Varela and Manaia, 2013). β-lactams, a class of antibiotics are easily broken down in the presence of water but have been found in the environment albeit at low concentrations and some b-lactams resistant genes have been detected in WWTPs (Bouki et al., 2013). High Tetracycline resistant gene prevalence has been found in all the processes that take place within a wastewater treatment plant that were assessed, suggesting treatment plants as potential sources for the dissemination of resistance into the environment (Auerbach et al., 2007). Antibiotic resistant bacteria are also abundant in activated sludge (Ge et al. 2013), and could enter the environment by the application of sludge as manure for growing crops. There is therefore the need to ensure removal of antibiotics and antibiotic resistant bacteria in wastewater to prevent their discharge into the environment. 2.6 Wastewater reuse Climate change, socio-economic transformations and increase in human population over the years have resulted in the concerns regarding the sustainability of water use for humans. Wastewater reuse in agriculture is now considered as a viable solution to reduce water scarcity. In 2010 the African Water Facility proposed an initiative for the effective treatment of wastewater and faecal sludge as it can be a valuable resource with potential for reuse and also can serve to finance and provide incentive for strong sanitation systems as well as reduce surface water contamination Currently, process water for industrial cooling, irrigation and aquifer recharge are some of the accepted and applied wastewater reuse activities (Maryam and Büyükgüngör 2019). Countries like Switzerland, Australia, Singapore, South Africa, Tunisia, Cyprus, 18 University of Ghana http://ugspace.ug.edu.gh Saudi Arabia, Qatar, and Kuwait run successful wastewater reuse projects (Maryam and Büyükgüngör 2019). In the United States of America, Texas, California and Arizona use reclaimed water for irrigation due to water scarcity. In addition, these states have adopted some wastewater reuse regulations (Aslan et al 2018). However, recycled water is routinely screened for faecal coliform bacteria levels but not antibiotic resistant indicator bacteria (Aslan et al., 2018). The concern with wastewater reuse is the contamination of the environment as well as the risk of acquiring infections due to exposure of antibiotic resistant bacteria and antibiotics from wastewater-irrigated produce. Although there have been many advancements in wastewater treatment technologies over the years, there are concerns on wastewater discharge and reuse safety (Aslan et al., 2018). Many personal care products and pharmaceutical products have been detected 30 cm below soil where turf- grass fields were irrigated with treated effluent ( Xu et al., 2009) Currently there are guidelines and critical values for indicator bacteria for effluent discharge and for reuse of wastewater and greywater for agriculture (EPA, 2010; WHO 2006) and other non-critical uses. However, these guidelines do not have recommended antibiotic resistant bacteria limits in wastewater for discharge into the environment nor reuse for irrigation and other purposes. In addition, most studies that have evaluated the suitability of wastewater for agriculture have mostly focused on indicator bacteria and not pathogenic bacteria. There is the need for the quantification of antibiotic resistance bacteria in addition to other indicator organisms to facilitate monitoring wastewater 19 University of Ghana http://ugspace.ug.edu.gh effluent discharge and use in agriculture in order to reduce the potential risks to protect human and environmental health. 2.7 Human health and ecological impacts of discharge of antibiotic resistant bacteria from wastewater treatment plants Enteric diseases such as cholera and diphtheria are attributed to poor sanitation, the use of contaminated water and poor hygiene practices. Depending on their origin, wastewater may contain microorganisms, organic compounds, metals and other substances. Non-degradable pharmaceutical contaminants like hormones and antibiotics found in wastewater persist in the water cycle and have become of greater concern over the years. Wastewater contains a rich diversity of microorganisms including pathogenic and non-pathogenic viruses and bacteria. The purpose of wastewater treatment is to remove contaminants and reduce the undesirable impacts on the aquatic environment and public health. Studies have demonstrated that these microorganisms (including antibiotic resistant bacteria) and other compounds may survive the treatment processes and can be detected in final effluents (Rizzo et al., 2013). Antibiotics present in wastewater may apply pressure on bacteria which resulting in the development of resistant genes, which persist in the environment and have harmful effects on humans and animals. The release of waste containing human bacteria into environments already laden with bacteria and are enriched in resistance elements increases the chances of acquiring novel resistance by pathogens that have clinical significance (Martinez, 2009; Zhang et al., 2009). Non- pathogenic and pathogenic bacteria may proliferate or acquire virulence or resistance genes. 20 University of Ghana http://ugspace.ug.edu.gh This is a major public health, as resistant bacteria and genes that are released into the environment could be transferred to humans through surface water used for irrigation, recreation and potable water production (Xi et al., 2009; Figueira et al., 2012). Vegetables irrigated with surface water sources that receive wastewater may transfer antibiotic resistance bacteria onto the human food chain. Multiple drug resistant Salmonella sp have been found in water used for irrigation of vegetables (Al-Jassim et al., 2015; Parvathi et al., 2011). Antibiotic-resistant Enterococcus sp has been detected in reclaimed water used for spray irrigation leaving farm workers exposed to Enterococci during spray irrigation activities (Carey 2013). In Europe 2011, an epidemic was caused by multiple antibiotic resistant enterohaemorrhagic E. coli on vegetables which came from contaminated irrigation water. Soil and water may also be contaminated by antibiotic resistance bacteria resulting in major changes in the population and physiology of microbiota present and as well as their activities when irrigated with wastewater (Martinez, 2009). Dalkmann et al. (2012) compared effects of wastewater irrigation in soils and found a rise in total microbiota and a rise in the abundance of antibiotic resistant gene in soil irrigated with wastewater in Mexico. This study seeks to investigate the presence of antibiotic resistant P. aeruginosa, E. coli and A. hydrophila in the Legon Sewage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods and the environmental health implications of the treatment plant discharge. 21 University of Ghana http://ugspace.ug.edu.gh 2.8 Tools for detecting antibiotic resistant bacteria in waste water and surface water To recognize hotspots for the possibility of spreading of antibiotic resistant genes and resistance bacteria, their relative abundance has to be determined. Two main approaches are used for determining antibiotic resistant bacteria and genes, namely culture-based and molecular-based methods for clinical and environmental samples. Environmental samples which include wastewater and surface water contain a myriad of bacterial species as compared to clinical bacteria, as such in order to achieve accurate results modifications need to be made which, can be applied to wastewater and surface waters antibiotic resistance surveillance testing. Microbial analysis of water is done using the membrane filtration method or spread plate method after which bacteria are isolated for further identification and then characterization of antibiotic resistance. Coliforms and other enteric bacteria are mostly cultured because they serve as indicators of microbial contamination (APHA, 2012). Isolates are identified, counted and confirmed using biochemical tests after which they are screened for their resistance to selected antibiotics. Common methods for determining resistance patterns are disc diffusion and micro dilution methods. The dilution method is based on determining the minimum concentration (MIC) of antibiotics that inhibit the tested isolate by using agar or broth dilutions. The disc diffusion method includes the determination of zones of inhibition on a plate covered with Mueller Hinton agar seeded with the isolate being tested. The zones of inhibitions are determined at the minimum inhibitory concentration for a specific antibiotic. Standardized performance values (e.g. from Clinical Laboratory Standards Institute (CLSI), 2017) are used in the distinction between organisms that are susceptible, resistant or intermediate. 22 University of Ghana http://ugspace.ug.edu.gh Studies have been done that involved modifications to the culture-based methods due to the laborious nature of culture-based methods. Selective media are augmented with antibiotics at varied concentrations for the enumeration of bacteria in environmental samples (Rizzo et al., 2013). Percentage resistance is then computed as the ratio between bacteria that grow with and without target antibiotic (Li et al., 2010; Novo and Manaia, 2010; Figueira et al., 2011). However, this addition of antibiotics to selective media has not been standardized. Mueller-Hinton agar is the ideal medium for routine antibiotic susceptibility testing because it has shown reproducibility for repeated antibiotic susceptibility and data has been collected with regard to its use in susceptibility tests (Hardy diagnostics, 1996). Molecular methods have also been used for the examination of antibiotic resistance genes in water and wastewater. Molecular methods such as Polymerase Chain Reaction (PCR), whole-genome sequencing deoxyribonucleic microarray, metagenomics, and whole genome sequencing have been used to detect presence of microorganisms, which are fastidious (McConnell 2016). They are used on the basis that an isolate carrying a gene for resistance implies resistance. Therefore, genetic based tests focus on the finding resistant genes or genetic elements that help in the transfer of genes in bacteria. There is lack of standardization with these molecular methods for detecting resistant genes (Rizzo et al., 2013). However, the use of genes in molecular tests have an advantage over culture-based methods because they are more useful in epidemiological studies that trace the spread of important resistance genes in wastewater-impacted environments. This is possible because molecular methods enable the detection of genetic markers that code for antibiotic resistance as well as virulence genes in tandem. Molecular and culture-based methods can be used together for analyzing pathogens and 23 University of Ghana http://ugspace.ug.edu.gh the genes that encode resistance in the target organism (McConnell, 2016). Figure 3 illustrates how both methods are complementary. Figure 2.3: Methods used in assessing antibiotic resistance genes and antibiotic resistance bacteria present in wastewater and surface water (Adapted from McConnell, 2016). 2.9 Current Knowledge on antibiotic resistant bacteria and antibiotic resistance genes in wastewater treatment plants and surface waters. Studies have shown that wastewater treatment plants have a significant role in the development and proliferation of antibiotic resistance, and can therefore serve as major reservoirs or hotspots (Szczepanowski et al., 2009; Rizzo et al., 2013; Luczkiewicz et 24 University of Ghana http://ugspace.ug.edu.gh al., 2010; Al-Jassim, et al., 2015; Kim et al., 2016; Rafraf et al., 2016; Nordgård, et al., 2017). Wastewater treatment plants are considered as hotspots because of the potential presence of substances like antibiotics, heavy metals and biocides which exert selective pressure on bacteria (Xi et al., 2009; Plosz et al., 2010; Michael et al., 2013) and the presence of other favorable microbial growth conditions (such as nutrients) during treatment. These conditions may favor horizontal transfer of resistance genes between bacteria present. Table 2.1 shows results from antibiotic resistance prevalence studies in wastewater treatment plant and the receiving environment. Studies in antibiotic resistant bacteria have mostly been done on bacteria that are used as water quality indicators, for example coliforms and enterococci (Sabate et al., 2008; Figueira et al., 2011; Amaya et al., 2012). Thus, coliforms and enterococci are the most investigated bacterial groups in wastewater treatment plants. There have also been studies on resistance in human pathogens such as Staphylococcus aureus and other gram-negative bacteria (Thompson et al., 2013). Standardization of methods for testing resistance in environmental bacteria is limited compared to clinical settings that have been standardized extensively. Currently, most studies on antibiotic resistance in have been focused on clinical settings, as such data on antibiotic resistant bacteria in wastewater and wastewater receiving environments is lacking especially in Ghana. Regulations or guidelines for the permitted or desirable maximum limits for contaminants like antibiotics, antibiotic resistant bacteria and antibiotic resistance genes that can be discharged into the environment and reused are inadequate. This study therefore seeks to help fill this knowledge gap by providing data on antibiotic resistance in treated effluent from the 25 University of Ghana http://ugspace.ug.edu.gh Legon Sewerage Treatment Plant and the receiving water body. This will provide evidence for decision making regarding improvement in treatment options in order to reduce exposure and human health risks. 26 University of Ghana http://ugspace.ug.edu.gh Table 2.1. Studies quantifying antibiotic resistance bacteria in urban wastewater treatment and receiving rivers Guadeloupe Enterobactereaceae Third generation cephalosporin (50.4%), Ciprofloxacin Guyomard-Rabenirin et al., (51.6%), Tetracycline (40.2%), Cotrimoxazole (34.1%) 2017 and Gentamicin (11.8%) Saudi Arabia Acinetobacter sp, Aeromonas sp, Legionella sp, Ampicillin (72%), Erythromycin (51%), Chloramphenicol Al-Jassim et al. (2015) Mycobacterium sp, Streptococcus sp, Neisseria (46%), Kanamycin (41%), Ceftazidime 32%), Tetracycline sp, Pseudomonas sp, , Enterococcus sp (21%), Meropenem (12%), Ciprofloxacin (6.6%) Ireland E. coli Ampicillin (12.5%), Streptomycin (10%), Galvin et al., (2010) Sulfamethoxazole (12.5%), Tetracycline (39%), Ciprofloxacin 0%), Cefoxitin (2.6%) Poland E. coli Ampicillin (34%), Piperacillin (24%), Tetracycline (23%), Luczkiewicz et al. (2010) Levofloxacin (15%), Nitrofurantoin (53%), Ciprofloxacin (10%), Trimethoprim/Sulphamethoxazole (11%), Erythromycin (44%), Ciprofloxacin (29%), Tetracycline (20%), USA Acinetobacter Trimethoprim (100%), Rifampin (77.5%), Zhang et al. (2009) Chloramphenicol (35%), Amoxicillin clavulanate (20%), Ciprofloxacin (11.3%) 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 The Study Area The study was carried out at the Legon Sewage Treatment Plant and the receiving Onyasia Stream located near the University of Ghana Botanical garden, Legon, and Accra. Accra is the capital city of Ghana, covering an area of 200 square kilometers with a population of about 2.5 million (World Population Review, 2020). Legon, a suburb of Accra located 12 kilometers northeast of the city center in the Accra Metropolitan District Low ridges characterize the landscape of Accra, 20 m above sea level separated by small river courses running in a southerly direction towards the sea. Accra has a gentle sloping terrain, which favors natural drainage by gravity. The Odaw River and its tributaries, the Nima, Onyasia, Dakobi and Ado Streams drain the catchment area. Currently, conventional sewerage network covers close to 15% of Accra with areas that are not covered using public toilets, septic tanks, pit latrines and pan latrines (Mohammed et al, 2017). The Legon, Kotoku and Mudor Sewage Treatment plants are currently the main plants functioning in Accra, The Legon Sewage Treatment Plant was chosen for the study because effluents from the plant are discharged directly into the Onyasia stream, which serves as an irrigation source for vegetable farms along the stream. In addition, the design of treatment plant and its operation influence the fate of resistant bacteria and resistant genes found in wastewater (Kim and Aga, 2007). Wastewater stabilization ponds have shown to enhance the transfer 28 University of Ghana http://ugspace.ug.edu.gh of resistance genes among bacteria when compared to other methods of treatment (Neudorf et al., 2017). Figure 3.1 shows a map of the study area. Figure 3.1. A map of study Area 29 University of Ghana http://ugspace.ug.edu.gh 3.1.1 Description of study site Legon Sewage Treatment plant The Legon Sewage Treatment plant was constructed and commissioned in 2012 as part of the Accra Sewerage Improvement Project (ASIP), to extend sewage treatment in order to improve sanitation and sewage treatment in Accra. The treatment plant uses waste stabilization ponds which consist of open basins including anaerobic, facultative and maturation ponds, and the effluent discharged directly into the Onyasia Stream. The Legon Sewage Treatment Plant consists of 3 anaerobic ponds, 3 facultative ponds and 6 maturation ponds with an outlet after which the treated effluent is discharged into the Onyasia Stream. The plant has the capacity to treat (9,000 m3) of sewage inflow per day and was expected to serve 33,000 residents (ASIP appraisal report) but is currently operating at 3606 m3 per day (Manager, Legon sewage treatment plant). The treatment plant treats wastewater from the University of Ghana, Presbyterian Senior High School, University of Professional Studies, Achimota Senior High School and Achimota Hospital. (African development fund, 2005). Volumes of liquid waste from these institutions are channelled through various pumping stations into a receiving tank at the treatment plant daily. The liquid waste first goes through a wide screen which removes large particles and other non–biodegradable materials. The liquid waste then flows into a distribution chamber and from there it flows into three grit channels each connecting to a treatment stream. Primary treatment occurs in the anaerobic pond, where suspended solids and soluble parts of organic matter Biological Oxygen Demand (BOD)) are removed. The secondary treatment which occurs in the facultative pond where most of the remaining BOD is removed with the help of 30 University of Ghana http://ugspace.ug.edu.gh heterotrophic bacteria and algae. The main function of the maturation pond which is a tertiary treatment process is to remove pathogens and nutrients. Figure 3.2 below shows a schematic diagram of the Legon Sewage Treatment plant with the different ponds. Figure 3.2: Schematic representation of the Legon Sewage treatment plant. (Source Legon Sewage Treatment Plant) Onyasia Stream Effluent from the Legon Sewage Treatment Plant is discharged into the Onyasia Stream. The Onyasia Stream runs within the Odaw catchment, stretching from Akuapem Mountains into the sea at Korle Gonno. 31 University of Ghana http://ugspace.ug.edu.gh The Onyasia stream serves as a source of irrigation water for nearby vegetable farms. Human activities such as farming, settlements, dumping of refuse, littering has resulted in the pollution of the stream. 3.2 Study design The study assessed the extent to which the Legon Sewage Treatment Plant contributes to the increase of antibiotic resistance in the Onyasia stream. The study employed a descriptive method that involved the assessment of antibiotic resistant E. coli, P. aeruginosa and A. hydrophila in wastewater and the receiving stream over a six-month period. Permission was sought from the manager of the Legon Sewage Treatment Plant prior to data collection. The study involved collection of wastewater from two points in the Legon Sewage Treatment Plant influent i.e. where the plant receives wastewater from the University of Ghana, Achimota Senior High School catchment area and effluent i.e. treated water in the final maturation point that discharges into the Onyasia stream. Three points along the Onyasia stream course were selected relative to the treatment plant discharge point; outfall i.e. the point of wastewater discharge into the stream, upstream i.e. 500 meters before the treatment plant discharge point (outfall) and downstream i.e. 500 meters down the treatment plant discharge point). 32 University of Ghana http://ugspace.ug.edu.gh Figure 3.3: Map showing sampling points EF: Effluent, US: Upstream; OF: Outfall; DS: Downstream E. coli, A. hydrophila and P. aeruginosa are the organisms chosen for this study. These bacteria were selected because they are pathogens that cause diseases in humans. The choice of bacteria is based on the fact that they are pathogens which cause human diseases 33 University of Ghana http://ugspace.ug.edu.gh and their resistance to antibiotics can pose a serious health threat to humans, also WHO listed P. aeruginosa and Enterobactereaceae some of the priority pathogens for antibiotic resistance research (Shrivastava et al., 2018). Surveillance of A. hydrophila in wastewater is also important due to their ubiquity in aquatic environments making it able for other bacteria to transfer resistance to this organism. Culture-dependent methods were used to quantify E. coli, A. hydrophila and P. aeruginosa resistant to selected antibiotics. 3.3 Field data collection Prior to sampling a transect walk was carried out in December 2017 in order to observe activities along the course of Onyasia stream. Sampling was done over a 6-month period between January 2018 and June 2018. Both wastewater influent and effluent were sampled. Wastewater and water sampling was done between 8am to 9 am and immediately transported to the laboratory for further analysis. All the samples were collected in triplicate and pooled together as one composite sample per sampling location. Sampling site, location, and the Global Position System (GPS) readings of the locations are presented in appendix VII. All sampling procedure followed standard protocols in the Standard Methods for Examination of Water and Wastewater (APHA, 2012). Below is a detailed description of how samples were collected. 34 University of Ghana http://ugspace.ug.edu.gh 3.3.1 Wastewater collection from treatment plant Influent and effluent samples from the treatment plant were collected aseptically in 500 millilitres (ml) Whirl Pak bags (eNasco, Fort Atkinson, WI) with a sampling pole. Influent and final effluent wastewater samples were collected from 0.2-0.3meters (m) below the surface. Samples were collected in triplicate from influent and mixed as a composite sample in a sterile bottle. Effluent samples were also collected in triplicate mixed as a composite sample in a sterile bottle. Samples were kept on ice and transported in a cooler box to the CSIR-Water Research Institute Microbiology Laboratory for analyses. All samples were analysed within 24 hours of sample collection. 3.3.2 Surface water collection from Onyasia stream Upstream, outfall and downstream water samples were taken 0.2-0.3m below the surface aseptically with a sampling pole into sterile 500ml bottles. Samples were collected in triplicate from each sampling location and mixed as a composite sample per sampling site in a sterile bottle. Samples were kept on ice transported in a cooler box to the CSIR-Water Research Institute Microbiology Laboratory for analyses. All samples were analysed within 24 hours of sample collection. 3.4 Laboratory analysis Laboratory analyses were conducted following protocols in the Standard Methods for Examination of Water and Wastewater (APHA, 2012) and Clinical Laboratory Standards Institute Guidelines (CLSI, 2017; CLSI, 2018). Bacterial analysis of the wastewater and water samples was determined by membrane filtration method. 35 University of Ghana http://ugspace.ug.edu.gh 3.4.1 Wastewater analysis Ten-fold serial dilutions of wastewater in phosphate-buffered saline solution was carried out. One ml of each dilution as well as 1ml undiluted influent wastewater was then filtered through a 0.45 μm filter membrane (Millipore, Billerica, MA) membrane filters by vacuum filtration. Membrane filters were put on prepared selective 47mm agar plate in sterile petri plates aseptically. Each sample was plated on Aeromonas agar base (OXOID) supplemented with ampicillin for the isolation of A. hydrophila, Cetrimide agar for isolation of P. aeruginosa, Chromo cult coliform agar (MERCK) for E. coli, and total coliforms and M-FC agar for faecal coliforms. Inoculated plates were incubated for 24 hours at 37°C for A. hydrophila , P. aeruginosa, E. coli, total coliforms and 44°C for faecal coliforms. Following incubation, characteristic colonies that grew on the plates were counted and recorded. Colonies were identified as dark green colonies with opaque centers and translucent peripheries for A. hydrophila, green fluorescing colonies for P. aeruginosa and blue colonies for E. coli and counted as colony forming units (CFU) per 100ml of the water sample. Counts were recorded as mean values. Five presumed A. hydrophila, P. aeruginosa, and E. coli colonies for each of the five sampling site sets were randomly selected and streaked by means of a sterile wire loop over nutrient agar for further purification. Isolates were sub cultured thrice and the purity confirmed by microscopy. Pure colonies were transferred onto Tryptic Soy agar (DIFCO) slants for further identification and antibiotic sensitivity testing. 36 University of Ghana http://ugspace.ug.edu.gh Physical and biochemical confirmatory tests such as gram reaction, catalase, indole, citrate, oxidase, triple sugar iron, glucose fermentation and sulphide indole motility were carried out to confirm isolates according to procedures outlined in Cheesbrough (2005). 3.4.2 Surface water analysis Ten-fold serial dilutions of surface water in phosphate-buffered saline solution was carried out. One ml of each dilution and well as 1ml and 10ml undiluted each of the undiluted surface water samples were separately filtered through gridded 0.45 μm (Millipore, Billerica, MA) membrane filters by vacuum filtration. Membrane filtration procedure was used to analyse surface water as described in section 3.4.2. Isolation and confirmatory tests were carried out for surface water as outlined in section 3.4.2. 3.4.4 Physical confirmatory test Gram reaction test was carried out for all isolates. A smear was prepared by placing a loop full of bacteria isolate on a slide applying heat. The slide was flooded with crystal violet for 1 minute. The slide was gently rinsed under an indirect stream of tap water for 2 seconds. Iodine was added to the slide for 1 minute and rinsed. The slide was then flooded with Ethyl alcohol (95%) for 15 seconds to decolorize the smear and flooded with a counter stain, safranin for 30 seconds. The slide was washed and dried with absorbent paper and then observed under a bright field microscope (Nikon eclipse 90i microscope) to determine to determine the morphology of cells. 37 University of Ghana http://ugspace.ug.edu.gh 3.4.5 Biochemical confirmatory tests Biochemical tests were carried out on 24-hour culture plates, including oxidase and catalase for A. hydrophila and P. aeruginosa motility, citrate and glucose fermentation for A. hydrophila, Indole, and Triple Sugar Iron agar tests for E. coli. 3.4.5.1 Catalase test A loopful of bacteria isolate was placed on a dry slide. A drop of hydrogen peroxide solution (3%) was put on the culture. Rapid bubble formation after 5-10 seconds indicated positive for catalase test. 3.4.5.2 Indole test A loopful of bacteria isolate was inoculated into 4 millilitres (ml) of tryptone water and incubated for 24 hours at 37°C. A drop of Kovacs reagent was added after incubation and observed after 20 minutes. A pink to red ring formation indicated a positive indole test. 3.4.5.3 Citrate test A tests tube with 4 ml of Simmons Citrate agar was lightly inoculated by touching the tip of a straight wire loop to a 24 hour bacteria culture and incubated at 37°C for a period of 24 hours. Blue colour formation indicated a citrate positive and green colour citrate negative. 3.4.5.4 Oxidase test Bacteria isolates were smeared on filter papers impregnated with oxidase reagent 1% (w/v) aqueous solution of oxidase reagent (tetramethyl-p phenylenediamine). Oxidase positive isolates were observed by the formation of a purple colour within 5 to 10 seconds. 38 University of Ghana http://ugspace.ug.edu.gh 3.4.5.5 Sulphide indole motility test A test tube with 5ml Sulphide Indole Motility (SIM) Medium was inoculated by stabbing the centre of the medium to a depth of half an inch. The test tube was incubated for 24 hours at 35°C. Hydrogen sulphide (H2S) production and motility was observed. After H2S and motility reaction was observed three drops of Kovac’s reagent was added to the solution in the test tube. Motility is seen as a diffused zone of growth arising from the inoculation line. The formation of a pink to red band ring on the top of the SIM after addition of Kovac’s reagent represented a positive for indole. 3.4.5.6 Glucose fermentation test A loopful of A. hydrophila isolate was inoculated in a 4 ml test tube of Phenol Red Glucose broth with a Durham tube and incubated for 24 hours at 37°C. Color change and acid production was observed. Positive glucose fermentation was seen as a change in the color of broth from purple to yellow. Gas production was observed by the formation of a bubble in the inverted Durham tube. 3.4.5.7 Triple sugar iron test A loopful E. coli isolate was inoculated by stabbing the butt of a 20 mL triple sugar iron test agar slant and then streaking upwards along the surface of the slant. The neck of the test tube was capped, incubated for 24 hours at 37°C and observed colour change; a yellow colouration indicated acid production due to fermentation of glucose, lactose or sucrose and a red colouration indicated alkaline production due to non-fermentation of the sugars; 39 University of Ghana http://ugspace.ug.edu.gh glucose, lactose or sucrose on butt and slant. Hydrogen Sulphide production was observed as black precipitate on the medium. (Cheesbrough, 2005). 3.4.6 Antibiotic sensitivity testing The antibiotic susceptibility test was carried out on five biochemically confirmed isolates taken randomly for each sample using the Kirby Bauer Disc Diffusion method (Bauer et al., 1966) on Mueller-Hinton agar as recommended by Clinical Laboratory Standards Institute guidelines (CLSI 2017; CLSI, 2018). Antibiotics used for antibiotic susceptibility test were selected based on CLSI recommendations (CLSI, 2017; CLSI, 2018). Antibiotics tested represent seven different classes of antibiotics including β lactams (Amoxicillin clavulanate), Monobactams (Aztreonam), Carbapenems (Imipenem), Aminoglycosides (Gentamicin), Tetracyclines (Tetracycline) and Quinolones (Ciprofloxacin), Cephalosporin (Cefuroxime and Ceftazidime). These antibiotics also constitute the major classes of drugs commonly used in the treatment of infections. Mueller Hinton agar was prepared according to manufacturer’s instructions and sterilized at 121°C for 15 minutes at 1 bar pressure in an autoclave. Thirty-one mL of Mueller Hinton Agar (MHA) was aseptically dispensed into 100mm plastic petri dishes and made to dry under a laminar flow hood. Cell suspensions of bacteria were prepared by transferring twenty-four hour colonies of each bacteria isolate into tubes with 5 ml of sterile saline (0.85%) water and turbidity adjusted to correspond to 0.5 McFarland standard. Within 15 minutes of inoculation, the bacterial suspension was then streaked on Mueller Hinton agar plates by means of a sterile cotton swab. Antibiotic discs for Gentamicin, Amoxicillin clavulanate, Tetracycline, Ciprofloxacin, Imipenem, Cefuroxime, Ceftazidime and Aztreonam (OXOID) were placed 40 University of Ghana http://ugspace.ug.edu.gh on inoculated plates with a sterile forcep. Inoculated Mueller Hinton plates were incubated for 16 hours at 35°C. Following incubation, a ruler was used to measure the zones of inhibition diameters. To determine the response i.e. resistant, intermediate resistance or susceptible of each isolate to test antibiotics, measured zone of inhibition were compared with CLSI zone diameter interpretive charts. The percentage for an antibiotic resistance prevalence was calculated as the proportion of the number of strains resistant to the antibiotic over the total number of strains tested, multiplied by 100. 3.4.6.1 Multiple resistance patterns Multiple antibiotic resistance may be regulated by multiple resistance mechanisms in both related an unrelated strain (Hayford, 2016). Isolate resistance to different antibiotic groups were identified. Binomial resistance values, which was determined by the number of responses of each isolate, were tailed and sorted into five groups, which were isolates resistant to 0, 1, 2, 3, or ≥4 antibiotics. 3.5 Quality assurance All samples collected for laboratory analysis were transported to the laboratory on ice on the day of collection and analysed within 4 hours while observing strict aseptic techniques. Negative control was done by plating 0.1ml of sterile distilled water and incubated concurrently with cultural samples. This was done to ensure bacteria loads recovered from samples were not influenced by laboratory conditions. 41 University of Ghana http://ugspace.ug.edu.gh Reference organisms P. aeruginosa ATCC 29213 and E. coli ATCC 25922 were used as controls in accordance with CLSI guidelines to ensure that antibiotic disc diffusion process was consistent. Positive control was done by plating spiked reference P. aeruginosa ATCC 27853 and E. coli 25922 strains in sterile distilled water to test the efficacy of bacteriological media. Antibiotics were stored at -20°C for long term storage and tested for potency using reference P. aeruginosa ATCC 27853 and E. coli 25922 strains. All glassware used for bacteriological analyses were sterilized in a hot-air oven for 2hours at 170°C. All bacteriological media, Phosphate Buffered Saline, saline and pipette tips were sterilized in an autoclave at a temperature of 121°C and pressure of 1 bar for 15 min. All wire loops and forceps were flamed with a Bunsen burner before and after usage. The neck of media bottles was also flamed before and after dispensing. All laboratory analyses were conducted in a laminar flow hood to ensure aseptic environment. Seventy percent ethanol and ten percent bleach used to disinfect work areas before and after use. Gloves were worn for all laboratory procedures. 3.6 Data analysis Data processing was carried out with Statistical Package for Social Science (SPSS. Chicago, Illinois, USA) Version 23 and Microsoft Excel and analysed using Kruskal-Wallis One-way Analysis of Variance (ANOVA). The differences in bacterial levels across sampling sites as well as differences across months were determined using the Kruskal-Wallis one-way ANOVA with significance level set at P < 0.05. 42 University of Ghana http://ugspace.ug.edu.gh Bacterial isolate responses to antibiotic susceptibility disc diffusion were categorized as either susceptible or resistant i.e. resistant and intermediate resistance and assigned a binary value for each response: 0 for susceptible and 1 for resistant. Isolates and isolate responses were then grouped and significant associations by Chi-square analysis at significance level, p˂0.05 with 95% confidence interval was determined. Percentage resistance to antibiotics was calculated and results presented in tabular and graphical forms. 43 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1 Occurrence of total coliforms, E. coli, A. hydrophila, P. aeruginosa in treatment plant All E. coli isolates were confirmed as Gram-negative rods after performing gram reaction test. All isolates tested were positive for indole and triple sugar iron test. All Aeromonas hydrophila isolates were confirmed as Gram-negative rods after performing gram reaction test. All isolates tested were positive for glucose fermentation, motility and citrate tests. Isolates were negative for oxidase test. All Pseudomonas aeruginosa isolates were seen as pigmented greenish colonies with fluorescence when cultured on Cetrimide agar. Pseudomonas aeruginosa strains were identified as Gram-negative rods. All Pseudomonas aeruginosa isolates were oxidase positive. E. coli, A. hydrophila and P. aeruginosa were present in all wastewater and stream water samples. Table 4.1 presents level of E. coli, A. hydrophila, P. aeruginosa in the treatment plant. Mean count of E. coli in the treatment plant over the study period was 7.88 log 10 CFU/100ml in influent and 2.58 log 10 CFU/100ml in final effluent wastewater. Mean count of A. hydrophila was 5.57 log 10 CFU /ml in influent and 3.90 log 10 CFU /100ml in effluent. Mean count for P. aeruginosa was 6.44 log 10 CFU/100ml and 3.17 log10 CFU/100ml for influent and effluent wastewater respectively. Results show the mean abundance of bacteria decreased after treatment (effluent). Figures 4.1, 4.2 and 4.3 show bar chats of monthly E. coli, A. hydrophila and P. aeruginosa counts in the treatment plant. 44 University of Ghana http://ugspace.ug.edu.gh Kruskal-Wallis statistical test showed significant differences between occurrence of bacteria isolated from influent and effluent wastewater during the study period. Table 4.1 Levels of P. aeruginosa, E. coli, and A. hydrophila in influent and effluent wastewater Mean Log 10 CFU/100ml of bacteria Sample E.coli Standard P. aeruginosa Standard A. Standard site error error hydrophilla error Influent 7.88 0.17 6.44 0.24 5.57 0.03 Effluent 2.58 0.23 3.17 0.06 3.90 0.12 Table 4.2 represents the mean values of total coliform levels in both treated and untreated wastewater effluent over the period of study. The highest levels of total coliforms in influent and effluent samples was recorded in March. There was a significant decrease in mean total and faecal coliform counts in effluent after treatment (8.32 Log10 CFU/100ml and 7.72 Log10 CFU/100ml mean total coliform and faecal coliform for influent and 4.64 Log10 CFU/100ml and 3.22 Log10 CFU/100ml mean total and faecal coliform for effluent respectively). Figures 4.4 and 4.5 show bar chats of monthly levels. 45 University of Ghana http://ugspace.ug.edu.gh Table 4.2 Mean levels of total and faecal coliforms in influent and effluent wastewater. Mean Log10 CFU/100ml of bacteria Sample site Total Coliform Standard error Faecal Coliform Standard error Influent 8.32 0.09 7.92 0.20 Effluent 4.64 0.16 3.22 0.08 4.2 Occurence of E. coli, A. hydrophila, P. aeruginosa in Onyasia Stream E. coli was present in both influent and effluent wastewater samples. Mean counts of E. coli in the Onyasia Stream were 2.83 log 10 CFU/100ml, 3.58 log 10 CFU/100ml and 6.02 log 10 CFU/100ml in upstream, outfall and downstream water respectively (Table 4.3). Mean counts of A. hydrophila in upstream, outfall and downstream were 4.34 log 10 CFU/100ml, 4.68 log 10 CFU/100ml and 5.96 log 10 CFU/ml respectively (Table 4.3). Counts for P. aeruginosa in upstream, outfall and downstream water samples was 3.34 log10 CFU/100ml, 3.68 log10 CFU/100ml and 5.49 log 10 CFU/100ml respectively (Table 4.3). Results show the mean abundance of bacteria tended to increase downstream. 46 University of Ghana http://ugspace.ug.edu.gh Kruskal-Wallis one-way ANOVA statistical test showed significant differences between mean E. coli, A. hydrophila and P. aeruginosa counts in upstream and downstream sampling sites. Table 4.3 Levels of P. aeruginosa, E. coli, and A. hydrophila in the Onyasia stream Mean Log10 CFU/100ml of bacteria Sample site E.coli Standard P. aeruginosa Standard A. Standard error error hydrophilla error Outfall 3.58 0.25 3.68 0.18 4.68 0.09 Upstream 2.83 0.17 3.34 0.07 4.34 0.11 Downstream 6.02 0.34 5.49 0.26 5.96 0.23 4.3 Monthly variations in bacteria within sample sites 4.3.1 Treatment plant Figure 4.1 shows the monthly variations of E. coli in the treatment plant. The highest monthly level of E. coli in influent was 8.41 log 10 CFU/100ml recorded in March. May recorded the lowest monthly level of E. coli of 7.13 log 10 CFU/100ml. Effluent recorded the highest E. coli of 3.30 log 10 CFU/100ml in February and the lowest (2.0 log 10 CFU/100ml) in June. 47 University of Ghana http://ugspace.ug.edu.gh 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Influent Effluent Fig 4.1: A line chart showing levels of E. coli in treatment plant Figure 4.2 shows the monthly variations of P. aeruginosa in the treatment plant. The highest monthly level of P. aeruginosa in influent was 7.34 log 10 CFU/100ml, which was recorded in January. April recorded the lowest monthly level of P. aeruginosa of 5.48 log 10 CFU/100ml. Effluent recorded the highest P. aeruginosa of 3.30 log 10 CFU/100ml in 48 E coli levels (Log10 CFU/100mL) University of Ghana http://ugspace.ug.edu.gh February, March and April. The lowest P. aeruginosa count in effluent of 3.00 log 10 CFU/100ml recorded in January. 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Influent Effluent Fig 4.2: A line chart showing levels of P. aeruginosa in treatment plant Figure 4.3 depicts the monthly variations of A. hydrophila in the treatment plant. The highest monthly level of A. hydrophila in influent was 5.64 log 10 CFU/100ml, which was 49 P. aeruginosa levels (Log10 CFU/100mL) University of Ghana http://ugspace.ug.edu.gh recorded in April. April recorded the lowest monthly level of A. hydrophila of 5.43 log 10 CFU/100ml in influent. Effluent recorded the highest A. hydrophila of 4.24 log 10 CFU/100ml in February. The lowest effluent A. hydrophila count of 3.60 log 10 CFU/100ml was recorded in May and June. 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Influent Effluent Fig 4.3: A line chart showing levels A. hydrophila in treatment plant. 50 A. hydrophila levels Log10CFU/100mL)) University of Ghana http://ugspace.ug.edu.gh Figure 4.4 and 4.5 depict the monthly levels of total and faecal coliforms in the treatment plant. The highest monthly level of total coliform in influent was recorded in March (8.72log10 CFU/100ml) and the lowest recorded in June (8.11log10 CFU/100ml). The highest monthly level of total coliform counts in effluent was recorded in March at 5.57log10 CFU/100ml and the lowest recorded in June i.e. 4.23log10 CFU/100ml. March recorded the highest faecal coliform in influent at 8.45log10 CFU/100ml and the lowest in May at 7.20log10 CFU/100ml. The highest faecal coliform count was recorded in effluent was recorded in February (3.57log10 CFU/100ml) and the lowest recorded in May (3.00log10 CFU/100ml /100ml). 10.00 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Influent Effluent Fig 4.4: A line chart showing levels of total coliforms in treatment plant 51 Total coliform levels (Log10CFU/100ml) University of Ghana http://ugspace.ug.edu.gh 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Influent Effluent Fig 4.5: A line chart showing levels of faecal coliforms in treatment plant Kruskal-Wallis statistical test showed that the distribution of total coliforms, faecal coliforms, E. coli, P. aeruginosa and A. hydrophila over months in both influent and effluent was not significantly different at p>0.05. 52 Faecal coliform levels (Log10CFU/100ml) University of Ghana http://ugspace.ug.edu.gh 4.3.2 Onyasia Stream Figure 4.6 shows the monthly variations of E. coli in the Onyasia Stream. The highest monthly level of E. coli was 6.9 log 10 CFU/100ml, which was recorded downstream in March. The lowest monthly level of E. coli was 2.30 log 10 CFU/100ml, which was recorded upstream in June. 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Upstream Outfall Downstream Fig 4.6: A line chart showing levels E. coli in Onyasia Stream 53 E. Coli levels (Log10CFU/100mL) University of Ghana http://ugspace.ug.edu.gh Figure 4.7 depicts the monthly variations of A. hydrophila in the Onyasia stream water samples. The highest monthly level of A. hydrophila was 6.97 log 10 CFU/100ml, which was recorded downstream in February. The lowest monthly level of A. hydrophila was 3.88 log 10 CFU/100ml, which was recorded upstream in June. 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Upstream Outfall Downstream Fig 4.7: A line chart showing levels A. hydrophila in Onyasia Stream Figure 4.8 depicts the monthly variations of P. aeruginosa in the Onyasia stream water samples. The highest monthly level of P. aeruginosa was 6.20 log 10 CFU/100ml, which was 54 A. hydrophila levels (Log10CFU/100mL) University of Ghana http://ugspace.ug.edu.gh recorded downstream in January. The lowest monthly level of P. aeruginosa was 3.11 log 10 CFU/100ml, which was recorded upstream in June. 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 January February March April May June Months Upstream Outfall Downstream Fig 4.8: A line chart showing levels of P. aeruginosa in Onyasia Stream Kruskal-Wallis statistical test showed that distribution of E. coli, P. aeruginosa and A. hydrophila levels across months was not significantly different at p>0.05. 4.4 Field observations Prior to water quality assessment for antibiotic resistance, a field observation exercise was conducted along the environs of the Onyasia stream to observe and human activities that may influence the occurrence of anti-biotic resistance. The survey revealed vegetable farms 55 P. aeruginosa levels (Log10 CFU/100mL) University of Ghana http://ugspace.ug.edu.gh along the Onyasia stream course beyond the downstream sampling point which abstract water directly from the stream for irrigation. Plates 1 to 3 below are pictures of some of these observations Plate 1: A photograph showing lettuce farm located at downstream sampling site 56 University of Ghana http://ugspace.ug.edu.gh Plate 2: A photograph showing vegetable farm abstracting water for irrigation (note arrow showing water hose for abstraction) 57 University of Ghana http://ugspace.ug.edu.gh Plate 3: A photograph showing a farmer abstracting water for irrigation of vegetables It was also observed that there were sewage pipes connected from households discharge wastewater into the stream. This shows other possible sources of antibiotic resistance in the Onyasia stream. Plates 4 and 5 below are pictures of some of these observations. 58 University of Ghana http://ugspace.ug.edu.gh Plate 4: A photograph showing wastewater connections from a household into the Onyasia stream (note arrow showing PVC waste pipe) 59 University of Ghana http://ugspace.ug.edu.gh Plate 5: A photograph showing wastewater connections into the Onyasia stream (note arrow showing PVC waste pipe) 4.5 Determination of antibiotic resistant E. coli isolates 4.5.1 Occurrence of all antibiotic resistant E. coli isolates The number of all E. coli isolates expressing resistance to individual antimicrobial agents is indicated in Table 4.4. 60 University of Ghana http://ugspace.ug.edu.gh Sixteen percent of all isolates were found to be susceptible to all 7 antibiotics. All isolates showed low resistance prevalence to Imipenem (9%), Gentamicin (17%), Aztreonam (26%) and Ciprofloxacin (29%). Isolates showed high resistance prevalence to Amoxicillin clavulanate (57%), Cefuroxime (52%) and Tetracycline (49%). Table 4.4 Percentage number of all E. coli resistant to antibiotics tested Antibiotics Percentage(%) resistance and susceptibility of isolates Resistant Susceptible GN-10 17 83 AMC-20 57 43 TE-30 49 51 CIP-5 29 71 IMP-10 9 91 CXM-30 54 44 ATM-30 26 74 CAZ-30 28 72 TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 4.5.2 Occurrence of antibiotic resistant E. coli isolates across sampling sites Figures 4.9 and 4.10 show percentage occurrence of antibiotic resistance in effluent, outfall and upstream and downstream sampling sites for E. coli isolates. Resistance prevalence in effluent isolates was high for Amoxicillin clavulanate (50%), Cefuroxime (47%) and Tetracycline (37%). On the contrary, resistance in effluent isolates was low for ciprofloxacin (10%), Imipenem (17%), Aztreonam (20%) and Gentamicin (27%). 61 University of Ghana http://ugspace.ug.edu.gh Resistance rates in outfall isolates were high for Amoxicillin clavulanate (60%), Cefuroxime (50%) and Tetracycline (40%) but low for Ciprofloxacin (30%), Aztreonam (27%), Gentamicin (20%) and Imipenem (13%). Downstream had the highest resistance rates in all stream isolates with Amoxicillin clavulanate (83%), Cefuroxime (73%), Tetracycline (56%) and being the highest. Downstream isolates however recorded lower resistance rates for Aztreonam (40%), Ciprofloxacin (30%), Gentamicin (30%) and Imipenem (13%). Upstream isolates showed lower resistance rates compared to outfall and downstream isolates. Resistance prevalence rates for upstream isolates were 43%, 40%, 33%, 13%, and 10% for Amoxicillin clavulanate, Cefuroxime, Tetracycline, Gentamicin, Aztreonam and Ciprofloxacin respectively. All upstream isolates were susceptible to Imipenem. In general, isolate resistance to antibiotics tested was higher downstream compared to upstream sampling sites. Chi-square tests for isolate resistance by individual sampling site showed significant differences (p< 0.05) between upstream and downstream sampling sites for all seven antibiotics. Chi-square tests for isolate resistance by individual sampling site showed significant differences (p>0.05) between effluent and downstream sampling sites for all seven antibiotics. Figure 4.7 and 4.8 show prevalence of resistant E. coli in the treatment plant and stream. 62 University of Ghana http://ugspace.ug.edu.gh 90 80 70 60 50 40 30 20 10 0 GN-10 AMC-20 TE-30 CIP-5 IMP-10 CXM-30 ATM-30 Antibiotics Influent Effluent Figure 4.9: A bar chart showing percentage occurrence of resistant E. coli isolates in treatment plant TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 80 70 60 50 40 30 20 10 0 GN-10 AMC-20 TE-30 CIP-5 IMP-10 CXM-30 ATM-30 Antibiotic UPSTREAM OUTFALL DOWNSTREAM Figure 4.10: A bar chart showing percentage occurrence of resistant E. coli isolates in the Onyasia Stream TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 63 Percentage resistance of E. coli isolates % Number of resistant E.coli isolates University of Ghana http://ugspace.ug.edu.gh 4.5.3 Multi-drug resistance in E. coli isolates Out of the 150 confirmed isolates, 83% showed resistance to a minimum of 1 out of 7 tested antibiotic agent. Seventy percent of all isolates showed resistance to not less than 2 antibiotic agents. Thirty-two isolates (21 %) were resistant to 2 agents, 24 (16%) of isolates tested were resistant to 3 agents and 48(32%) of isolates tested were resistant to 4 or more agents. Out of 105 multi-drug resistant isolates collected (resistant to a minimum of 2 antibiotics), 22 isolates were obtained from effluent,18 isolates from upstream, 29 isolates from outfall and 30 from downstream. Table 4.5 and 4.6 show multi-drug resistance of E. coli isolates in the treatment plant and stream. Table 4.5: Multi-drug resistance of E. coli isolates in the treatment plant. Number of isolates with Resistance to n antibiotics: n= (%) n= (%) n= (%) n= (%) n≥ (%) 0 1 2 3 4 Sample site Influent 4 13 3 10 5 17 5 17 13 43 Effluent 8 27 6 20 4 13 4 13 8 27 n=0: number of isolates not resistant to all antibiotics tested; n=1: number of isolates resistant to 1 antibiotic; n=2; number of isolates resistant to 2 antibiotics, n=3: number of isolates resistant to 3 antibiotics; n=4: number of isolates resistant to 4 or more antibiotics 64 University of Ghana http://ugspace.ug.edu.gh Table 4.6: Multi-drug resistance of E. coli isolates in the O nya sia stream Sample site Number of isolates with Resistance to n antibiotics: n= (%) n= (%) n= (%) n= (%) n≥ (%) 0 1 2 3 4 Outfall 1 3.33 7 23.33 7 23.33 1 3.33 14 46.67 Upstream 12 40 1 3 8 27 8 27 1 3 Downstrea 0 0 3 10 8 27 7 23 12 40 m n=0: number of isolates not resistance to any of the antibiotics tested; n=1: number of isolates resistant to 1 antibiotic; n=2; number of isolates resistance to 2 antibiotics, n=3: number of isolates resistance to 3 antibiotics; n=4: number of isolates resistance to 4 or more antibiotics 4.6 Determination of antibiotic resistant A. hydrophila isolates 4.6.1 Occurrence of all antibiotic resistant A. hydrophila isolates The number of all A. hydrophila isolates expressing resistance to individual antimicrobial agents are indicated in Table 4.7. Fifteen percent of all isolates were susceptible to all 7 antibiotics. Sixty-eight percent of all isolates expressed resistance to Amoxicillin clavulanate out of the isolates collected followed by Cefuroxime (43%) and Tetracycline (31%) of all isolates. Lower resistance rates to Aztreonam, Ciprofloxacin and Imipenem in all isolates were at 27%, 22% and 17% 65 University of Ghana http://ugspace.ug.edu.gh respectively. Resistance to Gentamicin was found in fewer isolates, at rates of 9% for all isolates. Table 4.7 Percentage number of all A. hydrophila resistant to antibiotics tested Antibiotic Percentage(%) resistance and susceptibility of isolates Resistant Susceptible GN-10 9 91 AMC-20 68 32 TE-30 31 69 CIP-5 22 78 IMP-10 22 78 CXM-30 43 57 ATM-30 27 73 CAZ-30 20 80 TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 4.6.2 Occurrence of antibiotic of A. hydrophila isolates across sampling sites Figures 4.11 and 4.12 show percentage occurrence of antibiotic resistance in influent, effluent, outfall and upstream and downstream sampling sites in A. hydrophila isolates. Amoxicillin, Cefuroxime and Tetracycline resistance prevalence was high in effluent, outfall and downstream sampling sites. Upstream isolates expressed much lower resistance rates compared to other sampling sites. 66 University of Ghana http://ugspace.ug.edu.gh Resistance prevalence in effluent isolates was high for Amoxicillin clavulanate (97%) and Cefuroxime (50%), Aztreonam (37%) and Imipenem (33%). Resistance in effluent isolates was low for Tetracycline (23%), Gentamicin (17%), and Ciprofloxacin (13%). Resistance rates in outfall isolates were high for Amoxicillin clavulanate (77%) and Cefuroxime (37%). Resistance rates were low for Aztreonam (17%), Imipenem (16%), Ciprofloxacin (13%) and Gentamicin (7%) and in outfall isolates. Downstream had the highest resistance rates in all stream isolates with Amoxicillin clavulanate (80%), Cefuroxime (47%) Aztreonam (40%) and Tetracycline (33%). Downstream isolates however recorded lower resistance rates for Imipenem (27%), Ciprofloxacin (17%), and Gentamicin (10%). Upstream isolates showed lower resistance rates compared to outfall and downstream isolates, with resistance rates of 27%, 10% and 7% for Amoxicillin clavulanate, Tetracycline and Cefuroxime respectively. Upstream isolates were all susceptible to Imipenem, Gentamicin, Ciprofloxacin, and Aztreonam. Isolate resistance to antibiotics tested was higher in downstream isolates compared to upstream isolates. Chi-Square tests for isolate resistance by individual sampling site showed significant differences (p< 0.05) between upstream and downstream sampling sites for all antibiotics tested except Gentamicin. 67 University of Ghana http://ugspace.ug.edu.gh 120 100 80 60 40 20 0 GN-10 AMC-20 TE-30 CIP-5 IMP-10 CXM-30 ATM-30 Antibiotic Influent Effluent Figure 4.11: A bar chart showing percentage resistance of A. hydrophila isolates in treatment plant TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 90 80 70 60 50 40 30 20 10 0 GN-10 AMC-20 TE-30 CIP-5 IMP-10 CXM-30 ATM-30 Antibiotic UPSTREAM OUTFALL DOWNSTREAM Figure 4.12: A bar chart showing percentage resistant A. hydrophila isolates in the Onyasia Stream TET-30: Tetracycline 30 μg; AMC-20: Amoxicillin Clavulanate 20 μg; CIP-5: Ciprofloxacin 5μg; ATM-30: Aztreonam 30 μg; CXM-30: Cefuroxime- 30 μg: IMP-10: Imipenem 10μg; GN-10: Gentamicin 68 Percentage resistance of A. Hydrophila % Number of A. hydrophila isolates isolates University of Ghana http://ugspace.ug.edu.gh 4.6.3 Multi-drug Resistance in A. hydrophila isolates Out of the 150 isolates, 84% showed resistance to at least one of 7 tested antibiotic agent. Sixty-one (98 of total) of isolates showed resistance to not less than 2 antibiotic agents. Twenty-five (26%) isolates were resistant to 2 agents, 25 were resistant to 3 agents and 42 were resistant to 4 or more agents. Out of 98 multi-drug resistant isolates collected .i.e. those resistant to a minimum of 2 antibiotics, 27 isolates were obtained from influent wastewater, 27 isolates from effluent,1 isolate from upstream, 15 isolates from outfall and 22 from downstream. Table 4.8 and 4.9 shows tables of multiple resistant isolates per sampling site. Table 4.8: Multi-drug resistance of A. hydrophila isolates by sampling site in the treatment plant Sample site Number of isolates with Resistance to n antibiotics: n=0 (%) n=1 (%) n=2 (%) n=3 (%) n≥4 (%) Influent 1 3 2 7 3 10 1 3 23 77 wastewater Final 0 0 3 10 10 33 10 33 7 23 effluent n=0: number of isolates not resistant to all antibiotics tested; n=1: number of isolates resistant to 1 antibiotic; n=2; number of isolates resistant to 2 antibiotics, n=3: number of isolates resistant to 3 antibiotics; n=4: number of isolates resistant to 4 or more antibiotics 69 University of Ghana http://ugspace.ug.edu.gh Table 4.9: Multi-drug resistance of A. hydrophila isolates by sampling site in the Onyasia stream Sample site Number of isolates with Resistance to n antibiotics: n=0 (%) n=1 (%) n=2 (%) n=3 (%) n≥4 (%) Outfall 2 7 13 43 5 17 6 20 4 13 Upstream 19 63.33 10 33.33 1 3.33 0 0 0 0 Downstream 1 3 7 23 6 20 8 27 8 27 n=0: number of isolates not resistant to all antibiotics tested; n=1: number of isolates resistance to only one antibiotic; n=2; number of isolates resistance to two antibiotics, n=3: number of isolates resistance to three antibiotics; n=4: number of isolates resistance to four or more antibiotics 4.7 Determination of antibiotic resistant P. aeruginosa isolates 4.7.1 Occurrence of antibiotic resistant of all P. aeruginosa isolates The number of all P. aeruginosa isolates expressing resistance to individual antimicrobial agents are indicated in Table 4.7. Forty-four percent of all isolates were susceptible to all 5 antipseudomonal agents. Resistance rates were generally low for antipseudomonal agents. All Isolates expressed resistance to Aztreonam (37%), Ciprofloxacin (33%) and Gentamicin (22%). Resistance to Ceftazidime and Imipenem was lower compared to other antipseudomonal agents with 70 University of Ghana http://ugspace.ug.edu.gh resistance rates of 11% and 5% respectively. Figure 4.13 shows resistant profiles of all P. aeruginosa isolated 120 100 80 60 40 20 0 GN-10 CIP-5 IMP-10 ATM-30 CAZ-30 Antibiotics Resistant Susceptible Figure 4.13: A bar chart showing percentage occurrence of resistance of all isolated P. aeruginosa. ATM; Aztreonam 30 μg; CAZ: Ceftazidime 10 μg; CIP: Ciprofloxacin 5μg; GN: Gentamicin 10 μg; IPM: Imipenem 10 μg 4.7.2 Occurrence of antibiotic resistance P. aeruginosa isolates across sampling sites Figures 4.14 and 4.15 show isolate resistance responses by sampling site and antibiotics. High resistance is seen in final effluent wastewater, outfall and downstream isolates. Upstream isolates showed low resistance rate to antipseudomonal agents. Upstream 71 Percentage resistance of P. aeruginosa isolates University of Ghana http://ugspace.ug.edu.gh isolates were all susceptible to Imipenem and resistance to Ceftazidime was 3%. Resistance to Ciprofloxacin was the highest with resistance rates of 40%, 20%, and 13% at downstream, outfall and final effluent sampling sites respectively. After Ciprofloxacin, Aztreonam had the next highest resistance at 33%, 17% and 13% at downstream, outfall and final effluent sampling site respectively. In general, isolate resistance to antibiotics tested was higher downstream than upstream. Chi-square tests for isolate resistance by individual sampling site showed significant differences (p< 0.05) between resistance to Ciprofloxacin, Aztreonam and Gentamicin at all sampling sites for upstream and downstream isolates. 72 University of Ghana http://ugspace.ug.edu.gh 35 30 25 20 15 10 5 0 GN-10 CIP-5 IMP-10 ATM-30 CAZ-30 Antibiotic Influent Effluent Figure 4.14: A bar chart showing percentage resistance of P. aeruginosa isolates in treatment plant ATM; Aztreonam 30 μg; CAZ: Ceftazidime 10 μg; CIP: Ciprofloxacin 5μg; GN: Gentamicin 10 μg; IPM: Imipenem 10 μg 73 % Number of P. aeruginosa isolates University of Ghana http://ugspace.ug.edu.gh 60 50 40 30 20 10 0 GN-10 CIP-5 IMP-10 ATM-30 CAZ-30 Antibiotic UPSTREAM OUTFALL DOWNSTREAM Figure 4.15: A bar chart showing percentage occurrence of resistant P. aeruginosa isolates in the Onyasia Stream ATM; Aztreonam 30 μg; CAZ: Ceftazidime 10 μg; CIP: Ciprofloxacin 5μg; GN: Gentamicin 10 μg; IPM: Imipenem 10 μg 4.7.3 Multi-drug resistance in P. aeruginosa isolates Thirty-six percent (122 of total) of all 150 P. aeruginosa isolates showed resistance to at least 1 antibiotic agent. Twenty-one percent of all isolates showed resistance to not less than 2 antibiotic agents. Nineteen (13% of total) of all isolates were resistant to 2 agents, 9 (6% of total) were resistant to 3 agents and 4 (3% of total) were resistant to 4 or more 74 Percentage resistance of P. aeruginosa isolates University of Ghana http://ugspace.ug.edu.gh agents. Out of 32 multi-drug resistant isolates collected (resistant to at least 2 antibiotics), 6 isolates were obtained from effluent, 9 isolates from outfall and 12 from downstream sampling sites. In general, multiple resistance to antibiotics was highest in downstream sites, followed by outfall and effluent. Multiple resistant P. aeruginosa isolates were not recorded in the upstream sampling site. Table 4.10 and 4.11 records tables of multiple resistant isolates per sampling site. Table 4.10: Multi-drug resistance of P. aeruginosa isolates by sampling site. Sample Number of isolates with Resistance to n antibiotics: site n=0 (%) n=1 (%) n=2 (%) n=3 (%) n≥4 (%) Influent 1 3 2 7 3 10 4 13 20 67 Final 0 0 4 13 11 37 7 23 8 27 effluent n=0: number of isolates not resistant to all antibiotics tested; n=1: number of isolates resistant to 1 antibiotic; n=2; number of isolates resistant to 2 antibiotics, n=3: number of isolates resistant to 3 antibiotics; n=4: number of isolates resistant to 4 or more antibiotics 75 University of Ghana http://ugspace.ug.edu.gh Table 4.11: Multi-drug resistance of P. aeruginosa isolates by sampling site. Sample site Number of isolates with Resistance to n antibiotics: n=0 (%) n=1 (%) n=2 (%) n=3 (%) n≥4 (%) Outfall 2 7 12 40 8 27 4 13 4 13 Upstream 19 63 10 33 1 0 0 0 0 0 Downstrea 1 3 8 27 6 20 9 30 6 20 m n=0: number of isolates not resistant to all antibiotics tested; n=1: number of isolates resistance to only one antibiotic; n=2; number of isolates resistance to 2 antibiotics, n=3: number of isolates resistance to 3 antibiotics; n=4: number of isolates resistance to 4 or more antibiotics This study sought to investigate the presence of antibiotic resistant P. aeruginosa, E. coli and A. hydrophila in the Legon Sewage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods and the environmental health implications of the treatment plant discharge. In general, P. aeruginosa, E. coli and A. hydrophila was recorded in all sampling sites. Although the Legon Sewage Treatment Plant significantly reduced the levels of bacteria in influent wastewater, levels of bacteria in effluent wastewater was very high. Downstream 76 University of Ghana http://ugspace.ug.edu.gh bacterial levels was significantly high than upstream. Resistance to Amoxicillin Clavulanate, Cefuroxime and Tetracycline was the highest. Downstream isolates displayed the highest resistance and well as multiple resistance. 77 University of Ghana http://ugspace.ug.edu.gh DISCUSSION 5.0 Introduction Aquatic environments are considered suitable reservoirs for antibiotic resistance distribution, because of constant discharge of antibiotics and antibiotic resistant bacteria into the environment (Ramírez Castillo et al., 2013). Aquatic systems may therefore be contaminated by the release of sewage and serve as wastewater point sources and sources of antibiotics and resistant microflora. Most water bodies that receive these contaminants are often used in irrigating crops with and without treatment and this may pose potential health risks to humans and animals through the food chain (Zhang et al., 2009; Mazari- Hiriart et al., 2008). As such monitoring pathogens and their resistance patterns present in effluents and the receiving surface waters is important. Wastewater treatment plants are recognized as major hotspots and risk environments for the development and dissemination of antibiotic resistance in aquatic environments (Kummerer, 2009; Rizzo et al., 2013). Studies conducted in various countries have detected the presence of antibiotics in hospital effluents, municipal effluents and sewage treatment stations (Rizzo et al., 2013). These antibiotics in effluent contribute to the selection of antibiotic bacteria in the environment. In addition, antibiotic resistant bacteria in effluent may further transfer resistance to other non-infectious and infectious bacteria through mobile genetic elements like plasmid and transposons. In the present study, antibiotic resistance levels present in final effluents from the Legon Sewage Treatment Plant as well in the receiving Onyasia stream were assessed. 78 University of Ghana http://ugspace.ug.edu.gh 5.1 Occurrence of E. coli, A. hydrophila and P. aeruginosa E. coli, A. hydrophila and P. aeruginosa were recorded in all sampling sites. Abundance of E. coli, A. hydrophila and P. aeruginosa loads in effluent was lower compared to influent water; as such, there was improved wastewater quality after treatment. There was up to a fivefold reduction of mean bacterial loads. However, the levels exceed the Ghana Environmental Protection Agency (EPA) permissible limits of E.coli (10CFU/100ml and total coliform (400CFU/100ml) for wastewater discharge. This could be as a result of the high influx of bacteria in influent from the catchment population as such although there was reduction appreciable reduction in bacterial counts, levels remained high. Mean total and faecal coliform counts were much lower compared to previous studies carried out by Kwabla (2017) in the Legon Sewage Treatment Plant. Results of this study is consistent with a study conducted by Abuenyi (2010) which recorded E. coli values in final effluents from a treatment plant above Environmental Protection Agency’s permissible levels. Although wastewater treatment plants processes typically reduce the number of heterotrophic bacteria, high numbers of bacteria may still be discharged into surface water (Kwak et al., 2014). Comparatively E. coli counts in final effluent were much lower than P. aeruginosa and A. hydrophila. This may be due to the fact that E. coli has a shorter die-off period compared to P. aeruginosa and A. hydrophila. P. aeruginosa are able to colonize biofilms making their survival rate higher in water (Mena and Gerba, 2009). 79 University of Ghana http://ugspace.ug.edu.gh Mean A. hydrophila counts in influent wastewater and final effluent were much higher in this study compared to a study conducted by Hassani et al. (1992) which had mean counts of 5.63 × 104 and 1.04 × 103 for influent wastewater and final effluent respectively. Although the wastewater treatment plant tested was in the expected range for their ability to reduce the loads of bacteria in influent wastewater, E. coli, P. aeruginosa and A. hydrophila counts found close to the discharge point in the Onyasia Stream were high. E. coli, P. aeruginosa and A. hydrophila counts increased significantly from upstream to downstream sites in the Onyasia stream. Similarly, Lorenzo et al. (2018) reported an increase in E. coli from upstream to downstream in the Zenne River, Belgium. Downstream bacterial counts were higher than in final effluents samples. This suggests that the treatment plant is not the only source of E. coli, A. hydrophila and P. aeruginosa in the stream. This is consistent with field observations where wastewater was seen to be discharged into the Onyasia stream at downstream sampling points. 5.2 Antibiotic resistance in E. coli isolates Susceptibility of all E. coli isolates to antibiotics classes including β- lactam/β-lactamase inhibitors (Amoxicillin clavulanate), Cephalosporin (Cefuroxime and Ceftazidime), Monobactam (Aztreonam), Carbapenem (Imipenem), Aminoglycosides (Gentamicin), Tetracycline (Tetracycline) and Quinolones (Ciprofloxacin) were determined. Isolates displayed resistance to between one and seven antibiotic agents. Imipenem, Gentamicin, Aztreonam and Ciprofloxacin residence rates were low. High activity of 80 University of Ghana http://ugspace.ug.edu.gh Imipenem may be due to the fact that there is infrequent use clinically and in the community compared to other antibiotics tested. Imipenem is a group 2 Carbapenem used as a last resort antibiotic against particularly resilient Gram-negative pathogens. As such, Imipenem is not frequently prescribed which implies resistance to the agent would not be expected to be high. Aztreonam is also not frequently prescribed and the mode of prescription for Gentamicin is through injections, which is not preferred as such usage may be low in the population. Accordingly, a large proportion of the isolates collected from all sampling sites showed low resistance to Imipenem, Gentamicin, Aztreonam, and Ciprofloxacin. High resistance to Amoxicillin clavulanate, Cefuroxime and Tetracycline was observed in E. coli isolates from effluent and downstream isolates. Resistance to Amoxicillin clavulanate (50%), Cefuroxime (47%) and Tetracycline (37%) in effluent isolates were lower compared to downstream isolates. Resistance to tetracycline was lower compared to a study conducted in South Africa by Olayinka and Okoh (2017), which recorded higher Tetracycline resistance (74.1%) in final effluents from two waste treatment plants. Downstream isolates recorded the high resistance to Amoxicillin clavulanate (83%), Cefuroxime (73%) and Tetracycline (56%). A study conducted by Sullivan & Karthikeyan (2013), found a substantial occurrence of bacteria resistant to tetracycline and tetracycline resistant genes in downstream sediment and surface water samples from Carter’s Creek watershed, a watershed influenced by waste discharge and other anthropogenic activities. The study observed that the occurrence of Tetracycline resistance genes increased downstream of WWTPs. 81 University of Ghana http://ugspace.ug.edu.gh Resistance was higher in downstream compared to final effluent samples, which may suggest that there may be other sources of antibiotic resistant bacteria other than the Legon Sewage Treatment Plant in the Onyasia stream. Similarly, high levels of antibiotic resistant coliforms were reported downstream a wastewater treatment plant discharge in surface water (Goni-Urriza et al., 2000; Zhang et al., 2009; Akiyama and Savin, 2010; Guyomard- Rabenirina et al., 2017; Akiyama and Savin, 2010). In this study, a significant association (p < 0.05) was found to exist between the location of sampling sites relative to the waste treatment plant .i.e. upstream and downstream, and isolates expressing resistance to Amoxicillin clavulanate, Cefuroxime and Tetracycline. This result is similar to a study conducted by Laird (2016) which found an increase in E. coli resistant isolates downstream in an urbanized watershed impacted by wastewater treatment plants, which lends support to the hypothesis that WWTPs effluent may be contributing to the conveyance of antibiotic resistance bacteria downstream from discharge points. Upstream isolates expressed resistance to Amoxicillin clavulanate, Cefuroxime and Tetracycline although comparatively lower to other sites, further suggesting that there may be some unknown sources of antibiotics or resistance strains discharged further upstream of the treatment plant. Seventy percent of all E. coli isolates expressed resistance to not less than 2 antibiotics. This result is similar to a study by Odonkor and Addo (2012) which recorded 63% of multiple resistant E. coli strains in drinking water sources in Accra. Blaak et al. (2015) reported a high presence of multiple drug resistant E. coli in surface waters. 82 University of Ghana http://ugspace.ug.edu.gh E. coli isolates from upstream were resistant to 2 or more antibiotic agents, although only one isolate was resistant to more than 4 antibiotics. Fifty-three percent of E. coli isolates from final effluent from the treatment plant were resistant to not less than 2 antibiotic agents. Downstream recorded the highest number of isolates resistant to 2 or more antibiotics with 93% multiple resistant isolates, which is similar to results from a study by Blaak et al., 2015 which reported high multiple resistant strains downstream a WWTP. Multi-drug resistance was found to increase downstream the treatment plant for isolates resistant to ≥ 2, ≥ 3, and ≥ 4 agents. Studies have observed high rates in the development of multi-drug resistance in E. coli isolates in WWTPs (Korzeniewska et al., 2013; Amador et al., 2015). This is found to be primarily driven by the transfer of conjugative plasmids and are shown in other studies to persist and propagate through the environment once discharged. (Laird, 2016). Hence high resistance found in E.coli downstream. 5.3 Antibiotic resistance in A. hydrophila Ubiquitous bacteria, which are capable of colonizing different water types, are of particular importance when determining antimicrobial resistance dissemination in the environment. As a result of their ubiquity and resistance patterns, Aeromonas sp can grow in different sources of water; as such, they are important in assessing possible forms of antimicrobial resistance dissemination. Aeromonas sp have been shown to develop and spread antibiotic resistance in the aquatic systems (Figueira et al., 2011) rendering surveillance of resistance in this organism is therefore very imperative. Susceptibility of all A. hydrophila isolates to antibiotics classes, which include β- lactam/β- lactamase inhibitors (Amoxicillin clavulanate), Cephalosporin (Cefuroxime and 83 University of Ghana http://ugspace.ug.edu.gh Ceftazidime), Monobactam (Aztreonam), Carbapenem (Imipenem), aminoglycosides (Gentamicin), Tetracycline (Tetracycline) and Quinolone (Ciprofloxacin), were determined. Isolates exhibited resistance to between one and seven antibiotic agents. Among all the antibiotics, Gentamicin and Ceftazidime were active against 91% and 80 % of all A. hydrophila isolates. Also, susceptibility rates of all isolates to Aztreonam, Ciprofloxacin, Imipenem was 73 %, 78% and 78% respectively. High resistance to Amoxicillin clavulanate (68%), Cefuroxime (43%) and Tetracycline (31%) was observed in all isolates. Final effluent recorded resistance of 97% for Amoxicillin clavulanate, 50% for Cefuroxime and 23% Tetracycline. The rate of isolate resistance to tetracycline in effluent was found to be lower than expected when compared to similar research (Igbinosa and Okoh, 2012) which recorded 77.8% in effluent samples. Resistance to tetracycline was 10% and 33% upstream and downstream respectively. Tetracycline resistance has also been reported in Aeromonas sp isolated from a river that receives wastewater discharge (Goñi-Urriza, 2000). High Amoxicillin clavulanate and Cefuroxime resistance (80% and 47% respectively) was recorded downstream. Resistance rates were higher in downstream than in final effluent samples suggesting that there are other sources contributing to high resistance in the Onyasia stream other than the waste treatment plant. Other external sources may include untreated wastewater from households and abattoirs, which are directly discharged into the stream. Upstream isolates recorded lower Amoxicillin clavulanate (23%) and Cefuroxime (23%) resistance. A significant association (p < 0.05) was found to exist between the location of sampling sites relative to the waste treatment plant .i.e. upstream, downstream) and isolates 84 University of Ghana http://ugspace.ug.edu.gh expressing resistance to Amoxicillin clavulanate, Cefuroxime and Tetracycline. This result further corroborates effluents from wastewater treatment plant as contributors to the conveyance of antibiotic resistance bacteria downstream from discharge points (McConnell 2016). Sixty-one percent of all Aeromonas hydrophila isolates expressed resistance to 2 or more antibiotics, 99% of A. hydrophila isolates from final effluent from the treatment plant were resistant to 2 or more antibiotic agents. Fifty-six percent of A. hydrophila isolates in final effluent expressed resistance to 3 or more antibiotics. This result is similar to results from a study conducted by Figueira et al. (2011), which recorded high multiple resistance to at least three different antibiotic classes (Gentamycin, Tetracycline and Sulphamethoxazole/Trimethoprim) in Aeromonas punctata and Aeromonas media isolated from final effluents. The Downstream sampling site recorded the highest number of isolates resistant to 2 or more antibiotic with 73% multiple resistant isolates. Upstream recorded 3% isolates resistant to 2 or more isolates. Multi-drug resistance was found to increase downstream the treatment plant for isolates resistant to ≥ 2, ≥ 3, and ≥ 4 agents. Studies on antibiotic resistance in multiple resistance of Aeromonas sp using culture dependent methods in waste water and surface water is limited. 85 University of Ghana http://ugspace.ug.edu.gh 5.4 Antibiotic resistance in P. aeruginosa isolates P. aeruginosa is a ubiquitous member of the genus Pseudomonas. P. aeruginosa causes nosocomial infections in immunocompromised patients, which are often difficult to treat (Hayford, 2016). Treatment of these infections are limited to a few class of antibiotics usually referred to as antipseudomonal antibiotics due to the fact that Pseudomonas sp are resistant to common antibiotics (Hayford, 2016). There is the need for routine surveillance of the distribution, resistance pattern of P. aeruginosa as well as other pathogenic bacteria from wastewater in order to provide local data on resistance pathways especially the contribution from wastewater. Susceptibility of all P. aeruginosa isolates to antibiotics classes, which include Cephalosporin (Ceftazidime), Monobactam (Aztreonam), Carbapenem (Imipenem), Aminoglycosides (Gentamicin), and Quinolone (Ciprofloxacin) were determined. Forty- five percent of all P. aeruginosa isolates were susceptible to all antipseudomonal agents. Among all the antibiotics tested, Imipenem and Ceftazidime were active against 96% and 89 % of all P. aeruginosa isolates. The high activity of these antibiotics in P. aeruginosa may be due to infrequent use of these antibiotics community and clinical settings in Ghana. High resistance to Aztreonam (37%), Ciprofloxacin (33%) and Gentamicin 22%) was observed in all isolates, a result is found similar to a study conducted by Hayford (2016), which reported resistance of P. aeruginosa to Aztreonam (22%) in clinical and environmental samples in Accra. Ciprofloxacin resistance (54%) and Gentamicin resistance (60%) was also higher in Hayford, 2016 compared to this study. High resistance 86 University of Ghana http://ugspace.ug.edu.gh rate of P. Aeruginosa to Aztreonam have also been observed in Brazil (Pitondo-Silva et al., 2014). Final effluent recorded high resistance for Aztreonam (33%), Ciprofloxacin (13%) and Gentamicin (17%). This result is in contrast with a study conducted by Igbinosa et al. (2012) which found higher resistance rates for Gentamicin (22%) of P. aeruginosa isolated from effluent of a wastewater treatment plant in South Africa. Imipenem susceptibility was lower in this study when compared to Igbinosa et al., 2012, which recorded 100% susceptibility in P. aeruginosa isolates to Imipenem. Results of this study in contrasted with a study carried out by Basri et al. (2017) which recorded slightly higher resistance rates for ciprofloxacin (15%) in P. aeruginosa in effluents from a wastewater treatment plant. In this current study, effluent isolate resistance to gentamicin was higher compared to Basri et al. (2017) which recorded no resistance (0%) to P. aeruginosa in effluent of a wastewater treatment plant. There was an increase in the percentages of resistant strains downstream from the wastewater discharge for Aztreonam (50%) and Ciprofloxacin (40%). Resistance rates in effluents were lower than downstream samples, and similar resistant patterns were seen in effluent and downstream isolates. P. aeruginosa isolated from the upstream also showed some resistance to Aztreonam (20%) and Ciprofloxacin (3%) albeit at low rates. This observation suggests that the source of the resistance was located further upstream from the discharge and that it was not related to treatment plant discharge. However, possible sources of antibiotic resistance were not observed at the upstream sampling site of this study, the reason might also be attributed to 87 University of Ghana http://ugspace.ug.edu.gh natural resistance. Studies have shown that indigenous bacteria in unpolluted environments can also show some antibiotic resistance (Laird 2016). Nevertheless, a significant association (p < 0.05) was found to exist between the location of sampling site relative to the waste treatment plant (upstream, outfall and downstream) and isolates expressing resistance to Aztreonam and Ciprofloxacin. This shows that the discharge of treated wastewater contributes to antibiotic resistant P. aeruginosa in the receiving Onyasia stream. Studies have shown that multiple resistant strains persist and propagate in the aquatic environment once discharged (Laird, 2016). In this study, however resistance to Aztreonam, Ciprofloxacin, and Gentamycin was more frequently found in multi-drug resistant isolates, which implies that resistance to these antibiotic agents is mostly driven by similar mechanisms of defense coded by resistance genes to other agents, or that the acquisition of resistance to these agents usually occurs in tandem with other antibiotic resistances. High Quinolone-Aminoglycoside (Ciprofloxacin-Gentamicin) cross- resistance in this study is similar to results from Hayford, 2016 study which characterized multiple resistant P. aeruginosa from environmental sources in Kumasi, Ghana. Survey along the course of Onyasia stream sampling points did not show other possible sources of antibiotic resistance close to the upstream sampling point. On the contrary, it was observed that there were many wastewater connections directly from households, which possibly discharge untreated wastewater into the stream. Vegetable farms were found along the Onyasia stream and close to the downstream sampling points. The stream water was abstracted for irrigation in these vegetable farms. 88 University of Ghana http://ugspace.ug.edu.gh This finding has human health implications, as humans could be exposed to resistant strains of E. coli, Pseudomonas aeruginosa, Aeromonas hydrophila and other resistant organisms through the food chain or contact with contaminated water. Globally, antibiotic resistant bacteria have been found on fresh fruit and vegetables (O’ Flaherty et al., 2019). O’Flaherty et al. (2019) created a quantitative human exposure assessment model using scenario analysis to investigate the potential human exposure to antibiotic resistant E. coli through the consumption of lettuce irrigated with surface water. Results from their study showed the mean human exposure levels ranged between 1.00 × 10−2 and 1.35 × 106 colony forming units antibiotic resistant E. coli per 100 g of surface water irrigated lettuce for the different scenarios investigated. This finding has human health implications, as humans could be exposed to resistant strains of E. coli, P. aeruginosa, A. hydrophila and other resistant organisms through the food chain or contact with contaminated water due to the fact that the Onyasia stream serves as an irrigation water source. Generally, this study shows there is a significant increase in antibiotic resistance prevalence of E. coli, A. hydrophila and P. aeruginosa downstream of the treatment plant compared to upstream of the treatment plant. This finding is consistent with a study conducted by Guyomard-Rabenirina et al. (2017) which observed high resistant rates of E. coli downstream a wastewater treatment plant in Guadeloupe. High resistance downstream could also be attributed to wastewater discharge from households as observed in the field. Resistant strains were also found upstream, which suggest other sources of antibiotic 89 University of Ghana http://ugspace.ug.edu.gh resistance other than the treatment plant. Possible sources could be wastewater from households and abattoirs. Nonetheless, it seems likely that waste treatment plant discharge may be contributing to some degree to the spread of antibiotic resistant E. coli, A. hydrophila and P. aeruginosa. High resistance rates found in effluent isolates in this study is consistent with results recorded in a study conducted by Neudorf et al. (2017), which showed that wastewater stabilization ponds enriched the relative antibiotic resistance genes due to the long holding periods resulting in higher antibiotic resistance rates. This highlights the role of treatment plants in the spread of antibiotic resistance in the environment. 5.5 Suitability of treatment plant effluent for reuse in agriculture Monitoring wastewater for antibiotic resistance bacteria is important to understand the spread of resistance. Treated wastewater is recommended for use in agriculture in order to achieve sustainable management of water resources, which however this presents a health risk as wastewater treatment plants are unable to significantly reduce antibiotics, antibiotic resistant bacteria and genes which may end up in the food chain when contaminated produce are consumed. Mean E. coli, A. hydrophila and P. aeruginosa reduced significantly in effluent compared to influents showing improved wastewater quality after treatment, with up to a fivefold reduction of mean bacterial loads. 90 University of Ghana http://ugspace.ug.edu.gh According to the WHO standards for wastewater, excreta and greywater for agriculture, the effluent from the Legon Sewage Treatment Plant meets the requirement for unrestricted ( <105 E.coli/100 ml ) and unrestricted irrigation (<103 E.coli/100 ml). Kwabla (2017) recommended reuse of treated wastewater from the Legon Sewage Treatment Plant in light of the effluents meeting the WHO guidelines for the reuse of wastewater and grey water for agriculture. However, E. coli, and A. hydrophila resistance rates to Amoxicillin clavulanate, Cefuroxime, Tetracycline, Aztreonam and Imipenem, as observed in this study were high in effluent isolates even though bacterial counts were lower. Multiple antibiotic resistant isolates were also very high in effluent isolates. This has implications for wastewater reuse in agriculture as antibiotic resistance bacteria may enter the food chain. Results from this study is consistent with a study conducted by Aslan et al., 2018 in wastewater treatment effluents used an irrigation water source. Similar to this study, high resistance to antibiotics tested .i.e. Ampicillin, Sulfamethoxazole, Ciprofloxacin and Tetracycline was recorded in E. coli isolated from effluents. Studies have shown that consumers are at risk of being exposed to and consuming antibiotic resistant bacteria in raw produce irrigated with treated wastewater (Pina et al., 2018). There is also a major health risk to farm workers as they may be exposed to antibiotic resistant bacteria through contact with wastewater effluent. A study conducted by Goldstein (2013) detected Vancomycin Resistant Enterococci and Methicillin Resistant Staphylococcus aureus were found in 26% and 29% of spray irrigation workers respectively. 91 University of Ghana http://ugspace.ug.edu.gh High antibiotic resistance found in this study is of great concern and emphasises the importance of monitoring the quality of wastewater for irrigation purposes with respect to antibiotic resistance bacteria and the need to improve the wastewater treatment process to remove antibiotic resistant bacteria. 5.6 Implications for guidelines and standards for compliance in effluent quality discharge into the environment and agriculture. This study shows the presence of bacterial isolates with high resistance to antibiotics tested as well as multiple resistant E. coli, A. hydrophila and P. aeruginosa. Currently microbiological guidelines on wastewater discharge and the safe reuse of wastewater in agriculture exists for indicator microorganisms (Ghana EPA, 2010; WHO, 2006). However, these guidelines do not include critical values for antibiotic resistance bacteria for wastewater discharge or reuse in agriculture. Results from this study confirms the need for revision of these guidelines to include monitoring of antibiotic resistance bacteria due to the possible risk of antibiotic resistant bacteria entering the food chain from contaminated irrigation water. E. coli has been suggested for monitoring antibiotic resistance in wastewater and surface water (Gekenidis et al., 2018). This study sought to investigate the presence of antibiotic resistant P. aeruginosa, E. coli and A. hydrophila in the Legon Sewerage Treatment Plant and the receiving Onyasia stream using bacterial resistance data produced by culture-dependent methods and the environmental health implications of the treatment plant discharge. Findings show although there was a significant reduction in levels of P. aeruginosa, E. coli and A. 92 University of Ghana http://ugspace.ug.edu.gh hydrophila in effluent being discharged from the treatment plant as well high isolate resistant to antibiotics tested. The general objective of the study was achieved. This study contributes to the knowledge gap about antibiotic resistance in wastewater discharge and wastewater impacted environment in Ghana. 93 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1 CONCLUSIONS This study has provided a basis for the need for further treatment of wastewater before discharge and reuse as well as surveillance of antibiotic resistant bacteria in treated effluent and the receiving stream. The following major conclusions were reached during the conduct of this study.  Legon Sewage Treatment plant has been proven to significantly reduce bacterial loads after treatment. However, effluent quality did not meet Ghana EPA guideline limit for wastewater discharge.  Findings show treated wastewater from the Legon Sewage Treatment Plant meets the WHO guideline limit for unrestricted (<105 E.coli/100 ml) and unrestricted irrigation (<103 E.coli/100 ml). However, it contains E. coli, P. aeruginosa and A. hydrophila resistant to most of antibiotics tested as well as high multiple resistant bacteria.  Farm hands are therefore at risk of acquiring resistant infections during irrigation. Antibiotic resistant bacteria could also enter the food chain through effluent reuse for irrigation.  The relative location of sampling points upstream and downstream of the Legon Sewage Treatment Plant discharge points had a significant impact on the 94 University of Ghana http://ugspace.ug.edu.gh occurrence of antibiotic resistant P. aeruginosa, E. coli, and A. hydrophila as resistance was higher downstream compared to upstream.  This finding shows that the Legon Sewerage Treatment Plant potentially contributes to the spread and persistence of antibiotic resistant bacteria in the Onyasia stream.  Resistant E. coli, P. aeruginosa and A. hydrophila were found upstream of the treatment plant suggesting other sources of antibiotic resistance in the stream before treatment. As such results do not conclusively identify the Legon Sewage treatment plant discharge as the only source of increased rates of resistance seen in this study.  There are human health implications on the use of the Onyasia stream as an irrigation water source by farmers downstream as high bacterial resistance was detected in water samples.  Farmers who come in contact with the irrigation water as well as consumers of raw vegetables irrigated with contaminated water are at risk of exposure to resistant pathogens. 95 University of Ghana http://ugspace.ug.edu.gh 6.2 RECOMMENDATIONS To help preserve the quality of water and mitigate the spread of antibiotic resistance in the receiving Onyasia Stream, the efficacy of Legon Sewerage Treatment Plant needs to be improved.  There is a need for continuous surveillance of antibiotic resistance bacteria in Legon Sewerage Treatment Plant by the Ghana Environmental Protection Agency and the Water Research Institute as faecal bacteria in wastewater can be used as an early warning system to detect emerging resistance trends in the population, to provide insights into the burden of faecal carriage.  Culture based methods and molecular tools may be used in tandem to ensure more accurate characterization of the abundance and transfer of antibiotic resistance genes.  In order to help reduce the risk associated with exposure of antibiotic resistant bacteria in treated wastewater to farmers and through the food chain, farmers should be trained on good agricultural and handling practices.  The Onyasia Stream is used untreated for irrigation and can pose a significant health risk to farmers and consumers through direct contact with contaminated wastewater, breathing in aerosols and consumption of vegetables. The Ministry of Agriculture, should encourage farmers through education campaigns, to use 96 University of Ghana http://ugspace.ug.edu.gh protective equipment when handling stream water. Stream water should be used to irrigate vegetables not consumed raw.  The WHO Guidelines for the Safe Use of Wastewater, Excreta and Greywater: Wastewater Use in Agriculture and Ghana Environmental Protection Agency standards for wastewater discharge should be revised to include critical values for antibiotic resistant bacteria.  E. coli could serve as one of the organisms used an indicator for screening antibiotic bacteria in wastewater treatment plants. Further Research  Further research is needed to investigate health risks associated with wastewater harbouring antibiotic resistant bacteria used in agriculture, aquaculture, recreational and other non-critical uses.  A quantitative microbial risk assessment (QRMA) is needed to highlight the risks associated with consuming produce irrigated with treated wastewater from the treatment plant as well as water from the Onyasia Stream.  Further research is needed to be conducted to identify other possible sources of antibiotic resistant bacteria and antibiotics in the Onyasia Stream as some resistant bacteria were found upstream of the treatment. 97 University of Ghana http://ugspace.ug.edu.gh  Resistance profile of bacteria in surface water and sediment may vary, as such further research is needed on prevalence of resistant bacteria in the sediments of the Onyasia stream.  There is also the need for standardization of techniques for evaluation of antibiotic resistance in the environment. 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Applied Microbiology and Biotechnology, 82(3), 397–414. https://doi.org/10.1007/s00253-008-1829-z 110 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I AEROMONAS ISOLATION MEDIUM COMPOSITION Ingredients Grams/Litre Peptone, special 5.0 Yeast extract 3.0 L-Lysine hydrochloride 3.5 L-Arginine hydrochloride 2.0 Inositol 2.5 Lactose 1.5 Sorbose 3.0 Xylose 3.75 Bile salts 3.0 Sodium thiosulfate 10.67 Sodium chloride 5.0 111 University of Ghana http://ugspace.ug.edu.gh Ferric ammonium citrate 0.8 Bromothymol Blue 0.04 Thymol Blue 0.04 Agar 12.5 Final pH 7.0 ± 0.2 at 25°C 112 University of Ghana http://ugspace.ug.edu.gh APPENDIX II E. COLI ISOLATION MEDIUM COMPOSITION Ingredients Grams/Litre Chromocult® Coliform Agar Enzymatic Digest of Casein 1 Yeast Extract 2 NaCl 5 NaH2PO4 x 2 H2O 2.2 Na2HPO4 2.7 Sodium Pyruvate 1 Sorbitol 1 Tryptophane 1 Tergitol® 7 0.15 6-Chloro-3-indoxyl-beta-Dgalactopyranoside 0.2 5-Bromo-4-chloro-3-indoxyl-β-Dglucuronic acid 0.1 Isopropyl-beta-Dthiogalactopyranoside 0.1 113 University of Ghana http://ugspace.ug.edu.gh Agar-agar* 10 114 University of Ghana http://ugspace.ug.edu.gh APPENDIX III PSEUDOMONAS ISOLATION MEDIUM COMPOSITION Ingredients Grams / Litre Gelatin peptone 20.000 Magnesium chloride 1.400 Potassium sulphate 10.000 Cetrimide 0.300 Agar 15.000 115 University of Ghana http://ugspace.ug.edu.gh APPENDIX IV PLATES SHOWING SAMPLE COLLECTION AND ANALYSIS Plate 6: A photograph showing sample collection Plate 7: A photograph showing antibiotic sensitivity test being carried out using Kirby Bauer Disc diffusion method 116 University of Ghana http://ugspace.ug.edu.gh APPENDIX V PLATES SHOWING BACTERIAL CULTURE PLATES Plate 8: A photograph showing culture plates showing P. aeruginosa (left) and E. coli colonies (right) Plate 9: A photograph of culture plates showing A. hydrophila colonies 117 University of Ghana http://ugspace.ug.edu.gh Plate 10: A photograph showing antibiotic sensitivity tests of A. hydrophila. Note the zones of inhibition 118 University of Ghana http://ugspace.ug.edu.gh APPENDIX VI SOME BIOCHEMICAL TESTS CONDUCTED ON ISOLATES Plate 11: A set up showing Oxidase positive (Left) and Citrate positive (Right) tests 119 University of Ghana http://ugspace.ug.edu.gh Plate 12: A set up showing glucose fermentation (Left) and Sulphide Indole Motility (SIM) positive (Right) tests 120 University of Ghana http://ugspace.ug.edu.gh APPENDIX VII WATER SAMPLING SITES AND COORDINATES SAMPLE SITE COORDINATES SITE NUMBER 1 Treatment plant Influent N 05° 39’ 44.9’’ W 000° 11.28. 0’’ 2 Treatment plant effluent N 05° 39’ 56. 0’’ W 000° 11 28. 7’’ 3 Upstream (500 meters before the treatment N 05° 39’ 58. 8’’ W 000° plant discharge point) 11 20. 1’’ 4 Outfall (Discharge point) N 05° 40’ 00. 5’’ W 000° 11 27. 2’’ 5 Downstream(500 meters after the treatment N 05° 40’ 08. 4’’ W 000° plant discharge point) 11 31. 0’’ 121 University of Ghana http://ugspace.ug.edu.gh APPENDIX VIII BACTERIAL COUNTS, ANTIBIOTIC SENSITIVITY PATTERNS AND BIOCHEMICAL IDENTIFICATION Negative control (blanks) analysed alongside water and wastewater samples Blank MONTH Chromocult agar Aeromonas agar Cetrimide agar JANUARY >1 >1 >1 FEBRUARY >1 >1 >1 MARCH >1 >1 >1 APRIL >1 >1 >1 MAY >1 >1 >1 JUNE >1 >1 >1 122 University of Ghana http://ugspace.ug.edu.gh Levels of E. coli, A. hydrophila and P. aeruginosa in wastewater and surface water at the sampling sites. E.coli CFU/100ml SAMPLE ID January February March April May June Mean Mean log Influent 100,000,000 80,000,000 260,000,000 90,000,000 13,600,000 70,000,000 102,266,667 7.88 Effluent 100 2,000 1,300 560 200 100 710 2.58 Outfal 60,000 3,000 2,000 3,000 1,800 1,600 11,900 3.58 Upstream 2,000 1,800 1,000 430 300 200 955 2.83 Downstream 3,000,000 6,400,000 8,000,000 600,000 170,000 90,000 3,043,333 6.02 P. aeruginosa CFU/100ml Influent 22,000,000 3,400,000 3,100,000 300,000 2,900,000 2,300,000 5,666,667 6.44 Effluent 1,000 2,000 2,000 2,000 1,200 1,100 1,550 3.17 Outfal 30,000 6,000 5,000 3,000 2,200 2,000 8,033 3.68 Upstream 4,000 3,000 2,000 2,000 1,800 1,300 2,350 3.34 Downstream 1,600,000 1,100,000 900,000 98,000 74,000 74,000 641,000 5.49 A. hydrophila CFU/100ml Influent 372,000 376,000 270,000 440,000 400,000 400,000 376,333 5.57 Effluent 17,000 17,300 10,000 5,320 4,000 4,000 9,603 3.90 Outfall 87,000 58,400 58,000 50,000 40,000 20,000 52,233 4.68 123 University of Ghana http://ugspace.ug.edu.gh Upstream 32,000 34,000 28,000 30,000 15,000 7,600 24,433 4.34 Downstream 2,100,000 9,400,000 1,800,000 1,720,000 100,000 100,000 2,536,667 5.96 124 University of Ghana http://ugspace.ug.edu.gh Levels of total coliform in wastewater Total coliforms CFU/100ml Sample January February March April May June Mean Mean ID Log Influent 100000000 90000000 280000000 96000000 16000000 88000000 111666667 7.92 Effluent 1080 3200 2800 1650 1000 1280 1835 3.26 125 University of Ghana http://ugspace.ug.edu.gh A. hydrophila breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2018) INFERENCE GEN AMC TET CIP IPM CXM ATM Diameter of zones of inhibition S ≥ 15 18 15 21 16 18 21 R < 12 13 11 15 13 14 17 TET: Tetracycline 30 μg; AMC: Amoxicillin Clavulanate 20 μg; CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CXM: Cefuroxime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible E. coli breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2017) INFERENCE GEN AMC TET CIP IPM CXM ATM Diameter of zones of inhibition S ≥ 15 18 15 21 23 23 21 R < 12 13 11 15 19 14 17 TET: Tetracycline 30 μg; AMC: Amoxicillin Clavulanate 20 μg; CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CXM: Cefuroxime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible 126 University of Ghana http://ugspace.ug.edu.gh P. aeruginosa breakpoint values for interpretation of zone diameters as stated by European Committee on Antimicrobial Susceptibility Testing CLSI (2017) INFERENCE GN CIP IMP ATM CAZ Diameter of zones of inhibition S ≥ 15 21 19 22 18 R < 12 15 15 15 14 CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μ; IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible E. coli antibiotic sensitivity quality control analysis MONTH ISOLATE ANTIBIOTICS GN AMC TET CIP IPM CXM ATM JANUARY E. coli ATCC 25922 18 20 23 32 26 22 29 FEBRUARY E. coli ATCC 25923 19 20 24 32 26 22 28 MARCH E. coli ATCC 25924 19 19 23 31 26 22 28 127 University of Ghana http://ugspace.ug.edu.gh APRIL E. coli ATCC 25925 19 19 23 31 27 21 28 MAY E. coli ATCC 25926 19 20 23 31 27 21 28 JUNE E. coli ATCC 25927 19 20 24 31 26 21 28 TET: Tetracycline 30 μg; AMC: Amoxicillin Clavulanate 20 μg; CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CXM: Cefuroxime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible P. aeruginosa antibiotic sensitivity quality control analysis MONTH ISOLATE ANTIBIOTICS GN CIP IMP ATM CAZ JANUARY P. aeruginosa ATCC 29214 19 31 20 24 25 FEBRUARY P. aeruginosa ATCC 29215 19 31 21 24 25 MARCH P. aeruginosa ATCC 29216 20 31 21 24 24 APRIL P. aeruginosa ATCC 29217 20 32 20 23 25 MAY P. aeruginosa ATCC 29218 20 32 20 23 25 JUNE P. aeruginosa ATCC 29219 20 31 21 24 25 128 University of Ghana http://ugspace.ug.edu.gh JUNE P. aeruginosa ATCC 29220 20 30 21 23 24 CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CAZ: Ceftadzidime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg Susceptibility patterns of Aeromonas hydrophila isolates against the selected antibiotics Aeromonas hydrophila zones of inhibition measured in millimeters (mm) INFLUENT ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION G I AM I TE I CI I IM I CX I AT I CA I MULTIPLE N N C N T N P N P N M N M N Z N DRUG RESISTANCE JBAA 20 S 25 S 10 R 18 I 26 S 12 R 17 R 6 R 4 JBBA 19 S 11 R 6 R 21 S 25 S 17 I 20 I 7 R 3 JBCA 24 S 10 R 6 R 26 S 24 S 12 R 10 R 12 R 4 JBGA 18 S 6 R 7 R 27 S 22 S 6 R 21 S 17 R 4 JBQA 26 S 11 R 7 R 25 S 22 S 6 R 9 R 25 S 4 FBAA 14 R 13 R 11 R 20 I 13 R 26 S 32 S 31 S 4 FBBA 17 S 20 S 14 I 24 S 20 S 33 S 25 S 20 I 0 FBCA 14 R 16 I 6 R 25 S 20 S 15 S 31 S 18 I 2 FBFA 20 S 21 S 11 R 25 S 20 S 30 R 25 S 31 S 2 FBGA 18 S 7 R 11 R 20 I 21 S 25 S 19 I 22 S 2 MBA 18 S 16 I 12 I 20 I 13 R 24 R 17 I 7 R 3 MBB 14 R 13 R 12 I 20 I 13 R 20 R 22 S 17 R 4 MBC 17 S 6 R 6 R 10 R 23 S 6 R 17 S 7 R 4 MBD 18 S 6 R 6 R 8 R 23 S 6 R 22 R 17 R 4 MBE 19 S 22 S 6 R 10 R 25 S 6 R 22 R 17 R 4 ABA 20 S 24 S 7 R 8 R 25 S 6 R 20 I 11 R 4 ABB 19 S 25 S 6 R 10 R 25 S 6 R 19 I 8 R 4 129 University of Ghana http://ugspace.ug.edu.gh ABC 20 S 15 S 25 S 19 I 27 S 23 S 32 S 25 S 0 ABD 18 S 12 R 6 R 9 R 24 S 6 I 9 S 13 S 3 ABE 15 R 6 S 7 R 19 I 23 S 6 S 24 S 15 S 2 MBA 26 S 17 I 30 S 30 S 35 S 6 R 25 S 30 S 1 MBB 20 S 21 S 23 S 23 S 30 S 23 S 30 S 26 S 0 MBC 17 S 18 S 14 I 14 R 25 S 8 R 29 S 26 S 2 MBD 19 S 20 S 21 I 22 S 31 S 21 S 31 S 25 S 0 MBE 17 S 17 I 14 I 14 R 27 S 7 R 28 S 24 S 2 JBAA 17 S 6 R 6 R 10 R 23 S 6 R 17 S 7 R 4 JBBA 18 S 6 R 6 R 8 R 23 S 6 R 22 S 17 R 4 JBCA 19 S 22 S 6 R 10 R 25 S 6 R 22 S 17 R 4 JBDA 18 S 12 R 6 R 9 R 24 S 6 R 9 R 13 R 4 JBEA 15 S 6 R 7 R 14 R 20 S 6 R 16 R 15 R 4 ISOLATE ID EFFLUENT ANTIBIOTICS/INTERPRETATION G I AM I TE I CI I IM I CX I AT I CA I N N C N T N P N P N M N M N Z N JAA 23 S 15 I 15 I 27 S 26 S 6 R 17 R 25 S 2 JAB 24 S 18 I 15 I 29 S 27 S 9 R 20 I 24 S 1 JAC 20 S 14 I 11 R 30 S 29 S 6 R 32 S 21 S 2 FAA 29 S 17 I 20 S 30 S 30 S 7 R 21 S 30 S 1 FAB 18 S 14 I 11 R 25 S 31 S 28 S 22 S 10 R 2 FAC 17 S 14 I 11 R 26 S 18 S 31 S 13 R 31 S 2 AAE 20 S 16 I 20 S 30 S 20 S 6 R 22 S 25 S 1 JAD 21 S 14 R 17 S 30 S 23 S 6 R 21 S 13 R 3 FAB 20 S 7 R 23 S 15 R 20 S 25 S 23 S 12 R 3 FAD 19 S 13 R 25 S 30 S 19 S 27 S 30 S 30 S 1 MAA 18 S 11 R 20 S 30 S 21 S 25 S 31 S 31 S 1 MAB 13 R 13 R 25 S 26 S 17 S 25 S 18 I 27 S 2 MAC 15 R 11 R 23 S 30 S 19 S 22 S 21 S 28 S 2 MAD 16 S 7 R 25 S 27 S 18 S 30 S 17 I 32 S 1 MAE 17 S 9 R 20 S 20 I 8 R 13 R 21 S 17 R 4 130 University of Ghana http://ugspace.ug.edu.gh AAA 14 I 10 R 25 S 31 S 13 R 6 R 20 S 25 S 3 AAB 20 S 12 R 24 S 30 S 12 R 6 R 17 I 25 S 3 AAC 23 S 12 R 23 S 32 S 14 S 6 R 13 R 27 S 3 AAD 14 I 10 R 20 S 32 S 12 S 6 R 15 R 30 S 3 MAA 20 S 11 R 22 S 30 S 15 S 23 S 22 S 27 S 1 MAB 20 S 12 R 27 S 30 S 18 S 27 S 35 S 30 S 1 MAC 20 S 10 R 30 S 35 S 13 R 20 S 12 R 30 S 3 MAD 14 I 8 R 11 R 20 I 8 R 14 R 21 S 25 S 4 MAE 20 S 8 R 20 S 20 I 9 R 13 R 29 S 14 R 4 JAA 21 S 10 R 28 S 30 S 13 R 23 S 15 R 29 S 3 JAB 15 S 9 R 28 S 25 S 14 S 24 R 30 S 28 S 2 JAC 21 S 8 R 27 S 29 S 11 R 20 S 11 S 27 S 2 JAD 22 S 7 R 30 S 35 S 11 R 18 S 20 S 26 S 2 JAE 21 S 12 R 29 S 40 S 13 R 22 S 21 S 28 S 2 JAE 17 S 13 S 15 I 25 S 30 S 6 R 19 I 23 S 1 U P S T R E A M I S O L A T E S ISOLATE ID ANTIBIOTICS/INTERPRETATION G I AM I TE I CI I IM I CX I AT I CA I N N C N T N P N P N M N M N Z N JOAA 20 S 13 R 11 R 32 S 19 S 12 R 30 S 30 S 3 JOBA 15 S 20 S 21 S 30 S 18 S 25 S 32 S 30 S 0 JOCA 18 S 26 S 27 S 24 S 12 R 25 S 33 S 28 S 1 JOD 26 S 24 S 21 S 24 S 22 S 24 S 17 R 26 S 1 JOE 21 S 12 R 25 S 25 S 23 S 29 S 30 S 28 S 1 FOA 20 S 17 I 30 S 26 S 20 S 30 S 34 S 30 S 0 FOB 20 S 19 S 25 S 30 S 20 S 30 S 17 R 26 S 1 FOC 20 S 24 S 26 S 22 S 17 S 30 S 32 S 29 S 0 FOD 15 S 25 S 23 S 20 I 11 R 30 S 30 S 26 S 1 FOE 20 S 8 R 25 S 26 S 13 R 35 S 30 S 30 S 2 MOA 18 S 8 R 18 S 21 S 25 S 30 S 34 S 26 S 1 MOB 20 S 20 S 26 S 25 S 12 R 28 S 30 S 28 S 3 MOC 14 I 13 R 11 R 20 I 24 S 21 S 35 S 30 S 2 131 University of Ghana http://ugspace.ug.edu.gh MOD 22 S 6 R 35 S 17 I 18 S 6 R 25 S 26 S 2 MOE 13 I 9 R 25 S 18 I 19 S 28 S 33 S 28 S 1 AOA 16 S 13 R 27 S 35 S 22 S 14 R 35 S 28 S 2 AOB 18 S 15 I 28 S 32 S 10 R 6 R 30 S 30 S 2 AOC 15 S 9 R 28 S 35 S 20 S 6 R 30 S 30 S 2 AOD 6 R 6 R 10 R 28 S 17 S 6 R 32 S 27 S 4 AOE 20 S 11 R 29 S 34 S 24 S 7 R 20 I 28 S 2 MOA 17 S 11 R 28 S 33 S 25 S 29 S 34 S 26 S 1 MOB 19 S 14 I 28 S 34 S 27 S 25 S 35 S 28 S 0 MOC 18 S 11 R 30 S 38 S 14 S 6 R 17 R 30 S 3 MOD 18 S 12 R 27 S 34 S 27 S 6 R 23 S 30 S 2 MOE 18 S 16 I 30 S 38 S 14 S 9 R 17 R 28 S 2 JOA 22 S 11 R 15 I 25 S 26 S 30 S 30 S 30 S 1 JOB 21 S 14 I 17 S 30 S 28 S 30 S 35 S 29 S 0 JOC 23 S 11 R 19 S 25 S 22 S 23 S 30 S 29 S 1 JOD 20 S 14 I 17 S 32 S 30 S 20 S 31 S 32 S 0 JOE 24 S 12 R 20 S 25 S 22 S 13 R 30 S 17 R 3 OUTFAL ISOLATES ISOLATE ID ANTIBIOTIC/INTERPRETION G I AM I TE I CI I IM I CX I AT I CA I N N C N T N P N P N M N M N Z N JUAA 20 S 15 S 24 S 30 S 13 S 25 S 30 S 29 S 1 JUBA 19 S 20 S 27 S 34 S 20 S 30 S 35 S 27 S 0 JUCA 20 S 19 S 29 S 35 S 20 S 28 S 27 S 30 S 0 JUD 18 S 16 R 20 S 25 S 16 S 21 S 36 S 32 S 0 JUE 16 S 20 S 25 S 29 S 17 S 22 S 30 S 28 S 0 FUA 15 S 20 S 24 S 30 S 15 S 26 S 29 S 34 S 0 FUB 20 S 20 S 22 S 30 S 19 S 30 S 28 S 28 S 0 FUC 20 S 16 S 23 S 25 S 15 S 21 S 36 S 29 S 0 FUD 21 S 7 R 22 S 26 S 15 S 22 S 35 S 24 S 1 FUE 15 S 22 S 20 S 26 S 20 S 30 S 32 S 29 S 0 MUA 16 S 20 S 22 S 30 S 30 S 29 S 36 S 30 S 0 132 University of Ghana http://ugspace.ug.edu.gh MUB 20 S 24 S 24 S 35 S 35 S 21 S 32 S 26 S 0 MUC 21 S 16 S 25 S 29 S 22 S 22 S 28 S 27 S 0 MUD 19 S 18 S 29 S 29 S 20 S 25 S 27 S 32 S 0 MUE 22 S 20 S 30 S 31 S 18 S 29 S 32 S 31 S 0 AUA 16 S 12 R 25 S 30 S 18 S 29 S 30 S 27 S 1 AUB 17 S 13 R 28 S 29 S 25 S 30 S 30 S 30 S 1 AUC 18 S 15 S 25 S 32 S 29 S 30 S 29 S 27 S 0 AUD 16 S 14 R 25 S 32 S 17 S 29 S 35 S 30 S 0 AUE 17 S 16 S 30 S 25 S 26 S 31 S 35 S 28 S 0 MUA 21 S 15 R 30 S 40 S 20 S 30 S 40 S 35 S 0 MUB 20 S 6 R 35 S 23 S 26 S 6 R 35 S 32 S 2 MUC 21 S 14 S 28 S 35 S 21 S 30 S 35 S 28 S 0 MUD 20 S 14 S 32 S 29 S 26 S 7 R 30 S 25 S 1 MUE 21 S 14 S 28 S 35 S 21 S 30 S 35 S 28 S 0 JUAA 17 S 13 R 15 I 26 S 21 S 30 S 36 S 30 S 1 JUBA 17 S 15 S 34 S 41 S 20 S 30 S 40 S 29 S 0 JUCA 20 S 13 S 34 S 35 S 15 S 30 S 20 S 29 S 1 JUD 17 S 15 S 11 R 26 S 22 S 32 S 38 S 29 S 1 JUE 17 S 15 S 11 R 26 S 22 S 32 S 38 S 29 S 1 DOWNSTREAM ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION G I AM I TE I CI I IM I CX I AT I CA I N N C N T N P N P N M N M N Z N JDA 25 S 14 I 7 R 20 I 13 R 12 R 28 S 30 S 2 JDB 24 S 6 R 20 S 21 S 20 S 10 R 29 S 28 S 2 JDC 25 S 11 R 20 S 25 S 24 S 25 S 30 S 26 S 1 JDD 24 S 8 R 20 S 30 S 26 S 8 R 31 S 33 S 2 JDE 20 S 13 R 28 S 34 S 24 S 25 S 32 S 31 S 1 FDA 17 S 8 R 17 S 25 S 26 S 30 S 30 S 29 S 1 FDB 20 S 13 R 15 S 25 S 25 S 25 S 27 S 25 S 1 FDC 23 S 8 R 20 S 35 S 30 S 25 S 35 S 27 S 1 FDD 19 S 12 R 20 S 23 S 30 S 20 S 16 R 17 R 3 133 University of Ghana http://ugspace.ug.edu.gh FDE 20 S 7 R 26 S 25 S 23 S 30 S 35 S 30 S 1 MDA 22 S 9 R 18 S 23 S 20 S 6 R 16 R 31 S 3 MDB 22 S 10 R 25 S 30 S 25 S 25 S 32 S 16 R 2 MDC 20 S 12 R 25 S 32 S 20 S 30 R 14 R 28 S 3 MDD 15 S 11 R 11 R 33 S 23 S 6 R 30 S 29 S 3 MDE 18 S 6 R 26 S 21 S 14 R 10 R 25 S 31 S 2 ADA 20 S 10 R 9 R 33 S 20 S 28 S 28 S 30 S 2 ADB 18 S 11 R 10 R 30 S 20 S 22 S 14 R 27 S 3 ADC 18 S 12 R 9 R 31 S 13 R 30 S 25 S 25 S 3 ADD 19 S 11 R 8 R 26 S 20 S 18 S 16 R 30 S 3 ADE 19 S 10 R 8 R 17 I 14 R 26 S 28 S 25 S 2 MDA 15 S 10 R 10 R 18 I 13 R 25 S 27 S 24 S 3 MDB 20 S 9 R 33 S 40 S 21 S 30 S 25 S 27 S 1 MDC 25 S 19 S 28 S 27 S 40 S 10 R 16 R 31 S 2 MDD 16 S 17 S 24 S 40 S 30 S 30 S 25 S 27 S 0 MDE 24 S 20 S 29 S 35 S 34 S 11 R 16 R 32 S 2 JDA 15 S 18 S 13 I 25 S 15 R 6 R 15 R 24 S 2 JDB 16 S 12 R 13 I 20 I 15 R 6 R 17 R 17 R 3 JDC 21 S 13 R 25 S 30 S 18 S 8 R 20 I 20 I 1 JDD 12 R 20 S 22 S 22 S 22 S 9 R 18 I 17 R 3 JDE 12 R 20 S 26 S 21 I 13 R 8 R 20 I 20 I 3 TET: Tetracycline 30 μg; AMC: Amoxicillin Clavulanate 20 μg; CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CXM: Cefuroxime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible 134 University of Ghana http://ugspace.ug.edu.gh Susceptibility of P. aeruginosa isolates against the selected antibiotics P. aeruginosa Zones of inhibition measured in millimeters (mm) INFLUENT ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION MULTIPLE DRUG RESIST ANCE GN IN CIP IN IMP IN ATM IN CAZ IN JBA 20 S 27 S 35 S 27 S 29 S 0 JBB 23 S 16 I 27 S 30 S 31 S 0 JBC 23 S 16 I 27 S 25 S 30 S 0 JBD 18 S 26 S 20 S 30 S 14 I 0 JBE 6 R 6 R 30 S 20 S 15 I 2 FBA 17 S 7 R 30 S 23 S 35 S 1 FBB 18 S 8 R 29 S 27 S 14 I 1 FBC 18 S 30 S 23 S 24 S 30 S 0 FBD 8 R 6 R 30 S 15 R 24 S 3 FBE 20 S 6 R 30 S 29 S 27 S 1 MBA 19 S 30 S 18 S 20 I 25 S 1 MBB 18 S 13 R 20 S 8 R 14 R 3 MBC 18 S 29 S 24 S 20 I 22 S 0 135 University of Ghana http://ugspace.ug.edu.gh MBD 18 S 30 S 19 S 20 I 25 S 0 MBE 18 S 30 S 20 S 21 I 25 S 0 ABA 20 S 31 S 26 S 25 S 25 R 1 ABB 15 S 26 S 24 S 24 S 24 R 1 ABC 15 S 27 S 27 S 25 S 25 S 0 ABD 21 S 30 S 27 S 27 S 24 S 0 ABE 15 S 26 S 25 S 23 S 23 S 0 MABA 16 S 28 S 21 S 22 S 22 S 0 MABB 17 S 29 S 21 S 23 S 29 S 0 MABC 17 S 31 S 24 S 22 S 21 S 0 MABD 18 S 29 S 20 S 24 S 22 S 0 MABE 17 S 30 S 21 S 23 S 31 S 0 JBA 20 S 20 S 20 S 32 S 27 S 0 JBB 18 S 13 R 23 S 20 S 23 S 1 JBC 20 S 30 S 17 R 14 R 22 S 2 JBD 17 S 27 S 25 S 18 I 25 S 0 JBE 22 S 30 S 30 S 14 R 26 S 1 FINAL EFFLUENT ISOLATES 136 University of Ghana http://ugspace.ug.edu.gh ISOLATE ID ANTIBIOTICS/INTERPRETATION GN IN CIP IN IMP IN ATM IN CAZ IN JAA 19 S 30 S 20 S 23 S 25 S 0 JAB 24 S 35 S 25 S 26 S 23 S 0 JAC 20 S 34 S 24 S 27 S 29 S 0 JAD 18 S 23 S 20 S 23 S 23 S 0 JAE 19 S 30 S 24 S 27 S 22 S 0 FAA 20 S 15 R 28 S 10 R 25 S 1 FAB 21 S 26 S 29 S 30 S 24 S 0 FAC 20 S 25 S 25 S 31 S 27 S 0 FAD 19 S 35 S 25 S 26 S 28 S 0 FAE 15 S 26 S 26 S 23 S 28 S 0 MAA 15 S 15 R 20 S 22 S 25 S 1 MAB 20 S 14 R 21 S 10 R 28 S 2 MAC 16 S 25 S 21 S 18 I 22 S 0 MAD 15 S 29 S 23 S 22 S 23 S 0 MAE 15 S 25 S 23 S 21 I 20 S 0 AAA 27 S 30 S 24 S 21 I 28 S 0 137 University of Ghana http://ugspace.ug.edu.gh AAB 20 S 25 S 22 S 30 S 25 S 0 AAC 8 R 26 S 6 R 6 R 14 R 4 AAD 23 S 30 S 8 R 10 R 29 S 2 AAE 8 R 25 S 21 S 25 S 27 S 1 MAA 13 R 30 S 24 S 20 I 24 S 1 MAB 25 S 35 S 20 S 21 I 23 S 0 MAC 14 I 30 S 21 S 22 S 26 S 0 MAD 28 S 35 S 20 S 22 S 21 S 0 MAE 14 I 30 S 21 S 21 I 28 S 0 JAA 18 S 23 S 20 S 22 S 22 S 0 JAB 19 S 30 S 24 S 23 S 29 S 0 JAC 20 S 13 R 28 S 22 S 21 S 1 JAD 21 S 26 S 29 S 23 S 22 S 0 JA E 20 S 25 S 25 S 22 S 20 S 0 OUTFAL ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION GN IN CIP IN IMP IN ATM IN CAZ IN JOA 15 S 30 S 25 S 33 S 30 S 0 138 University of Ghana http://ugspace.ug.edu.gh JOB 19 S 34 S 24 S 30 S 31 S 0 JOC 19 S 33 S 19 S 31 I 25 S 0 JOD 16 S 34 S 29 S 29 S 24 S 0 JOE 29 S 31 S 27 S 27 S 29 S 0 FOA 31 S 32 S 21 S 28 S 27 S 0 FOB 30 S 35 S 29 S 22 S 30 S 0 FOC 31 S 38 S 27 S 24 S 32 S 0 FOD 17 S 37 S 26 S 29 S 31 S 0 FOE 17 S 37 S 26 S 28 S 29 S 0 MOA 15 S 34 S 22 S 21 I 33 S 0 MOB 13 S 25 S 16 S 18 I 25 S 1 MOC 12 R 17 I 19 S 20 I 24 S 1 MOD 15 S 19 I 22 S 21 I 21 S 0 MOE 15 S 25 S 21 S 22 S 23 S 0 AOA 16 S 13 R 25 S 22 S 24 S 1 AOB 18 S 15 R 23 S 10 R 17 I 2 AOC 16 S 14 R 23 S 11 R 17 I 2 AOD 11 R 24 S 23 S 21 I 20 S 1 139 University of Ghana http://ugspace.ug.edu.gh AOE 17 S 30 S 23 S 20 I 20 S 0 MOA 14 I 27 S 23 S 23 S 21 S 0 MOB 11 R 34 S 24 S 21 I 26 S 1 MOC 16 S 34 S 26 S 24 S 25 S 0 MOD 10 R 34 S 24 S 20 I 27 S 1 MOE 16 S 30 S 25 S 24 S 25 S 0 JOA 17 S 30 S 23 S 22 S 23 S 0 JOB 8 R 10 R 10 R 14 R 13 R 4 JOC 17 S 30 S 23 S 15 R 23 S 1 JOD 15 S 6 R 6 R 6 R 13 R 4 JO E 15 S 15 R 20 S 20 I 20 S 1 UPSTREAM ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION GN IN CIP IN IMP IN ATM IN CAZ IN JUSA 25 S 31 S 24 S 23 S 24 S 0 140 University of Ghana http://ugspace.ug.edu.gh JUSB 21 S 30 S 30 S 23 S 26 S 0 JUSC 23 S 31 S 23 S 24 S 25 S 0 FUSA 18 S 28 S 30 S 27 S 27 S 0 FUSB 26 S 33 S 26 S 30 S 29 S 0 FUSC 22 S 31 S 25 S 31 S 31 S 0 FUSD 18 S 30 S 29 S 24 S 25 S 0 FUSE 29 S 39 S 23 S 30 S 26 S 0 FUSF 25 S 36 S 22 S 29 S 25 S 0 FUSG 21 S 30 S 30 S 25 S 29 S 0 MUSA 15 S 30 S 19 S 20 I 24 S 0 MUSB 16 S 33 S 21 S 23 S 25 S 0 MUSC 17 S 29 S 22 S 22 S 25 S 0 MUSD 16 S 32 S 24 S 20 I 23 S 0 MUSE 16 S 35 S 24 S 22 S 25 S 0 AUSA 18 S 30 S 23 S 23 S 26 S 0 AUSB 18 S 30 S 25 S 20 I 29 S 0 AUSC 17 S 30 S 24 S 22 S 25 S 0 AUSD 17 S 30 S 25 S 23 S 31 S 0 141 University of Ghana http://ugspace.ug.edu.gh AUSE 17 S 31 S 24 S 22 S 25 S 0 MUSB 17 S 30 S 23 S 20 I 20 S 0 MUSC 18 S 29 S 28 S 22 S 25 S 0 MUSD 17 S 30 S 23 S 23 S 20 S 0 MUSE 18 S 27 S 30 S 24 S 24 S 0 JUSA 17 S 25 S 21 S 14 R 23 S 1 JUSB 15 S 26 S 23 S 22 S 15 I 0 JUSC 16 S 25 S 19 S 22 S 24 S 1 JUSD 20 S 26 S 28 S 17 I 25 S 0 JUSE 25 S 31 S 27 S 25 S 28 S 0 MUSA 11 R 24 S 23 S 22 S 25 S 1 DOWNSTREAM ISOLATES ISOLATE ID ANTIBIOTICS/INTERPRETATION GN IN CIP IN IMP IN ATM IN CAZ IN JDSA 19 S 14 R 22 S 25 S 25 S 1 142 University of Ghana http://ugspace.ug.edu.gh JDSB 20 S 16 R 26 S 26 S 23 S 1 JDSC 15 S 30 S 24 S 27 S 25 S 0 JDSD 23 S 11 R 20 S 23 S 27 R 2 JDSE 12 R 11 R 31 S 10 R 29 S 3 FDSA 15 S 30 S 32 S 28 S 25 S 0 FDSB 15 S 25 S 23 S 26 S 23 S 0 FDSC 23 S 11 R 14 R 23 R 27 R 4 FDSD 24 S 32 S 36 S 20 S 25 S 0 FDSE 12 R 12 R 34 S 15 R 25 S 3 MDSA 23 S 11 R 20 S 15 R 25 S 2 MDSB 15 S 32 S 25 S 8 R 25 S 1 MDSC 15 S 26 S 19 S 20 I 25 S 0 MDSD 10 R 30 S 21 S 10 R 19 S 2 MDSE 12 R 30 S 22 S 11 R 20 S 2 ADSA 16 S 26 S 23 S 20 I 23 S 0 ADSB 18 S 10 R 26 S 26 S 11 R 2 ADSC 15 S 31 S 23 S 23 S 26 S 0 ADSD 16 S 29 S 24 S 21 I 27 S 0 143 University of Ghana http://ugspace.ug.edu.gh ADSE 10 R 9 R 27 S 25 S 11 R 3 MDSA 27 S 8 R 31 S 15 R 25 S 2 MDSB 24 S 15 R 23 S 13 R 25 S 2 MDSC 19 S 13 R 25 S 30 S 26 S 1 MDSD 22 S 31 S 30 S 14 R 25 S 1 MDSE 20 S 20 S 30 S 30 S 30 S 0 JDSA 12 R 30 S 24 S 21 I 23 S 1 JDSB 11 R 30 S 26 S 23 S 22 S 1 JDSC 15 S 25 S 21 S 21 I 20 S 0 JDSD 16 S 28 S 20 S 21 I 25 S 0 JDSE 12 R 30 S 26 S 23 S 26 S 1 TET: Tetracycline 30 μg; AMC: Amoxicillin Clavulanate 20 μg; CIP: Ciprofloxacin 5μg; ATM: Aztreonam 30 μg; CXM: Cefuroxime 30 μg: IMP: Imipenem 10μg; GN: Gentamicin 10 μg; R: Resistant; I: Intermediate S: Susceptible 144 University of Ghana http://ugspace.ug.edu.gh Biochemical and physical confirmatory tests for E. coli isolates BIOCHEMICAL TEST AND PHYSICAL TESTS ISOLATE ID GRAM ⁻/+ INDOLE TSI SLANT BUTT GAS H2S JBAA ⁻ ⁺ ACID ACID ⁺ ⁻ JBBA ⁻ ⁺ ACID ACID ⁺ ⁻ JBCA ⁻ ⁺ ACID ACID ⁺ ⁻ JBGA ⁻ ⁺ ACID ACID ⁺ ⁻ JBQA ⁻ ⁺ ACID ACID ⁺ ⁻ FBAA ⁻ ⁺ ACID ACID ⁺ ⁻ FBBA ⁻ ⁺ ACID ACID ⁺ ⁻ FBCA ⁻ ⁺ ACID ACID ⁺ ⁻ FBFA ⁻ ⁺ ACID ACID ⁺ ⁻ FBGA ⁻ ⁺ ACID ACID ⁺ ⁻ MBA ⁻ ⁺ ACID ACID ⁺ ⁻ MBB ⁻ ⁺ ACID ACID ⁺ ⁻ MBC ⁻ ⁺ ACID ACID ⁺ ⁻ MBD ⁻ ⁺ ACID ACID ⁺ ⁻ MBE ⁻ ⁺ ACID ACID ⁺ ⁻ ABA ⁻ ⁺ ACID ACID ⁺ ⁻ ABB ⁻ ⁺ ACID ACID ⁺ ⁻ ABC ⁻ ⁺ ACID ACID ⁺ ⁻ ABD ⁻ ⁺ ACID ACID ⁺ ⁻ ABE ⁻ ⁺ ACID ACID ⁺ ⁻ MBA ⁻ ⁺ ACID ACID ⁺ ⁻ MBB ⁻ ⁺ ACID ACID ⁺ ⁻ MBC ⁻ ⁺ ACID ACID ⁺ ⁻ 145 University of Ghana http://ugspace.ug.edu.gh MBD ⁻ ⁺ ACID ACID ⁺ ⁻ MBE ⁻ ⁺ ACID ACID ⁺ ⁻ JBAA ⁻ ⁺ ACID ACID ⁺ ⁻ JBBA ⁻ ⁺ ACID ACID ⁺ ⁻ JBCA ⁻ ⁺ ACID ACID ⁺ ⁻ JBDA ⁻ ⁺ ACID ACID ⁺ ⁻ JBEA ⁻ ⁺ ACID ACID ⁺ ⁻ JAA ⁻ ⁺ ACID ACID ⁺ ⁻ JAB ⁻ ⁺ ACID ACID ⁺ ⁻ JAC ⁻ ⁺ ACID ACID ⁺ ⁻ FAA ⁻ ⁺ ACID ACID ⁺ ⁻ FAB ⁻ ⁺ ACID ACID ⁺ ⁻ FAC ⁻ ⁺ ACID ACID ⁺ ⁻ AAE ⁻ ⁺ ACID ACID ⁺ ⁻ JAD ⁻ ⁺ ACID ACID ⁺ ⁻ FAB ⁻ ⁺ ACID ACID ⁺ ⁻ FAD ⁻ ⁺ ACID ACID ⁺ ⁻ MAA ⁻ ⁺ ACID ACID ⁺ ⁻ MAB ⁻ ⁺ ACID ACID ⁺ ⁻ MAC ⁻ ⁺ ACID ACID ⁺ ⁻ MAD ⁻ ⁺ ACID ACID ⁺ ⁻ MAE ⁻ ⁺ ACID ACID ⁺ ⁻ AAA ⁻ ⁺ ACID ACID ⁺ ⁻ AAB ⁻ ⁺ ACID ACID ⁺ ⁻ AAC ⁻ ⁺ ACID ACID ⁺ ⁻ AAD ⁻ ⁺ ACID ACID ⁺ ⁻ MAA ⁻ ⁺ ACID ACID ⁺ ⁻ MAB ⁻ ⁺ ACID ACID ⁺ ⁻ MAC ⁻ ⁺ ACID ACID ⁺ ⁻ 146 University of Ghana http://ugspace.ug.edu.gh MAD ⁻ ⁺ ACID ACID ⁺ ⁻ MAE ⁻ ⁺ ACID ACID ⁺ ⁻ JAA ⁻ ⁺ ACID ACID ⁺ ⁻ JAB ⁻ ⁺ ACID ACID ⁺ ⁻ JAC ⁻ ⁺ ACID ACID ⁺ ⁻ JAD ⁻ ⁺ ACID ACID ⁺ ⁻ JAE ⁻ ⁺ ACID ACID ⁺ ⁻ JAE ⁻ ⁺ ACID ACID ⁺ ⁻ JOAA ⁻ ⁺ ACID ACID ⁺ ⁻ JOBA ⁻ ⁺ ACID ACID ⁺ ⁻ JOCA ⁻ ⁺ ACID ACID ⁺ ⁻ JOD ⁻ ⁺ ACID ACID ⁺ ⁻ JOE ⁻ ⁺ ACID ACID ⁺ ⁻ FOA ⁻ ⁺ ACID ACID ⁺ ⁻ FOB ⁻ ⁺ ACID ACID ⁺ ⁻ FOC ⁻ ⁺ ACID ACID ⁺ ⁻ FOD ⁻ ⁺ ACID ACID ⁺ ⁻ FOE ⁻ ⁺ ACID ACID ⁺ ⁻ MOA ⁻ ⁺ ACID ACID ⁺ ⁻ MOB ⁻ ⁺ ACID ACID ⁺ ⁻ MOC ⁻ ⁺ ACID ACID ⁺ ⁻ MOD ⁻ ⁺ ACID ACID ⁺ ⁻ MOE ⁻ ⁺ ACID ACID ⁺ ⁻ AOA ⁻ ⁺ ACID ACID ⁺ ⁻ AOB ⁻ ⁺ ACID ACID ⁺ ⁻ AOC ⁻ ⁺ ACID ACID ⁺ ⁻ AOD ⁻ ⁺ ACID ACID ⁺ ⁻ AOE ⁻ ⁺ ACID ACID ⁺ ⁻ MOA ⁻ ⁺ ACID ACID ⁺ ⁻ 147 University of Ghana http://ugspace.ug.edu.gh MOB ⁻ ⁺ ACID ACID ⁺ ⁻ MOC ⁻ ⁺ ACID ACID ⁺ ⁻ MOD ⁻ ⁺ ACID ACID ⁺ ⁻ MOE ⁻ ⁺ ACID ACID ⁺ ⁻ JOA ⁻ ⁺ ACID ACID ⁺ ⁻ JOB ⁻ ⁺ ACID ACID ⁺ ⁻ JOC ⁻ ⁺ ACID ACID ⁺ ⁻ JOD ⁻ ⁺ ACID ACID ⁺ ⁻ JOE ⁻ ⁺ ACID ACID ⁺ ⁻ JUAA ⁻ ⁺ ACID ACID ⁺ ⁻ JUBA ⁻ ⁺ ACID ACID ⁺ ⁻ JUCA ⁻ ⁺ ACID ACID ⁺ ⁻ JUD ⁻ ⁺ ACID ACID ⁺ ⁻ JUE ⁻ ⁺ ACID ACID ⁺ ⁻ FUA ⁻ ⁺ ACID ACID ⁺ ⁻ FUB ⁻ ⁺ ACID ACID ⁺ ⁻ FUC ⁻ ⁺ ACID ACID ⁺ ⁻ FUD ⁻ ⁺ ACID ACID ⁺ ⁻ FUE ⁻ ⁺ ACID ACID ⁺ ⁻ MUA ⁻ ⁺ ACID ACID ⁺ ⁻ MUB ⁻ ⁺ ACID ACID ⁺ ⁻ MUC ⁻ ⁺ ACID ACID ⁺ ⁻ MUD ⁻ ⁺ ACID ACID ⁺ ⁻ MUE ⁻ ⁺ ACID ACID ⁺ ⁻ AUA ⁻ ⁺ ACID ACID ⁺ ⁻ AUB ⁻ ⁺ ACID ACID ⁺ ⁻ AUC ⁻ ⁺ ACID ACID ⁺ ⁻ AUD ⁻ ⁺ ACID ACID ⁺ ⁻ AUE ⁻ ⁺ ACID ACID ⁺ ⁻ 148 University of Ghana http://ugspace.ug.edu.gh MUA ⁻ ⁺ ACID ACID ⁺ ⁻ MUB ⁻ ⁺ ACID ACID ⁺ ⁻ MUC ⁻ ⁺ ACID ACID ⁺ ⁻ MUD ⁻ ⁺ ACID ACID ⁺ ⁻ MUE ⁻ ⁺ ACID ACID ⁺ ⁻ JUAA ⁻ ⁺ ACID ACID ⁺ ⁻ JUBA ⁻ ⁺ ACID ACID ⁺ ⁻ JUCA ⁻ ⁺ ACID ACID ⁺ ⁻ JUD ⁻ ⁺ ACID ACID ⁺ ⁻ JUE ⁻ ⁺ ACID ACID ⁺ ⁻ JDA ⁻ ⁺ ACID ACID ⁺ ⁻ JDB ⁻ ⁺ ACID ACID ⁺ ⁻ JDC ⁻ ⁺ ACID ACID ⁺ ⁻ JDD ⁻ ⁺ ACID ACID ⁺ ⁻ JDE ⁻ ⁺ ACID ACID ⁺ ⁻ FDA ⁻ ⁺ ACID ACID ⁺ ⁻ FDB ⁻ ⁺ ACID ACID ⁺ ⁻ FDC ⁻ ⁺ ACID ACID ⁺ ⁻ FDD ⁻ ⁺ ACID ACID ⁺ ⁻ FDE ⁻ ⁺ ACID ACID ⁺ ⁻ MDA ⁻ ⁺ ACID ACID ⁺ ⁻ MDB ⁻ ⁺ ACID ACID ⁺ ⁻ MDC ⁻ ⁺ ACID ACID ⁺ ⁻ MDD ⁻ ⁺ ACID ACID ⁺ ⁻ MDE ⁻ ⁺ ACID ACID ⁺ ⁻ ADA ⁻ ⁺ ACID ACID ⁺ ⁻ ADB ⁻ ⁺ ACID ACID ⁺ ⁻ ADC ⁻ ⁺ ACID ACID ⁺ ⁻ ADD ⁻ ⁺ ACID ACID ⁺ ⁻ 149 University of Ghana http://ugspace.ug.edu.gh ADE ⁻ ⁺ ACID ACID ⁺ ⁻ MDA ⁻ ⁺ ACID ACID ⁺ ⁻ MDB ⁻ ⁺ ACID ACID ⁺ ⁻ MDC ⁻ ⁺ ACID ACID ⁺ ⁻ MDD ⁻ ⁺ ACID ACID ⁺ ⁻ MDE ⁻ ⁺ ACID ACID ⁺ ⁻ JDA ⁻ ⁺ ACID ACID ⁺ ⁻ JDB ⁻ ⁺ ACID ACID ⁺ ⁻ JDC ⁻ ⁺ ACID ACID ⁺ ⁻ JDD ⁻ ⁺ ACID ACID ⁺ ⁻ JDE ⁻ ⁺ ACID ACID ⁺ ⁻ Biochemical and physical confirmatory tests for Aeromonas hydrophila BIOCHEMICAL TEST AND PHYSICAL TESTS ISOLATE ID GRAM -/+ MOTILIY CITRATE GLUCOSE OXIDASE JBAA ⁻ + + + + JBBA ⁻ + + + + JBCA ⁻ + + + + JBGA ⁻ + + + + JBQA ⁻ + + + + FBAA ⁻ + + + + FBBA ⁻ + + + + FBCA ⁻ + + + + FBFA ⁻ + + + + FBGA ⁻ + + + + MBA ⁻ + + + + MBB ⁻ + + + + MBC ⁻ + + + + MBD ⁻ + + + + 150 University of Ghana http://ugspace.ug.edu.gh MBE ⁻ + + + + ABA ⁻ + + + + ABB ⁻ + + + + ABC ⁻ + + + + ABD ⁻ + + + + ABE ⁻ + + + + MBA ⁻ + + + + MBB ⁻ + + + + MBC ⁻ + + + + MBD ⁻ + + + + MBE ⁻ + + + + JBAA ⁻ + + + + JBBA ⁻ + + + + JBCA ⁻ + + + + JBDA ⁻ + + + + JBEA ⁻ + + + + JAA ⁻ + + + + JAB ⁻ + + + + JAC ⁻ + + + + FAA ⁻ + + + + FAB ⁻ + + + + FAC ⁻ + + + + AAE ⁻ + + + + JAD ⁻ + + + + FAB ⁻ + + + + FAD ⁻ + + + + MAA ⁻ + + + + MAB ⁻ + + + + MAC ⁻ + + + + 151 University of Ghana http://ugspace.ug.edu.gh MAD ⁻ + + + + MAE ⁻ + + + + AAA ⁻ + + + + AAB ⁻ + + + + AAC ⁻ + + + + AAD ⁻ + + + + MAA ⁻ + + + + MAB ⁻ + + + + MAC ⁻ + + + + MAD ⁻ + + + + MAE ⁻ + + + + JAA ⁻ + + + + JAB ⁻ + + + + JAC ⁻ + + + + JAD ⁻ + + + + JAE ⁻ + + + + JAE ⁻ + + + + JOAA ⁻ + + + + JOBA ⁻ + + + + JOCA ⁻ + + + + JOD ⁻ + + + + JOE ⁻ + + + + FOA ⁻ + + + + FOB ⁻ + + + + FOC ⁻ + + + + FOD ⁻ + + + + FOE ⁻ + + + + MOA ⁻ + + + + MOB ⁻ + + + + 152 University of Ghana http://ugspace.ug.edu.gh MOC ⁻ + + + + MOD ⁻ + + + + MOE ⁻ + + + + AOA ⁻ + + + + AOB ⁻ + + + + AOC ⁻ + + + + AOD ⁻ + + + + AOE ⁻ + + + + MOA ⁻ + + + + MOB ⁻ + + + + MOC ⁻ + + + + MOD ⁻ + + + + MOE ⁻ + + + + JOA ⁻ + + + + JOB ⁻ + + + + JOC ⁻ + + + + JOD ⁻ + + + + JOE ⁻ + + + + JUAA ⁻ + + + + JUBA ⁻ + + + + JUCA ⁻ + + + + JUD ⁻ + + + + JUE ⁻ + + + + FUA ⁻ + + + + FUB ⁻ + + + + FUC ⁻ + + + + FUD ⁻ + + + + FUE ⁻ + + + + MUA ⁻ + + + + 153 University of Ghana http://ugspace.ug.edu.gh MUB ⁻ + + + + MUC ⁻ + + + + MUD ⁻ + + + + MUE ⁻ + + + + AUA ⁻ + + + + AUB ⁻ + + + + AUC ⁻ + + + + AUD ⁻ + + + + AUE ⁻ + + + + MUA ⁻ + + + + MUB ⁻ + + + + MUC ⁻ + + + + MUD ⁻ + + + + MUE ⁻ + + + + JUAA ⁻ + + + + JUBA ⁻ + + + + JUCA ⁻ + + + + JUD ⁻ + + + + JUE ⁻ + + + + JDA ⁻ + + + + JDB ⁻ + + + + JDC ⁻ + + + + JDD ⁻ + + + + JDE ⁻ + + + + FDA ⁻ + + + + FDB ⁻ + + + + FDC ⁻ + + + + FDD ⁻ + + + + FDE ⁻ + + + + 154 University of Ghana http://ugspace.ug.edu.gh MDA ⁻ + + + + MDB ⁻ + + + + MDC ⁻ + + + + MDD ⁻ + + + + MDE ⁻ + + + + ADA ⁻ + + + + ADB ⁻ + + + + ADC ⁻ + + + + ADD ⁻ + + + + ADE ⁻ + + + + MDA ⁻ + + + + MDB ⁻ + + + + MDC ⁻ + + + + MDD ⁻ + + + + MDE ⁻ + + + + JDA ⁻ + + + + JDB ⁻ + + + + JDC ⁻ + + + + JDD ⁻ + + + + JDE ⁻ + + + + 155 University of Ghana http://ugspace.ug.edu.gh Biochemical and physical confirmatory tests for P. aeruginosa isolates BIOCHEMICAL TEST AND PHYSICAL TESTS ISOLATE ID GRAM -/+ OXIDASE CATALASE JBAA ⁻ + + JBBA ⁻ + + JBCA ⁻ + + JBGA ⁻ + + JBQA ⁻ + + FBAA ⁻ + + FBBA ⁻ + + FBCA ⁻ + + FBFA ⁻ + + FBGA ⁻ + + MBA ⁻ + + MBB ⁻ + + MBC ⁻ + + MBD ⁻ + + MBE ⁻ + + ABA ⁻ + + ABB ⁻ + + ABC ⁻ + + 156 University of Ghana http://ugspace.ug.edu.gh ABD ⁻ + + ABE ⁻ + + MBA ⁻ + + MBB ⁻ + + MBC ⁻ + + MBD ⁻ + + MBE ⁻ + + JBAA ⁻ + + JBBA ⁻ + + JBCA ⁻ + + JBDA ⁻ + + JBEA ⁻ + + JAA ⁻ + + JAB ⁻ + + JAC ⁻ + + FAA ⁻ + + FAB ⁻ + + FAC ⁻ + + AAE ⁻ + + JAD ⁻ + + FAB ⁻ + + FAD ⁻ + + 157 University of Ghana http://ugspace.ug.edu.gh MAA ⁻ + + MAB ⁻ + + MAC ⁻ + + MAD ⁻ + + MAE ⁻ + + AAA ⁻ + + AAB ⁻ + + AAC ⁻ + + AAD ⁻ + + MAA ⁻ + + MAB ⁻ + + MAC ⁻ + + MAD ⁻ + + MAE ⁻ + + JAA ⁻ + + JAB ⁻ + + JAC ⁻ + + JAD ⁻ + + JAE ⁻ + + JAE ⁻ + + JOAA ⁻ + + JOBA ⁻ + + 158 University of Ghana http://ugspace.ug.edu.gh JOCA ⁻ + + JOD ⁻ + + JOE ⁻ + + FOA ⁻ + + FOB ⁻ + + FOC ⁻ + + FOD ⁻ + + FOE ⁻ + + MOA ⁻ + + MOB ⁻ + + MOC ⁻ + + MOD ⁻ + + MOE ⁻ + + AOA ⁻ + + AOB ⁻ + + AOC ⁻ + + AOD ⁻ + + AOE ⁻ + + MOA ⁻ + + MOB ⁻ + + MOC ⁻ + + MOD ⁻ + + 159 University of Ghana http://ugspace.ug.edu.gh MOE ⁻ + + JOA ⁻ + + JOB ⁻ + + JOC ⁻ + + JOD ⁻ + + JOE ⁻ + + JUAA ⁻ + + JUBA ⁻ + + JUCA ⁻ + + JUD ⁻ + + JUE ⁻ + + FUA ⁻ + + FUB ⁻ + + FUC ⁻ + + FUD ⁻ + + FUE ⁻ + + MUA ⁻ + + MUB ⁻ + + MUC ⁻ + + MUD ⁻ + + MUE ⁻ + + AUA ⁻ + + 160 University of Ghana http://ugspace.ug.edu.gh AUB ⁻ + + AUC ⁻ + + AUD ⁻ + + AUE ⁻ + + MUA ⁻ + + MUB ⁻ + + MUC ⁻ + + MUD ⁻ + + MUE ⁻ + + JUAA ⁻ + + JUBA ⁻ + + JUCA ⁻ + + JUD ⁻ + + JUE ⁻ + + JDA ⁻ + + JDB ⁻ + + JDC ⁻ + + JDD ⁻ + + JDE ⁻ + + FDA ⁻ + + FDB ⁻ + + FDC ⁻ + + 161 University of Ghana http://ugspace.ug.edu.gh FDD ⁻ + + FDE ⁻ + + MDA ⁻ + + MDB ⁻ + + MDC ⁻ + + MDD ⁻ + + MDE ⁻ + + ADA ⁻ + + ADB ⁻ + + ADC ⁻ + + ADD ⁻ + + ADE ⁻ + + MDA ⁻ + + MDB ⁻ + + MDC ⁻ + + MDD ⁻ + + MDE ⁻ + + JDA ⁻ + + JDB ⁻ + + JDC ⁻ + + JDD ⁻ + + JDE ⁻ + + 162 University of Ghana http://ugspace.ug.edu.gh 163