University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES NASOPHARYNGEAL CARRIAGE AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF STAPHYLOCOCCUS AUREUS AMONG CHILDREN UNDER FIVE YEARS IN ACCRA BY MARY-MAGDALENE OSEI (10599052) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN MICROBIOLOGY JULY, 2018 University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Mary-Magdalene Osei, declare that the work presented in this thesis is the result of my own research work carried out in the Research Laboratory of the Department of Medical Microbiology, School of Biomedical and Allied Health Sciences (SBAHS), Korle-Bu, under the supervision of Dr. Nicholas T.K.D. Dayie (SBAHS) and Dr. Japheth A. Opintan (SBAHS) and that all references cited in this work have been duly acknowledged. Sign………………………………………… Date…………………………………… Mary-Magdalene Osei (Student) Sign………………………………………... Date……………………………………. Dr. Nicholas T.K.D. Dayie (Main Supervisor) Sign………………………………………. Date……………………………………. Dr. Japheth A. Opintan (Co-Supervisor) i University of Ghana http://ugspace.ug.edu.gh DEDICATION Firstly, to the Almighty God, the source of all abundant blessings for His endless love Secondly, to my beloved family Mr. Anthony Kwaku Osei (Father- deceased) Miss Edith Akutor Klu (Mother) Mr. Joseph Clamas Kwasi Osei (Brother) Mrs. Elizabeth Adwoa Sakyibea Osei-Owusu (Sister) ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I would like to acknowledge my supervisors, Dr. Nicholas T.K.D Dayie and Dr. Japheth A. Opintan, both Senior Lecturers at the Department of Medical Microbiology, School of Biomedical and Allied Health Science (SBAHS), University of Ghana (UG), who have been selflessly involved with editing the scripts and their guidance in several ways. 1 would also like to acknowledge the kind efforts of Dr. Simon Kwaku Attah (SBAHS, UG), and Dr. Appiah-Korang Labi (Korle-Bu Teaching Hospital) for their assistance in editing the scripts and their inspiration. I wish to appreciate Dr. Kwamena Sagoe (Head, Department of Medical Microbiology), Mrs. Evelyn Dzomeku (Senior Administrative Assistant) and Mrs. Emelia Nettey (Administrative Assistant) for their encouragement in diverse ways. Funding of this work was obtained from ORID (University of Ghana, Legon), through the effort of Dr. Nicholas T.K.D. Dayie and the Health Associated Infection (HAI) Ghana Project through the effort of Dr. Japheth A. Opintan. Furthermore, I wish to acknowledge the assistance received from Miss Elizabeth Y. Tettey, Mrs. Georgina Tetteh-Ocloo, Mr. Prince Pappoe, Miss Marjorie Quashie and Mr. Derrick Opoku at various stages of the project. Finally, I would like to appreciate the role of my family, for standing by me in prayers and in many other ways. May the Almighty God richly bless you all. iii University of Ghana http://ugspace.ug.edu.gh ABSTRACT BACKGROUND: Staphylococcus aureus (S. aureus), although found predominantly in the anterior nares of the human population, can also colonise the nasopharynx. There are concerns regarding a potential increase in S. aureus carriage and disease after the introduction of the Pneumococcal Conjugate Vaccines (PCVs) in Africa. Hence, the need for a better understanding of the nasopharyngeal carriage prevalence of S. aureus, antimicrobial resistance profile and possible virulence genes that may assist the organism to be more infectious. AIM: To investigate S. aureus and methicillin resistant S. aureus (MRSA) nasopharyngeal carriage among children under five years in the conjugate vaccine era in Accra. METHOD: This cross-sectional study used 410 archived nasopharyngeal swab samples collected from September to December 2016 from children under five years old from selected schools in the Accra Metropolis. In this current study, S. aureus was isolated and identified using standard bacteriological methods. Further characterisation such as antimicrobial susceptibility testing (AST) according to 2017 Clinical and Laboratory Standard guidelines (CLSI), mecA and LukF- PV (Panton-Valentine Leucocidin (PVL) genes were screened by polymerase chain reaction (PCR). Data on resistance of S. aureus to standard antibiotics were generated and analysed using descriptive statistics. RESULTS: The predominant bacteria isolated were Coagulase Negative Staphylococci representing 47.3% (194/410), followed by S. aureus, 23.2% (95/410); Diphtheriodes, 5.4% (22/410); Micrococcus species, 3.7% (15/410); Klebsiella pneumoniae, 3.2% (13/410); Moraxella species and Citrobacter species had 1.5% (6/410) each; Escherichia coli, Enterobacter species and Pseudomonas species had 0.9% (2/410). The mecA mediated MRSA carriage prevalence was 2.1% (2/95) and it was found in females aged 36 months (3 years) and 52 months (4 years) old. Females recorded a higher carriage prevalence in comparison to males, but this difference was not statistically significant [51.6% (49/95) vs. 48.8% (46/95), p=0.533]. Of all the age groups, individuals in the age range of 37-48 months (3.1-4.0 years) recorded the highest S. aureus carriage prevalence of 32.6% (31/95). Resistance of S. aureus isolates to various antibiotics were as follows: penicillin, 97.9% (93/95); amoxiclav, 20% (19/95); tetracycline, 18.9% (18/95); iv University of Ghana http://ugspace.ug.edu.gh erythromycin, 5.3% (5/95); ciprofloxacin, 2.1% (2/95) and gentamicin, 1.1% (1/95). None of the isolates were resistant to cotrimoxazole, clindamycin, linezolid, and teicoplanin. No inducible clindamycin resistance was observed with erythromycin resistance strains. Three {3.2% (3/95)} of the isolates were multidrug resistant (MDR), of which 66.7% (2/3) was MRSA and 33.3% (1/3) was Methicillin Susceptible S. aureus (MSSA). Panton-Valentine Leucocidin (LukF-PV) gene was only associated with 58% (55/95) of the MSSA. CONCLUSION: It is concluded that, a considerable proportion of school children under five years sampled from Accra harboured S. aureus in the nasopharynx and a low prevalence of mecA gene mediated MRSA. Proper antibiotic stewardship programmes are critical to limit the clinical impact of MRSA. Penicillin non-susceptibility and resistance to other beta-lactam antimicrobials were observed. Susceptibility to cotrimoxazole, clindamycin, linezolid and teicoplanin were observed coupled with low multidrug resistance among the isolates. High nasopharyngeal carriage prevalence of MSSA harbouring LukF-PV mediated PVL gene may serve as a major risk of contracting severe infection. It is recommended that holistic health intervention targeting MSSA may be required during pneumococcal vaccination campaigns. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT DECLARATION ............................................................................................................................. i DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENT ............................................................................................................. iii ABSTRACT ................................................................................................................................... iv TABLE OF CONTENT ................................................................................................................. vi LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................................... xii CHAPTER ONE ............................................................................................................................. 1 1.0 INTRODUCTION .................................................................................................................... 1 1.1 Background .......................................................................................................................... 1 1.2 Problem Statement .............................................................................................................. 2 1.3 Rationale of the study ......................................................................................................... 3 1.4 Aim of the study................................................................................................................... 4 1.5 Specific Objectives of the study.......................................................................................... 4 CHAPTER TWO ............................................................................................................................ 6 2.0 LITERATURE REVIEW ......................................................................................................... 6 2.1 Microbiota of the nasopharynx .......................................................................................... 6 vi University of Ghana http://ugspace.ug.edu.gh 2.2 Nasopharyngeal carriage of Staphylococcus aureus and its Clinical Significance ........ 6 2.3 Staphylococcus aureus and Streptococcus pneumoniae co-colonisation of the Nasopharynx ............................................................................................................................ 10 2.4 Biology and Classification of Staphylococcus aureus ..................................................... 11 2.4.1 Pathogenesis and Virulence Determinants of Staphylococcus aureus.................... 14 2.5 Identification and typing of Staphylococcus aureus ....................................................... 17 2.5.1 Phenotypic typing of Staphylococcus aureus ............................................................ 20 2.5.2 Genotypic typing of Staphylococcus aureus.............................................................. 21 2.6 Antimicrobial Susceptibility Profile of Staphylococcus aureus ..................................... 24 2.6.1 Development of resistance to Penicillinase-resistant penicillins among Staphylococcus aureus in Africa and Ghana ..................................................................... 24 2.6.2 Mechanism of Antibiotic Resistance of Staphylococcus aureus .............................. 27 2.6.3 Effect of some Fluoroquinolones and Cephalosporins on methicillin resistant Staphylococcus aureus ......................................................................................................... 33 2.6.4 Inducible Clindamycin Resistance in Staphylococcus aureus ................................. 34 2.6.5 Factors that influence antimicrobial Resistance ...................................................... 37 CHAPTER THREE ...................................................................................................................... 39 3.0 MATERIALS AND METHODS ....................................................................................... 39 3.1 Study area .......................................................................................................................... 39 3.3 Sampling site ...................................................................................................................... 39 vii University of Ghana http://ugspace.ug.edu.gh 3.4 Sample size determination ................................................................................................ 39 3.5 Study design ....................................................................................................................... 40 3.6 Nasopharyngeal specimen and processing ...................................................................... 41 3.6.1 Identification of bacterial isolates ............................................................................. 41 3.6.2 Antimicrobial Susceptibility Testing ........................................................................ 42 3.7 DNA Extraction for Molecular Screening of Staphylococcus aureus ........................... 46 3.7.1 Multiplex Polymerase Chain Reaction (PCR) screening for MecA, Pvl and Spa genes ...................................................................................................................................... 46 3.8 Statistical Analysis............................................................................................................. 49 3.9 Ethical Approval ............................................................................................................... 49 CHAPTER FOUR ......................................................................................................................... 50 4.0 RESULTS ............................................................................................................................... 50 4.1 Demographics of the Study Population ........................................................................... 50 4.2 Nasopharyngeal Staphylococcus aureus carriage ........................................................... 50 4.3 Antimicrobial susceptibility profile of the Staphylococcus aureus isolates .................. 54 4.4 Multidrug Resistance ........................................................................................................ 60 4.5 Molecular screening results .............................................................................................. 60 CHAPTER FIVE .......................................................................................................................... 61 5.0 DISCUSSION, CONCLUSIONS, LIMITATION AND RECOMMENDATIONS .............. 61 5.1 Discussion ........................................................................................................................... 61 viii University of Ghana http://ugspace.ug.edu.gh 5.2 Conclusions ........................................................................................................................ 65 5.3 Limitations ......................................................................................................................... 65 5.4 Recommendations ............................................................................................................. 66 REFERENCES ............................................................................................................................. 67 APPENDIX I ................................................................................................................................ 96 DNA Extraction ....................................................................................................................... 96 APPENDIX II ............................................................................................................................... 98 Gel Preparation (2% Agarose Gel) and Electrophoresis .................................................... 98 APPENDIX III ............................................................................................................................ 100 Polymerase Chain Reaction (PCR) Master Mix Preparation ........................................... 100 APPENDIX 1V ........................................................................................................................... 102 Gels ......................................................................................................................................... 102 ix University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Phenotypic methods for differentiation of Staphylococcus aureus from Coagulase Negative Staphylococcus. ............................................................................................................. 13 Table 2: Comparison of typing methods for characterising Staphylococcus aureus. .................. 19 Table 3: Prevalence of beta-lactams and non-beta-lactams antimicrobial resistance profile of S. aureus in Africa. ........................................................................................................................... 26 Table 4: Mode of action and antimicrobial resistance mechanisms of Beta-Lactams and non-Beta- Lactam antimicrobials in Staphylococcus aureus ......................................................................... 29 Table 5: Phenotypic groupings and their features of D-test......................................................... 45 Table 6: Primers used for the Multiplex PCR in the detection of Spa, Pvl and mecA genes. ...... 48 Table 7: Bacterial pathogens isolated from the nasopharyngeal swab samples. ......................... 52 Table 8: Carriage prevalence of Staphylococcus aureus by age group in children ≤5years attending nursery and kindergarten facilities in Accra. ................................................................................ 53 Table 9: Antimicrobial susceptibility profile of Staphylococcus aureus isolates. ...................... 55 Table 10: Antimicrobial resistance profile of S. aureus (MRSA) and (MSSA) .......................... 56 Table 11: Susceptibility profile of phenotypic Double Disc Diffusion Test (D-test) among erythromycin resistance strains. .................................................................................................... 58 Table 12: Assessment of erythromycin resistance among MRSA and MSSA. ........................... 59 Table 13: components with their various concentrations and volumes used to prepare the master mix .............................................................................................................................................. 100 Table 14: Thermocycling conditions for the PCR ..................................................................... 101 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Flowchart indicating the laboratory test used to identify S. aureus. ............................ 18 Figure 2: Phenotypic clindamycin resistance in S. aureus. ......................................................... 36 Figure 3: A picture of the Researcher on the bench. .................................................................... 42 Figure 4: A picture of the Becton Dickinson Phoenix SpecTM Nephelometer used. ................... 43 Figure 5: Agarose gel electrophoresis pattern for amplification product of Spa, mecA and Pvl genes using multiplex PCR. .......................................................................................................... 47 Figure 6: Agarose gel electrophoresis showing the patterns of mecA, Pvl and Spa genes from a multiples PCR. ............................................................................................................................ 102 Figure 7: Agarose gel electrophoresis showing the patterns of Pvl and Spa genes from a multiplex PCR. ............................................................................................................................................ 102 xi University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS ABR Antibiotic resistance AMC Amoxiclav AMR Antimicrobial Resistance AST Antimicrobial Susceptibility Test ATCC American type culture collection β Beta BSI Blood stream infection CC Clindamycin CDC Center for Disease Control cgMLST core genome Multilocus Sequencing typing CIP Ciprofloxacin CLSI Clinical and Laboratory Standard Guidelines COT Co-trimoxazole DNA Deoxynucleic acid DNase Deoxyribonuclease D-test Double disc diffusion test E Erythromycin erm gene associated with MLSB FOX Cefoxitin GEN Gentamicin HAI Health associated infection H2O water H2O2 Hydrogen peroxide HIV Human immune virus ICU Intensive care unit L Lincosamide xii University of Ghana http://ugspace.ug.edu.gh LukF-PV gene associated with Panton-Valentine Leucocidin M Macrolide MecA gene associated with MRSA MecC gene associated with MRSA MDR Multidrug resistance MHA Muller Hinton agar MLS Macrolide Lincosamide Streptogramin MLSB Macrolide Lincosamide Streptogramin B MLST Multilocus Sequencing typing MOH Ministry of Health MRSA Methicillin Resistance Staphylococcus aureus CA-MRSA Community-acquired Methicillin Resistance Staphylococcus aureus MS Macrolide Streptogramin MSSA Methicillin Susceptible Staphylococcus aureus msr gene associated with efflux pump of macrolide n Number NGS Next Generation Sequencing NP Nasopharyngeal NPS Nasopharyngeal swab ORID Office of Research, Innovation and Development OF Oxidative Fermentation PBPs (2a) Penicillin-Binding Protein (2a) PBS Phosphate buffered saline PCR Polymerase chain reaction PCV Pneumococcal Conjugate Vaccine PEN Penicillin G PFGE Pulsed-field gel electrophoresis PVL Panton-Valentine Leucocidin xiii University of Ghana http://ugspace.ug.edu.gh PYR Pyrolidonylaminopeptidase RNA Ribonucleic acid tRNA amino-acyl transferase S Streptogramin SBAHS School of Biochemical and Allied Health Sciences SDS-PAGE Sodium dodecyl-sulphate polyacrylamide Spa Staphylococcal Protein A SPSS Statistical package for the social sciences SLV Single locus variant SCCmec Staphylococcal cassette chromosome mec ST Sequence type STGG Skim milk glucose glycerol TBE Tris/Borate/EDTA TET Tetracycline Tet M and Tet O genes associated with Tetracycline resistance THF Tetrahydrofolic acid TSST-1 Toxic Shock Syndrome Toxin 1 µm/µl micrometer/microliter UG University of Ghana VP Voges Proskauer WGS Whole genome sequencing WHO World Health Organisation xiv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Bacterial colonisation in young children with developing immune systems may cause many childhood diseases. Transmission and carriage prevalence of S. aureus have been reported in populations where people live in close contact with one another, for example, children in schools (Thapa et al., 2017). The human nasopharynx and oropharynx are colonised by a wide array of microorganisms from commensal bacteria to potential pathogens and higher risk of transmission of these pathogens are among children (Thapa et al., 2017). Examples of these organisms are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus which are usually commensals but occasionally can cause respiratory or invasive infectious diseases (Leiberman et al., 1999). Staphylococcus aureus (S. aureus), although found predominantly in the anterior nares of approximately 20–80% of the human population, can also colonise the nasopharynx often in biofilms, which serve as a reservoir for local and invasive disease (Wertheim et al., 2005; van den Bergh et al., 2012; Bosch et al., 2013; Brown et al., 2014a; Esposito et al., 2014). Nasopharyngeal colonization of S. aureus has been identified as the most important risk factor for subsequent invasive infections among children with young immune systems (Brown et al., 2014a; Reddinger et al., 2018). Those with underlying illnesses and healthy persons are at risk of obtaining diverse endocarditis, osteomyelitis, meningitis, skin and soft tissue infections, bacteremia, and pneumonia due to S. aureus (Assefa et al., 2013). An estimated 20- 30% of humans are potential carriers of S. aureus in the nasopharynx , though carriage prevalence differ with geographic setting, age, sex and seasonality (Bojang et al., 2017). In current years, the epidemiology of S. aureus infection has been altered with methicillin resistant S. aureus (MRSA) 1 University of Ghana http://ugspace.ug.edu.gh being progressively recognized in the community settings (Grundmann et al., 2010a). In view of this, researchers globally are concerned with the understanding of the genetic relatedness of the pathogen across diverse geographic provinces and care settings (Veenhoven et al., 2003; Grundmann et al., 2010a). Staphylococcus aureus and Streptococcus pneumoniae (S. pneumoniae) are mutual colonisers of the upper respiratory tract and can cause invasive disease (Brogden et al., 2005; Pettigrew et al., 2008; Xu et al., 2012; Thevaranjan et al., 2016). However, the introduction of pneumococcal conjugate vaccines (PCVs) has been found to alter the microbial flora in the nasopharynx of vaccinated persons and their associates (Davis et al., 2013; Shak et al., 2013). Some studies suggest a reverse relationship between Staphylococcus aureus and Streptococcus pneumoniae in the nasopharynx and furthermore, suggested that S. aureus might be replacing vaccine pneumococcal serotypes as the dominant nasopharyngeal coloniser post PCV introduction (Bogaert et al., 2004; Regev-Yochay et al., 2004; Quintero et al., 2011; Huang and Chen, 2015). This has heightened the fear that, there will be a noticeable upsurge in S. aureus disease after the introduction of PCVs in Africa (Nzenze, 2015). 1.2 Problem Statement S. aureus is one of the main pathogens linked with hospitalization, and causes high morbidity and mortality among children under five in third world countries (Assefa et al., 2013). Studies have shown that S. aureus colonisation increases the menace of S. aureus infection, and indeed carriage of S. aureus in the nasopharynx is related to subsequent S. aureus infection (Wertheim et al., 2004). A variant of S. aureus, methicillin resistant S. aureus (MRSA), accounts for a substantial amount of S. aureus infections, projected as high as 74.1% of all hospital acquired and 30.1% of all community developed infections (Song et al., 2017). Methicillin resistant S. aureus is associated 2 University of Ghana http://ugspace.ug.edu.gh with multidrug resistance and is of grave public health importance (van Duin and Paterson, 2016). A recent report estimated that, MRSA caused 80,000 invasive infections and more than 11,000 deaths in 2011 in United States of America (USA) (Dantes et al., 2013). In a recent outbreak of MRSA in Ghana, three neonates lost their lives in the intensive care unit (ICU) of the paediatric ward of the Korle-Bu Teaching Hospital (www.newsghana.com.gh; Retrieved 26th July, 2018). 1.3 Rationale of the study Respiratory tract infections are among the leading causes of death in children and adults worldwide (WHO, 2007). Staphylococcus aureus is a significant source of respiratory tract infections and its carriage in the nasopharynx has been recognized as an imperative risk factor for following invasive infections (Pizzutto et al., 2017). Studies have shown that the inverse correlation between S. pneumoniae and S. aureus is significant for the carriage of vaccine-type S. pneumoniae strains (Wiertsema et al., 2011; Reiss-Mandel and Regev-Yochay, 2016; Navne et al., 2017). This has raised concerns that removal of vaccine type pneumococci from the nasopharynx could facilitate colonization of S. aureus and other respiratory organisms (Wiertsema et al., 2011; Reiss-Mandel and Regev-Yochay, 2016; Navne et al., 2017). Introduction of PCV-13 is associated with notable changes in the flora of the nasopharynx hence there is a need to explain whether these changes are obstinate, and affect the pattern of respiratory and invasive diseases in different geographical regions (Navne et al., 2017). Bogaert et al., (2004) reported that S. aureus-related acute otitis media has been observed in higher proportions among those who are PCV vaccinated and a possible mechanism for the observed changes may be as a result of the dynamic nature of the nasopharyngeal microbial composition, where the balance may be skewed due to a PCV-related 3 University of Ghana http://ugspace.ug.edu.gh clearance of vaccine-type (VT) pneumococci which leaves the nasopharyngeal niche vacant to be occupied by other opportunistic microbes (Weinberger et al., 2011). In May 2012, Ghana introduced PCV 13 in the Expanded Immunization Programme as part of efforts to lessen the problem of pneumococcal infection in children less than five years (Ministry of Health, 2014). The epidemiology of pneumococcal carriage prevalence, before and after PCV 13 in Ghana, has been well described (Denno et al., 2002; Leimkugel et al., 2005; Hauri, 2007; Donkor et al., 2010; Dayie et al., 2013; Donkor E. S., 2013; Donkor et al., 2013). However, little is known about nasopharyngeal carriage, antibiotic susceptibility patterns and occurrence of virulence genes of S. aureus among children under five post PCV 13 vaccination. Since carriage of S. aureus increases the risk of S. aureus infections, it is important to assess the nasopharyngeal carriage prevalence of S. aureus and to monitor if carriage of MRSA bacteria is increasing in the community. In addition, a better understanding of the antibiogram will offer useful information for coherent beneficial and preventive policies. 1.4 Aim of the study To investigate S. aureus and MRSA nasopharyngeal carriage among children under five years in the conjugate vaccine era in Accra. 1.5 Specific Objectives of the study To determine the: 1. prevalence of nasopharyngeal carriage of S. aureus and MRSA among children under five years in Accra. 4 University of Ghana http://ugspace.ug.edu.gh 2. antimicrobial susceptibility profile of S. aureus isolated from children under five years in Accra. 3. occurrence of Panton-Valentine Leucocidin (PVL) virulence genes among S. aureus isolated from children under five years in Accra. 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Microbiota of the nasopharynx In children the nasopharyngeal flora becomes established during the first year of life (Peterson et al., 2016). The most common pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Staphylococcus aureus are normal and transient residents of the nasopharyngeal (NP) niche, where they are embedded in a complex microbiota of generally presumed harmless commensals (Blaser and Falkow, 2009). The human microbiome in general is assumed beneficial to the host due to stimulation and maturation of immune systems, promotion of mucosal structure and function and providing actual ‘colonization resistance’ against pathogen invasion (Blaser and Falkow, 2009). Although colonization by the “potential pathogens” of the NP microbiome is mainly asymptomatic, progression towards upper respiratory tract infections, pneumonia or even sepsis and meningitis may occur (Garcia-Rodriguez, 2002; Bogaert et al., 2004). 2.2 Nasopharyngeal carriage of Staphylococcus aureus and its Clinical Significance Bacterial establishment is thought to be a prerequisite for an individual to become infected, but bacterial colonisation does not normally result in infection (Devine et al., 2015). Therefore, bacterial carriage may be observed to detect before and after changes following the operation of a preventative vaccination approach (Tocheva et al., 2011). Furthermore, carriage of a single or a number of bacteria species can be observed for changes attributed to age, health status, ethnicity, many other environmental factors and geographical location, (Coughtrie et al., 2014; Warnke et 6 University of Ghana http://ugspace.ug.edu.gh al., 2014). Respiratory bacteria can be perceived using relatively non-invasive means such as a nasopharyngeal swab (Gladstone et al., 2012; Satzke et al., 2013) or nose swab, implying that a larger number of patients can be enlisted to strengthen the results of a study. Staphylococcus aureus is a commensally carried Gram-positive bacterium that can act as an opportunistic respiratory pathogen in susceptible individuals. Carriage of S. aureus in the nasopharynx is positively associated with non-invasive and invasive infections, compared to those who do not carry S. aureus (Brown et al., 2014b). Nasopharyngeal colonisation with S. aureus has been identified as a risk factor for subsequent S. aureus bacteraemia in children, adult population and risk groups such as HIV-infected individuals, sickle cell patients and those on chemotherapy (Xu et al., 2012; Van Nguyen et al., 2014). S. aureus nasopharyngeal carriage prevalence is common among pre- school children, with wide genotype variety and noteworthy prevalence of strains producing enterotoxins and exfoliative toxins. Data from the Center for Disease Control and Prevention presented that up to 60 % of all health care related S. aureus infections are due to MRSA (Hidron et al., 2008). In a nosocomial investigation study of blood stream infections (BSI), researchers stated that the second most common BSI pathogen is S. aureus (Wisplinghoff et al., 2004). In 2014, Van Nguyen et al conducted a research in Vietnam, where nasal and nasopharyngeal samples were collected and screened. Staphylococcus aureus prevalence of nose only carriers, throat only carriers, and nose and throat carriers were 88/1016 (8.7%), 141/1016 (13.9%) and 73/1016 (7.2%) respectively. This revealed that there is high carriage of S. aureus in the nasopharynx than nasal. Eighty-five (85) of the participants were children less than five years. Twenty-six (26) samples were positive for S. aureus giving a nasopharyngeal carriage of 30.6%. The research concluded that S. aureus nasopharyngeal carriage is present in about one-third of the 7 University of Ghana http://ugspace.ug.edu.gh northern Vietnamese population, and is more predominant among children. Risk factors for carriage have been identified in the community, both for MSSA and MRSA (Van Nguyen et al., 2014). Patients with no risk factors can acquire community acquired methicillin resistant S. aureus (CA-MRSA) infections. Even though it has been stated that CA-MRSA strains would not persist in the hospital setting due to their susceptibility to agents other than beta-lactams, it is now apparent that CA-MRSA clones have the likelihood of obtaining new resistance traits and may become resistant to other classes of antimicrobial agents through plasmids (Varga et al., 2012). Statistical information from Antimicrobial Investigation Programmes have also stated the growing rates of MRSA among S. aureus isolated from Intensive Care Unit (ICU) patients throughout the world (Jones et al., 2004; Rosenthal et al., 2012). Furthermore, investigators have establish that over the last decade, MRSA strains have surpassed and replaced MSSA strains as the leading cause of staphylococcal infections, which in turn have become more prevalent (Boswihi and Udo, 2018). Of the 830 nasopharyngeal samples collected from healthy pre-school children in Belgium, 286 (34%) samples yielded S. aureus-positive cultures. Staphylococcus aureus carriage rate was shown to be significantly higher among children >4 years of age (Blumental et al., 2013a). In Iran revealed that out of five hundred (500) samples collected from children who had no risk factors for colonization by S. aureus, a total of 148 (29.6%) children were colonized by S. aureus. Out of 260 males, 94 (36.2%) and of 240 females and 54 (22.5%) cases were carriers of S. aureus with a significant association (P value = 0.001). Six (4.1%) of the 148 S. aureus isolated from children were MRSA strains (Iraj Sedighi - Hamedan, et al., 2011). A research was conducted by Tavares et al., (2010) in Portugal, where nasopharyngeal samples were screened among healthy children up to 6 years of age, attending day care centers from the year 2006, 2007 and 2009. Seventeen percent (17%) of the children carried S. aureus where the mean age of the participants was 3.5 8 University of Ghana http://ugspace.ug.edu.gh years and 52.4% were males. Staphylococcus aureus carriage ranged from 13.2% to 21.6% depending on the sampling year and origin of isolates. Carriage of S. aureus was found to be significantly associated with age ranging from 6.3% among children aged less than two years and steadily increasing up to 27.5% among those aged six years. Within the last 20 years, many reports have described the association of such recurrent infections with the occurrence of small colony variants of S. aureus, a special phenotype with attenuated virulence, thereby facilitating intracellular survival and evasion of the immune system (Proctor et al., 2006). The prevalence of S. aureus in the nasopharynx in a study conducted in the Netherlands is quite stable between 20% and 30% in children less than five years of age but increases to 40–50% and remains stable from age 6 to 12 years of age after which it gradually decreases down to approximately 25% at age 18 (Bogaert et al., 2004). In Sub-Saharan Africa, there is a varied multiplicity among S. aureus lineages that colonise and communicate a disease to the population with diverse genetic backgrounds (Schaumburg et al., 2014). A research was conducted in the Gambia where 1264 healthy children were recruited. The overall prevalence of S. aureus nasopharyngeal carriage in these children was 25.9% (Bojang et al., 2017). Subsequently, a study was conducted among children for whom vaccination data were available from rural Gambia at birth and followed up to one year. The overall S. aureus carriage was 30.6% ( Kwambana et al., 2011; Bottomley et al., 2015). Another research conducted in Western Gambia where among newborns nasopharyngeal carriage of S. aureus peaked at day 6 (238 out of 377, 63.1%) (Roca et al., 2017). In Ethiopia where 234 nasopharyngeal samples were collected from healthy young children, 10.3% (24/234) carried S. aureus (Assefa et al., 2013). 9 University of Ghana http://ugspace.ug.edu.gh In Ghana, Sampane-Donkor et al., (2017) reported S. aureus nasopharyngeal carriage of 22% from children <5 years in HIV individuals. Eibach et al., (2017) revealed that out of 544 children screened in Kumasi, carriage prevalence of S. aureus was 22.1%). Adiku et al., (2015) reported that, out of 108 nasopharyngeal samples obtained from children under five years in Accra, 14.8% were S. aureus. Specimens for this research were collected in 2001 before the introduction of the pneumococcal vaccine in Ghana. 2.3 Staphylococcus aureus and Streptococcus pneumoniae co-colonisation of the Nasopharynx Streptococcus pneumoniae produces hydrogen peroxide (H2O2) which has a bactericidal effect in the inhibition of S. aureus and other respiratory pathogens in the nasopharynx (McLeod and Gordon, 1922; Regev-Yochay et al., 2006). Staphylococcus aureus catalase expression contributes to its ability to colonize and survive in the presence of S. pneumoniae (Regev-Yochay et al., 2008). The relationship between S. aureus and S. pneumoniae in the nasopharynx is very complex. The inverse correlation between carriage of S. aureus and carriage of vaccine-type S. pneumoniae in young children has been seen throughout many geographical areas, and through the years, including post-pneumococcus conjugate vaccine implementation (Reiss-Mandel and Regev- Yochay, 2016). Carriage rates of both S. pneumoniae and S. aureus are dynamic. These two strains do not reside alone in the nasopharynx; their presence or absence can be affected by other species and other competitive factors, or external interventions such as vaccination or antibiotic use. Interaction between the two bacteria may be reduced due to vaccination, but the emergence of new strains and the evolution of existing strains makes it difficult to predict the implications (Regev- Yochay et al., 2006; Reiss-Mandel and Regev-Yochay, 2016). Since the mechanism of interaction between the two species is not yet fully understood, it is impossible to predict whether the 10 University of Ghana http://ugspace.ug.edu.gh implementation of newer vaccines will result in further serotype replacement, a rise in S. aureus carriage, or a rise in a different species altogether. Clearly, further studies are required, both on the epidemiological effects of PCV vaccination, and on the mechanisms of this interaction (Regev- Yochay et al., 2008; Reiss-Mandel and Regev-Yochay, 2016). 2.4 Biology and Classification of Staphylococcus aureus Staphylococcus aureus is a Gram-positive bacterium that consist of small spherical (1-2 µm) cocci- shaped cells that tend to be arranged in clusters and are described as “grape-like”. This organism belongs to the Phylum Firmicutes, Class Bacilli, Order Bacillales, and Family Staphylococcaceae. They are basically non-motile, non-spore forming and usually non-capsulated ( Fedtke et al., 2004; Greenwood et al., 2012). On agar media, these organisms can grow in up to 10% salt, circular in size, 2–3 mm in diameter, have smooth and shiny surface as well as opaque colonies (Greenwood et al., 2012; Taylor and Unakal, 2017). These organisms can grow aerobically or anaerobically (facultative anaerobes) at temperatures between 18oC and 40oC. Haemolytic zones are frequently observed around the colonies on 5% sheep blood agar (Taylor and Unakal, 2017). The main distinctive diagnostic features of S. aureus are; the production of an extracellular enzyme, coagulase, which converts plasma fibrinogen into fibrin, aided by an activator present in plasma; production of thermostable nucleases that breakdown Deoxyribonucleic acid (DNA) and production of a surface-associated protein known as clumping factor or bound coagulase that reacts with fibrinogen (Greenwood et al., 2012). Based on the production coagulase, which is the clotting factor, staphylococci are divided into coagulase positive and coagulase negative (Taylor and Unakal, 2017). The more virulent species is S. aureus both in human and animals, while coagulase negative staphylococci such as S. epidermidis, S. saprophyticus and S. haemolyticus are generally 11 University of Ghana http://ugspace.ug.edu.gh less associated with severe diseases. Mannitol fermentation is used to distinguish S. aureus from S. epidermidis ( Fedtke et al., 2004; Taylor and Unakal, 2017). Staphylococcus aureus is a well-established coloniser that can be carried asymptomatically for short periods of time on epithelial surfaces, and for relatively longer periods on mucous membranes. Extended carriage suggests the development of a collective tolerance between the host and the bacterium. Infections, however, occur as a result of the disturbance of this balance (Vanbelkum et al., 2009). Furthermore, other phenotypic features used to distinguish S. aureus and coagulase negative staphylococcus are listed in Table 1 below. 12 University of Ghana http://ugspace.ug.edu.gh Table 1: Phenotypic methods for differentiation of Staphylococcus aureus from Coagulase Negative Staphylococcus. Phenotypic characteristics Staphylococcus aureus Coagulase Negative Staphylococcus Morphology on blood agar Gram staining + + Catalase + + Coagulase + - DNase (deoxyribonuclease) + - VP (Voges Proskauer) + + Pyrolidonyl aminopeptidase - + (PYR) Key: +, positive; -, negative. (Taylor and Unakal, 2017; Greenwood et al., 2012) 13 University of Ghana http://ugspace.ug.edu.gh 2.4.1 Pathogenesis and Virulence Determinants of Staphylococcus aureus Staphylococcus aureus infections are one of the most common bacterial infection in humans and are the causative agents of multiple human infections (Tong et al., 2015). Infections of S. aureus depend on the strains involved and the site of infection. These bacteria can cause invasive infections and/or toxin-mediated diseases (Tong et al., 2015). Infections range from minor skin problems to endocarditis, a life-threatening infection of the inner lining of the heart (endocardium). Signs and symptoms of diseases caused by this organism vary widely, depending on the location and severity of the infection. Skin infections include; boils (furuncles), impetigo, cellulitis, staphylococcal scalded skin syndrome. Infections from food poisoning causes nausea and vomiting, diarrhoea, dehydration and low blood pressure (Greenwood et al., 2012; Tong et al., 2015). Septicaemia due to S. aureus affect the internal organs such as the brain, heart or lungs, bones and muscles, surgically implanted devices, such as artificial joints or cardiac pacemakers. Toxic shock syndrome causes high fever, nausea and vomiting, a rash on your palms and soles that resembles sunburn, confusion, muscle aches, diarrhea, and abdominal pain. Septic arthritis symptoms may include joint swelling, severe pain in the affected joint and fever (Greenwood et al., 2012; Tong et al., 2015). The pathophysiology of S. aureus infection depends greatly on the strain or type of S. aureus. Evasion mechanism depends on the host immune response which include the production of an antiphagocytic capsule, sequestering of host antibodies or antigen masking by Protein A (Staphylococcal Protein A-Spa), biofilm development, intracellular survival, and blocking chemotaxis of leukocytes. Bacterial cell wall-associated proteins such as fibrinogen-binding proteins, clumping factors, and teichoic acids facilitate binding of the bacteria to extracellular matrix proteins and fibronectin in infectious endocarditis. Also, staphylococcal superantigens (TSST-1 or toxic shock syndrome toxin 1) are important virulence factors in 14 University of Ghana http://ugspace.ug.edu.gh infectious endocarditis, sepsis, as well as toxic shock syndrome. Pneumonia infections are associated with the bacterial producing Panton-Valentine Leukocidin (PVL), Spa, and alpha- haemolysin. Prosthetic device infections are often mediated by the ability of S. aureus strains to form biofilms as well as communicate using quorum sensing in a bacterial cell density-dependent manner (Puah et al., 2016; Taylor and Unakal, 2017). An important virulence factor which enables Staphylococcus aureus to evade host immune responses is Staphylococcal Protein A (Spa). Genotypes known as “spa-types”, based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains (Votintseva et al., 2014). Protein A of Staphylococcus aureus is a pathogenic factor whose encoding gene, spa, shows a variation in length in different strains (Shakeri et al., 2010). Recent work implemented protein A (Spa) as a vaccine antigen (Kim et al., 2010). In a study conducted in Belgium, spa typing of S. aureus strains showed 82 spa types. The TSST- 1 gene was the most prevalent, found in 70 S. aureus isolates (24%) and was equally distributed among MRSA and MSSA. In contrast, the PVL gene was carried by only three MSSA. The prevalence of PVL carriage in this healthy community of children was 2 (0.6%). Exfoliative toxin genes (TSST-1) were detected in 10 (3.5%) MSSA strains (Blumental et al., 2013b). In S. aureus related infections in tourists returning from Africa have advocated that African S. aureus might have a diverse genetic background and may also be more virulent than isolates from Europe (Denis et al., 2005; Beilouny et al., 2008; Schleucher et al., 2008; Zanger et al., 2012). Reports of fatal S. aureus pneumonia and convoluted skin and soft tissue infection in travelers 15 University of Ghana http://ugspace.ug.edu.gh returning from Africa were frequently associated with isolates producing Panton-Valentine Leukocidin (PVL) (Denis et al., 2005; Beilouny et al., 2008; Schleucher et al., 2008; Zanger et al., 2012). Panton-Valentine Leucocidin can lyse granulocytes and is associated with skin and soft tissue infections (Loeffler et al., 2009; Shallcross et al., 2013). Panton-Valentine Leukocidin is a distinctive virulence factor associated with a highly aggressive and often fatal form of community- acquired infections. Empiric treatment should be adapted to the type of infection and the resistance profile present in each country or region (Rojo et al., 2010). One special feature of S. aureus infections is their chronic and recurrent nature despite appropriate antibiotic treatment (Kahl et al., 2016). Field studies accomplished in Africa in the last twenty years have showed PVL-positive S. aureus infection in tourists. This has made Africa to be considered as a PVL-endemic region with high rates of PVL-positive isolates, mainly (MSSA), extending from 17% to 74% (Breurec et al., 2011). The explanations for this high prevalence are unidentified but researchers say it might be connected to different interaction of the host, the microorganism, the humid environment of tropical Africa and different complement 5a receptors, which have been recognized as PVL targets (Li et al., 2012; Spaan et al., 2013; Wang et al., 2013). This is in sharp difference to Europe, where the prevalence of PVL-positive isolates is low (0.9–1.4%) (von Eiff et al., 2004). As applied in Europe, the development of multi-country systems for the study of S. aureus infections and classification of strains will additionally improve the understanding of the peculiarities of the epidemiology and contribution of African strains to global S. aureus infections and spread (Grundmann et al., 2010b; Herrmann et al., 2013). In Ghana, out of 120 S. aureus screened, 123 isolates fitted into 35 diverse spa-types and 19 sequence types (ST) with the three most predominant spa-types. Panton-Valentine Leucocidin was 16 University of Ghana http://ugspace.ug.edu.gh 58% and TSST-1 was 14% of the S. aureus isolates (Eibach et al., 2017). Egyir et al., (2014) identified two community MRSA isolates in Ghana. These isolates were PVL-negative, displayed spa type (t5132) associated with ST508 and carried Staphylococcal cassette chromosome mec (SCCmec) type V. 2.5 Identification and typing of Staphylococcus aureus Investigation based on accurate identification and characterization of pathogens is important in any geographical location (Fournier et al., 2014) hence, it is essential to identify and type S. aureus which could be important for clinical and epidemiological surveillance and also for the appropriate selection of antimicrobials for treatment (Boswihi and Udo, 2018). The fundamental criteria for identifying S. aureus are colonial morphology on agar (especially on blood agar where most species show haemolysis), Gram staining, coagulase test, Pyrolidonyl aminopeptidase (PYR) and deoxyribonuclease (DNase). Figure 1 below illustrates how S. aureus can be identified in the laboratory. 17 University of Ghana http://ugspace.ug.edu.gh Key to figure 1: F, Fermentation and O, Oxidation metabolism. Figure 1: Flowchart indicating the laboratory test used to identify S. aureus. Source: https://www.pinterest.com. Retrieved on 20th May, 2018. Table 2 below describes the various methods used to type Staphylococcus aureus as well as their advantages and disadvantages. 18 University of Ghana http://ugspace.ug.edu.gh Table 2: Comparison of typing methods for characterising Staphylococcus aureus. Type of Methods Merits Demerits References methods available Phenotype Biotyping It is reproducible, easy to perform and It has poor discriminatory power (Rabello et al., 2005; interpreted. Most strains of S. aureus can be Alni et al., 2016). isolated using this method. Phenotype Phage typing It is fairly reproducible. It is discriminatory, care of biologically (Mehndiratta et al., active phages which are available only at 2010). reference centres and the technique is demanding. Many strains are non-typeable. Phenotype Antimicrobial This procedure is easy to perform and Diverse strains of S. aureus may possess (https://www.seimc.org. susceptibility interpreted, with fair amount of similar resistance pattern causing a Retrieved on 21st May, testing reproducibility. reduction in the discrimination power. 2018). Phenotype Protein typing Nearly all strains are typeable and procedures Patterns identified are complex, associations (https://www.seimc.org. have good reproducibility and easy to between multiple strains and explanation Retrieved on 21st May, interpret. becomes difficult. Requires specialised 2018). equipment and a high level of technical expertise. Genotyping Pulsed-field gel Reliable tool for comparing methicillin- Results can be challenging to compare (Bannerman et al., (fingerprint- electrophoresis resistant Staphylococcus aureus (MRSA) between laboratories and to interpret. PFGE 1995; van Belkum et al., based) (PFGE) isolates to known epidemic clones. is low quantity, not appropriate for long- 1998; Hallin et al., term epidemiological studies and assesses a 2007). limited amount of the microbial genome. Needs an exceptional equipment and a high level of technical expertise Genotyping Multilocus The sequence fragments of seven costly and its relatively low discriminatory (Feil et al., 2003; (PCR- based) sequencing typing housekeeping genes which has a low power that prevent its use for local Basset et al., 2011). (MLST), mutation rate brands MLST as the most epidemiology. appropriate for long-term and worldwide epidemiological studies Genotyping Whole genome Mapping of genome varied variations offers It is costly and bioinformatics expertise is (Harris et al., 2010; (PCR based) sequencing ideal resolution to infer phylogenetic required in order to assemble a whole Bertelli and Greub, (WGS) relatedness. It has been used in evolutionary genome sequence 2013; Price et al., 2013; studies, outbreak investigations, and Goldberg et al., 2015). phylogeographic distribution analyses. 19 University of Ghana http://ugspace.ug.edu.gh 2.5.1 Phenotypic typing of Staphylococcus aureus Phenotypic typing of S. aureus detects the characteristics expressed by the microorganism and is based on properties like incubation atmosphere/temperature, shape, size and assets that can be measured without reference to the genome (https://www.seimc.org. Retrieved on 21st May, 2018). In addition, biochemical characteristics such as catalase is used to distinguish staphylococci from streptococci. Furthermore, other parameters such as oxidation, fermentation, antigenic and antimicrobial susceptibility profiles are considered part of the phenotypic typing of S. aureus (https://www.seimc.org. Retrieved on 21st May, 2018). 2.5.1.1 Biotyping of Staphylococcus aureus Biotyping S. aureus makes use of the arrangement of the metabolic activities of the isolate, colonial morphology and environmental tolerances. This method can be achieved manually or using automated systems. Examples are fermentation of mannitol and haemolysis on blood agar (Rabello et al., 2005; Alni et al., 2016). 2.5.1.2 Phage typing of Staphylococcus aureus Strains of S. aureus can be classified by their pattern of resistance or susceptibility to a standard set of bacteriophages. This depends on the occurrence or nonexistence of specific receptors on the bacterial surface that are used by the virus to bind to the bacterial cell wall (Mehndiratta et al., 2010). 20 University of Ghana http://ugspace.ug.edu.gh 2.5.1.3 Antimicrobial susceptibility testing of Staphylococcus aureus This technique involves subjecting S. aureus to a set of antibiotics. Isolates that are different in their susceptibilities are considered as diverse strains. The identification of new or uncommon pattern of antibiotic resistance among isolates cultured from different patients is often the first sign of an outbreak. All strains are typeable. The susceptibility pattern of isolates taken over a period of time that signifies that the same strain may differ for one or more antibiotics due to acquisition of resistance (https://www.seimc.org. Retrieved on 21st May, 2018). 2.5.1.4 Protein typing of Staphylococcus aureus Protein typing depends on major or minor variations in the range of proteins made by different strains. The proteins, glycoproteins or polysaccharides are extracted from a culture of the strain, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained to compare with those of other strains (Xia and Wolz, 2014). Similar organisms display common protein patterns. A different technique called immunoblotting where electrophoresed products are transferred to nitrocellulose membrane and then exposed to antisera raised against a specific strain can also be used. The bound antibodies are then identified by enzyme-labelled anti- immunoglobulins (Christie and Dokland, 2012). These methods are currently employed for epidemiological studies of S. aureus (https://www.seimc.org. Retrieved on 21st May, 2018). 2.5.2 Genotypic typing of Staphylococcus aureus In current years, several tools have become accessible for typing of S. aureus, ranging from fingerprint based methods such as pulsed-field gel electrophoresis (PFGE), to PCR-based methods 21 University of Ghana http://ugspace.ug.edu.gh such as multilocus variable number tandem repeat analysis, to sequence-based methods such as multilocus sequencing typing (MLST), and most lately, to whole genome sequencing (WGS) (Joseph and Read, 2010). 2.5.2.1 Pulsed-Field Gel Electrophoresis (PFGE) Deoxyribonucleic acid (DNA) fingerprinting by Pulsed-field gel electrophoresis (PFGE) was a reliable tool for comparing methicillin-resistant Staphylococcus aureus (MRSA) isolates to known epidemic clones but its use is reduced (Frickmann et al., 2012). Pulsed-field gel electrophoresis (PFGE) has been the ‘gold standard’ genotyping method for MRSA for over a decade, and it has been used widely for local outbreak investigation, long-term surveillance of MRSA infections at regional and national levels and for international comparisons (Struelens et al., 2009). This procedure is built on bacterial DNA restriction endonucleases that digest and is with comparatively few restriction sites that generate less, but much bigger fragments than those produced conventionally using a constant field agarose gel electrophoresis (Francis et al., 2005). The location of electric field is changed periodically (“pulsed”) permitting the DNA fragments embedded in the agarose plugs to be separated by size (Miller et al., 2005). In PGFE analysis the chromosomal DNA composed have a restriction pattern with a well-defined fragments that enables the analysis and comparison of multiple isolates (Fey et al., 2003; Odutola et al., 2013). This technique has been used extensively for the epidemiologic surveillance of nosocomial and community acquired MRSA isolates (Vandenesch et al., 2003; Mulvey et al., 2005). 22 University of Ghana http://ugspace.ug.edu.gh 2.5.2.2 Multilocus Sequencing Typing (MLST) Multilocus sequencing typing (MLST) is a type of DNA sequencing where there is a short sequence recurrences of the polymorphic X region of the protein A gene including a variable number of 21- to 27-base pair (bp) sequences for S. aureus (Ruppitsch et al., 2006; Blumental et al., 2013a). This process is attaining approval among investigators, and it is predominantly used for the study of long-term population relatedness and for a better understanding of the emergence and evolution of MRSA clones S (Trindade et al., 2003). The seven loci representing housekeeping genes for S. aureus are amplified by PCR (Berglund et al., 2005). The PCR product is then sequenced and compared to known alleles held at the MLST Web site (http://www.mlst.net) to obtain an allelic profile. This allelic profile consists of a string of seven numbers which can be compared with MLST database on the internet, unifying and regulating epidemiology data collected all over the world. This method is very important and it gives a better understanding of the overall epidemiology of MRSA infections giving evidence on strain lineage (Maiden et al., 2013). 2.5.2.3 Whole Genome Sequencing (WGS) Whole-genome sequencing (WGS) can provide excellent resolution in global and local epidemiological investigations of S. aureus outbreaks (Cunningham et al., 2017). SCCmec is another method used for typing MRSA through the description of resistance as a result of movable or mobile genetic component. The occurrence of (MRSA) is clarified by the acquisition of the mecA gene, which is located on the staphylococcal cassette chromosome mec (SCCmec) (Basset et al., 2013). In 2007, a new S. aureus strain harboring mecA gene homologue, mecC (formerly mecALGA251), was found in humans and animals (Porrero et al., 2014). Currently, WGS-based 23 University of Ghana http://ugspace.ug.edu.gh typing has been achieved through next generation DNA sequencing (NGS) skill; this methodology permits the discovery of single nucleotide polymorphisms and core genome MLST (cgMLST) among investigative approaches (Harris et al., 2010; Bertelli and Greub, 2013; Goldberg et al., 2015). Whole-genome sequencing (WGS) is a rapidly advancing technology, and increasingly affordable benchtop sequencers could be in use in the routine clinical laboratory within the next decade (Metzker, 2010). It may soon be practical to sequence specimens directly in a matter of hours, resulting in enormous diagnostic improvements and creating new challenges for the routine laboratory (Didelot et al., 2012). One key application is likely to be antimicrobial resistance prediction from the genome sequence (“resistance genotype”), analogous to in silico multiplex PCR for a large number of known resistance genes (Gordon et al., 2014). 2.6 Antimicrobial Susceptibility Profile of Staphylococcus aureus Staphylococcus aureus antimicrobial susceptibility profile is used to demonstrate resistance and susceptible patterns to several antimicrobials and is usually carried out to determine which antimicrobial would be most successful in treating S. aureus infections in vivo (Alves et al., 2018) 2.6.1 Development of resistance to Penicillinase-resistant penicillins among Staphylococcus aureus in Africa and Ghana Penicillin resistance is due to the production of penicillinase, which inactivates the antibiotic. The emergence of penicillin resistance in S. aureus stimulated the development of new antibiotics such as streptomycin, tetracycline, erythromycin, and chloramphenicol in the 1950s (Davies & Davies, 2010) In the 1970s, some strains of S. aureus have emerged resistant to the penicillinase-stable penicillins (cloxacillin, dicloxacillin, methicillin, nafcillin, and oxacillin) (Dien et al., 2014). 24 University of Ghana http://ugspace.ug.edu.gh Penicillinase-resistant penicillins (flucloxacillin, dicloxacillin) are still the prime drugs for the treatment of severe methicillin-susceptible S. aureus (MSSA) infections, but first generation cephalosporins (cefazolin, cephalothin and cephalexin), clindamycin, lincomycin and erythromycin are also important in treating less severe MSSA infections. Severe MRSA infections should be treated with vancomycin and patient who are allergic to vancomycin teicoplanin should be used. Multi-resistant MRSA (mrMRSA) strains can be managed with a combination therapy of rifampicin and fusidic acid, because single use of these drugs increases resistance (Rayner & Munckhof, 2005). The resistance is the result of a supplemental penicillin binding protein (PBP 2a) encoded by the chromosomal mecA gene. The continuous search for antibiotics active against penicillin-resistant S. aureus led to the development of methicillin. Methicillin resistance occurs due to the acquisition of mecA or mecC (homolog of mecA) gene by previous susceptible strains (Harkins et al., 2017). However, in Ghana the antimicrobials listed in the 2017 standard treatment guidelines for treating staphylococcus aureus infections are flucloxacillin, cloxacillin, erythromycin, amoxicillin, amoxiclav, mupirocin and clindamycin (Standard Treatment Guideline of Ghana, 2017). Table 6 below describes the prevalence of beta-lactams and non-beta-lactams resistance of Staphylococcus aureus in Africa. 25 University of Ghana http://ugspace.ug.edu.gh Table 3: Prevalence of beta-lactams and non-beta-lactams antimicrobial resistance profile of S. aureus in Africa. Country Period Sample type Age of Beta-lactams, FOX E CC COT GEN TET CIP study study % subjects PEN AMC % % % % % % % (years) Tanzania 2011 Nasal swabs ≤ 5 - 7.9 10.5 14.0 16.7 65.8 27.2 23.7 4.4% (Moyo et al., 2014) South 2012- Nasopharyngeal infants 95 - 1.7 1.3 3.0 2.4 4.0 2.4 - Abdulgader, Africa 2013 swabs 2016 Nigeria 2009 Clinical All age 88.2 - 16.2 11.8 8.8 72.1 14.7 55.9 29.4 Shittu et al., groups 2011 Ghana 2015 Hospital beds - - 13.9 - 13.0 - 11.0 - - 9 Saba et al., 2017 Ethiopia 2016- Nasopharyngeal ≤ 11 84.1 - 33 7.4 26.1 51.1 - 21.6 27.3 Mulu et al., 2017 swabs 2018 Keys to table 6: PEN- Penicillin G, AMC- Amoxiclav, FOX- Cefoxitin, E- Erythromycin, CC- Clindamycin, COT- Cotrimoxazole, GEN- Gentamicin, TET- Tetracycline, CIP- Ciprofloxacin. 26 University of Ghana http://ugspace.ug.edu.gh Staphylococcus aureus develops resistance to antimicrobials by different mechanisms. These mechanisms include limiting uptake of the drug, modification of the drug target, enzymatic inactivation of the drug, and active efflux of the drug. Depending on the antimicrobial involved, the bacteria may use one or several of these resistance mechanisms. In particular, the localization of resistance genes on transferable genetic elements such as plasmids and transposons facilitates horizontal transfer of resistance between bacteria (van Hoek et al., 2011). 2.6.2 Mechanism of Antibiotic Resistance of Staphylococcus aureus Staphylococcus aureus has adapted to evolution in the antimicrobial era the ability of this organism to develop resistance strategies to existing and new antimicrobial they are exposed to through mutations or alterations of proteins, plasmids and transposons (Pantosti et al., 2007). 2.6.2.1 Mechanism of Beta-lactam Resistance The beta-lactamase enzymes possessed by some S. aureus strains hydrolysis the penicillin ring of drug causing the organism to became resistant to penicillin. This lead to the development of penicillin drugs such as methicillin that can resist the action of beta-lactamase producing strains for the treatment of staphylococcal infections. However, S. aureus strains resistant to methicillin agents soon appeared (Barber, 1961). There are three known mode of actions associated with S. aureus resistant to methicillin. These are beta-lactamases hyperproduction (McDougal and Thornsberry, 1986), alteration of normal Penicillin Binding Proteins (PBPs) (Tomasz et al., 1989), and the presence of an acquired penicillin-binding protein 2a (PBP2a) (Ubukata et al., 1985). However, most clinical isolates use the latter resistant mechanism. Staphylococcus aureus strains have four normal PBPs fixed on the cytoplasmic membrane which participate in the cross linking 27 University of Ghana http://ugspace.ug.edu.gh of the peptidoglycan of the bacterial cell wall. These normal PBPs have activity similar to serine proteases and have high affinity for beta-lactams agents and when this binding occurs, the PBPs are not able to function in the assembly of cell wall, causing bacterial death. PBP2a (Chambers, 1997). Penicillin-binding protein 2a produced by some strains of S. aureus has little affinity for beta-lactam antibiotics and therefore is capable of replacing the biosynthetic functions of the normal PBPs even in the presence of the beta-lactams, thereby preventing cell lysis. Organism isolated that contains the PBP2a mediated resistance mechanism are clinically resistant to all available β-lactams, including penicillins, cephalosporins, β-lactam/β-lactamase inhibitor combinations, monobactams, and carbapenems (Fasola and Peterson, 1992; Chambers, 1997). Table 4 below describes the mode of action and antimicrobial resistance mechanism of beta- lactams and non-beta-lactam antimicrobials in Staphylococcus aureus. 28 University of Ghana http://ugspace.ug.edu.gh Table 4: Mode of action and antimicrobial resistance mechanisms of Beta-Lactams and non-Beta-Lactam antimicrobials in Staphylococcus aureus Antimicrobial class Antimicrobial type Mode of action Mode(s) of resistance References β-lactams Penicillin Penicillin-Binding Protein (PBP) Alterations in the target penicillin- (Greenwood et Amoxiclav on bacteria cell wall. binding proteins. al., 2012) Cephalosporins Cefoxitin Inhibit PBP of PBP2a of MRSA. Mutation at the allosteric site of (Greenwood et PBP2a interfere with drug binding. al., 2012) Macrolide Erythromycin Binds to the bacterial 50S Modification of the 50S subunit in (Retsema and Fu, ribosome subunit and inhibit the area of the peptidyl transferase 2001) protein synthesis or to an efflux pump. Lincosamide Clindamycin Bind to the 50S ribosomal Modification of the ribosomal (Greenwood et subunit binding site al., 2012) Sulphonamides and diaminopyrimidines Co-trimoxazole Inhibit enzyme systems involved Metabolic pathway inhibitors (Reygaert, 2013) in the bacterial synthesis of tetrahydrofolic acid (THF) Aminoglycosides Gentamicin Bind to 30S Ribosomal Subunit Inhibit Protein Synthesis (Reygaert, 2013) Tetracyclines Tetracycline Prevent binding of amino-acyl Resistance is often due to the (Nguyen et al., transfer RNA (tRNA) to the acquisition of new genes, Tet M and 2014; ribosome Tet O Greenwood et al., 2012) Fluoroquinolones Ciprofloxacin Targets are the essential bacterial Alterations in the target enzymes (Drlica and Zhao, enzymes DNA gyrase and DNA (DNA gyrase and topoisomerase 1997; Jacoby, topoisomerase IV IV) and of changes in drug entry 2018) and efflux Linezolid Oxazolidones Preventing the formation of the Inhibit Protein Synthesis (Reygaert, 2013) ribosomal initiation complex. Teicoplanin, Vancomycin Glycopeptides Bind to bacterial cell wall Inhibit Cell Wall Synthesis (Reygaert, 2013) 29 University of Ghana http://ugspace.ug.edu.gh 2.6.2.1.1 Methicillin resistant Staphylococcus aureus Methicillin-resistant Staph. aureus (MRSA) is a major public health issue in hospitals and the community (Greenwood et al., 2012). Globally, a study conducted in Vietnam where ten (10) isolates representing 11.8% were MRSA positive (van Nguyen et al., 2014). Also, in a research conducted in Belgium, fourteen (14) strains isolated from 11 children (3% of the cohort) were identified as MRSA positive. In the healthy paediatric community, there is relatively high rate of MRSA carriage (3%), but MRSA isolates were not associated with PVL and mainly belonged to commonly hospital-associated clones (Blumental et al., 2013b). Methicillin resistant S. aureus (MRSA) strains were remarkably resistant to ciprofloxacin, erythromycin and clindamycin. Among MSSA carriers, carriage of isolates resistant to, at least, one antibiotic (in addition to penicillin) was more common in the children (Blumental et al., 2013a). However, in a study conducted by Iraj Sedighi - Hamedan, et al., (2011) in Iran, none of MRSA and MSSA was found to be resistant to vancomycin and clindamycin. Three (0.5%) of the 6 strains of MRSA and 7 (4.9%) of the 142 MSSA strains were resistant to erythromycin. Hence concluded that, the rate of colonization by S. aureus was high in children attending day-care centres whereas colonization with MRSA was not common in these children. These researchers recommended that, Clindamycin or trimethoprim–sulfamethoxazole could be used in mild to moderately severe diseases caused by CA-MRSA. According to another study conducted in Portugal by Tavares et al., (2010), among 365 isolates of S. aureus identified, resistance rates were found to be ; 88% for penicillin, 14% for erythromycin, 6% for clindamycin, 2% for tetracycline and <1% for oxacillin, rifampicin, ciprofloxacin, and Cotrimoxazole. Three (3) MRSA strains were isolated in this study. These strains had properties of CA-MRSA, such as low-level resistance to oxacillin and limited resistance to non-beta-lactams. Two (2) CA-MRSA were related to USA700 (ST72-IV): one was 30 University of Ghana http://ugspace.ug.edu.gh ST72-IVc, spa type t148; the other was ST939-IVa (ST939 is a single locus variant (SLV) of ST72), spa type t324. The third strain was related to USA300 (ST8-IV) being characterized by ST931 (SLV of ST8)-VI, spa type t008. The three MRSA strains were PVL-negative, but all carried LukE-LukD leukocidin, haemolysins gamma, gamma variant and beta, and staphylococcal enterotoxin sel. The researchers concluded that, S. aureus isolated from nasopharyngeal samples, suggest that, in Portugal, the prevalence of CA-MRSA carriage in healthy young children remains extremely low favoring the exclusion of this group as a reservoir of such isolates. In some sub-Saharan African countries, studies have shown that study subjects from communities and hospitals have been colonised with both MSSA and MRSA (Falagas et al., 2013). One striking feature of African MSSA in urban areas is the high level of resistance to penicillin (73.7–100%) (Ramdani-Bouguessa et al., 2006; Kolawole et al., 2013), co-trimoxazole (15–89.1%) (Mariem et al., 2013; Seni et al., 2013), and tetracycline (21.8–92%) ( Djoudi et al., 2013; Conceição et al., 2014). Statistical information on the treatment and use of antibiotics are scarce for urban populations and absent for remote populations. The most commonly recommended antibiotics in urban settings (percentage of encounters) were co-trimoxazole (40%), amoxicillin (11.8%) and metronidazole (8.6%) in Gambian children aged less than five years (Risk et al., 2013). African S. aureus isolates are categorized by a high proportion of resistance to penicillin, tetracycline, and co-trimoxazole, which reflects the frequent use of these drugs for treatment. Resistance detected against penicillin was 95.2% (n = 438), oxacillin 1.9% (n = 9), gentamicin 0.2% (n = 1), levofloxacin 1.5% (n = 7), erythromycin 4.4% (n = 20), clindamycin (1.5% (n = 5), tetracycline 47.6% (n = 219) and cotrimoxazole 46.7% (n = 215) (Schaumburg et al., 2014). Research conducted in Ethiopia showed that of 24 S. aureus isolates from the nasopharynx of children under 31 University of Ghana http://ugspace.ug.edu.gh five, 24 (100%) were resistant to ampicillin, 20 (83.3%) to amoxicillin, and 8 (33.3%) to tetracycline. S. aureus were also resistant (16.7%) and intermediate resistant (8.3%) to erythromycin. However, all isolates of S. aureus were susceptible to methicillin, vancomycin, ciprofloxacin, gentamicin and Augmentin. Moreover, almost all isolated S. aureus 22 (91.7%) showed multidrug resistance (resistance to more than one antibiotic) (Assefa et al., 2013). In Ghana, Sampane-Donkor et al., (2017) reported that S. aureus resistance was the highest for penicillin (100%), followed by tetracycline (80.8%), cefuroxime (73.1%), erythromycin (38.5%), ciprofloxacin (19.2%), gentamicin (23.8%) and then cefoxitin (7.7%). Four (3.4%) of the twenty- six S. aureus isolates were MRSA. In the study, prevalence of multidrug resistance was found to be 16.7% (9/36) for S. aureus. Eibach et al., (2017), in a research carried out at Kumasi in Ghana, reported two (2%) isolates of methicillin-resistant S. aureus (MRSA) that were multidrug resistant. They also reported a high level of resistance against penicillin (96.7%) and tetracycline (52.5%) in a study. Gentamicin, trimethoprim/sulfamethoxazole, clindamycin and erythromycin recorded 99.2%, 97.5%, 94.2% and 86.7% sensitivity respectively. All the isolates were sensitive to linezolid and teicoplanin. Of all the S. aureus isolates, 1.7% (n=2) were MRSA. The MRSA strains were resistant to penicillins, cefoxitin and tetracycline. High prevalence of PVL (57.5%) was also noted among the S. aureus. Egyir et al., (2014), conducted a research in the community, where 124 S. aureus were isolated. Hundred and thirteen (113) representing 91% were resistant to penicillin and 25% to tetracycline. Resistance to erythromycin, fusidic acid, norfloxacin and cefoxitin was less than 5%. All isolates were susceptible to clindamycin, trimethoprim- sulphamethoxazole, gentamicin, rifampicin, mupirocin, and linezolid. Seven (6%) isolates were MDR, with penicillin-tetracycline-norfloxacin (n= 5) being the predominant resistance profile. 32 University of Ghana http://ugspace.ug.edu.gh Only two (2) isolates (1.6%; 2/124) were resistant to cefoxitin and confirmed to be MRSA by mecA PCR. Research conducted in Ghana, concluded that, the knowledge of Antibiotic resistance (ABR) is high among prescribers, however a gap in the knowledge and perception of optimal antibiotic prescription practices exist among prescribers. The researchers found out that, amoxicillin is the first line presumptively prescribed antibiotic hence could attribute to the increase in resistance. And also there is the need for a formal source of information on ABR to support prescriber’s antibiotic prescription practices (Asante et al., 2017). 2.6.3 Effect of some Fluoroquinolones and Cephalosporins on methicillin resistant Staphylococcus aureus Crowcroft et al., (1999), investigated the correlation between the incidence of MRSA and the use of different classes of antimicrobials in hospitals. It was found out that, incidence of nosocomial MRSA increased with increasing use of ceftazidime, amoxicillin with clavulanic acid and quinolones. The researchers concluded that, the correlation between antimicrobial use and MRSA are complex, and that, interventions should be aimed at promoting more rational prescribing patterns supported by adequate experimental and epidemiological evidence. Advice for preventing and controlling MRSA has focused mainly on hygienic measures and precautions to avoid cross- transmission. Dziekan et al., (2000), reported in a study that, intensity of care, number of transfers (from one health care to another), and fluoroquinolone therapy were independently associated with acquisition of MRSA and concluded that, epidemiological support to recent molecular studies suggested that, fluoroquinolone use may increase the transmissibility of MRSA in hospitals. Analysis of the 5 years data set showed that temporal variations in MRSA incidence followed temporal variations in the use of fluoroquinolones, third-generation cephalosporins, macrolides 33 University of Ghana http://ugspace.ug.edu.gh and amoxicillin/clavulanic acid (Aldeyab et al., 2008). Exposure to levofloxacin or ciprofloxacin is a significant risk factor for the isolation of MRSA, but not MSSA (Weber et al., 2003). Monnet et al., (2004) observed a dynamic temporal relationship between MRSA macrolide use, third- generation cephalosporin use, and fluoroquinolone use and suggested that, the use of antimicrobial drugs to which MRSA outbreak strains are resistant may be an important factor in perpetuating an outbreak. Thouverez et al., (2003), suggested that, when selection pressure exerted by an antibiotic is insufficient (that is, below a threshold level), fitness advantages performs a predominant role in the dissemination of MRSA clones. The balance between the selection pressure exerted by antibiotics and the disadvantage of lower replication rates of resistant strains in the absence of antibiotics complicates the biological model of clonal dissemination of epidemic MRSA strains. 2.6.4 Inducible Clindamycin Resistance in Staphylococcus aureus Clindamycin is a lincosamide which is a substitute choice for mild to moderate Staphylococcal infections especially in penicillin allergic patients (Lakshmi and Saikumar, 2018). Macrolide (M), lincosamide (L), and streptogramin (S), (MLS) ribosomal methylase (erm genes) are responsible for the resistance mechanism in phenotypic MLSB accountable for constitutive and inducible resistance and efflux pump (msr genes) is also responsible for phenotypic M (Palavecino, 2004). When MLSB resistance is constitutive, staphylococci are resistant to erythromycin a macrolide and clindamycin a lincosamide. Inducible resistant strains are resistant to erythromycin and inducibly resistant to clindamycin. Strains of S. aureus that uses the efflux pump mechanism are resistant to erythromycin and susceptible to clindamycin. Induction phenomenon can be confirmed by means of a disc diffusion test by the introduction of a 2 μg clindamycin disc and a 15 μg erythromycin disc [Double disc diffusion test (D-test)] spaced 15–26 mm apart. Inducible clindamycin resistance 34 University of Ghana http://ugspace.ug.edu.gh (positive D-test) suggests the presence of an erm gene that could result in constitutive clindamycin resistance and clinical failure (Ji, 2014). Figure 2 below shows susceptible test for erythromycin and clindamycin in double disc diffusion test. 35 University of Ghana http://ugspace.ug.edu.gh Figure 2: Phenotypic clindamycin resistance in S. aureus. Source: http://jcm.asm.org/content/51/12/4196/F1.large.jpg. Retrieved on 13th July,2018. S. aureus organism showing normal susceptible test for erythromycin and clindamycin (fig. 2a). S. aureus organism showing resistance to both erythromycin and clindamycin disc. This shows a constitutive resistance and should be described as resistant to both erythromycin and clindamycin drugs (fig. 2b). When the S. aureus organism do not show flattening of the clindamycin zone it should be described as clindamycin susceptible, D-test negative (fig. 2c). S. aureus organism that shows flattening of the side of the clindamycin inhibition zone close to the erythromycin disc, is usually described as D-shaped. This implies that the S. aureus strain has an inducible gene causing the organism to show inducibly resistance and should be described as clindamycin resistant, D- test positive (fig. 2d). Note: all zones of inhibition must be compared to standard charts used by every geographical location. 36 University of Ghana http://ugspace.ug.edu.gh Detection of inducible clindamycin resistance is very important in CA-MRSA because clindamycin is one of the antibiotics recommended to treat CA-MRSA infections and clinical laboratories are advised to perform the D test in macrolide resistant MRSA isolates (Palavecino, 2004; CDC, 2012). In a study in Iran among children under five years, three of the 6 strains of MRSA and 7 of the 142 MSSA strains were resistant to erythromycin, D-test conducted on all of the erythromycin resistance strains indicated positive. The researchers recommended that, for every erythromycin resistance strain, D-test should always be carried out for detection of inducible clindamycin resistance. (Iraj Sedighi - Hamedan, et al., 2011). 2.6.5 Factors that influence antimicrobial Resistance Antibiotic use is one of the most important determinants of antibiotic resistance, thus antibiotic stewardship programmes that promote judicious use of antibiotics are urgently needed and could prove to be more cost effective than targeted screening based on risk factors, isolation of the carriers and decolonization (Nsofor, 2015). Internationally, there is an increasing concern over antimicrobial resistance (AMR), which is presently estimated to account for more than 700,000 deaths per year worldwide (http://www.who.int/ antimicrobial resistance. Retrieved on 31st, May, 2018). More than a third of the countries on the continent of Africa did not have current AMR data published in the public domain and only a few of those AMR data were surveillance data. A high level of drug resistance exists to frequently prescribed antibiotics on the African continent. The standardization and quality of the microbiological identification and susceptibility testing methods need to be enhanced to allow national and international organizations to monitor the extent of the 37 University of Ghana http://ugspace.ug.edu.gh AMR problem. All of the identified areas of concern need urgent attention by the global health community in order to halt the public health threat associated with the spread of AMR (Tadesse et al., 2017). The rise and spread of AMR impends the effective control and treatment of various bacterial diseases worldwide (Tadesse et al., 2017). The successes gained in reducing mortality and morbidity through early use of antibiotics, based on empiric guidelines will be in serious threat if proper actions are not taken to control AMR. Availability of routine and research data on pathogen susceptibilities is an important step towards designing targeted strategies to tackle the global AMR crisis (Mazumder et al., 2014; Gera et al., 2016). 38 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study area Archived swabs used in this study were collected in the Accra metropolis of the Greater Accra region of Ghana which is entirely urban. This metropolis falls within the coastal belt of Ghana which has humid and warm climatic condition. Accra has the highest population density compared to other districts in Ghana. According to 2010 population and housing census, the inhabitants of Accra was 1,665,086, signifying 42% of the region’s total inhabitants (www.statsghana.gov.gh/docfiles; Retrieved 10th May, 2018). The archived swabs were collected from a list of nurseries and kindergartens within the metropolis obtained from Ghana Education Service. A total of Seven (7) schools were randomly selected, from Kaneshie, Mamprobi, Korle- Gonno and Palladium (Ashiedu Keteke). 3.3 Sampling site Archived Swabs kept in skim milk tryptone glucose glycerol (STGG) and stored in -80 °C freezer obtained from a cross sectional survey conducted from September to December 2016, from children under five years (< 5) in the Accra Metropolis community in Greater Accra region was used in this study. 3.4 Sample size determination The minimum sample size was determined using the formula: N= Z2 (P) (1-P) 39 University of Ghana http://ugspace.ug.edu.gh (ERROR)2 Description: N= minimum sample size Z= 1.96 which is the standard score for the confidence interval of 95%. E= margin of error at 5% (Standard value of 0.05) P= Estimated Staphylococcus aureus prevalence in nasopharynx 25.2% or 0.252 = ((Ebruke et al., 2016). The minimum sample size (N) = (1.96)2 (0.252) (1- 0.252) / (0.05)2 N = 289.65 = 290 The minimum sample size determined was 289.65, however 410 samples in total was used. 3.5 Study design Only one nasopharyngeal swab (NPS) was collected from the study participants who were children less than five years of age. Using calcium alginate swabs (Fisher Brand ®, USA) a total of 410 NPS were collected and directly inoculated into vials containing skim milk tryptone glucose glycerol (STGG) transport medium (Oxoid, Basingstoke, UK). Inoculated swabs were transported to the Department of Medical Microbiology. Inoculated vials were stored at- 80 °C. S. pneumoniae was isolated and antimicrobial susceptibility testing and serotyping were analysed using standard techniques on the samples. For the purpose of the current study, Staphylococcus aureus was isolated, antimicrobial susceptibility testing and molecular techniques were used to analyse the samples. 40 University of Ghana http://ugspace.ug.edu.gh 3.6 Nasopharyngeal specimen and processing Archived swabs in STGG stored in -80 °C freezer were brought out to thaw to room temperature and vortexed. A drop of the specimen in STGG were put in Sterile Tryptic soy broth (Oxoid, Ltd Basingstoke, UK) and incubated for 48 hours at 37 °C. After 48-hour incubation, a loop full of the broth was plated on sterile Blood agar supplemented with 5% sheep blood, MacConkey and Mannitol Salt Agar (Oxoid, Ltd Basingstoke, UK) and incubated aerobically for 18-24 hours. 3.6.1 Identification of bacterial isolates Microorganisms were identified using conventional methods (colony morphology, Gram stain, catalase test, and 4-24 hours’ tube coagulase test in rabbit-citrate-plasma (Becton and Dickinson ®; Heidelberg, Germany) and growth on mannitol salt agar. A Gram positive small to large yellow colonies from mannitol salt agar (Oxoid, Ltd Basingstoke, UK) was taken and sub cultured onto blood agar (Oxoid, Hampshire, England) plate. The blood agar plate was then incubated aerobically for 18 to 24 hours at 37oC. A large, round, golden-yellow colony, often with β- hemolysis on the blood agar plates was taken. Using a sterile plastic inoculating loop, a small amount of organism was picked up and catalase test was done. Coagulase test was performed to differentiate S. aureus from other Staphylococcus species. Gram-positive cocci that produced catalase and coagulase and fermented mannitol were identified as S. aureus. Gram negative rods cultured on MacConkey and other Gram negative cocci and positive rods were recognized using Gram stain, oxidase, urea, citrate, triple iron sugar, indole. 41 University of Ghana http://ugspace.ug.edu.gh Figure 3: A picture of the Researcher on the bench. 3.6.2 Antimicrobial Susceptibility Testing Antibiotic susceptibility tests were performed using Kirby Bauer’s disc diffusion method inoculated according to Clinical and Laboratory Standards Institute (CLSI) guidelines 2017 on the S. aureus isolates. A 0.5 McFarland equivalent suspension of organisms was incubated on Muller- Hinton agar (MHA) (Oxoid, Hampshire, England) (MHA) plate as designated in the CLSI recommendations (Patel and Clinical and Laboratory Standards Institute, 2017). Within 15 minutes after adjusting the turbidity of the inoculum to 0.5 McFarland suspension using Becton Dickinson Phoenix Spec TM Nephelometer (BD Phoenix Spec TM Nephelometer), a sterile cotton swab was dipped into the adjusted suspension. The dried sterile surface plates Muller-Hinton agar were 42 University of Ghana http://ugspace.ug.edu.gh inoculated by streaking the swab over the entire sterile agar surface to achieve confluent growth. After 15 minutes the various antimicrobial discs were positioned on the lawn of bacterial isolates using sterile forceps. Inoculated Muller Hinton agar were incubated aerobically, within 15 minutes after the application of the discs for 18-24 hours at 37°C. Figure 4: A picture of the Becton Dickinson Phoenix SpecTM Nephelometer used. Antimicrobials used for the antimicrobial susceptibility testing were Erythromycin (15μg), Clindamycin (2μg), Penicillin (10 Units), Tetracycline (30μg), Cefoxitin (30μg), Cotrimoxazole (1.25/23.75μg), Ciprofloxacin (5μg), Amoxiclav (20/10μg), Teicoplanin (30μg), Linezolid (30μg) and Gentamicin (10μg), all of BD BBLTM Sensi-Disc Antimicrobial Susceptibility Test Disc. MRSA were screened using Cefoxitin (30μg) disc by disc diffusion technique (Patel and Clinical and Laboratory Standards Institute, 2017). Cefoxitin zones of inhibition greater or equal to 22mm and less than 22mm were considered phenotypically to be MSSA and MRSA respectively. The results were construed according to CLSI 2017 guidelines. Staphylococcus aureus ATCC 25923 (American Type Culture Collection) was taken as the positive control strain. Multidrug resistance (MDR) which is defined as resistance of an organism to three or more different classes of antimicrobials was used to determine the MDR strains of S. aureus (Magiorakos et al., 2012), 43 University of Ghana http://ugspace.ug.edu.gh Isolates that were susceptible to Clindamycin and resistant to Erythromycin were confirmed for inducible resistance by the use of Double Disc-zone test (D-zone test). Clindamycin and Erythromycin disc were closely placed at a distance 15- 26mm from each other on the MHA plates. After 18-hours of incubation at 37ºC, plates were checked. Flattening of inhibition zone (D- shaped) around clindamycin was considered as inducible clindamycin resistance. The test permits for identification of three different phenotypes: a) Inducible MLSB (Macrolide-Lincosamide-Streptogramin B) phenotype: iMLSB S. aureus isolates shows resistance to Erythromycin (zone size≤13 mm) and sensitive to Clindamycin (zone size ≥21 mm) giving a D shaped zone of inhibition around Clindamycin with flattening near Erythromycin disc (D test positive). b) Constitutive MLSB phenotype: cMLSB S. aureus isolates shows resistance to both Erythromycin (zone size ≤13 mm) and Clindamycin (zone size ≤14mm). c) Methicillin-sensitive {MS (Macrolide Streptogramin)} phenotype: S. aureus isolates shows resistance to Erythromycin (zone size ≤13 mm) and sensitive to Clindamycin (zone size≥21 mm) (D test negative). Table 5 describes the categories in which the D-test are characterised. 44 University of Ghana http://ugspace.ug.edu.gh Table 5: Phenotypic groupings and their features of D-test Phenotypes Resistance CC E Double disc diffusion test phenotype result result description D+ Inducible MLSB S R Flattened, D shaped clear zone around CC disc close to resistant E disc D- MS S R susceptible zone around CC disc and resistant zone around E R Constitutive MLSB R R Resistant zones around CC and E discs S No Resistance S S Susceptible clear zones around discs S =Sensitive, R =Resistant, CC = Clindamycin, E =Erythromycin, MLSB = Macrolide- Lincosamide-Streptogramin B, MS= Macrolide Streptogramin 45 University of Ghana http://ugspace.ug.edu.gh 3.7 DNA Extraction for Molecular Screening of Staphylococcus aureus DNA was extracted from 95 S. aureus isolates using commercial kit from Zymo Research Quick- DNATM Fungal/ Bacterial Mini-prep and the manufacturer’s instructions was followed judiciously. The bacteria were plated overnight on Mannitol Salt agar. Pure isolated colonies were put in 50µl of Phosphate Buffered Saline (PBS) in Eppendorf tubes. The isolates in the Eppendorf tubes were stored in -80 freezer until DNA was extracted. 3.7.1 Multiplex Polymerase Chain Reaction (PCR) screening for MecA, Pvl and Spa genes Multiplex PCR was performed to detect MecA, Spa and Pvl genes. This was done following the method described by Larsen et al., (2008). Each PCR reaction contained 10 mM MecA primers, 10 mM Spa primers and 10 mM Pvl primers. Multiplex PCR Master Mix (New England BioLabs One Tag Quick-Load 2× Master Mix with Standard Buffer) and 1 lL of DNA template preparation. Amplification was performed in a MJ Research PTC-200 Peltier Thermal Cycler. The PCR was visualized using 2% Agarose and band size was compared to DNA marker (100 bp ladder from New England BioLabs). The positive controls used were Methicillin-resistant S. aureus ATCC 33591 which is positive for mecA and Spa genes, Methicillin-susceptible S. aureus ATCC 25923 which is also positive for Spa and Pvl genes and a negative control which was Nuclease free water. Figure 6 below shows the distribution of Spa, mecA and Pvl genes determined by PCR. 46 University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 7 8 9 1 0 Spa, 180- 600bp mecA, 162bp Pvl, 83bp Figure 5: Agarose gel electrophoresis pattern for amplification product of Spa, mecA and Pvl genes using multiplex PCR. Lane: 1 is the 100-bp ladder; lane: 2, Nuclease free H2O control (Negative control); Lane: 3, methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25923 (positive control for MSSA); and Lane: 4, methicillin- resistant Staphylococcus aureus (MRSA) ATCC 33591 (positive control for MRSA); Lane 5-10, Staphylococcus aureus isolates. Table 6 below are a list of primers used for the polymerase chain reaction (PCR). 47 University of Ghana http://ugspace.ug.edu.gh Table 6: Primers used for the Multiplex PCR in the detection of Spa, Pvl and mecA genes. Gene Primers Sequence (5’ – 3’) Size (bp) References Mec A Mec A P4 5-՜TCCAGATTACAACTTCACCAGG-3՜ 162 (Oliveira and Lencastre, 2002) Mec A P7 5՜-CCACTTCATATCTTGTAACG-3 Spa Spa 113F 5՜-TAAAGACGATCCTTCGGTGAGC-3՜ 180-600 (Harmsen et al., 2003) Spa 1514R 5-՜CAGCAGTAGTGCCGTTTGCTT-3 lukF-PV PvlF 5-՜GCTGGACAAAACTTCTTGGAATAT-3՜ 83 (Deurenberg et al., 2004) (Pvl) PvlR 5՜-GATAGGACACCAATAAATTCTGGATTG-3 48 University of Ghana http://ugspace.ug.edu.gh 3.8 Statistical Analysis All data was stored on a password protected computer that could be accessed by only research personnel. In addition, data was entered into Microsoft Excel spreadsheet and further transported into the SPSS software version 22 and analysed. Descriptive statistics such as percentages and proportions were used to calculate the S. aureus carriage prevalence per age groups and gender. Antibiotic resistance rates were determined. Test of association of S. aureus carriage with factors such as age and gender using independent sample Chi- square analysis. All data collected were entered into Microsoft Excel and descriptive analysis done using SPSS version 22. 3.9 Ethical Approval Ethical approval for the previous study was obtained from the Ethical and Protocol Review Committee of the College of Health Sciences, University of Ghana. With Protocol Identification Number: CHS-Et/M.9- P 4.3/ 2015-2016. 49 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 Demographics of the Study Population A total of four hundred and ten (410) children under five years of age were recruited from seven (7) schools into the previous study, which had focused on the epidemiology of S. pneumoniae nasopharyngeal colonisation among the study participants. Of the seven (7) schools, two (2) each were government and private schools, and the remaining three were mission schools. The same specimen was used for the isolation of S. aureus. The study participants comprised of 51.2% (210/410) males and 48.8% (200/410) females. The mean age for the children sampled were 38.8 months. All the 410 participants, had been vaccinated with the Pneumococcal Conjugate Vaccine 13 (PCV 13) at age 6, 10 and 14 weeks from birth, through the Ministry of Health/Ghana Health Service Expanded Programme on immunisation. In the previous study, their claims of vaccination had been verified from inspection of their vaccination cards. In total, only sixty-one (61) out of the total 410 children were ≤ 2 years. 4.2 Nasopharyngeal Staphylococcus aureus carriage Staphylococcus aureus and methicillin resistant S. aureus (MRSA) nasopharyngeal carriage prevalence among study participants were 23.2% (95/410) and 2.1% (2/95) respectively. Females recorded a higher S. aureus carriage prevalence of 51.6% (49/95) as opposed to the males 48.4% (46/95), but this difference was not statistically significant [p=0.533]. The youngest S. aureus carrier was six (6) months old, whiles the oldest was sixty (60) months old. When S. aureus carriage was stratified by age group, the age group of 37-48 months (3.1-4.0 years) recorded the 50 University of Ghana http://ugspace.ug.edu.gh highest carriage prevalence 32.6% (31/95). The two (2) MRSA were found in females aged 36 months (3 years) and 52 months (4 years). Isolation, identification and characterisation of streptococcus pneumoniae present in these samples had already been carried out. Table 7 below describes the prevalence of pathogens isolated from the nasopharyngeal swab samples. Table 8 below describes the carriage prevalence distribution of S. aureus among age groups in months, years and gender. 51 University of Ghana http://ugspace.ug.edu.gh Table 7: Bacterial pathogens isolated from the nasopharyngeal swab samples. Bacterial pathogen Number Prevalence (%) Coagulase negative Staphylococci 194 47.3 Staphylococcus aureus 95 23.2 Diphtheriodes 22 5.4 Micrococcus species 15 3.7 Moraxella species 6 1.5 Klebsiella pneumoniae 13 3.2 Citrobacter species 6 1.5 Escherichia coli 2 0.9 Enterobacter species 2 0.9 Pseudomonas species 2 0.9 52 University of Ghana http://ugspace.ug.edu.gh Table 8: Carriage prevalence of Staphylococcus aureus by age group in children ≤5years attending nursery and kindergarten facilities in Accra. Age Age Number of Carriage group Group Children prevalence of (months) (years) S. aureus Males Females Total (%) 0-12 0-1 2 3 5 (1.2) 3 (3.2%) 13-24 1.1-2 31 25 56 (13.7) 14 (14.7%) 25-36 2.1-3 86 86 172 (42.0) 28 (29.5%) 37-48 3.1-4 70 59 129 (31.5) 31 (32.9%) 49-60 4.1-5 21 27 48 (11.7) 19 (20%) Total 210 200 410 95 53 University of Ghana http://ugspace.ug.edu.gh 4.3 Antimicrobial susceptibility profile of the Staphylococcus aureus isolates Resistance of S. aureus to the antimicrobials tested decreased across penicillin G (97.9%), amoxiclav (20%), tetracycline (18.9%), erythromycin (5.3%), cefoxitin and ciprofloxacin (2.1% each) and gentamycin (1.1%). All the isolates were susceptible to cotrimoxazole, clindamycin, linezolid, and teicoplanin and susceptibility pattern of the following antimicrobials tested were: (penicillin G) 2.1%, amoxiclav (80%), tetracycline (81.1%), erythromycin (94.7%), cefoxitin and ciprofloxacin (97.9% each) and gentamicin (98.9%). The two methicillin resistant Staphylococcus aureus (MRSA) were resistant to penicillin G amoxiclav, tetracycline and cefoxitin. With the methicillin susceptible Staphylococcus aureus (MSSA) the resistant patterns were penicillin G (97.8%), amoxiclav (18.3%), tetracycline (17.3%), erythromycin (5.4%), ciprofloxacin (2.2%) and gentamicin (1.1%). Details of the distribution of antibiotic resistance are presented in Table 9. Table 9 below shows the susceptible and resistance patterns of the antimicrobials used in the study. Table 10 below describes the antimicrobial resistance profile of S. aureus among MRSA and MSSA. 54 University of Ghana http://ugspace.ug.edu.gh Table 9: Antimicrobial susceptibility profile of Staphylococcus aureus isolates. Antibiotics Susceptible Resistance N (%) N (%) Cotrimoxazole 95 100 0 0 Clindamycin 95 100 0 0 Linezolid 95 100 0 0 Teicoplanin 95 100 0 0 Penicillin G 2 2.1 93 97.9 Amoxiclav 76 80.0 19 20 Tetracycline 77 81.1 18 18.9 Erythromycin 90 94.7 5 5.3 Cefoxitin 93 97.9 2 2.1 Ciprofloxacin 93 97.9 2 2.1 Gentamicin 94 98.9 1 1.1 55 University of Ghana http://ugspace.ug.edu.gh Table 10: Antimicrobial resistance profile of S. aureus (MRSA) and (MSSA) Antimicrobial Number of resistance isolates (%) Total among MRSA and MSSA MRSA MSSA Penicillin G 2 (100) 91 (97.8) 91 Amoxiclav 2 (100) 17 (18.3) 19 Tetracycline 2 (100) 16 (17.2) 18 Erythromycin 0 5 (5.4) 5 Cefoxitin 2 (100) 0 2 Ciprofloxacin 0 2 (2.2) 2 Gentamicin 0 1 (1.1) 1 56 University of Ghana http://ugspace.ug.edu.gh Of the 95 S. aureus isolates, 5 were MSSA that were erythromycin resistant and clindamycin susceptible, and showed phenotypic D-test phenomenon of Macrolide Streptogramin phenotype. No inducible clindamycin resistance was observed among the erythromycin resistant strains. The two MRSA and 93 of the MSSA were susceptible to both erythromycin and clindamycin. Results of the D-test, and demonstration association of clindamycin, erythromycin, MSSA and MRSA are presented in Tables 11 and 12 respectively. Table 11 below shows the phenotypic D-test categories of the erythromycin resistant strains and Table 12 shows assessment of erythromycin resistant strains among MRSA and MSSA. 57 University of Ghana http://ugspace.ug.edu.gh Table 11: Susceptibility profile of phenotypic Double Disc Diffusion Test (D-test) among erythromycin resistance strains. Susceptibility pattern (Phenotype) Number of Isolates Percentages MS* phenotype (E* resistant and CC* sensitive with D 5 100% test negative) Inducible MLS *B phenotype (E* resistant and CC* 0 0% sensitive with D test positive) Constitutive MLSB phenotype (E* resistant and CC* 0 0% resistant) E*= Erythromycin; CC*=Clindamycin; MS*= Macrolide Streptogramin; MLS *B = Macrolide- Lincosamide-Streptogramin B 58 University of Ghana http://ugspace.ug.edu.gh Table 12: Assessment of erythromycin resistance among MRSA and MSSA. Susceptibility pattern (Phenotype) MRSA (n=2) MSSA (n=93) MS* phenotype (E* resistant and CC* susceptible with D test 0 5 negative) Inducible MLS *B phenotype (E* resistant and CC* 0 0 susceptible with D test positive) Constitutive MLS *B phenotype (E* resistant and CC* 0 0 resistant) E* sensitive and CC* susceptible 2 93 E*= Erythromycin; CC*=Clindamycin; MS*= Macrolide Streptogramin; MLS *B =Macrolide- Lincosamide-Streptogramin B 59 University of Ghana http://ugspace.ug.edu.gh 4.4 Multidrug Resistance Multidrug resistance (MDR) was detected in 3.2% (3/95) of the isolates, which exhibited resistance to tetracycline, penicillin, and amoxiclav. Of the three (3) MDR isolates, 66.7% (2/3) were MRSA, and 33.3% (1/3) was MSSA. 4.5 Molecular screening results The two (2) MRSA isolates were PCR positive for mecA mediated gene and 58% (55/95) were positive for PVL mediated by [lukF-PV (Pvl)] gene. The two positive MRSA isolates were PVL negative. All 95isolates were PCR positive for the Spa gene. 60 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION, CONCLUSIONS, LIMITATION AND RECOMMENDATIONS 5.1 Discussion The nasopharynx is a home to an array of bacteria among which some are normal flora and others pathogens (Siegel and Weiser, 2015). The introduction of a vaccine to control the growth of a specific bacteria may create a vaccine pressure that leads to disharmony, favouring the growth of other bacteria species that are not included in the vaccine (Ahl, 2013). In May 2012, Ghana introduced PCV 13 in the Expanded Programme on immunisation as part of the effort to lessen the burden of pneumococcal disease in children ≤ 5years (MOH, 2014). Studies have shown that where PCV 13 has been introduced, there is a marked increase in colonisation of non-vaccine bacteria species in the nasopharynx (Odutola et al., 2013; Biesbroek et al., 2014; Nzenze, 2015; Navne et al., 2017). It is therefore imperative to monitor the impact of the pneumococcal vaccine on the normal flora in the nasopharynx especially for S. aureus and its antimicrobial susceptibility patterns in order to contribute to effective treatment. The overall S. aureus carriage prevalence of 23. 2% stated in healthy children in this study is comparable to what has been reported previously in Ghana, 22.1% (Eibach et al., 2017), and in other countries: Gambia, 25.9% (Bojang et al., 2017b); Gambia, 20% (Kwambana et al., 2011b); Portugal, 21.6% (Tavares et al., 2010) and Netherlands, 23.2% (Bogaert et al., 2004). Sampane- Donkor et al., (2017) reported carriage prevalence of 22.0% among HIV children under five in Ghana. However, the carriage prevalence of post vaccination in healthy children reported in this 61 University of Ghana http://ugspace.ug.edu.gh study is slightly lower than what was reported in Iran, 29.6% (Iraj Sedighi - Hamedan, et al., 2011); Belgium, 34% (Blumental et al., 2013a); Vietnam, 30.6% (Van Nguyen et al., 2014) and Gambia, 30.6% (Bottomley et al., 2015). Nonetheless, the carriage prevalence observed in this study was higher than that reported in Ethiopia (10.3%). In that study Assefa et al., (2013) used a smaller sample size of 234 hence, this might account for the difference in the carriage prevalence reported. Before the introduction of PCV 13 in Ghana, Adiku et al., (2015) reported a 14.8% nasopharyngeal carriage of S. aureus in children under five years. However, 5 years after PCV 13 has been introduced in Ghana, this study has observed a marked increase of S. aureus in the nasopharynx (23.2%). This finding is consistent with what is reported in areas wherever PCV 13 has been introduced (Devine et al., 2015; Reiss-Mandel and Regev-Yochay, 2016). The phenomenon could be attributable to the removal of the vaccine serotypes of S. pneumoniae which regulate the normal flora in the nasopharynx (Bogaert, 2004; Devine et al., 2015). This regulation is carried out through the production of H2O2 which is bactericidal to S. aureus (McLeod and Gordon, 1922; Regev- Yochay et al., 2006; Reiss-Mandel and Regev-Yochay, 2016). There are 94 serotypes of S. pneumoniae but only 13 serotypes are present in the current vaccine. Studies showed that vaccine- type strains are those found to correlate with S. aureus carriage, and this elevated much concern in that the introduction of the vaccine would indirectly lead to a rise in S. aureus carriage and infection (Brogden et al., 2005; Pettigrew et al., 2008; Lee et al., 2009; Reiss-Mandel and Regev- Yochay, 2016). In this study, high resistance to penicillin (98%), amoxiclav (20%) and tetracycline (18.9%) were observed. The high penicillin non-susceptibility observed in this study is consistent with what has been reported previously in Ghana, (100%) by Sampane-Donkor et al., (2017). This high 62 University of Ghana http://ugspace.ug.edu.gh prevalence might be due to the fact that, HIV individuals are exposed to antimicrobials as well as resistant microbes owing to their health status (WHO, 2018). Penicillin and ampicillin belonging to the same group of Beta-Lactams had a resistance prevalence of 98% (Egyir et al., 2014) and 100% (Assefa et al., 2013) respectively. This observation may be due to the fact that penicillin and ampicillin have been on the market for a long time, is inexpensive and could easily be obtained over the counter (though prescription drugs) hence, may have been misused greatly. One outstanding feature of African MSSA in urban areas is the high level of resistance to penicillin (73.7–100%) (Ramdani-Bouguessa et al., 2006; Kolawole et al., 2013). Resistance to amoxiclav (20%) observed in this study was higher than what has been reported previously by Risk et al., (2013) in the Gambia. The difference could be due to the use of amoxiclav as the first line of presumptively prescribed antibiotic in Ghana (Asante et al., 2017). It is noteworthy to say that, there has been a marked reduction in tetracycline resistance (18.9%) in this study as compared to a higher resistance of 82% reported previously by Newman et al., (2011). In addition, 100% susceptibility of S. aureus to cotrimoxazole, clindamycin, linezolid and teicoplanin was observed, which agrees with findings from preceding studies carried out in Ghana (Egyir et al., 2014; Eibach et al., 2017) and Iran (Iraj Sedighi-Hamedan, et al., 2011). This could be due to low or no exposure of the study participants to cotrimoxazole and clindamycin and also because Linezolid and Teicoplanin drugs are not easily accessible and affordable. Iraj Sedighi-Hamedan, et al., (2011) recommended that clindamycin or cotrimoxazole (trimethoprim–sulfamethoxazole) could be used in mild to moderately severe diseases caused by CA-MRSA. Double disc diffusion test (D-test) was negative with Erythromycin resistant strains in this study, which is in contrast with the report by Iraj Sedighi-Hamedan, et al., (2011). It is concluded that with erythromycin resistant strains of S. aureus, D-test should always be carried out for the detection of inducible clindamycin resistance. 63 University of Ghana http://ugspace.ug.edu.gh The 3.2% multidrug resistance (MDR) perceived in this study, is lower than that previously reported in Ghana (6.0%) by Egyir et al., (2014) in the community and (16.7%) by Sampane- Donkor et al., (2017) among HIV children. This could be due to frequent exposure of this group to antimicrobial agents as prophylaxis (Sampane-Donkor et al., 2017). The 2.1% (n=2) MRSA identified in this study is comparable to what has been reported before by other researchers in Ghana 3.5% (n= 4) (Sampane-Donkor et al., 2017), 2.0% (n= 2) (Eibach et al., 2017) and 1.6% (n= 2) (Egyir et al., 2014). The finding in this study showed that, community carriage of MRSA in Ghana is low. This could be ascribed to the low intake of antimicrobial agents such as fluoroquinolones (levofloxacin or ciprofloxacin). Exposure to levofloxacin or ciprofloxacin is a noteworthy risk factor for the isolation of MRSA, but not for MSSA (Weber et al., 2003) and third generation cephalosporins (ceftazidime) in the community setting in Ghana. This is because they are expensive and are usually prescribed for acute infections (Newman et al., 2011; Egyir et al., 2014). Cumulative occurrence of MRSA with increasing use of ceftazidime, co-amoxiclav and fluoroquinolones (Crowcroft et al., 1999), Dziekan et al., (2000) also showed that fluoroquinolone use was an sovereign risk factor for MRSA. Usage of the afore-mentioned antimicrobial agents have been shown to correlate with an increase in MRSA prevalence ( Thouverez et al., 2003; Monnet et al., 2004; Aldeyab et al., 2008). Sub-Saharan Africa is now painstaking to be a Panton-Valentine Leucocidin (PVL)-endemic province with high rates of PVL-positive isolates, mainly Methicillin Susceptible S. aureus (MSSA), ranging from 17% to 74% (Breurec et al., 2011). In this present study, 58% of genes that code for PVL was observed and were also found in MSSA. This high prevalence of Panton- Valentine Leucocidin among MSSA is consistent with the 58% prevalence reported previously in 64 University of Ghana http://ugspace.ug.edu.gh Kumasi, Ghana (Eibach et al., 2017). Nonetheless, the finding in this study is in sharp distinction to what was reported in Europe (0.9-1.4%) ( von Eiff et al., 2004; Blumental et al., 2013a). The Panton-Valentine Leucocidin gene was not present in the two MRSA perceived in this study. The finding in this study is consistent with what was reported by Egyir et al., (2014) in Ghana. 5.2 Conclusions In conclusion, the study found that nasopharyngeal carriage prevalence of S. aureus was found among a considerable proportion of school children under five years post PCV-13 vaccination in Accra, Ghana. In addition, low mecA gene-mediated methicillin resistant S. aureus (MRSA) was observed. High Penicillin non-susceptibility and resistance to other Beta-Lactam and non-Beta- lactam antimicrobials have been observed. The Staphylococcus aureus isolated were completely susceptible to cotrimoxazole, clindamycin, linezolid and teicoplanin. There was no inducible clindamycin resistance among the erythromycin resistant strains and a considerable proportion of Panton-Valentine Leucocidin mediated LukF-PV gene (58%) was observed among Methicillin- susceptible S. aureus (MSSA) which is an epidemiological marker for severe S. aureus infections. 5.3 Limitations This study could not screen for other virulence genes such as Toxic Shock Syndrome Toxin - 1(TSST-1), perform spa types and sequence typing on the S. aureus isolates which could have helped to know the prevalence of the virulence genes of the spa and sequence types circulating in the geographical area. 65 University of Ghana http://ugspace.ug.edu.gh 5.4 Recommendations Based on the outcomes in this study, it is recommended that:  An extension of this study to other risk groups and continuous surveillance is imperative.  Further analysis should be done on these isolates to ascertain their Spa types and sequence types.  Holistic health intervention targeting MSSA may be required during pneumococcal vaccination campaigns.  Proper antibiotic stewardship programmes are critical to limit the clinical impact of MRSA. 66 University of Ghana http://ugspace.ug.edu.gh REFERENCES Abdulgader, S.M.A.A. 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Retrieved on 20th May, 2018. https://www.google.com/maps/@5.5497081,-0.2330194,14z. Retrieved on 20th May. 2018. http://microbesinfo.com/2016/04/d-test-a-test-for-detection-of-inducible-clindamycin-resistance- detection-in-staphylococcus-aureus/. Retrieved on 20th May, 2018. https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc- procedimientomicrobiologia1a.pdf. Retrieved on 21st May, 2018. http://www.who.int/antimicrobial-resistance/en. Retrieved on 31st May, 2018. http://www.mlst.net. Retrieved on 13th July, 2018. http://jcm.asm.org/content/51/12/4196/F1.large.jpg. Retrieved on 13th July,2018. http:/newsghana.com.gh. Retrieved on 26th July, 2018. 95 University of Ghana http://ugspace.ug.edu.gh APPENDIX I DNA Extraction Zymo Research Quick-DNATM Fungal/Bacterial Mini-prep kit was used for the DNA extraction. Procedure For maximum performance, beta-mercaptoethanol (user supplied) is added to the Genomic lysis buffer to a final dilution of 0.5% (v/v) that is, 500µl per 100ml. 1. 50-100mg (wet weight) bacterial cell that have been re-suspended in up to 200µl of water or isotonic buffer [example Phosphate buffer saline (PBS)] to a ZR BashingBeadTM Lysis Tube (0.1mm and 0.5mm). Add 750µl BashingBeadTM Buffer to the tube. 2. Bead beater fitted is secured with a 2ml tube holder assembly and process at maximum speed for ≥ 5 minutes. Note: Processing time required varies depending on the device and application and therefore should be evaluated on a case by case basis. For example, processing time can be as little as 3 minutes when using high-speed cell disrupters (example, the portable TerralyzerTM Sample Processor, FastPrep® - 24 similar) or as long as 20 minutes when using lower speeds (Disrupter GenieTM, or standard benchtop vortexes). 3. Centrifuge the ZR BashingBeadTM Lysis Tube (0.1 & 0.5mm) in a microcentrifuge at 10,000×g for 1 minute. 4. 400µl supernatant is transferred to a Zymo-SpinTM III-F in a Collection Tube and centrifuge at 8,000×g for 1 minute. 96 University of Ghana http://ugspace.ug.edu.gh 5. 1,200µl of Genomic Lysis Buffer is added to the filtrate in the Collection Tube from step 4. 6. 800µl of the mixture from step 5 is transferred to the Zymo-SpinTM IIC Column3 in the Collection Tube and centrifuge at 10,000×g for 1 minute. 7. Discard the flow through from the Collection Tube and repeat step 6. 8. 200µl DNA Pre-Wash Buffer is added to the Zymo-SpinTM IIC Column in a new Collection Tube and centrifuge at 10,000×g for 1 minute. 9. 500 µl g-DNA Wash Buffer is added to the Zymo-SpinTM IIC Column and centrifuge at 10,000×g for 1 minute. 10. Zymo-SpinTM IIC Column is transferred to a clean 1.5ml microcentrifuge tube and add 100 µl (35µl minimum) DNA Elution Buffer directly to the column matrix and then centrifuged at 10,000×g for 30 seconds to elute the DNA. Ultra-pure DNA is now ready for PCR. 97 University of Ghana http://ugspace.ug.edu.gh APPENDIX II Gel Preparation (2% Agarose Gel) and Electrophoresis A multi-purpose Agarose (From Cleaver Scientific Ltd AG-100 LE Agarose) was used. Procedure 1. Weigh 2 grams of agarose powder into a beaker. 2. Measure 100ml of 1X TBE (Tris/Borate/EDTA) and add to the 2 grams of agarose powder. 3. Boil till powder completely melts and dissolves. 4. Set the tray on a stable and flat surface and fix the comb in place. 5. Allow the agar to cool approximately 70-60o C and add 0.05mg/l Ethidium Bromide (EtBr) and mix well. 6. Pour it into the tray with the well comb in pace and allow to solidify. Pour gently to avoid bubbles. Loading the samples and running the agarose gel electrophoresis 1. Add loading buffer to each of the DNA sample. 2. Once the gel has solidified, place the electrophoresis gel in the gel tank or electrophoresis unit. 3. Fill the tank with 1X TBE (Tris/Borate/EDTA) until it covers the gel. Note: Add EtBr to the buffer. Ethidium Bromide is a positive charge and will run opposite to the DNA. This will help to bands to have an intense differentiation. 4. Carefully load a molecular weight ladder (100 bp ladder from New England BioLabs was used) in the first lane. Note: When loading the samples in the well, maintain a positive pressure to prevent bubbles. 98 University of Ghana http://ugspace.ug.edu.gh 5. Carefully load the samples in the additional wells of the gel. 6. Run the gel for 80-150 V until the dye line is approximately 75-80% way down the gel for one hour or one and half hours depending on gel concentration and voltage. For 2% agarose gel, 120V and for 70 minutes. Note: For the electrodes, black is negative and red is positive. DNA is negatively charged and will run towards the positively charged electrode. 7. Turn OFF power, disconnect the electrode from the power source and carefully remove the gel from the gel box or tank. 8. Using ultra violet light, visualise the DNA fragments. The DNA fragments are usually referred to as “bands” due to their appearance on the gel. 99 University of Ghana http://ugspace.ug.edu.gh APPENDIX III Polymerase Chain Reaction (PCR) Master Mix Preparation Table 13: components with their various concentrations and volumes used to prepare the master mix Components 25µl Reactions Conventional Multiplex One Tag Quick-Load 2× Master mix 12.5µl 12.5µl 12.5µl with standard buffer 10µM Forward primer 0.5 µl 0.5 µl 0.5 µl ×6=3 µl 10 µM Reverse primer 0.5 µl 0.5 µl 0.5 µl ×6=3 µl Template DNA Variable 0.5 µl 5 µl Nuclease-free water To 25µl 6.5 µl 1.5 µl Table 12 above describes the components and the respective concentrations used in preparing the master mix for the polymerase chain reaction (PCR). Protocol for PCR A. Mix individual components prior to use. B. Assembly all reaction components on ice. C. Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin. D. Transfer reaction quickly to a preheated thermocycler (94o C). 100 University of Ghana http://ugspace.ug.edu.gh Table 14: Thermocycling conditions for the PCR Step Temperature Time Initial Denaturation 94oC 30 seconds 30 cycles 94oC 15-30 seconds 45-68oC 15-60 seconds 68oC 1 minute/kb Final Extension 68oC 5 minutes Hold 4-10oC Table 13 above describes the various cycling temperatures and duration for the polymerase chain reaction. 101 University of Ghana http://ugspace.ug.edu.gh APPENDIX 1V Gels MecA Figure 6: Agarose gel electrophoresis showing the patterns of mecA, Pvl and Spa genes from a multiples PCR. Source: from the study Spa genes Pvl genes Figure 7: Agarose gel electrophoresis showing the patterns of Pvl and Spa genes from a multiplex PCR. Source: from the study 102