i UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY THE EFFECT OF DIFFERENT CARBOHYDRATES AND AMINO ACIDS ON THE ACTIVITY OF SELECTED ANTIMICROBIAL COMPOUNDS AGAINST MYCOBACTERIUM SMEGMATIS BY MICHAEL ACHEAMPONG DEBRAH (10517023) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN MOLECULAR BIOLOGY DECEMBER 2021 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I, Michael Acheampong Debrah (Department of Biochemistry, Cell, and Molecular Biology, University of Ghana, Legon-Accra), hereby declare and certify that this thesis was done by me under the supervision of Dr. Patrick Kobina Arthur and Dr. Vincent Amarh. All references have been duly cited. Michael Acheampong Debrah (10517023) Dr. Patrick Kobina Arthur (Supervisor) Dr Vincent Amarh (Co-supervisor) University of Ghana http://ugspace.ug.edu.gh iii ABSTRACT Antibiotics play a significant role in the medical treatment of bacterial diseases. Bacteria respond to antibiotic stress by several means including the initiation of stress responses that promote recruitment of resistance determinants or physiological changes that compromise antimicrobial activity. The overdependence and misuse of these drugs have led to the emergence of resistance in microorganisms which complicates patient management and as such, different strategies are being sought to alleviate this threat that such resistant pathogens pose. One such strategy is chemo- sensitization where compounds are used to potentiate the effect of antibiotics. This study investigates the effect of selected carbohydrates and amino acids on the efficacy of antimicrobial across different classes, against Mycobacterium smegmatis strains to provide insight into key cellular processes associated with the functioning of antibiotics, and whose modulation would be key in sustaining antibiotic action and preventing resistance development. Seventeen (17) compounds across fifty-three (53) triple-combinations were screened in an experimental set-up from which 29 combinations were identified that robustly break resistance and 24 that robustly induce resistance. The top two (2) conditions that break resistance (SOR (3X) and MAL (3X)) led to the activation of fifteen (15) antibiotics and the top two (2) that induce resistance (LAC (3X) and SIA+ADO (1.5X)) led to the loss of efficacy of twelve (12) antibiotics. The top two (2) compounds in each category (resistance breaking & resistance inducing) were then prioritized for gene expression analysis looking at a landscape of stress response regulons in both Mycobacterium smegmatis (wt) and multidrug-resistant strains. The resistant breaking compounds induced genes such as vapC that’s responsible for toxicity in the organism, which correlates with its resistant breaking property but also the expression of virulence genes, that is lsr is in contrast. University of Ghana http://ugspace.ug.edu.gh iv Table of Contents DECLARATION......................................................................................................................................... ii ABSTRACT ................................................................................................................................................ iii Table of Contents ....................................................................................................................................... iv List of Figures ............................................................................................................................................ vii List of Tables ............................................................................................................................................... x List of Abbreviations ................................................................................................................................ xii DEDICATION........................................................................................................................................... xv ACKNOWLEDGEMENT ........................................................................................................................ xvi 1.0 INTRODUCTION ................................................................................................................................. 1 1.1 BACKGROUND ..................................................................................................................................... 1 1.2 Aim ...................................................................................................................................................... 3 1.3 Specific Objectives .............................................................................................................................. 3 2.0 LITERATURE REVIEW .................................................................................................................... 5 2.1 DISCOVERY AND CHALLENGES REGARDING ANTIMICROBIALS .......................................................... 5 2.2 ANTIMICROBIAL ADJUVANTS & MECHANISM OF ACTION ................................................................. 6 2.2.1 REGULATION OF MICROBIAL CELLULAR METABOLISM ............................................. 7 2.2.2 INFLUENCING CELL MEMBRANE PERMEABILITY.......................................................... 9 2.2.3 INHIBITION OF EFFLUX PUMPS ......................................................................................... 10 2.2.4 SELECTIVE PRESSURE FOR NATURAL GUT FAUNA ..................................................... 11 2.3 ADAPTIVE AMR AND STRESS RESPONSE ........................................................................................... 12 3.0 MATERIALS AND METHODS ....................................................................................................... 14 3.1 BACTERIAL STRAINS & CULTURE CONDITIONS ................................................................................. 14 3.2 PREPARATION OF ANTIBIOTICS AND PHENOTYPIC COMPOUNDS ................................................... 15 3.3 DETERMINATION OF THE EFFECTS OF PHENOTYPIC COMPOUNDS ON ANTIBIOTIC ACTIVITY & MORPHOLOGY ........................................................................................................................................ 17 3.4 DETERMINATION OF THE EFFECTS OF PHENOTYPIC COMPOUNDS ON EFFLUX AND BIOFILM ACTIVITY .................................................................................................................................................. 19 University of Ghana http://ugspace.ug.edu.gh v 3.5 ANALYSIS OF STRESS RESPONSE EXPRESSION PATTERNS WITH ADJUVANTS .................................. 20 4.0 RESULTS ............................................................................................................................................ 23 4.1 RESULTS OF THE EFFECT OF DIFFERENT CARBOHYDRATES AND AMINO ACIDS ON THE ANTIMICROBIAL ACTIVITY OF ANTIFUNGAL AND ANTIBACTERIAL COMPOUNDS ................................. 23 4.1.1 EFFECT OF SINGLE PHENOTYPIC COMPOUNDS ON ANTIBIOTIC ACTIVITY .......... 23 4.1.2 EFFECT OF STRUCTURAL TRIPLE PHENOTYPIC COMPOUND COMBINATIONS ON ANTIBIOTIC ACTIVITY .................................................................................................................. 26 4.1.3 EFFECT OF FUNCTIONAL TRIPLE PHENOTYPC COMPOUND COMBINATIONS ON ANTIBIOTIC ACTIVITY .................................................................................................................. 29 4.1.4 RANKING SYSTEMS FOR THE ANTIBIOTIC RESISTANCE BREAKING AND INDUCING EFFECT ......................................................................................................................... 35 4.1.5 SINGLE PENOTYPIC COMPOUND RANKING BY RESISTANCE-MODIFYING EFFECT AGAINST MYCOBACTERIA .......................................................................................................... 37 4.1.6 STRUCTURAL AND FUNCTIONAL TRIPLE PHENOTYPIC COMPOUND COMBINATION RANKING BY RESISTANCE-MODIFYING EFFECT AGAINST MYCOBACTERIA ............................................................................................................................. 42 4.1.7 DIRECT COMPARISON AND EVALUATION OF SINGLE PHENOTYPIC COMPOUNDS TESTED AT 1X AND 3X DOSES TO GUIDE THE SELECTION OF TOP FOR FURTHER STUDIES ............................................................................................................................................ 51 4.1.8 EFFECTS OF SINGLE PHENOTYPIC COMPOUND ON CELL AND COLONY MORPHOLOGY ................................................................................................................................ 54 4.1.9 EFFECTS OF TRIPLE COMPOUND COMBINATIONS ON CELL AND COLONY MORPHOLOGY ................................................................................................................................ 62 4.2 RESULTS ON THE EFFICACY OF SELECTED COMPOUNDS AGAINST THE EFFLUX AND BIOFILM ACTIVITY ................................................................................................................................................................ 75 4.2.1 EFFECTS OF SINGLE COMPOUND ON CELLULAR TRANSPORT; ACCUMULATION IN MYCOBACTERIA ....................................................................................................................... 75 4.2.2 EFFECTS OF TRIPLE COMPOUND COMBINATION ON CELLULAR TRANSPORT; ACCUMULATION IN MYCOBACTERIA ...................................................................................... 78 4.2.3 EFFECTS OF SINGLE COMPOUND ON CELLULAR TRANSPORT; EFFLUX IN MYCOBACTERIA ............................................................................................................................. 81 4.2.4 EFFECTS OF TRIPLE COMPOUND COMBINATION ON CELLULAR TRANSPORT; EFFLUX IN MYCOBACTERIA ....................................................................................................... 83 4.2.5 EFFECTS OF SINGLE COMPOUND ON CELLULAR TRANSPORT; ADHESION INHIBITION IN MYCOBACTERIA ................................................................................................ 85 4.2.6 EFFECTS OF TRIPLE COMPOUND COMBINATION ON CELLULAR TRANSPORT; ADHESION INHIBITION IN MYCOBACTERIA ........................................................................... 88 University of Ghana http://ugspace.ug.edu.gh vi 4.2.7 EFFECTS OF SINGLE COMPOUND ON CELLULAR SURVIVAL; ADHESION DISRUPTION IN MYCOBACTERIA ............................................................................................... 90 4.2.8 EFFECTS OF TRIPLE COMPOUND COMBINATIONS ON CELLULAR SURVIVAL; ADHESION DISRUPTION IN MYCOBACTERIA ......................................................................... 93 4.2.9 EFFECTS OF SINGLE COMPOUND ON CELLULAR SURVIVAL; BIOFILM INHIBITION IN MYCOBACTERIA ................................................................................................ 95 4.2.10 EFFECTS OF TRIPLE COMPOUND COMBINATION ON CELLULAR SURVIVAL; BIOFILM INHIBITION IN MYCOBACTERIA ............................................................................... 97 4.2.11 EFFECTS OF SINGLE COMPOUND ON CELLULAR SURVIVAL; BIOFILM DISRUPTION IN MYCOBACTERIA ............................................................................................... 99 4.2.12 EFFECTS OF TRIPLE COMPOUND COMBINATION ON CELLULAR SURVIVAL; BIOFILM DISRUPTION IN MYCOBACTERIA ........................................................................... 101 4.3 CORRELATION TABLES OF INTERACTION ASSAY WITH PHENOTYPIC ASSAYS ................................ 103 4.4 RESULTS ON THE EXPRESSION PATTERNS OF STRESS RESPONSE GENES IN TREATED CELLS ......... 117 5.0 DISCUSSION .................................................................................................................................... 124 REFERENCES ........................................................................................................................................ 134 APPENDIX .............................................................................................................................................. 142 List of Supplementary Figures ............................................................................................................... 142 List of Supplementary Tables ................................................................................................................ 144 University of Ghana http://ugspace.ug.edu.gh vii List of Figures Figure 4.0: Antibiotic activity profile for M. smegmatis treated with modifier compounds. ..................... 26 Figure 4.1: Antibiotic activity profile for M. smegmatis WT with structurally grouped modifier compounds. ................................................................................................................................................ 28 Figure 4.2: Antibiotic activity profile for M. smegmatis WT with functionally grouped modifier compounds. ................................................................................................................................................ 32 Figure 4.3: Antibiotic activity profile for eMsA with functionally grouped modifier compounds.. .......... 33 Figure 4.4: Antibiotic activity profile for eMsB with functionally grouped modifier compounds. ............ 34 Figure 4.5: Antibiotic activity profile comparing 1X and 3X of the phenotypic compounds among the three strains.. ............................................................................................................................................... 57 Figure 4.6: Effect of single PCs (dalcitol, myo-inositol, sialicin, lactose and tryptophane) on colony and cell morphology of Ms wt.. ......................................................................................................................... 59 Figure 4.7:.Effect of single PCs (sorbose, malonic acid, rhamnose, sodium pyruvate, maltose and tryptophane) on colony and cell morphology of Ms wt .............................................................................. 61 Figure 4.8: Effect of single PCs (xylose, cellobiose, raffinose, inulin, gluconic acid and galactose) on colony and cell morphology of Ms wt ........................................................................................................ 65 Figure 4.9: Effect of selected resistance-breaking FTC compounds (SOR (3X), MAL (3X), SOR+TRY+MALT,, MYO+MAL (1.5X) and ADO+CEL+INU) on colony and cell morphology of Ms wt.. .............................................................................................................................................................. 67 Figure 4.10: Effect of selected resistance-inducing FTC compounds (LAC (3X), SIA+ADO (1.5X), TRY (3X), RAF (3X), and ADO+XYL+RAF) on colony and cell morphology of Ms wt ................................. 69 Figure 4.11: Effect of selected resistance-breaking FTC compounds (SOR (3X), MAL (3X), SOR+TRY+MALT,, MYO+MAL (1.5X) and ADO+CEL+INU) on colony and cell morphology of eMsA.. ......................................................................................................................................................... 71 Figure 4.12: Effect of selected resistance-inducing FTC compounds (LAC (3X), SIA+ADO (1.5X), TRY (3X), RAF (3X), and ADO+XYL+RAF) on colony and cell morphology of eMsA .................................. 73 University of Ghana http://ugspace.ug.edu.gh viii Figure 4.13: Effect of selected resistance-breaking FTC compounds (SOR (3X), MAL (3X), SOR+TRY+MALT,, MYO+MAL (1.5X) and ADO+CEL+INU) on colony and cell morphology of eMsB.. ......................................................................................................................................................... 75 Figure 4.14: Effect of selected resistance-inducing FTC compounds (LAC (3X), SIA+ADO (1.5X), TRY (3X), RAF (3X), and ADO+XYL+RAF) on colony and cell morphology of eMsB.. ................................ 78 Figure 4.15: Flourescence values for ethidium bromide accumulation in Ms wt with single compounds.. 81 Figure 4.16: Flourescence values for ethidium bromide accumulation in Ms wt with triple compound combinations.. ............................................................................................................................................. 83 Figure 4.17: Flourescence values for ethidium bromide efflux in (A)Ms wt. (B) eMsA and (C) eMsB. with single compounds.. ............................................................................................................................. 85 Figure 4.18: Fluorescence values for ethidium bromide Efflux in (A)Ms wt. (B) eMsA and (C) eMsB. with triple compound combinations ............................................................................................................ 88 Figure 4.19: Absorbance values for adhesion inhibition in (A)Ms wt. (B) eMsA and (C) eMsB with single compounds .................................................................................................................................................. 90 Figure 4.20: Absorbance values for adhesion inhibition in (A)Ms wt. (B) eMsA and (C) eMsB. with triple compound combinations. 10 ul of 10 mg/ul of each PC in the combination were added ........................... 93 Figure 4.21: Absorbance values for adhesion disruption in (A)Ms wt. (B) eMsA and (C) eMsB. with single compounds........................................................................................................................................ 95 Figure 4.22: Absorbance values for adhesion disruption in (A)Ms wt. (B) eMsA and (C) eMsB. with triple compound combinations ............................................................................................................................. 97 Figure 4.23: Absorbance values for biofilm inhibition in (A)Ms wt. (B) eMsA and (C) eMsB with single compounds. ................................................................................................................................................. 99 Figure 4.24: Absorbance values for biofilm inhibition in (A)Ms wt. (B) eMsA and (C) eMsB. with triple compound combinations ........................................................................................................................... 101 Figure 4.25: Absorbance values for biofilm disruption in (A)Ms wt. (B) eMsA and (C) eMsB. with single compounds. ............................................................................................................................................... 103 University of Ghana http://ugspace.ug.edu.gh ix Figure 4.26: Absorbance values for biofilm disruption in (A)Ms wt. (B) eMsA and (C) eMsB. with triple compound combinations ........................................................................................................................... 120 Figure 4.27: Expression analysis of vapB, lsr and vapC genes ................................................................ 121 Figure 4.28: Expression analysis of mshB, end and uspC genes .............................................................. 123 Figure 4.29: Expression analysis of 2758B, acr and relA genes ............................................................. 1235 University of Ghana http://ugspace.ug.edu.gh x List of Tables Table 3.0: List of antibiotics and their working amounts used in this work Error! Bookmark not defined. Table 3.1: Groupings of the seventeen (17) biomolecules .......................... Error! Bookmark not defined. Table 3.2: PCR Thermocycler conditions ................................................................................................... 22 Table 4.0: List of Structural Triple Combination and their groups ............................................................. 27 Table 4.1: List of functional Triple Combination and the antibiotics they affect ....................................... 30 Table 4.2: The various classes of antibiotics used in the experiment ......................................................... 35 Table 4.3 Resistant-breaking treatment of single compounds against M. smegmatis (Rank 1-6): ............ 39 Table 4.4: Resistant-breaking treatment of single compounds against M. smegmatis (Rank 7-17) ........... 40 Table 4.5: Resistant-inducing treatment of single compounds against M. smegmatis (Rank 1-5) ............. 41 Table 4.6: Resistant-inducing treatment of single compounds against M. smegmatis (Rank 6-15) ........... 43 Table 4.7: Resistant-breaking treatment of compounds (STC) against M. smegmatis (Rank 1-9) ........... 44 Table 4.8: Resistant-inducing treatment of compounds (STC) against M. smegmatis (Rank 1-10) ......... 45 Table 4.9: Resistant-breaking treatment of compounds (FTC) against M. smegmatis (Rank 1-10) ......... 46 Table 4.10: Resistant-breaking treatment of compounds (FTC) against M. smegmatis (Rank 11-20) ...... 47 Table 4.11: Resistant-breaking treatment of compounds (FTC) against M. smegmatis (Rank 21-30) ...... 48 Table 4.12: Resistant-inducing treatment of compounds (FTC) against M. smegmatis (Rank 1-7) .......... 49 Table 4.13: Resistant-inducing treatment of compounds (FTC) against M. smegmatis (Rank 8-20) ........ 50 Table 4.14: Resistant-inducing treatment of compounds (FTC) against M. smegmatis (Rank 21-34) .... 104 Table 4.15: Correlation table of single compounds against Ms wt ........................................................... 106 Table 4.16: Correlation table of single compounds against eMsA ........................................................... 109 Table 4.17: Correlation table of single compounds against eMsB ........................................................... 111 Table 4.18: Correlation table of selected FTC against Ms wt ................................................................... 113 Table 4.19: Correlation table of selected FTC against eMsA ................................................................... 115 Table 4.20: Correlation table of selected FTC against eMsB ................................................................... 117 Table 4.21: List of stress response genes with their function ................................................................... 123 Table 4.22: Summary of expression analysis of stress response genes .................................................... 123 University of Ghana http://ugspace.ug.edu.gh xi University of Ghana http://ugspace.ug.edu.gh xii List of Abbreviations ADO Adonitol AMPs Antimicrobial peptides AMR Antimicrobial resistance CELL Cellobiose CV Crystal Violet DAL Dulcitol eMsA erythromycin-resistant Mycobacterium smegmatis A eMsB erythromycin-resistant Mycobacterium smegmatis B ESKAPE E- Enterococcus faecium, S- Staphylococcus aureus, K- Klebsiella pneumonia, A: Acinetobacter baumannii, P: Pseudomonas aeruginosa, and E: Enterobacteriaceae EtBr Ethidium Bromide FTC Functional Triple Combination GAL Galactose GLU Gluconic Acid HIV Human Immunodeficiency Syndrome INU Inulin LAC Lactose MAL Malonic Acid MALT Maltose University of Ghana http://ugspace.ug.edu.gh xiii MDR Multidrug resistant MRSA methicillin-resistant Staphylococcus aureus Ms wt M. smegmatis wild type Mtb Mycobacterium tuberculosis MYO Myo-inositol NaP Sodium Pyruvate NPT non- phosphotransferase OD Optical Density PBS Phosphate Buffered Saline PC Phenotypic compounds RAF Raffinose RB Resistance Breaking RHA(M) Rhamnose RI Resistance Inducing RNA Ribonuclease activity RT-qPCR Reverse transcriptase-quantitative polymerase chain reaction SIA Sialicin SOR(B) Sorbose STC Structural Triple Combination University of Ghana http://ugspace.ug.edu.gh xiv TB Tuberculosis TRY Tryptophan XYL Xylose ZoI Zone of Inhibition University of Ghana http://ugspace.ug.edu.gh xv DEDICATION I dedicate this work to all members of Chemical Systems Biology Lab. University of Ghana http://ugspace.ug.edu.gh xvi ACKNOWLEDGEMENT My foremost gratitude is to God for seeing me through the MPhil training. Secondly, to my able and industrious supervisors, Dr. Patrick Kobina Arthur and Dr. Vincent Amarh, whose patience, guidance, mentoring, and supervision shaped and sharpened my research skills, I say I am very grateful. My next appreciation goes to all members of PAKARLab from 2018 to 2021 for their support and encouragement especially Dr. Ethel Blessie, Benaiah Annertey Abbey, Samuel Akwasi Acheampong, Gwendolyn Nita Amarquaye, Isaac Ogbe, Gifty Sowen, and Zerah Mingle for their various contributions. Finally, I am thankful to the West African Centre for Cell Biology of Infectious Pathogens for funding this project. Gratitude also to staff and faculty of the Department of Biochemistry, Cell and Molecular Biology for the supportive training and research environment. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 BACKGROUND Microorganisms are microscopic-sized organisms that include bacteria, archaea, fungi, and protists. These organisms can either be pathogenic or non-pathogenic. In the 20th Century, during the pre-antibiotic era, microbial infections accounted for a significant percentage of morbidities and mortalities (Dougherty & Pucci, 2014). The discovery of penicillin by Alexander Fleming began an antibiotic era, a period of redemption for human health. This addressed challenges with sepsis in advanced procedures like invasive surgery and chemotherapy subsequently increasing life expectancy (Silver, 2011; Tacconelli et al., 2018). During the years 1950 through 1970 described as the “Golden age”, many new antibiotic classes having different cellular targets were discovered. Humans seemed to be winning the evolutionary race against microbial infection till resistance to these antibiotics among microbes emerged (Hutchings et al., 2019). Antimicrobial resistance (AMR) is the evolution of microbes to nullify the effect of antimicrobials. Microbes employ different mechanisms to survive the killing or growth inhibitory effects of antimicrobials. The resistance gained by microbes may be acquired; that is gene transfer among organisms through vectors such as transposons, plasmids, bacteriophages, and integrons, for which plasmids are a major vector for the transfer of resistance genes among bacteria and associated with most resistant phenotypes (C Reygaert, 2018). The resistance may also be inherent through genetic (chromosomal) mutations resulting in cross-resistance (Li & Webster, 2018). Several factors can give rise to antimicrobial-resistant phenotypes such as the extent of expression of resistant genes by microorganism and their ability to endure antimicrobial pressure, just to state a few (Li & Webster, 2018). Microbes also make use of other biochemical types of resistance mechanisms to University of Ghana http://ugspace.ug.edu.gh 2 elude the effect of antimicrobials such as altering antibiotic targets, biofilm formation, enzymatic degradation, efflux pump overexpression, and so on (Džidić et al., 2008). AMR, now considered a global health concern, continues to increase primarily because of the non- compliance of health professionals and individuals in adhering to antimicrobial drug prescriptions (Ayukekbong et al., 2017). The problem is exacerbated by the widespread exploitation of antibiotics in aquaculture and farming practices (Manyi-Loh et al., 2018) and a drastic decrease in the discovery and development of new antimicrobial agents against an increasing rate in microbial evolution (Ragheb et al., 2019). The incidence and mortality rate as a result of AMR keeps increasing globally, approximately 700,000 individuals die annually from AMR infections; a number which has been extrapolated to increase to 10 million by 2050 exceeding cancer, which is the leading cause of global mortality (Neill, 2014). Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a global health threat and was the leading worldwide cause of mortality by an infectious disease prior to the COVID-19 pandemic and also the leading cause of death among HIV/AIDS individuals. Over 10 million TB cases and 1.5 million deaths were estimated in 2018(MacNeil et al., 2020) (. The prevalence of TB decreased when anti-mycobacterial drugs such as isoniazid (INH), rifampicin (RIF), pyrazinamide (PZD), ethambutol (EMB) were produced fifty years ago (Chakraborty & Rhee, 2015). Resistance to these anti-tuberculosis drugs occurred soon after their introduction, greatly increasing the difficulty of tuberculosis chemotherapy(Ahmad Khan et al., 2017). This has necessitated the understanding of the molecular mechanism underlying the resistance evolved by Mtb. Studies have implicated metabolism undertones in antibiotic resistance microbes, especially the ones involved in biofilm formation and latency. According to this view, microbe sensitivity to antibiotics (phenotypic resistance) can be modulated by alterations in microbe’s metabolism, signifying an association between microbial metabolism and antibiotic resistance. University of Ghana http://ugspace.ug.edu.gh 3 Antimicrobial drugs that have lost their efficacy because of AMR, can be revived through the utilization of adjuvants (metabolically relevant non-antibiotic compounds that improve the activity of antibiotics by breaking resistance or by enhancing bacterial susceptibility). Some examples include β-lactamase inhibitor compounds used in medical practices. The synchronal use of antimicrobials and adjuvants aside from enhancing efficacy, also provides a lower toxicity level since less amount of the antibiotic and adjuvant are used (Gill et al., 2015; Sanhueza et al., 2017). A lot of studies conducted recently have proven the increase in efficacy of antimicrobial agents when combined with certain adjuvants such as amino acids and carbohydrates (Bernal et al., 2013). This current study seeks to examine the effect of selected carbohydrates and amino acids on the bioactivity of different antimicrobial agents and how they modulate Mycobacterium smegmatis, C. albicans, and E. coli phenotypically and genetically. 1.2 Aim To investigate the effect of certain carbohydrates and amino acids on the antibiotic profile of Mycobacterium smegmatis (WT) and two multi-drug resistant strains, Erythromycin-resistant Mycobacterium smegmatis A & B. 1.3 Specific Objectives 1. Determine the effect of different carbohydrates and amino acids on the antimicrobial activity of antifungal and antibacterial compounds using phenotypic drug screening assay on Mycobacterium smegmatis and the two MDR strains, C. albicans, and E. coli. 2. Determine the efficacy of selected compounds against the efflux and biofilm activity of, and Mycobacterium smegmatis (WT) and two multi-drug resistant strains, Erythromycin- resistant Mycobacterium smegmatis A & B. University of Ghana http://ugspace.ug.edu.gh 4 3. Analyze the expression patterns of stress response genes in cells treated with growth conditions that increase and decrease drug resistance in Mycobacterium smegmatis and the two MDR strains. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER TWO LITERATURE REVIEW 2.1 DISCOVERY AND CHALLENGES REGARDING ANTIMICROBIALS Alexander Fleming’s serendipitous discovery of penicillin some decades ago transformed the course of medicine. The advent of antibiotics and other antimicrobial drugs has served for years as the only effective therapeutic option for the elimination of microbial infection (Dougherty & Pucci, 2014; Silver, 2011). These infections had previously translated to high morbidity and mortality before the discovery of antibiotics. However, the continuous use and misuse of these agents have resulted in the emergence of microbes that are no longer sensitive to these antibiotics (Jackson et al., 2018). The inefficacy of most antimicrobial drugs is a result of over-dependence and inappropriate use of these drugs (Mobarki et al., 2019). AMR can generally be categorized into three main types: acquired (inheritable), inherent (intrinsic), and adaptive (Alekshun & Levy, 2007). Acquired AMR develops from bacteria’s ability to obtain and express additional genetic material excluding genes already present within their primary genome. Mobile genetic elements which cary these new genes include plasmids, transposons, or some other yet-to-be-identified carrier in the immediate environment of the bacteria or other bacteria. This method of transfer and incorporation of new genetic elements is described as horizontal gene transfer (HGT) and resulting resistance may or may not be passed on to offspring. Intrinsic resistance on the other hand, describes an evolutionary change caused by the interactions of the microbe with its environment. The environment serves as a habitat for natural antimicrobials which interact with bacteria. Bacteria tend to harbour inherent genetic elements of resistance as a result of these natural interactions (Smith, 2017). These resistance patterns in the bacteria evolve University of Ghana http://ugspace.ug.edu.gh 6 over many years within the bacteria’s primary genome and can be directly transferred to new offspring during reproduction (Dcosta et al., 2011). Finally, adaptive AMR describes more tolerance-focused mechanisms bacteria have taken to withstand the effect of antibiotics. It is characterized by a quick development of resistance and fast reversibility to the non-resistant phenotype when the antibiotic is removed from the medium. Adaptive AMR is highly driven by bacterial exposure to subinhibitory concentrations of antibiotics or gradually increasing antimicrobial concentrations. (Sandoval-Motta & Aldana, 2016). 2.2 ANTIMICROBIAL ADJUVANTS & MECHANISM OF ACTION In the event that a compound is able to create an environment or trigger signaling pathways that convince these transiently resistant cells exhibiting adaptive AMR properties of the absence of a still-present antibiotic, this leads to a rapid transformation of bacterial cells back to sensitive phenotypes and consequently exposes them to antibiotic concentrations that would otherwise have been non lethal to the resistant phenotype. The compound is described to have potentiated the action of the antibiotic. Such compounds are described as adjuvants, molecules which on their own have none or marginal antibiotic effects, but synergistically potentiate the efficacy of an antibiotic. (Ejim et al., 2011; Sanhueza et al., 2017; Wright, 2016; Yang et al., 2017). The concept of one drug-one target in infectious disease has waned over the years because of the evolution of resistance (Worthington & Melander, 2012). Combination therapies involving adjuvants are now increasingly employed in clinical settings to deal with microbial infections that tend to be resistant to available antibiotics. This concept has long been applied for the treatment of some diseases such as cancer and HIV-AIDS (Bock & Lengauer, 2012). There are generally two forms of these combinatory therapies currently; combinations of two or more antibiotics (Drug-Drug combinations) or a drug and an adjuvant. University of Ghana http://ugspace.ug.edu.gh 7 The effects of combinations present three outcomes; Additivity (wherein there is no overall change as a result of the combination), antagonism (wherein the action of one agent acts against the effects of the other producing an overall negative result), and synergism (the action of one agent potentiates the activity of the other agent, increasing its overall efficacy). Most combination therapies are therefore geared towards synergistic outcomes (Wright, 2016). Antibiotics in separate classes are usually combined to tackle single drug-resistant strains to reduce the chances of antibiotic resistance to the combination. Common drug combinations are between aminoglycosides or quinolone drugs and beta-lactams (Tamma et al., 2012). However, drug-drug combinations are usually not preferred because it promotes the emergence of multi-drug resistance (MDR). However, adjuvant-drug (AD) combinations are preferred because it either breaks resistance or compliments antibiotic effect thereby reducing the development of resistance against such combinations (Gill et al., 2015; Wright, 2016). A combination of amoxicillin which is a Beta- lactam antibiotic and clavulanic acid, an inhibitor to beta-lactamase serving as the adjuvant is one common example of an AD combination (Worthington & Melander, 2012). The mechanisms by which antibiotic adjuvants potentiate the activity of antibiotics against resistant microbes have not been widely studied. Some of the currently known mechanisms include enhancing the intake of antibiotic agents through the bacteria plasma membrane, altering bacterial physiological/metabolic state, and efflux pump inhibition (Wright, 2016). Unlike antibiotics, adjuvants are not necessarily required to target specific components of the bacteria but rather potentiate the activity of the antibiotic against its target (Gill et al., 2015). 2.2.1 REGULATION OF MICROBIAL CELLULAR METABOLISM The metabolic state of bacteria determines their sensitivity to antibiotic therapy and certain metabolic patterns match with AMR. Molecules that can change the metabolic state of resistant bacteria to that of a sensitive one could reverse the antibiotic susceptibility profile (Maugeri et al., University of Ghana http://ugspace.ug.edu.gh 8 2019). In a study conducted by Claudi and colleagues in 2014, antibiotic therapy against fast- dividing Salmonella spp cells was more effective as compared to non-dividing or slow-growing cells and hence the latter cells were able to tolerate the antibiotic stress to form persisters. They concluded that the growth pattern of a microbe in infection can influence the efficacy of the antibiotic used. Therefore, the reversibility of the growth pattern can influence the activity of antimicrobial drugs (Claudi et al., 2014). Molecules that can increase the metabolic or growth rate of microbes can serve as antibiotic adjuvants. Studies in this area have identified some carbohydrate moieties to influence metabolic rate systems found within microbes and as such can be employed as antimicrobial adjuvants. Myoinositol, a carboxylic sugar was found to enhance the proliferation of Corynebacterium glutamicum when provided exogenously by serving as the source of energy and carbon to the Corynebacterium spp (Krings et al., 2006). In contrast to this observation, galactose intake and breakdown in Saccharomyces cerevisiae is lower as compared to fructose and glucose and as such decreases yeast proliferation (De Jongh et al., 2008). The bacterial metabolic milieu and antibiotic sensitivity pattern of Escherichia coli can be modified by the addition of exogenous small molecules such as nitric oxide, indole, prebiotics, vitamin-like nutrients, and so on (Allison et al., 2011). Allison and colleagues concluded in their research that the chemotherapeutic potential of aminoglycosides is enhanced against E. coli when exogenic metabolites especially of carbon sources are added. Moreover, the bactericidal activity of tobramycin against Pseudomonas aeruginosa is potentiated significantly in the presence of mannitol (Allison et al., 2011). Myoinositol, a vitamin-like nutrient has been reported to ameliorate the host’s defense system to eradicate resistant E. coli when used either alone or as an adjuvant with florfenicol (Chen et al., 2015). University of Ghana http://ugspace.ug.edu.gh 9 Microbial intracellular enzymes require optimum conditions within their immediate environment for proper physiological functioning (Robinson, 2015). Malonic acid, an organic acid, inhibits endodontic pathogens by decreasing intracellular pH. Undissociated molecules found within the cytosol of the pathogen-free up hydrogen ions (protons) in the presence of organic acids such as malonic acid (Ballal et al., 2011). Certain amino acids, such as tryptophan, proline, and arginine have been reported to be effective in the elimination of bacterial pathogens (Mishra et al., 2018; Zhu et al., 2014). The immune system secretes certain peptides with antimicrobial activity to eliminate pathogenic bacteria. These antimicrobial peptides (AMPs) consisting preponderantly of proline or tryptophan can eradicate pathogenic microbes by targeting metabolic pathways (Zhu et al., 2014) and hence are an auspicious source of potentiators of antibiotics. The robustness of these peptides is because of the aromaticity of tryptophan sidechains which is capable of quickly forming hydrogen bonds with the plasma membrane of these microbes (Bacalum et al., 2019). Aside from the hydrogen bonding, some literature has also reported that the bulky nature of tryptophan’s indole-sidechains can insert deeply into the lipid bilayer’s hydrocarbon core and interfere with the lipid acyl group interactions (Mishra et al., 2018). 2.2.2 INFLUENCING CELL MEMBRANE PERMEABILITY The impermeable membrane barrier of bacteria especially gram-negative bacteria serves as a major hurdle in the field of drug discovery (Peng et al., 2015). In a previous study, Peng and colleagues used exogenic glucose and alanine to revive the activity of kanamycin against resistant Edwardsiella tarda. A mixture of glucose and alanine as adjuvants to kanamycin altered the permeability of the plasma membrane increasing drug intake and eliminating resistant strains (Peng et al., 2015). University of Ghana http://ugspace.ug.edu.gh 10 Cellobiose lipids obtained from the yeast Cryptococcus spp displayed antifungal activity against Candida tropicalis by damaging their cell membrane. The fatty acid composition of the cellobiose residue is acetylated and contains hydroxyl groups that confer its membrane damaging activity (Kulakovskaya et al., 2014). 2.2.3 INHIBITION OF EFFLUX PUMPS Efflux pumps are membrane-bound proteins present in the cell membrane and originally evolved for use in expelling out cellular wastes as well as other toxic compounds and can generally be classified into five groups based on their composition, source of energy and substrate, several transmembrane regions: the Major Facilitator superfamily (MFS), small multidrug resistance family (SMR), which is a member of the much bigger drug/metabolite transporter superfamily (DMT), resistance-nodulation-cell division (RND) family, multidrug and toxic compound extrusion family (MATE) and the Adenosine Triphosphate (ATP)- Binding cassette families. Resistant microbes have adapted the capability of quickly expelling drugs or chemicals Using these efflux pumps which have a poly-substrate specificity (Sun et al., 2014). This decreases the intracellular concentrations of these antibiotics, thereby enhancing the probability of resistance mutations at sub-therapeutic doses of the antibiotic (Sun et al., 2014). Studies have proven that hindering the action of these pumps will lead to an increase in drug uptake to break antimicrobial resistance. Villagra and colleagues determined the effect of non- phosphotransferase (non-PTS) carbohydrate (xylose) on the antimicrobial resistance pattern of Salmonella Typhimurium. They concluded there is the possibility of an increase in the production of carbohydrate permeases which could modify the bacteria’s inner-membrane protein constituent. Their data also showed a resistance-breaking effect as a result of an alteration of the efflux pumps mediating the resistance by the carbohydrate permeases (Villagra et al., 2012). University of Ghana http://ugspace.ug.edu.gh 11 Ying Gong and colleagues conducted a study to determine the synergistic effect of a non- antifungal drug (NBP) and fluconazole (FLC) against fluconazole-resistant Candida albicans. Their data demonstrated an increase in fluconazole uptake and a decrease in the efflux activity by the fungi, leading to increased sensitivity of the fungal cells to FLC. Reverse Transcriptase-PCR analyses suggested a down-regulation of the expression of C. albicans drug resistance genes CDR1 and CDR2 transporter genes by the synergistic effect of the NBP and FLC (Ying Gong et al., 2019). 2.2.4 SELECTIVE PRESSURE FOR NATURAL GUT FAUNA Prebiotics are food ingredients that have beneficial functions in the human gut system; they are non-digestible and affect the activity of specific gut bacteria (Doran & Verran, 2007; Mandal et al., 2016). Inulin is an example of a prebiotic that ensures the survival of lactobacilli cells and induces their antimicrobial activity (Balthazar et al., 2018). Inulin can be found in a lot of foods like bananas, garlic, onions, and so on. It is classified under fructans, which are non-digestible carbohydrates (James, 2014). Aside from inulin being utilized mostly by gut lactobacilli spp, there have been some reports of its usage by some strains of Streptococci spp found in the oral cavity (Doran & Verran, 2007). A recent study conducted by Önal Darilmaz and colleagues against Escherichia coli implicated inulin as a key player in the antibacterial activity of a combination of inulin (prebiotic) and Lactobacillus fermentum (probiotic). The synergy between inulin and L. fermentum led to an increase in oxalate-mediated degradation of the E. coli cells (Önal Darilmaz et al., 2019). This data was consistent with a previous study conducted by Kareem and colleagues where they found a synergistic effect of inulin and Lactobacillus plantarum with inhibitory activity against various microbial pathogens (Kareem et al., 2014). University of Ghana http://ugspace.ug.edu.gh 12 From these, it is apparent that adjuvants exist as a wide variety, from simple sugars, amino acids, and fatty acids to other organic or inorganic molecules, and when used in combination with antibiotics can revert resistance to built-in naturally sensitive microbes or sensitize microbes that are intrinsically resistant to antimicrobial drugs (Bernal et al., 2013). Such combination therapies represent an untapped potential to repurpose obsolete antibiotics with limited or diminished antibiotic activity (Taylor et al., 2012) and increase our repertoire of antimicrobial drugs (Sanhueza et al., 2017). 2.3 ADAPTIVE AMR AND STRESS RESPONSE There is a general melding between adaptive AMR and stress response in bacteria because the general trigger for adaptive resistance, in this case the antibiotic, is also considered a stressor. Stress response describes a number of complex and dynamic process that involves the production of protective molecules (e.g. antibiotic degrading enzymes), changes in metabolism and behavior, induce the expression of multidrug efflux pumps or modify the permeability of the bacterial cell envelope, which can decrease the intracellular concentration of antibiotics and confer temporary resistance. Other stress response mechanisms, such as the induction of SOS response or stringent response, can cause temporary or reversible changes in bacterial DNA replication, transcription, and translation, leading to the transient inhibition of cell growth and metabolism. Taken together, these act toward enhancing bacterial ‘fitness’ against adverse conditions, but also impose a ‘fitness cost’ on growth and reproduction under non-stressful conditions (Johnsen et al., 2009). For instance, in Pseudomonas aeruginosa, mutations in stress response genes can mitigate the fitness cost of fluoroquinolone resistance by improving bacterial growth and survival under oxidative stress. Similarly, in Salmonella enterica, mutations in stress response genes can increase the fitness of multidrug-resistant strains under low-nutrient conditions. These findings suggest that stress response can play a key role in modulating the trade-offs between resistance and fitness cost, University of Ghana http://ugspace.ug.edu.gh 13 and may contribute to the spread and persistence of resistant bacteria in different environments (Agnello et al., 2016; Fàbrega & Vila, 2013). It is important to note however, that not all adaptive AMR mechanisms are related to stress response. One such example of temporary resistance observed in bacteria that is independent of stress response is the phenomenon of “persistence” or “tolerance” to antibiotics. Persister cells are a subpopulation of bacteria that exhibit a transient and non-inheritable resistance to antibiotics, even in the absence of any obvious stressor or environmental cue. Persister cells are thought to arise through stochastic processes of genetic or phenotypic variation, and can be characterized by a slowed or arrested metabolism, reduced growth rate, and altered expression of genes involved in stress response, metabolism, and virulence. Persister cells have been identified in various bacterial species and clinical isolates, and are believed to play a significant role in the persistence and recurrence of chronic infections, as well as in the emergence and spread of antibiotic resistance. The exact mechanisms underlying persister formation and persistence are not yet fully understood, but are believed to involve complex interactions between genetic, biochemical, and physiological factors (Lewis, 2010). Overall, the relationship between adaptive AMR mechanisms, fitness cost and stress response is a complex and dynamic process that depends on multiple factors, including the type and severity of stress, the genetic and environmental context, and the availability of resources. By understanding the mechanisms and trade-offs involved in this interplay, we can develop new strategies for controlling AMR, such as targeting stress response pathways to enhance the fitness cost of resistance, or manipulating environmental conditions to reduce the selective pressure for resistance. University of Ghana http://ugspace.ug.edu.gh 14 CHAPTER THREE MATERIALS AND METHODS 3.1 BACTERIAL STRAINS & CULTURE CONDITIONS The model organisms used for this experiment were Mycobacterium smegmatis mc2155 (Ms wt acquired from ETH-Zurich, Switzerland) and two other multi-drug resistant (MDR) strains, erythromycin-resistant Mycobacterium smegmatis A and B (eMsA and eMsB). These multi-drug Mycobacteria spp were obtained from the Laboratory for Chemical Systems Biology of Infectious Disease and kept on a 7H10 slant at 4°C. Before use, each organism was sub-cultured from the slants onto agar plates and incubated at 37°C for 48 h. The cells were acid-fast stained to serve as a quality control before seeding them into 7H9 broth to make starter cultures. These were incubated with shaking at 180 rpm. Seed cultures were made from the starter cultures at optical density (OD) of 0.1 and incubated for another 48 h for growth until the mid-exponential growth phase was obtained before every bioassay. The OD reading taken at an absorbance of 600nm was adjusted to 0.7 as the working OD. Some limited drug screening assays were done using the C. albicans, and E. coli. The 7H9 media constituted monopotassium phosphate (1 g/L), magnesium sulphate (0.05 g/L), ammonium sulphate (0.5 g/L), calcium chloride (0.0005 g/L), disodium phosphate (2.5 g/L), copper sulphate (0.001 g/L), biotin (0.0005 g/L), L-glutamic acid (.5 g/L), ferric ammonium citrate (0.04 g/L), and pyridoxine (0.001 g/L). The broth was prepared by weighing 0.1305 g of 7H9 broth base into a clean and sterile conical flask containing 25 ml of distilled water. This solution was complimented with 0.02125 NaCl, 62.5 uL (0.25%) of 20% Tween 80, and 110 uL (0.44%) glycerol and autoclaved at 121°C for 15 min. The components of 7H10 media used include; L-glutamic acid (0.5 g/L), ammonium sulphate (0.5 g/L), sodium citrate (0.4 g/L), magnesium sulphate (0.025 g/L), zinc sulphate (0.001 g/L), ferric ammonium citrate (0.04 g/L), monopotassium phosphate (1.5 g/L), disodium phosphate (1.5 g/L), University of Ghana http://ugspace.ug.edu.gh 15 malachite green (0.00025 g/L), sodium citrate (0.4 g/L), calcium chloride (0.0005 g/L), copper sulphate (0.001 g/L) and agar (15 g/L). Per manufacturer’s instructions, 1.9g of Middlebrook 7H10 agar base was weighed into a sterile conical flask containing 100 ml of distilled water to make a 1.9% solution. This solution was augmented with 0.085% NaCl and 0.5% dextrose. 3.2 PREPARATION OF ANTIBIOTICS AND PHENOTYPIC COMPOUNDS The selected standard antimicrobial drugs used were, Ampicillin, Amoxicillin, Vancomycin, Isoniazid, Ethambutol, Ethionamide, Pyrazinamide, Methicillin, Rifampicin, Linezolid, Tetracycline, Chloramphenicol, Erythromycin, Streptomycin, Cycloserine, Gentamycin, Paromomycin, 5- Fluorouracil, Clindamycin, and Moxifloxacin. Stock and working concentrations of each compound were prepared and kept in the freezer (0°C) for storage. These Antimicrobial drugs were pipetted based on the working concentrations as shown on the table.: University of Ghana http://ugspace.ug.edu.gh 16 Table 3.0: List of antibiotics and their working amounts used in this work Antibiotic Abbreviation Working amounts (ug) Rifampicin Rif 10 Tetracycline Tet 20 Streptomycin Strep 30 Isoniazid INH 20 Erythromycin Ery 40 Ampicillin Amp 40 Moxifloxacillin Moxi 1 Linezolid Lin 40 Vancomycin Van 5 Ethambutol Emb 10 Pyrazinamide PZD 40 Amoxacillin Amx 40 Chloramphenicol Chlo 40 Cycloserine Cyser 20 Gentamycin Gen 10 Metronidazole Met 30 Paromomycin Para 20 5-Fluorouracil 5-FU 1 Clindamycin Clind 40 Ethionamide Eth 40 The list of the phenotypic compounds is in the Table below. Table 3.1: Groupings of the seventeen (17) biomolecules 1. DI- AND TRI-SACCARIDES ❖ LACTOSE (4) ❖ MALTOSE ❖ RAFFINOSE ❖ CELLOBIOSE ❖ INULIN 2. FIVE (5) AND SIX (6)-CARBON ALCOHOLS ❖ DULCITOL ❖ ADONITOL 3. FIVE (5) AND SIX (6)-CARBON MONOSACCHARIDES ❖ SORBOSE ❖ GALACTOSE University of Ghana http://ugspace.ug.edu.gh 17 ❖ RHAMNOSE ❖ XYLOSE 4. ACIDIC GLYCOSIDES ❖ GLUCONIC ACID 5. AMINO ACID ❖ TRYPTOPHAN 6. SHORT CHAIN FATTY ACID ❖ MALONIC ACID ❖ PYRUVATE 7. SIX (6)-CARBON COMPOUNDS AND ALCHOLIC GLYCOSIDES ❖ MYOINOSITOL ❖ SALICIN A mass of 10 mg of each compound was weighed into a 15 ml falcon tube containing 10 ml of 7H9 broth for Mycobacterium culturing. The mixture was filter sterilized with a Whatman filter and a syringe. The compounds were stored in the refrigerator. 3.3 DETERMINATION OF THE EFFECTS OF PHENOTYPIC COMPOUNDS ON ANTIBIOTIC ACTIVITY & MORPHOLOGY As previously described, seed and starter cultures were set up from slants and adjusted to their working OD. The 17 single compounds were tested in the first set of assays performed. After this, 10 structural triple combinations were generated based on the structure and diversity of the phenotypic compounds. From the results obtained, another set of 34 functional triple combinations (FTC) was generated based on the antibiotics the phenotypic compounds affected by breaking or inducing resistance. A volume of 100 ul of each phenotypic compound (1 ug/ul) was spread on agar plates and allowed to dry. The cells at the working OD were spread on the modified agar plates and known amounts of antibiotics pipetted onto sterile filter discs and allowed to dry were placed on the inoculated plates. The zones of inhibition created by the antibiotics were measured after incubating the assay plates at 37°C for 48 hr. A control was set up where no phenotypic compound was spread on the plates and all experiments were set up in duplicates. University of Ghana http://ugspace.ug.edu.gh 18 A triple combination system of the phenotypic compounds (PC) was established depending on their structure and effect on antibiotic activity. The structural triple combination (STC) had ten groups while the functional triple combination (FTC) had 34 groups, each group containing a combination of 3 PCs. The activity of the compounds was grouped into resistance breaking (compounds that increased activity of the antibiotic) and resistance inducing (compounds that reduced the activity of the antibiotic compound compared to the control). For the phenotypic assays, the 17 single compounds and the top 5 resistant breaking of the 35 FTC, as well as the top 5 resistant inducing combinations of the FTC, were used. The effect of the FTC was ranked by considering the number of different antibiotics they affected. The organism was sub-cultured from the slants onto agar plates and incubated at 37°C for 48 h. The cells were acid-fast stained to serve as a quality control before seeding them into 7H9 broth to make starter cultures. These were incubated with shaking at 180 rpm. Seed cultures were made from the starter cultures at optical density (OD) of 0.1 and incubated for another 48 h until the mid-exponential growth phase was obtained before every bioassay. The OD reading taken at an absorbance of 600nm was adjusted to 0.7 as the working OD. A volume of 100 ul of each phenotypic compound (1 ug/ul), both single PCs and triple PC combinations, was spread on agar plates and allowed to dry. For colony morphology, 10 ul of the 0.7 OD of cells was spotted twice on one half the plate. A 10-4 dilution was prepared from the stock cells (OD of 0.7) and 100 ul was spread on the other half of the plate. The plates were incubated for 48 h and images of the plates were captured. After, a single colony was picked from the spread and acid-fast stained to determine the effect of the various treatments. University of Ghana http://ugspace.ug.edu.gh 19 3.4 DETERMINATION OF THE EFFECTS OF PHENOTYPIC COMPOUNDS ON EFFLUX AND BIOFILM ACTIVITY To assess accumulation, the three strains of M. smegmatis were cultured in 7H9 broth for 48 hr at 37°C in a shaking incubator at a speed of 180 rpm. The OD was adjusted to 0.8 and the cultures were centrifuged at 13, 000 rpm for 3 min and the cell pellet was washed twice with 0.8% Phosphate Buffered Saline (PBS). The pellets were resuspended in 0.8% PBS and the OD adjusted to 0.4. A volume of 3 ul ethidium bromide (EtBr) with a concentration of 3 ug/ul was added to all the cell suspensions. An amount of 990 ul of cells containing EtBr at an OD of 0.4 was pipetted into 2 ml Eppendorf tubes and 10 ul of each PC at a concentration of 20 mg/ul were added to each reaction tube to give a total amount of 0.1 mg. Verapamil at the same amount was added to one of the tubes to serve as the positive control. an amount of 10 ul of 0.8% PBS was added to one reaction set up to serve as a negative control and all the reactions for the PC plus the controls were set up in triplicates. The reaction tubes were incubated for one hour in a shaking incubator after which 100 ul of the content of each reaction tube was pipetted into a 96-well plate. Fluorescence of EtBr at excitation and emission wavelengths at 530 at 585 (nm) respectively were read within time intervals of 30, 60, and 120 min using a Varioskan microplate reader. For the induction of efflux pump activity, 0.4% glucose was added to each reaction tube containing EtBr cells. This was done by preparing a 0.8% PBS solution containing the 0.4% glucose solution. The protocol as described in the accumulation assay was followed. That is, the reaction setup was incubated for an hour and 100 ul of each tube were aliquoted into a 96 well plate and fluorescence read at 0, 15, 30, 60, and 120 min. Finally, adhesion and biofilm assays were done as described by Sandberg et al (2008) with slight modification. M. smegmatis mc2155, eMsA, and eMsB cultures were incubated for 48 hr and their OD600 readjusted to 0.5. University of Ghana http://ugspace.ug.edu.gh 20 To determine whether the PC inhibits adhesion and biofilm formation, two different assays were set up for each aspect. A volume of 990 ul of the overnight cultures (OD = 0.5) was aliquoted into a 2 ul Eppendorf tube in both setups. To each reaction tube, 10 ul of PC (10 ug/ ml) was added. For the PC combinations, the volume of cells used was adjusted to 970 ul to make up for the additional volume of PCs used. A volume of 200 ul from each reaction tube was aliquoted into 96 well plates and incubated at 60 rpm for 24 h for adhesion and 72 h for biofilm. After the stated period of incubation for the respective assay, the cultures in the 96 well plate were washed off with 0.8% PBS and allowed to air dry. A volume of 20 ul of 1% crystal violet (CV) was added to each well and allowed to stand for 15 min. The plates were washed off with distilled water to remove any unbound CV to cells. A volume of 20 ul of 95% ethanol was added to each well to solubilize the CV. The absorbance was read at 595 nm with the Varioskan plate reader. The inhibitory effect of the PC against cell adhesion and biofilm formation was determined. The inhibition assays are then followed by disruption assays in which a volume of 200 ul of Mycobacterial culture of OD = 0.5 was aliquoted into 96 well microtiter plates. The plates were incubated with shaking at 60 rpm for 24 h and 72hr to study adhesion and biofilm respectively. After the incubation period, the plates were washed twice with distilled water and replaced with 200 ul of 7H9 containing PC at an amount of 0.1 mg. The two setups were incubated for 2 h with shaking at 60 rpm. The microtiter plates were washed twice with distilled water and 20 ul of 95% ethanol was added. The absorbance was read at 595 nm. Signified compounds that disrupted adhered cells and biofilm formation. 3.5 ANALYSIS OF STRESS RESPONSE EXPRESSION PATTERNS WITH ADJUVANTS The two top hit treatment conditions that were resistant breaking and resistance inducing were used in the setup for the RNA extraction. Thus, there was a control (no PC added), two resistance- inducing combinations (LAC (3X) and SIA+ADO (1.5X)), and two resistance breaking University of Ghana http://ugspace.ug.edu.gh 21 combinations (SOR (3X) and MAL (3X)). The top two resistant breaking and resistant inducing combinations were chosen based on the ranking order where these compounds had the highest effect on the number of antibiotics they affected. The three M. Smegmatis strains were cultured as described in section 3.1, a seed plate was made from stored culture slants by streaking and incubating overnight. The cells were acid-fast stained before subculturing into 50 ml 7H9 broth and incubated in a shaking incubator for 72 h. This first culture (day 1 culture) was not treated. Fresh 50ml 7H9 broths containing 0.1 mg of the various PCs were prepared and seeded using the first culture to obtain a day 2 culture. The starting OD of the day 2 culture was set at 0.05 and incubated for another 72 h. For the extraction, 50 ml falcon tubes were pre-weighed, and the treated cell cultures were transferred into them. The tubes were centrifuged at 13,000 rpm for 5 minutes at 4℃. The pellet obtained was left to dry in the hood and the tubes measured. The difference in the weights measured is the weight of the cells pelleted. A kit was used for the extraction, the ZR Fungal/Bacterial MiniPrep kit from Zymo Research. The weight of the cells ranged from 400 – 600 mg. To each falcon tube, 2 ml of RNA lysis buffer was added to resuspend the cells into solution. Of this, 1 ml was pipetted into the tubes containing the bashing bead and vortexed for 10 minutes. The tubes were then centrifuged at 15,000 rpm for 1 minute. A volume of 400ul of the lysate (supernatant) was carefully transferred into the Zymo- SpinTM IIICG column placed in collection tubes and centrifuged at 14,000 rpm for 30 secs. This step was repeated with the same column to obtain a total volume of 800 ul of flow through. To this collection, an equal amount of 96% ethanol was added and mixed thoroughly and 600 ul pipetted into the Zymo-Spin™ IICR in a collection tube. This was centrifuged at the previous speed and for the same period. The flow-through was discarded. To the column, 400ul of the RNA Prep Buffer was added and the flow-through discarded. The column was washed twice by adding 700 ul and 400 ul of RNA Wash Buffer and centrifuged at each point for 30 secs and 2 minutes University of Ghana http://ugspace.ug.edu.gh 22 respectively. The columns were transferred into sterile 1.5 ml Eppendorf tubes and 10 ul of warm Dnase/Rnase-Free Water was added and eluted time for the same column to give a final volume of 30 ul of extracted RNA. The RNA extracted was then quantified and its purity checked using a nanodrop. Extracted RNA samples were stored at -24 0C before running the RT-qPCR the following day. Stored RNA samples were diluted to 25 ng/ul using Dnase/Rnase-Free Water. The Luna® Universal One-Step RT-qPCR kit was the kit used for the RT-qPCR experiments. For a 1X reaction of 20 ul RT-qPCR reaction mix, 10ul of the Luna Universal One-Step Reaction Mix (2X), 1ul of the Luna WarmStart® RT Enzyme Mix (20X), 0.8ul each of the 10 uM forward and reverse primers and nuclease-free water added to top up to the required volume. For each primer pair, 20ul of each triplicate per treatment was prepared for 10 selected stress response genes. The assay was performed on ice. The plates were sealed carefully using a transparent plate sealer and centrifuged in a microcentrifuge for 1 min. The Applied Biosystems real-time machine was programmed using the thermocycling protocol as stated in the Luna® Universal One-Step RT-qPCR Kit. Table 1.4: PCR Thermocycler conditions Cycle Step Temperature Time Cycles Reverse Transcription 55 0C 10 minutes 1 Initial Denaturation 95 0C 1 minute 1 Denaturation 95 0C 10 seconds 40 Extension 60 0C 1 minute Melt Curve 60 – 95 0C various 1 The generated data were then analyzed. University of Ghana http://ugspace.ug.edu.gh 23 CHAPTER FOUR RESULTS 4.1 RESULTS OF THE EFFECT OF DIFFERENT CARBOHYDRATES AND AMINO ACIDS ON THE ANTIMICROBIAL ACTIVITY OF ANTIFUNGAL AND ANTIBACTERIAL COMPOUNDS 4.1.1 EFFECT OF SINGLE PHENOTYPIC COMPOUNDS ON ANTIBIOTIC ACTIVITY The ability of modifier compounds to augment or suppress the antibiotic activity of standard in- use antibiotics in three different strains of mycobacteria was set up for 17 different compounds. The phenotypic interaction assays using single compounds against Ms wt showed most of the compounds used had an accumulated activity lower than the control. All the compounds except cellobiose decreased the antibiotic activity either by decreasing the activity of the individual antibiotics or reducing the number of antibiotics that had activity. Most of the compounds could be said to be resistance-inducing activity. Contrary to all the phenotypic modifying compounds used, cellobiose had the highest accumulated antibiotic impact relative to the control. In the presence of cellobiose, it was observed that there was an increase in the antibiotic activity of vancomycin, linezolid, and gentamicin. It is also noteworthy to point out that cycloserine which is inactive in the control got revived in the presence of cellobiose. Unlike the other antibiotics, Moxifloxacin had a reduction in activity when cellobiose was added. Since most of the antibiotic’s activity increased (13 out of 20 antibiotics) after cellobiose modification, it can be said to be resistant-breaking. Maltose had the least accumulated activity on the antibiotics used, having changed 8 out of 20 antibiotics. In the presence of maltose, amoxicillin, vancomycin, ethambutol, and chloramphenicol all had a reduction in their antibiotic activity. 5-fluorouracil being the only exception, had a slight increase in activity. Maltose is largely expressed as resistant-inducing activity (Fig 4.0A). University of Ghana http://ugspace.ug.edu.gh 24 Results from testing the individual phenotypic compounds against eMsA showed that all the compounds used had an accumulated activity higher or equal to the control. Most of the compounds aside from increasing the activity of individual antibiotics revived the activity of the other antibiotics that were not active in the control. Most of the compounds can be classified as resistant-breaking. Cellobiose, just like in Ms wt had the highest accumulated bioactivity. In the presence of this compound, not only did it increase 5-fluorouracil slightly but revived the activities of ampicillin, amoxicillin, erythromycin, gentamicin, and ethionamide. Contrary to this, it reduced slightly the activity of paromomycin and streptomycin. Cellobiose can be classified as a resistant breaking agent. Adonitol having the least accumulated impact did not have much effect on the antibiotics, with ethionamide being the only antibiotic to be revived while chloramphenicol, streptomycin, and 5-fluorouracil were slightly reduced (Fig 4.0B). Against eMsB, most of the phenotypic compounds had accumulated activities higher than that of the control (10 out of 17 compounds). Hence most of the compounds can be said to be resistant- breaking compounds. Inulin modified media had the highest effect on antibiotics used against eMsB. Amoxicillin, ampicillin, vancomycin, and chloramphenicol all had their activities revived in the presence of inulin. Streptomycin, gentamicin, and 5-fluorouracil had a decrease in activity. Sorbose had the least accumulated activity among the compounds and fell below the control line. This compound can be said to be a resistant inducing agent which caused Tetracycline to lose its activity while streptomycin, gentamicin, and 5-fluorouracil had a decline in their bioactivity. Cellobiose, which has consistently been the highest in both wt and eMsA, was not the most potent here with eMsB but was among the resistant-breaking compounds (Fig 4.0C). This interaction study, though designed to address the antimicrobial resistance phenomenon, turns out to demonstrate the significantly different cellular metabolism in the two MDR strains. Also provides a path forward for a more detailed study of the basis of the high level of resistance University of Ghana http://ugspace.ug.edu.gh 25 acquired by these two strains. The same analysis also applied to E. coli and Candida albicans and surprisingly, E. coli did not respond at all while C. albicans responded to an extent similar to that of the Mycobacteria cells and provide background data for the future. 0 50 100 150 200 250 300 350 o n e o f n h ib it io n s (m m ) nteraction Assay of single compounds against s ( T) Amp 40 Amx 40 an 40 10 mb 10 D 40 oxi 0.5 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 10 th 18 0 50 100 150 o n e o f n h ib it io n ( m m ) nteraction Assay of single compounds against e sA Amp 40 Amx 40 an 40 10 mb 10 D 40 oxi 0.5 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 10 th 18 0 50 100 150 o n e o f n h ib it io n ( m m ) nteraction Assay of single compounds against e s Amp 40 Amx 40 an 40 10 mb 10 D 40 oxi 0.5 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 10 th 18 350 350 University of Ghana http://ugspace.ug.edu.gh 26 Figure 4.0: Antibiotic activity profile for M. smegmatis treated with modifier compounds. 7H10 agar plates were treated by spreading 100ul of 1mg/ml of the various PCs. A working OD of 0.7 of cells was prepared and spread on the modified plates. Discs impregnated with the working amounts of antibiotics were placed on the inoculated plate and incubated for 48 h. The zones of inhibition of the active antibiotics were measured and a plot of measured zones of inhibition under controlled conditions and in the presence of each modifier compound for (A) Ms wt (B) eMsA and (C) eMsB 4.1.2 EFFECT OF STRUCTURAL TRIPLE PHENOTYPIC COMPOUND COMBINATIONS ON ANTIBIOTIC ACTIVITY To increase the activities observed with the individual compounds, a triple combination of the compounds based on structural similarities was generated. Following from the primary effects of the modifier compounds on the antibiotic activity profiles across the organisms, compounds were grouped based into different structural classes; Di and tri-saccharides (lactose-4, maltose, raffinose, cellobiose, inulin), 5&6 carbon alcohols (dulcitol, adonitol), 5&6 carbon sugars (sorbose, galactose, rhamnose, xylose), Acid alcohol (gluconic acid), Amino acid (tryptophan), Fatty acid (malonic acid and pyruvate) and lastly 6 carbon compounds (myo-inositol and sialicin). The triple 26eaction26onns used were representatives of each class and across groups. Ten structural triple combinations were created to test against the wt and two resistant mutant strains (Table 3.3). From the assay where the STC compounds were used to test Ms wt, all the ten triple combinations had cumulative activity lower than the control. These combinations can collectively be more resistant inducing, even though some had resistant breaking activities with individual antibiotics (Fig 4.1A). Against eMsA, five out of the structural triple combination compounds had activities slightly higher than the control, while the other half were slightly below the control. Also, the triple University of Ghana http://ugspace.ug.edu.gh 27 combination Raf+Cel+Inu had the highest effect, affecting 9 out of 20 antibiotics as compared to the control (Fig 4.1B). The interaction assay of STC compounds with antibiotics against eMsB showed similar observations as seen in eMsA where all the compounds had effects equal to or slightly lower than the control (Fig 4.1C). The overall effects of this STC work were not as interesting as that of the single compounds. For this reason, the combinations were not pursued further in this study. Table 2.0: List of Structural Triple Combination and their groups 1st criterium; three (3) compounds in each group 1. Raffinose Cellobiose Inulin 2. Dulcitol Adonitol D-gluconic acid 3. L-(-)-sorbose L-rhamnose Xylose 2nd criterium; one (1) compound in each group 4. Raffinose Adonitol L-rhamnose 5. Cellobiose D-gluconic acid Tryptophan 6. D-gluconic acid Malonic acid Myoinositol 7. Tryptophan D-gluconic acid Galactose 3rd criterium; two (2) compounds from one group 8. Malonic acid Sodium-pyruvate D-gluconic acid 9. Myo-inositol Sialicin Tryptophan 10. Dulcitol Adonitol Tryptophan University of Ghana http://ugspace.ug.edu.gh 28 Figure 4.1: Antibiotic activity profile for M. smegmatis WT with structurally grouped modifier compounds. The disc diffusion method as described above was used to obtain the plot of measured zones of inhibition under controlled conditions and in the presence of structurally grouped modifier compounds for (A) Ms wt, (B) eMsA, and (C) eMsB. On an agar plate 100ul of each PC in the STC was spread on it before inoculation. 0 50 100 150 o n e o f n h ib it io n ( m m ) nteraction Assay of ST of compounds with antibiotics against e sA 0 50 100 150 200 250 300 350 o n e o f n h ib it io n ( m m ) nteraction Assay of ST of compounds with antibiotics against s T Amp 40 Amx 40 an 40 10 mb 10 D 40 oxi 0.5 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 10 th 18 350 350 University of Ghana http://ugspace.ug.edu.gh 29 4.1.3 EFFECT OF FUNCTIONAL TRIPLE PHENOTYPC COMPOUND COMBINATIONS ON ANTIBIOTIC ACTIVITY Concurrently, following the investigation of the role of structural similarities and presence of specific functional groups in potentiating the activity of the antibiotics, the modifier compounds are then organized based on the antibiotic(s) which they affect and grouped in triples toward a combined synergistic effect resulting in 34 different functional triple combinations. The compounds were grouped into triple sets based on the individual activities of the single compounds on the antibiotics. In the assays where the functional triple combination compounds were used against the wt strain, most of the compounds’ cumulative activities were higher than the control. This observation demonstrates a higher resistant-breaking effect when the compounds were combined based on their functional similarities. For example, Myo+Rham+Raf is the triple combination with the highest effect compared to the control. Even though the number of antibiotics impacted by this combination and control are the same, the combination increased the activity of the antibiotics (Fig 4.2A and B). The eMsA bioassay showed that some of the compounds (13/34) had cumulative activities higher than the control while most had cumulative activities lower than the control (18/34). In the presence of Rham (3X), 10/20 antibiotics got their activity changed compared to the control. Most of them had an increase in activity suggesting Rham(3X) is resistant breaking (Fig 4.3A and B). Against eMsB, most of the compounds (23/34) had cumulative activities higher than that of the control as compared to 6/24 compounds having activities lower than the control and Myo+Sia+Lac had the highest accumulative antibiotic impact, changing 8 out of 20 antibiotics. Even though Tryp+Malt+Raf also changed the activity of 8 out of 20, the effect on the individual antibiotics was higher in the former than the latter (Fig 4.4A and B). University of Ghana http://ugspace.ug.edu.gh 30 Variations in antibiotic activity were more pronounced in both resistant strains in terms of both gain and loss of activity under different treatment combinations. Fourteen of thirty-seven different triple combinations used showed an overall increase in antibiotic activity as plotted of a cumulative stacking of zones of inhibition measured from active antibiotics out of a total of 20 antibiotics used. The list for the FTC generated is as shown below: Table 4.1: List of functional Triple Combination and the antibiotics they affect Functional Triple Combination (FTC) Dose The amount for each (mg) Effect Mal+Rha+NaP 1X 0.1 (+) Increases activity of Ampicillin, Amoxicillin, and Vancomycin of eMsA and B Cel+Inu+Glu 1X 0.1 (+) Increases activity of Ampicillin, Amoxicillin, and Vancomycin of eMsA and B Myo+Sia+Lac 1X 0.1 (-) Decreases activity of Isoniazid against WT Sor+Try+Mal 1X 0.1 (-) Decreases activity of Isoniazid against WT Ado+Xyl+Raf 1X 0.1 (-) Decreases activity of Isoniazid against WT Dal+Myo+Raf 1X 0.1 (-) Decreases activity of Moxifloxacin against WT and eMsB Myo+Rha+Raf 1X 0.1 (-) Decreases activity of Moxifloxacin against WT and eMsB 3Lac 3X 0.3 (-) Decreases activity of Rifampicin Malt+Raf+Gal 1X 0.1 (+) Increases activity of Tetracycline against eMsA Sor+Mal+Glu 1X 0.1 (-) Decreases activity of Tetracycline eMsB University of Ghana http://ugspace.ug.edu.gh 31 Lac+Try+Mal 1X 0.1 (+) Increases activity of Chloramphenicol against eMsB Ado+Raf+Cel 1X 0.1 (+) Increases activity of Chloramphenicol against WT 1.5Rha+1.5Inu 1.5X 0.15 (+) Increases activity of Chloramphenicol against eMsB (-) Decreases activity of Chloramphenicol against MS 3Myo 3X 0.3 (+) Increases activity of Erythromycin against eMsA and eMsB Lac+Try+NaP 1.5X 0.15 (+) Increases activity of Erythromycin against eMsA 1.5Xyl+1.5Glu 1.5X 0.15 (+) Increases activity of Erythromycin against WT 1.5Sia+1.5Ado 1.5X 0.15 (+) Increases activity of Erythromycin against WT 1.5Malt+1.5Cel 1.5X 0.15 (+) Increases activity of Erythromycin against eMsA 3Glu 3X 0.3 (+) Increases activity of Erythromycin against WT and eMsA Sia+NaP+Raf 1X 0.1 (+) Increases activity of Streptomycin against eMsA Try+Malt+Raf 1X 0.1 (+) Decreases activity of Streptomycin against eMsA Sor+Rha+Malt 1X 0.1 (-) Decreases activity of Paromomycin of eMsA and B 1.5Myo+1.5Mal 1.5X 0.15 (-) Decreases activity of 5-Fluorouracil of WT and eMsB 3Myo 3X 0.3 (-) Decreases activity of 5-Fluorouracil of eMsB 3Mal 3X 0.3 (-) Decreases activity of 5-Fluorouracil of WT 3Inu 3X 0.3 (+) Increases activity of Clindamycin of WT Ado+Cel+Glu 1X 0.1 (+) Increases activity of Ethionamide of eMsA University of Ghana http://ugspace.ug.edu.gh 32 Figure 4.2: Antibiotic activity profile for M. smegmatis WT with functionally grouped modifier compounds. The plot of measured zones of inhibition under controlled conditions and in the presence of functionally grouped modifier compounds for Mycobacterium smegmatis wild-type. Modified agar plates were prepared by spreading the FTC on the plate and allowed to dry. Cells of working OD 0.7 were used for inoculation and antibiotic-impregnated discs were placed. The plates were incubated for 48h and the ZOI was measured. Fig 3a 0 100 200 300 400 500 o n e o f in h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against s T Amp 40 Amx 40 an 40 20 mb 10 D 40 oxi 1 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 0 100 200 300 400 500 o n e o f in h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against s T Amp 40 Amx 40 an 40 20 mb 10 D 40 oxi 1 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 University of Ghana http://ugspace.ug.edu.gh 33 Figure 1.3: Antibiotic activity profile for eMsA with functionally grouped modifier compounds. The plot of measured zones of inhibition under controlled conditions and in the presence of functionally grouped modifier compounds for erythromycin-resistant Mycobacterium smegmatis A. Fig 3b 0 50 100 150 200 o n e o f in h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against e sA Amp 40 Amx 20 an 20 40 mb 50 D 40 oxi 1 if 5 in 10 Tet 30 hlo 30 ry 20 Strep 15 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 0 50 100 150 200 o n e o f n h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against e sA Amp 40 Amx 20 an 20 40 mb 50 D 40 oxi 1 if 5 in 10 Tet 30 hlo 30 ry 20 Strep 15 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 500 500 University of Ghana http://ugspace.ug.edu.gh 34 Figure 4.4: Antibiotic activity profile for eMsB with functionally grouped modifier compounds. The plot of measured zones of inhibition under controlled conditions and in the presence of functionally grouped modifier compounds for erythromycin-resistant Mycobacterium smegmatis B. Fig 3c 0 50 100 150 200 o n e o f in h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against e s Amp 40 Amx 40 an 40 20 mb 10 D 40 oxi 1 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 0 50 100 150 200 o n e o f in h ib it io n ( m m ) nteraction Assay of functional triple combination of compounds with antibiotics against e s Amp 40 Amx 40 an 40 20 mb 10 D 40 oxi 1 if 10 in 5 Tet 20 hlo 40 ry 40 Strep 30 yser 20 et 30 Gen 10 ara 20 5 fu 1 lind 40 th 40 500 500 University of Ghana http://ugspace.ug.edu.gh 35 4.1.4 RANKING SYSTEMS FOR THE ANTIBIOTIC RESISTANCE BREAKING AND INDUCING EFFECT The overall efficacy of the resistant-breaking and resistant-inducing phenotypic compounds against the three strains of M. smegmatis (wt, eMsA, and eMsB) were ranked based on certain derived parameters. The parameters used were; 1. The number of antibiotic classes affected (# classes) 2. The number of antibiotics that gained activity, from zero to a number (# 0-n), the number of antibiotics that lost activity, from a number to zero (# n-0) 3. The quantum of change of the zone of inhibition (ZoI) 4. The sum of these parameters to give an overall value for ranking. The number of antibiotic classes (Table 4.2) affected by each resistant-breaking or resistant- inducing compound was recoded. Different classes of antibiotics were used for this study to understand the activity of the compounds. This parameter shows whether the compounds have specific or broad-spectrum efficacy on the antibiotics. Most of the compounds had an effect on multiple antibiotics, which shows their broad effect while a few had a specific effect on particular antibiotics. In this study, most of the antibiotics had an effect against the Ms wt just a few of the antibiotics had no activity (cycloserine, pyrazinamide, and ethionamide). Contrary to this most of the compounds were inactive against the mutant strains (eMsA and eMsB) and just a few had activity (rifampicin, moxifloxacin, gentamicin, paromomycin, and 5-fluorouracil). One noteworthy feature was the ability of the compounds to be able to either revive these compounds or make them lose their activity. The number of antibiotics whose ZoI moved from a particular number to zero (n-0) in the presence of resistant-breaking compounds was recorded. Similarly, the number of antibiotics whose activity got revived, that is moved from zero to a number (0-n) in the presence of resistant-inducing compounds were recorded. University of Ghana http://ugspace.ug.edu.gh 36 The magnitude of the change of the zone of inhibition was recorded by summing the difference obtained, increase for resistant breaking, and decrease in resistant inducing treatment. Some of the changes were increased or decreased and others were either from zero to a number or from a number to zero. The quantum of change here measures the extent of change of antibiotic activity in the presence of the phenotypic compounds. These three parameters (# of classes, # 0-n/n-0, and quantum of change) were summed up to get the overall value. This total value obtained was used to rank the resistant breaking and resistant inducing compounds in descending order, from the highest to the lowest. An initial ranking was done using the number of antibiotics affected by each phenotypic compound. These numbers were placed in a bracket of each phenotypic compound and were used for the initial ranking. Table 4.2: The various classes of antibiotics used in the experiment Antibiotic Class Example of Antibiotics 1 Aminoglycoside Gentamycin, Streptomycin, Paramomycin 2 Fluoroquinolones Moxifloxacin 3 Glycopeptide Vancomycin 4 Macrolide Erythromycin 5 Penicillin Amoxicillin, Ampicillin 6 Antimycobacterial Rifampicin, Isoniazid, Pyrazinamide, Cycloserine, Ethambutol 7 Tetracycline Tetracyline 8 Amphenicol Chloramphenicol 9 Nitroimidazole Metronidazole 10 Lincosamide Clindamycin 11 Thioamine Ethionamide 12 Antimetabolite 5-Fluorouracil 13 Oxazolidinone Linezolid University of Ghana http://ugspace.ug.edu.gh 37 4.1.5 SINGLE PENOTYPIC COMPOUND RANKING BY RESISTANCE-MODIFYING EFFECT AGAINST MYCOBACTERIA Each modifier compound was assessed based on its resistance-breaking and resistance-inducing effect. The resistance breaking effect either is a return of antibiotic activity or an increase in the extent of antibiotic activity as measured by zones of inhibition in millimeters and vice versa for the resistance-inducing effect. Two ranking systems were applied to each of the 17 compounds as presented in tables (Tables 4.3-4.6); A general rank based on the previously defined resistance- breaking or resistance inducing potential of the compound, and an overall rank representing a total of the number of different classes of antibiotics affected, the total loss or gain of activity to and from a reference point 0 and the quantum change in the antibiotic zone of inhibition (ZoI). The top 5 resistance-breaking compounds by general rank were inulin, cellobiose, raffinose, rhamnose, and lactose respectively and by overall rank; inulin, cellobiose, raffinose, lactose, and sodium pyruvate whilst the top 5 resistance inducing compounds by general rank were maltose, sorbose, rhamnose, sialicin, lactose and by overall rank rhamnose, maltose, sorbose, lactose, and sialicin. University of Ghana http://ugspace.ug.edu.gh 38 Table 4.3: Resistant-breaking treatment of single compounds against M. smegmatis (Rank 1-6) S STA T A G T AT T F S G DS verall ranking ank Treatment rganism esistant reaking classes 0 n uantum change of o verall 1 1 nulin(8) s lind 0 18 8 10 .5 121.5 e sA an 0 15 mb 0 8 Gen 0 15.5 e s Amp 0 1 Amx 0 13 an 0 11 hlo 0 11 2 2 ellobiose( ) s yser 0 5 82.5 4.5 e sA Amp 0 13 Amx 0 14 ry 0 14 th 0 11 e s Amp 0 10.5 hlo 0 11 3 3 affinose( ) e sA Tet 0 11 80 3 yser 0 10 Gen 0 13 e s Amp 0 15 Amx 0 14 an 0 .5 hlo 0 .5 4 hamnose(5) e sA Amp 0 10 4 5 58.5 .5 Gen 0 12 e s Amp 0 14.5 an 0 .5 hlo 0 12.5 4 5 actose(5) e sA Amx 0 14.5 4 5 5 4 ry 0 11 Gen 0 14.5 e s Amp 0 14 hlo 0 11 5 Sodium yruvate(5) e sA Amx 0 12 5 5 1 1 mb 0 10 in 0 8 ry 0 11 Gen 0 20 Table 1a University of Ghana http://ugspace.ug.edu.gh 39 Table 4.4: Resistant-breaking treatment of single compounds against M. smegmatis (Rank 7-17) S STA T A G T AT T F S G DS verall ranking ank Treatment rganism esistant reaking classes 0 n uantum change of o verall Galactose(5) e sA Amx 0 15 5 5 5 .5 .5 0 11 Tet 0 ry 0 14.5 Gen 0 10 8 Dalcitol (4) e sA ry 0 10 3 4 53 0 e s Amp 0 15 an 0 ry 0 1 8 altose(4) s an 10.5 21 4 3 0 in 11.5 23 e sA Gen 0 22 e s Amp 0 1 10 alonic Acid(4) e sA Amp 0 13 4 4 52 0 an 0 12 Gen 0 15 e s hlo 0 12 11 11 yo inositol(3) e sA Amp 0 12 2 3 41 4 Amx 0 15 Gen 0 14 14 12 Adonitol(3) e sA th 0 11 3 3 35 41 e s Amp 0 11 hlo 0 13 13 13 ylose(3) e sA Amp 0 13 2 3 3 42 Gen 0 13.5 e s Amp 0 10.5 12 14 Tryptophane (3) e sA Amx 0 12 3 3 38.5 44.5 ry 0 12 Gen 0 14.5 15 15 Gluconic Acid(3) e sA Amp 0 10 2 3 34 3 th 0 10 e s Amp 0 14 1 1 Sorbose(2) e sA Amp 0 12 2 2 32 3 Gen 0 20 1 1 Sialicin(1) e sA Gen 0 22.5 1 1 22.5 24.5 University of Ghana http://ugspace.ug.edu.gh 40 Table 4.5: Resistant-inducing treatment of single compounds against M. smegmatis (Rank 1-5) S STA T D G T AT T F S G DS verall ranking ank Treatment rganism esistant reaking classes n 0 uantum change of o verall 2 1 altose(4) s 14 0 3 3 4 .5 55.5 Amx 2 13 e sA ara 12 0 e s ara .5 0 3 2 Sorbose(3) s 14 0 3 3 3 42 e sA ara 12 0 e s Tet 10 0 1 3 hamnose(3) s hlo 32 0 3 3 55.5 1.5 Amx 2 13 e s ara .5 0 5 4 Sialicin(2) s 14 0 2 1 2 32 Amx 2 12 4 5 actose(2) s Amx 2 13 2 1 35 38 if 21 0 University of Ghana http://ugspace.ug.edu.gh 41 Table 4.6: Resistant-inducing treatment of single compounds against M. smegmatis (Rank 6-15) S STA T D G T AT T F S G DS verall ranking ank Treatment rganism esistant reaking classes n 0 uantum change of o verall yo inositol(2) s 14 0 2 1 2 .5 30.5 e s 5fu 24 10.5 Tryptophane(2) s 14 0 2 1 21.5 24.5 e s Strep 14.5 8 ellobiose(1) s 14 0 1 1 14 1 14 alonic Acid(1) e s Tet 10 0 1 1 10 12 8 10 Sodium yruvate(1) s Amx 2 11 1 0 1 1 11 11 Dalcitol (1) s Amx 2 13.5 1 0 13.5 14.5 12 Adonitol(1) s 14 0 1 1 14 1 12 13 affinose(1) e s oxi 12 0 1 1 12 14 13 14 Gluconic Acid(1) e s Tet 10 0 1 1 10 12 14 15 Galactose(1) e s Tet 10 0 1 1 10 12 Table 2b University of Ghana http://ugspace.ug.edu.gh 42 4.1.6 STRUCTURAL AND FUNCTIONAL TRIPLE PHENOTYPIC COMPOUND COMBINATION RANKING BY RESISTANCE-MODIFYING EFFECT AGAINST MYCOBACTERIA Triple compound combination effects were assessed based on their resistance-breaking and resistance-inducing effect. Again, the resistance breaking effect either being a return of antibiotic activity or an increase in the extent of antibiotic activity as measured by zones of inhibition in millimeters and vice versa for the resistance-inducing effect. Two ranking systems were applied to generate a different number of combinations based on their resistance-breaking and resistance- induction respectively as presented in tables (Tables 4.7-4.8); A general rank based on the previously defined resistance-breaking or resistance inducing potential of the compound, and an overall rank representing a total of the number of different classes of antibiotics affected, the total loss or gain of activity to and from a reference point 0 and the quantum change in the antibiotic zone of inhibition (ZoI). The top 5 resistance-breaking compounds by general rank were Raf+Cel+Inu, Mal+NaP+Glu, Cel+Glu+Try, Sor+Rha+Xyl, and Dal+Ado+Glu respectively and by overall rank; Raf+Cel+Inu, Sor+Rha+Xyl, Mal+NaP+Glu, Cel+Glu+Try, and Dal+Ado+Glu whilst the top 5 resistance inducing compounds by general rank were Raf+Cel+Inu, Sor+Rha+Xyl, Dal+Ado+Try, Myo+Sia+Try, Mal+NaP+Glu and by overall rank Raf+Cel+Inu, Myo+Sia+Try, Dal+Ado+Try, Sor+Rha+Xyl, and Mal+NaP+Glu. University of Ghana http://ugspace.ug.edu.gh 43 Table 4.7: Resistant-breaking treatment of compounds (STC) against M. smegmatis (Rank 1-9) S STA T A G T AT T F ST T A T AT DS verall anking ank Treatment rganism esistant reaking classes 0 n uantum change of o verall 1 1 af el nu (3) e sA Amp 0 15 3 2 40 45 nh 0 14 ara 20 3 2 al a Glu (2) e sA Amp 0 14 2 2 2 33 Tet 0 15 4 3 el Glu Try (2) e sA Amp 0 14.5 2 2 24.5 28.5 Tet 0 10 2 4 Sor ha yl (2) e sA Amp 0 15 2 1 30 33 Amx 0 15 5 5 Dal Ado Glu (2) e sA Amp 0 11 2 2 23 2 ys 0 12 Dal Ado Try (1) e s ara 0 .5 1 1 .5 11.5 8 Glu al yo (1) e sA Amp 0 15 1 1 15 1 8 yo Sia Try (1) e sA Amx 0 1 1 1 1 1 Try Glu Gal (1) e sA Amp 0 1 1 1 1 1 University of Ghana http://ugspace.ug.edu.gh 44 Table 4.8: Resistant-inducing treatment of compounds (STC) against M. smegmatis (Rank 1-10) S STA T D G T AT T ST T A T AT DS verall anking ank Treatment rganism esistant nducing classes n 0 uantum change of o verall 1 1 af el nu (5) s lind 11 0 5 2 4 81 an 22 11 Amx 2 12 e s if 2 11 hlo 20 0 4 2 Sor ha yl (4) s lind 11 0 4 2 45 51 ry 22 11 Amx 2 12 e sA ara 0 3 3 Dal Ado Try (4) s lind 11 0 4 2 45.5 51.5 Amx 2 12 ry 22 10.5 e sA ara 0 2 4 yo Sia Try (4) s lind 11 0 4 1 5 1 Strep 2 13 Amx 2 13 e s if 2 11 5 5 al a Glu (3) s lind 11 0 3 2 43 48 Amx 2 11 e sA hlo 1 0 Dal Ado Glu (3) s Amx 2 13 3 1 34 38 lind 11 0 e sA oxi 1 8 Glu al yo (2) s lind 11 0 2 1 2 2 Amx 2 11 8 el Glu Try (2) s lind 11 0 2 2 20 24 e sA ara 0 af Ado ha (2) s lind 11 0 2 2 20 24 e sA ara 0 10 Try Glu Gal (2) e sA 5Fu 13 0 2 2 30 34 oxi1 0 University of Ghana http://ugspace.ug.edu.gh 45 Table 4.9: Resistant-breaking treatment of compounds (FTC) against M. smegmatis (Rank 1-10) S STA T A G T AT T F T A T AT DS verall anking ank Treatment rganism esistant reaking classes 0 n uantum change of o verall 1 1 Sorb (3 ) (5) e sA Amp 0 13.5 4 4 81 8 Amx 0 13 ry 0 24.5 e s Tet 0 Gen 15 3 5 2 al (3 ) (4) e sA an 0 1 3 3 3 e s an 0 18 Tet 0 12 hlo 0 1 3 3 Sorb Try alt (4) e sA an 0 14.5 4 3 .5 3.5 ry 0 15 e s Tet 0 13 Gen 15 3 1 4 yo al (1.5 ) (4) e s Amp 0 11 3 4 43.5 50.5 Amx 0 11.5 Tet 0 10 hlo 0 11 1 5 Ado el Glu (4) s et 0 11 4 4 42 50 e s Tet 0 et 0 11 th 0 11 2 yl Glu (1.5 ) (4) e sA Amx 0 1 4 3 8.5 5.5 an 0 .5 ry 0 21 Gen 15.5 34.5 14 yo ha af (3) s ry 24 48 3 2 4 .5 51.5 e sA Amx 0 12 an 0 10.5 8 al ha a (3) e sA Amx 0 1 3 2 0 5 an 0 13 Gen 15 30 4 el nu Glu (3) s 5 Fu 10.5 28.5 3 2 5 0 e sA Amx 0 1 ry 0 30 11 10 yo Sia ac (3) e sA an 0 12.5 3 3 50.5 5 .5 e s 0 28 Tet 0 10 Table 5a University of Ghana http://ugspace.ug.edu.gh 46 Table 4.10: Resistant-breaking treatment of compounds (FTC) against M. smegmatis (Rank 11- 20) S STA T A G T AT T F T A T AT DS verall anking ank Treatment rganism esistant reaking classes 0 n uantum change of o verall 11 Sorb al Glu (3) s if 8 24 3 2 58 3 e sA Amx 0 14 e s hlo 0 20 15 12 Ado af el (3) e sA an 0 12.5 3 3 45 51 ry 0 21 e s Tet 0 11.5 13 ham nu (1.5 ) (3) e sA an 0 14 3 2 0 5 e s Amp 0 12 Gen 15 34 14 Glu (3 ) (3) e sA Amp 0 15 3 2 1.5 .5 ry 0 24 e s Gen 15 3 .5 10 15 Sia a af (3) s lind 0 20 3 3 51.5 5 .5 e sA Amx 0 14 ry 0 1 .5 12 1 Sorb ha alt (3) s et 0 15 3 3 48 54 lind0 18 e sA an 0 15 2 1 Dal (3 ) (2) e s Amp 0 8.5 2 2 20 24 Tet 0 11.5 22 18 alt el (1.5 ) (2) e s Tet 0 11.5 2 2 28 32 hlo 0 1 .5 18 1 Sia Ado (1.5 ) (2) e s Tet 0 11.5 2 1 42 45 Gen 15 30.5 1 20 ac Try a (2) e sA ry 0 21.5 2 2 33.5 3 .5 e s Tet 0 12 Table 5b University of Ghana http://ug