ISOLATION AND MOLECULAR CHARACTERIZATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM POULTRY AND POULTRY FARM WORKERS IN KANO, NIGERIA

ABSTRACT

The increasing rate of drug resistance associated with methicillin resistant Staphylococcus aureus is not only a problem in the clinical sector but also in livestock disease treatment and management. Methicillin resistant Staph. aureus is now a leading cause of staphylococcal infections in human and animals. In view of the present serious problem of resistance to antibiotics from Staph. aureus, the present study was undertaken to investigate the incidence of methicillin resistance in Staph. aureus. The study was carried out in Kano State, Nigeria to evaluate the incidence of methicillin resistant Staph aureus from poultry and poultry farm workers. Cloacae and nostril of 1200 poultry birds selected randomly in 12 farms from the three senatorial zones of Kano State and 60 nostrils of poultry farm workers were screened for the presence of Staph aureus using standard microbiological techniques. Antibiotic susceptibility pattern was determined using disc agar diffusion (DAD) method. Vancomycin resistance was determined using vancomycin agar screening method. Molecular studies of 16SrRNA, nuc, mecA and PVL gene were carried out using multiplex PCR, the PCR may permit sufficient sensitivity and specificity for the direct detection of Staph. aureus.Twenty two isolates were tested for Panton Valentine Leukocidin (PVL) using PCR. Ninety eight isolates (8.2%) were confirmed and characterized as Staph aureus, sixty six of the isolates were from broiler and 32 from layers. Cloacae yielded high number of Staph aureus than nostril. The result of antibiotic susceptibility test showed general resistance to β-lactam antibiotics; ampicillin and oxacillin and oxytetracycline at 71.4% each, chloramphenicol (61.2%) and sulfamethaxazole/trimethroprim (51 %). However higher percentage of sensitivity was recorded against vancomycin (74.5 %), AugmentinR and cefoxitin (69.4 %), ciprofloxacin (64.3 %), gentamicin 60.2% and neomycin 54.1%. Thirty percent (30.6 %) of the Staph aureus isolates were phenotypically identified as methicillin resistant using cefoxitin 30 µg., and one from poultry farm workers. Determination of multiple antibiotics resistance index showed that 80 (81.6 %) were resistant to 3 or more antibiotics MARI ≥ 0.3 and 5.1 % had MARI 0.3. Eighty three point three percent of the MRSA (83.3 %) were multidrug resistant. Eighty nine point eight percent (83.3 %) of the total staphylococcal (89.8 %) isolates produced β-lactamase and 19/30(63.3%) phenotypic MRSA were β-lactamase hyper producers. Vancomycin resistance determined using vancomycin screening agar showed that 90% of the MRSA were vancomycin resistant (VRSA). The molecular analysis of the isolate using PCR showed that all the isolates were Staph aureus of 800bp. PCR showed a correlation between phenotype and recovery of MRSA and genotypic detection of mecA gene. The prevalence of mecA mediated methicillin resistance in Staph aureus is high as 40.7% of the total MRSA isolate carried mecA gene. The amplified product of Staph aureus mecA gene showed a correlation between the staphylococcal penicillin binding protein (PBP2a). Sixteen (53.3%) were PBP2a positive. Only one isolate from farm worker had mecA gene. Twenty two isolates were tested for Panton Valentine Leukocidin (PVL) but only 14 (63.36%) were positive. Analysis showed that PVL was not associated with truly community acquired as all PVL positive isolates in this study were from poultry. Further analysis showed that 3 of the seven housekeeping genes (pta, gmk, and yqil) were present. 35% expressed Spa typing at variable regions.

Multi-locus sequence typing of 8 isolates selected showed that the main sequence type (ST398) of livestock associated methicillin resistant Staph. aureus (LA-MRSA) was not present in the poultry used in this study. The multi-drug resistant nature of most of the isolates, and the high resistance level to especially β-lactam antibiotics is a sign of misuse and overuse of the agents in the environment (poultry farms). These call for rapid and accurate detection of multi drug resistant methicillin resistant Staph. aureus, and drawing up of guidelines for the prompt, effective and appropriate use of antibiotic therapy and for control of CA-MRSA and LA-MRSA.

TABLE OF CONTENTS

Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Abstract vi
Table of Contents vii
List of Tables xiv
List of Figures xv
List of Plates xvi
List of Appendices xvii

CHAPTER ONE
1.0 Introduction 1
1.1 Statement of Research Problems 4
1.2 Justification of the Research 6
1.3 Aim and Objectives 8
1.4 Research Hypothesis 9
1.4.1 Null Hypothesis (H0) 9
1.4.2 Alternate Hypothesis (Ha) 9

CHAPTER TWO
2.0 Literature Review 10
2.1 The Genus Staphylococcus 10
2.1.1 Differentiation of Staphylococci From Micrococci 12
2.1.2 Differentiation of Staphylococcus aureus from other Coagulase Negative Staphylococci 14
2.2 Staphylococcus aureus 14
2.2.1 Culture Characteristics and Laboratory Identification 15
2.2.2 Carriage of Staph. aureus 17
2.2.3 Pathogenesis of Staph. aureus 19
2.2.4 Virulence factors in Staph. aureus 22
2.3 History of Antimicrobial Chemotherapy 25
2.3.1 Development of Chemotherapeutic Agents 27
2.4 Antibiotics: Definition 29
2.4.1 Sources of Antibiotics 30
2.4.2 The Beta-Lactam Antibiotics 31
2.4.2.1 Mechanism of action of the penicillins and cephalosporins 35
2.4.3 Aminoglycosides 36
2.4.4 Tetracyclines 37
2.4.5 Trimethoprim-Sulfamethoxazole 38
2.4.6 Quinolones 39
2.5 Bacterial Drug Resistance 41
2.5.1 Origin of Drug Resistance 44
2.5.1.1 Non Genetic or inherent 44
2.5.2 Genetic origin 45
2.5.2.1Chromosomal Resistance 46
2.5.2.2 Extra Chromosomal 47
2.5.2.3 Transfer of Genetic Information 49
2.6 Biochemical Mechanism of Resistance 50
2.6.1 Enzymatic inactivation of the antibiotic 50
2.6.2 Alteration of the Target Site 52
2.6.3 Decreased concentration of Drug at the target site 54
2.6.4 Failure to metabolize the Drugs 56
2.6.5 Metabolic Bypass 56
2.7 Antibiotic Resistance in Staph. aureus 56
2.7.1 Multidrug resistant Staph. aureus 58
2.7.2 Staphylococcal Cassette Chromosome 60
2.8 Methicillin resistance Staph. aureus 63
2.8.1 History and epidemiology 63
2.8.2 Evolution of MRSA 64
2.8.3 Mechanism of Methicillin Resistance 65
2.8.4.1 Altered penicillin Binding Protein (PBP2a) 67
2.8.4.2 Regulation of PBP2a expression 67
2.9 Detection of Methicillin Resistant Staph. aureus 69
2.10 Hospital Acquired methicillin resistant Staph. aureus (HA-MRSA) 71
2.11 Community-Associated Methicillin Resistant Staph. aureus (CA-MRSA) 72
2.12 Livestock-Associated Methicillin Resistant Staph. aureus 75

CHAPTER THREE
3.0 Materials and Methods 78
3.1 The Study Area 78
3.1.1 The Study Population 78
3.2 Materials 79
3.2.1 Culture Media 79
3.2.2 Chemical Reagents 79
3.2.3 Antibiotic Disc 80
3.2.4 Plasma 80
3.2.5 Equipment 80
3.3 Methods 81
3.3.1 Sample Collection Areas 81
3.3.2 Sample Collection 81
3.3.3 Preliminary Identification of Organism 81
3.3.4 Isolation and purification of Staph.aureus 81
3.3.4.1 Simple Staining 81
3.3.4.2 Gram Staining 82
3.3.4.3 Growth on Selective Media 82
3.3.5 Biochemical and Confirmatory Tests 83
3.3.5.1 Catalase Test 83
3.3.5.2 Coagulase Test 83
3.3.5.3 Deoxyribonuclease (DNAse) test 84
3.3.6 Staph. Agglutination Test 84
3.3.7 Microgen Staph ID Test 85
3.3.8 Test for β-lactamase Production 86
3.3.8.1 Starch Solution 86
3.3.8.2 Nitrocefin Test (β-lactamase typer producers) 86
3.3.9 Antibiotic Susceptibility Test 87
3.3.10 Determination of Methicillin (Oxacillin) resistance using cefoxitin disc 88
3.3.11 Determination of Multiple Antibiotic Resistance (MAR) Index 88
3.3.12 Screening for Vancomycin resistance 88
3.3.14 Test for Penicillin-Binding Protein (PBP2a) 89
3.4 Molecular analysis 90
3.4.1 DNA extraction 90
3.4.2 Polymerase Chain Reaction for Detection of 16SrRNA, nuc gene and mecA gene 91
3.4.3 Detection of PVL (luks-lukf) gene by PCR 94
3.4.4 Spa Typing 95
3.4.5 Multilocus Sequence Typing (MLST) 96
3.4.6 Staphylococcal Cassette Chromosome (SCCmec) Typing. 98
3.5 Statistical Analysis 100

CHAPTER FOUR
4.0 Result 101
4.1 Sample Population 101
4.2 Biochemical and Confirmatory Test 103
4.2.1 Screening of Isolates Using Staph.Agglutination Test 106
4.2.2 Identification of Isolates Using Microgen Staph ID 106
4.3 Distribution of Staph. aureus according to Geopolitical zones 108
4.4 Recovery of Staph aureus from Broilers and Layers 109
4.5 Staph. aureus isolated from poultry farm workers. 113
4.6 Antibiotic Susceptibility Profile 114
4.7 Identification of Hetero Resistant Strains 118
4.8 Test Forβ – Lactamase Production 120
4.9 Determination of Phenotypic Methicillin Resistance Using Cefoxitin 121
4.10 Detection of Vancomycin Resistance 121
4.11 Detection of Penicillin binding Protein (PBP2a) 121
4.12 Determination of β-lactamase Hyper Production 121
4.13 Multidrug Resistance (MDR) And Multiple Antibiotic Resistance Index 122
4.14 Molecular Analysis 127
4.14.1: Genomic DNA of Staph. aureus Isolates 128
4.14.2 Detection of 16SrRNA, mecA and nuc gene 129
4.14.3: Detection of mecA gene 131
4.14.4: Detection of Nuc and yqil (one of the seven housekeeping genes ) 133
4.14.5 Spa typing of Staph. aureus 134
4.14.6 PVL Detection 136
4.14.7 Detection of the seven housekeeping genes 138
4.14.8 DNA sequencing 140

CHAPTER FIVE

5.0 Discussion 143

CHAPTER SIX
6.1 Summary 158
6.2 Conclusions 160
6.3 Contribution to Knowledge 161
6.4 Recommendations 162
REFERENCES 163
APPENDICES 197

CHAPTER ONE

1.0 INTRODUCTION

Staphylococcus aureus, a Gram positive coccus is a frequent cause of skin infections, such as boils and pimples. Since the 1970s the usual treatment for these infections has been penicillins and penicillinase resistant antibiotics such as methicillin. Today however, this treatment is likely to fail. In 2003, well over 60% of the Staph.aureus strains isolated in hospitals were resistant to this antibiotic (Eugene et al., 2009). Methicillin resistance in this bacterial species represents a threat to human health. Originally, methicillin resistant Staph.aureus (MRSA) was a nosocomial pathogen, but in the 1990s, MRSA spread into communities worldwide (Lee, 2003). About a third of healthy individuals carry Staph. aureus on their skin and nose (Grundmann et al., 2010). It is also a common cause of wound and urinary tract infections (Sina et al., 2011).

Carriage of Staph.aureus Sequence Type (ST) 398 has primarily been reported as occurring among persons in contact with livestock, including swine and cattle (Wulf et al., 2008; Smith et al., 2009).This association has given rise to the characterization of this strain as live stock associated (Wulf and Voss, 2008).

Pigs have been shown to be major reservoir of methicillin resistant Staph. aureus multi-locus sequence type 398 (Leonard and Markey, 2008). It has also shown potential for zoonotic transmission (Huijsdens et al., 2006). This clonal complex associated with disease in livestock has also been implicated in human infection (Witte et al., 2007). It is known to cause diseases in poultry, feed and companions animals (Person et al., 2009; Hunter et al., 2011).

Methicillin resistant Staph.aureus (MRSA) is becoming increasingly recognized among persons in the community without established risk factors (Kluytman et al., 2006; Faria et al., 2005). MRSA in animal disease have not, until now been considered a source of infection to humans, although transmission appears to be primarily between animals, undistinguished isolates have been found in their human contacts, particularly those with occupational exposure (Armand et al., 2005; Khanna et al., 2008). MRSA CC398 was first detected in 4 pigs and one healthy pig farmer in France (Aubury et al., 2004). Clinical infection was described in the daughter of a pig farmer in the Netherlands in 2004 (Voss et al., 2005). That study also showed that 23% of pig farmers in a small survey in the same region were seropositive for MRSA CC 398.

Staph.aureus is perhaps the most notorious of all the bacterial pathogens associated with human infection. In 1942 the year penicillin G was introduced, some resistant strains of Staph. aureus were found (Kunin, 2000). Staph. aureus is the first bug to battle penicillin in 1967 due to its ability to produce β-lactamase. Resistant semisynthetic penicillins in the early 1960s provided temporary respite which ended with the emergence of methicillin resistant Staph. aureus, discovered shortly after methicillin became available for clinical use (Miall et al., 2001; Fluit et al., 2001).

MRSA is of concern not only because of its resistance to methicillin but also because it is generally resistant to many other chemotherapeutic agents (Vidhani et al., 2003). Multi-drug resistant Staph.aureus evolved following acquisition of discrete preformed antimicrobial resistance genes by horizontal gene transfer and resistance determinants generated by chromosomal mutation which poses great challenges in treatment of staphylococcal infections (Jesen and Lyon, 2009).

Methicicilrin resistant Staph.aureus (MRSA) strain carries a large heterologous mobile genetic element, staphylococcal cassette chromosome (SCC) which includes the central element of methicillin resistance, the mecA gene (Ito et al., 1999). Until now, six SCCmec type (I-VI) have been identified in Staph. aureus which are defined by combination of the mec gene complex class with the ccr allotype (Ito et al., 2001; Oliveira et al., 2006). SCC mec typing has been established as an important addendum to the characterization and identification of MRSA clones and is routinely used in many laboratories. Several strategies have been developed for SCC mec type (Okuma et al., 2002) and their broad application has led to the detection of several variants or subtype of the major SCC type (Olivera and de Lancastre 2002; Ito et al., 2003). Community acquired methicillin resistant Staph. aureus (CA-MRSA) has a characteristic staphylococcal cassette chromosome type IV (SCCmec IV) gene, lacking in non β–lactam determinant and possessing distinct necrotizing toxin, Panton valentine leukocidin (PVL).

Methicillin resistant Staph.aureus (MRSA) isolates in livestock have gained particular attention during recent years (Wulf and Voss, 2008). The identification of livestock associated MRSA in food producing animals has raised questions regarding the presence of MRSA in food of animal origin. Several studies were conducted in different parts of the world to screen food of animal origin intended for human consumption for the presence of MRSA and also to identify the MRSA types present (de Boer et al., 2009). MRSA are now regarded as a major cause of hospital and community acquired infection worldwide and the problem is exacerbated by the emergence of multi-drug resistant MRSA (MDR-MRSA). Such isolates demonstrated a reduced susceptibility to almost all clinically available antibiotics and are often only susceptible to glycopeptides and investigational drugs (Lopez et al., 2005).

1.1 STATEMENT OF RESEARCH PROBLEMS

Methicillin resistant Staph.aureus is a major cause of hospital and community infections that are becoming increasingly difficult to combat because methicillin resistant Staph. aureus resist almost all currently available antibiotics (Blum, 1995). Community acquired MRSA is the major cause of skin and soft tissue infections (SSTIs) that has become increasingly problematic due to its high virulence and the ease with which they spread in the community (Reyes et al., 2011). The presence of livestock associated MRSA in farmers constitutes a major threat to public health and health care system (Golding et al., 2010). Methicillin resistant Staph.aureus prevalence in humans is strongly associated with prevalence in animals and intensity of contact with animals positive for methicillin resistant Staph. aureus (Graveland et al., 2010). Zoonotic transmission of this clone of MRSA (ST398) has also been documented especially among farm workers and their family (Lee, 2003).

MRSA has been isolated from food of animal origin such as dairy products, beef, chicken (Normonno et al., 2007) and although food borne transmission is plausible (Lee, 2003). Methicillin resistant Staph.aureus (MRSA) has also been detected in several species and animal derived products (Leonard and Markey, 2008). An emerging subtype of methicillin resitant Staph.aureus, a clonal complex CC398, is associated with animals, particularly pigs (Armand et al., 2005). MRSA is a significant cause of nosocomial and community morbidity and mortality which over two decades have become a worldwide problem exacerbated by the emergence of multi drug resistant methicillin resistant Staph. aureus (Aires et, al. 1998).

The leading role of MRSA in these infections is associated with its resistance to most currently available antibiotics resulting in treatment failure (Enright et al., 2002). The panton valentine leukocidin (PVL) encoded by LukF-LUkS genes associated with CA-MRSA are responsible for the wide spread of skin and soft tissue infection (David et al., 2007). No antibiotic resistance marker has distinguished a species more than what methicillin resistance has for Staph. aureus. Methicillin resistance indicates resistance to all β-lactam antibiotics, including cabarpenems. MRSA isolates are increasingly resistant to multiple non β-lactam antibiotics, with reports of strains not susceptible to vancomycin creating a lot of problems in clinical settings for the successful chemotherapy of infections.

Although the mechanisms, risk factors and other information about emergence of methicillin resistant Staph. aureus in animal are rather poorly understood, the close contact between human and various animal species and antimicrobial use in animals presumably facilitate the emergence and spread of MRSA (Wesse et al., 2010).

Here in Nigeria, several studies conducted across the country showed that methicillin resistant Staph. aureus has been isolated and is a common cause of hospital and community acquired infections with varying prevalence (Sina et al., 2011; Adegoke and Komolafe, 2009). Also, nasal carriage of Staph.aureus amongst students has also been reported (Abdulhadi et al., 2008).

There have however, been little studies on the prevailing resistance of poultry farm isolates to commonly used antibiotics, especially in the northern part of the country particularly in Kano. Therefore it is the aim of this study to isolate and identify such resistant strains and carryout their molecular characterization.

1.2 JUSTIFICATION OF THE RESEARCH

The occurrence of methicillin resistant staph.aureus not only in livestock but also in food of animal origin might represent a relevant issue with regard to human health and food safety for consumption. The MRSA isolated from human skin and soft tissue infections were mainly the MRSA (ST398) seen in cattle and poultry (Dullweber, 2010; Mulders et al., 2010).

The emergence and involvement of a distinct clone complex of MRSA (ST 398) associated with livestock in human disease in many countries suspected to have arisen from the increasing use of antibiotics in animal feeds, especially in poultry give a good reason for the study since poultry farming is common in Nigeria.

This study will provide compelling epidemiological and microbiological evidence that persons living with chicken or working on farms will be at increased risk of being colonized or infected with LA-MRSA. It is important to identify the origin of the isolates and their dissemination on the farm, and to evaluate the potential health hazard.

Since MRSA (ST 398) is implicated in human disease and has been reported in poultry in different parts of the world, they may also be present in poultry farms in Nigeria and this study will ascertain the presence and prevalence of MRSA (ST 398) in poultry farms in Kano State, Nigeria. Persons working on farms or living with the chickens are at a high risk of contacting the MRSA and transmitting it to their family and community at large, and may likely cause staphylococcal infection. Poultry farming in rural community is practiced at small scale level, in addition to the large poultry farms scattered all over the country; it is possible that these animals carry methicillin resistant Staph. aureus (Olayinka et al., 2010).

The presence of methicillin resistant Staph.aureus has been reported in Nigeria (Olayinka et al., 2010) but there is no report of LA-MRSA of clonal complex ST398 in human infections in communities and hospital settings in the country. Besides, people working on the farms have little or no knowledge about the MRSA and are at high risk of contacting the bacteria through handling, and cleaning of the farm and this could easily be transferred to community. Thus molecular analysis of the isolates and a comprehensive analysis of their antimicrobial resistance properties would provide relevant information for health workers in the country and plan preventive and therapeutic measures in combating this emerging infection and minimizing risk factors. Transmission of MRSA ST398 from zoonotic reservoir to human could exacerbate presence of MRSA. There is absolute need to study this clonal complex 398 in other food producing animals not only poultry in the country. It is hoped that information from this research work will increase awareness among health care professionals that animals are a possible source of MRSA infection and that the potential for animal to human transmission exists.

1.3 AIM AND OBJECTIVES

AIM: To isolate and molecularly characterize MRSA isolates from poultry and poultry farm

workers in Kano State.

Specific Objectives

1. To isolate Staph. aureus from chicken nares and cloacae (broilers and layers) and from the farm workers.

2. To screen the isolates for antimicrobial susceptibility using DAD.

3. To identify methicillin resistant isolates using cefoxitin.

4. To use latex agglutination method to determine the presence of PBP2a.

5. To determine those MRSA isolates that harbor mecA gene using PCR method

6. To screen the isolates for staphylococcal cassette chromosome (SCCmec) typing and lukF-lukS gene by multiplex PCR.

7. To determine multilocus sequence typing (MLST) for the clonal complex for confirmation of LA-MRSA.

8. To subject all MRSA isolates to Spa sequence typing.

1.4 RESEARCH HYPOTHESIS

1.4.1 Null Hypothesis (H0)

Methicillin resistant Staph.aureus (MRSA) will not be isolated from both poultry and poultry farm workers in Kano State, Nigeria.

1.4.2 Alternate Hypothesis (Ha)

Methicillin resistant Staph.aureus (MRSA) will be isolated from both poultry and poultry farm workers in Kano State, Nigeria.