ABSTRACT
Pseudomonas aeruginosa is an ubiquitous, Gram-negative, rod-shaped, monoflagellated bacterium, capable of causing serious infections in immunocompromised host. It is one of the most difficult multidrug resistant microorganisms capable of producing metallo-β-lactamase (MBL). Though a notorious organism, its identification should not be limited to pigment production alone, as some strains do not produce pigments. This study was aimed at evaluating the increasing prevalence of multidrug resistance profile and MBL production in P. aeruginosa isolated from nasal cavity of cattle from Kara market, Ogun state, Nigeria.
In this study, a total of 570 samples were randomly collected from the nasal cavity of healthy male and female cattle using sterile swab sticks. They were tested for the presence of P. aeruginosa on cetrimide agar, observing the agar plates for growth and pigmentation. Further biochemical test and molecular analysis were carried out to further confirm their identity as P. aeruginosa. The antibiogram was determined using ceftazidime, cefroxime, gentamicin, ciprofloxacin, ofloxacin, augumentin, nitrofurantouin, ampicillin, imipenem and meropenem discs. Carbapenems (meropenem and imipenem) discs combined with EDTA were used to test for the production of MBL in P. aeruginosa.
A total of 439 samples were found to be positive for Pseudomonas out of the 570 samples collected. Of these positive samples, 229 isolates were from female cattle (31.0% showing pigment production and 68.9% non-pigment producers) and 210 isolates were from the male cattle (48.57% showing pigment production and 51.43% non-pigment producers). Isolates obtained from the female cattle showed 69.01% (pigment producers) and 82.28% (non-pigment producers) multidrug resistance while 75.49% (pigment producers) and 84.26% (non-pigment producers) were multidrug resistant among the isolates from the male cattle. In determining MBLs production, 47.84% were positive for MBL production using the IMP+EDTA and 75.50% were positive for MBL using the MER+EDTA.
From this study, it was concluded that multidrug resistant P. aeruginosa is present in the nasal cavity of cattle and its presence could be detrimental to the cattle, cattle herders, beef handlers and consumers. The level of resistance was high in both pigment and non-pigment producing isolates, though the resistance of non-pigment producing P. aeruginosa was higher. Drugs of choice to be considered first for the treatment of infections caused by multidrug resistant P. aeruginosa are gentamicin, ofloxacin, ciprofloxacin and meropenem. Nitrofurantoin is expensive and not easily purchased hence they are to be considered as the last drug of choice at extreme cases.
Keywords:Pseudomonas.aeruginosa, Carbapenem, resistance, Metallo-beta-lactamase, Cattle.
Word Count: 394
TABLE OF CONTENTS
Content Page
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Abstract v
Table of Contents vi
List of Tables vii
List of Figures viii
List of Plates ix
CHAPTER ONE: INTRODUCTION
1.1 Background to the Study 1
1.1.1 Pseudomonas aeruginosa 1
1.1.2 Multidrug resistant Pseudomonas aeruginosa 2
1.1.3 Carbapenems 2
1.1.4 Metallo-beta-lactamase 3
1.2 Statement of the Problem 3
1.3 Objective of the Study 4
1.4 Research Questions 5
1.5 Significance of the Study 5
1.6 Justification for the Study 5
CHAPTER TWO: REVIEW OF LITERATURE
2.1 Pseudomonas aeruginosa 6
2.2 Mechanism of Multidrug Resistance of P. aeruginosa 7
2.2.1 Production of Antibiotic-inactivating Enzymes (Beta-lactamases) 7
Content Page
2.2.2 Decreased Permeability of the Outer Membrane 8
2.2.3 Efflux Pumps System 8
2.2.4 Biofilms formation 8
2.3 Infections Caused by P. aeruginosa 9
2.3.1 Central Nervous System infections 10
2.3.2 Ear Infections including External Otitis 10
2.3.3 Eye Infections 10
2.3.4 Bone and Joint Infections 11
2.3.5 Urinary Tract Infections 11
2.3.6 Gastrointestinal Infections 11
2.4 Pigmented and Non-pigmented P. aeruginosa 11
2.5 Caberpenem 12
2.5.1 Mechanism of Action 13
2.5.2 Microbiological Activity 13
2.5.3 Mechanisms of Resistance against Caberpenems 13
2.5.4 Types of Carbapenem 14
2.6 Metallo-Beta-Lactamase 16
2.6.1 Verona Integron-encoded Metallo-β-lactamase (VIM) 16
2.6.2 Imipenemase (IMP) 17
2.6.3 New Delhi Metallo-β-lactamase (NDM) 17
2.7 Imipenem 17
2.7.1 Mechanisms of Resistance 17
2.8 Meropenem 18
Content Page
CHAPTER THREE: METHODOLOGY
3.1 Research Design 20
3.2 Population 20
3.3 Sample size and sampling Technique 20
3.4 Sample Processing 21
3.5 Identification of the Isolates 22
3.5.1 Gram Staining 22
3.5.2 Catalase Test 22
3.5.3 Molecular Analysis 22
3.6 Antibiotics Sensitivity Test 24
3.7 Phenotypic Screening for Metallo-β-Lactamase (MBL) 25
3.8 Statistical Analysis 25
CHAPTER FOUR: DATA ANALYSIS, RESULTS AND
DISCUSSION OF FINDINGS
4.1 Result 26
4.1.1 Source Distribution of Isolates 26
4.1.2 Molecular Identifications of the Isolates 30
4.1.3 Antibiotic Sensitivity Testing 32
4.1.4 Multidrug Resistance (MDR) Profile 37
4.1.5 Carbapenem Susceptibility, Resistance and Intermediate Profile 39
4.1.6 Metallo-beta-lactamase Profile 41
4.2 Discussion 43
4.2.1 Introduction 43
Content Page
4.2.2 Distribution and attribute of P. aeruginosa 43
4.2.3 Metallo-beta-lactamase Production 44
4.2.4 Antibiotic Sensitivity Testing 44
CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS
5.1 Summary 46
5.2 Conclusion 47
5.3 Recommendations 47
5.4 Contribution to Knowledge 47
REFERENCES 49
APPENDIX 61
LIST OF TABLES
Table Page
4.1: Distribution of carbapenem susceptibile, resistant and intermediate susceptible
Profiles of the isolated Pseudomonas aeruginosa from cattle 40
LIST OF FIGURES
Figure Page
4.1: Source distribution of Pseudomonas isolated from nasal cavity of cattle 27
4.2: Varying Pigment Production by P. aeruginosa isolated from the nasal
cavity of Female and Male Cattle 28
4.3: Distribution of susceptibility profile of pigment producing P.aeruginosa
from the female animals 33
4.4: Distribution of susceptibility profile of non-pigment producing P.aeruginosa
from the female animals 34
4.5: Distribution of susceptibility profile of pigment producing P.aeruginosa
from the male animals 35
4.6: Distribution of susceptibility profile of non-pigment producing P.aeruginosa
from the male animals 36
4.7: Multidrug resistance profile of P. aeruginosa isolated from the nasal cavity
of female and male cattle 38
4.8: Distribution of MBL production in P. aeruginosa isolated from cattle 42
LIST OF PLATES
Plate Page
4.1: 18-24 hours growth of Pigment and Non-pigment producing
Pseudomonas sp. Cultured on Cetrimide Agar 29
4.2:Electrophoregram of PCR product obtained
using PGSS primers for Pseudomonas species 31
CHAPTER ONE
INTRODUCTION
1.1 Background to the Study
Cattle are the most common and largest domesticated animal in the world (Bollongino,2012). They are reared for meat, milk and other dairy products. According to an estimate from 2011, there are about 1.4 billion cattle in the world. In 2009, cattle became one of the first livestock animals to have a fully mapped genome(Brown, 2009). They are herbivores because they feed on grasses, legumes and roughage. They are also known as ruminants because they have one stomach with four compartments. In Nigeria (West Africa), commercial beef cattle production is common especially in the Northern part of the country (Bollongino, 2012).
Pseudomonas aeruginosa (P. aeruginosa) is a bacterium capable of causing serious infections in cattle e.g. mastitis. It is found in the milk of cattle because it requires few nutrients to grow and multiply. Water supplies, contaminated drugs and infusion equipment are the major sources of this organism. It has also been isolated from waste feed, soil, manure and animal skin. Cows that are immunologically compromised due to other infectious diseases or are nutritionally deficient are also more susceptible to P. aeruginosa infections. The bacterium is resistant to antibiotics (John & Roger, 2011). In 2005, Haydar was able to isolate some bacteria causing pneumonia from the nasal cavity of healthy cattle especially in animals suffering from defects in their immune status or stressed (Haydar, 2005). About 2.43% of the isolates were Pseudomonas species. This showed that Pseudomonas aeruginosa can be an infectious agent in cattle and can be transmitted as zoonotic infection (Haydar, 2005).
- Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative, rod-shaped, monoflagellated bacterium ranging from about 1-5 µm long and 0.5-1.0 µm wide (Lederberg, 2000). They are ubiquitous microorganism that can be isolated from soil, water, humans, animals, plants, sewage, and hospitals (Lederberg, 2000). Itis an opportunistic human pathogen which often colonizes immunocompromised patients such as those with cystic fibrosis, cancer or AIDS (Botzenhardt et al., 1993). Itis the second leading cause of Gram-negative nosocomial infections (Carmeli et al., 1999) carrying a 40-60% mortality rate (Fick, 1993). It has a natural resistance mechanism to many antibiotics because of a resistance transfer plasmid, extra genetic material carried in the cells with genes that code for proteins that destroy antibiotic substances (Madigan & Martinko 2006).
1.1.2 Multi-drug resistantPseudomonas aeruginosa
Multi-drug resistant (MDR) P. aeruginosa are organisms resistantto one antimicrobial agent in three or moreantipseudomonal antimicrobial classes(carbapenems, fluoroquinolones, penicillins/cephalosporins and aminoglycosides)(Magiorakos et al., 2011). Multi-drug resistance in P. aeruginosa arises from low outer membrane permeability, multidrug efflux systems which accounts for its intrinsic mechanisms of resistance, enzyme production, target mutations (Kotra et al., 2000) and biofilm formation (Carmeli et al., 2002). In addition to these factors, other bacterial exoproducts contributing to multidrug resistance in P. aeruginosa arelipopolysaccharides and elastase which induce harmful pathogenesis resulting in tissue destruction. Apart from enabling motility, the flagellum of P. aeruginosa plays an indirect role in membrane permeabilization and surfactant protein-mediated bacterial clearance (Zhang, 2007). MDR P. aeruginosa are very problematic because of its inherent resistance to many drug classes andability to acquire resistance to all effectiveantimicrobial agents (Gad et al., 2007).
1.1.3 Carbapenems
Carbapenems are β-lactam group of drugs that are usually used as antibiotics of last resort for treating infections due to multiple-resistant Gram-negative bacilli. Often times, the stable response of P. aeruginosa to extended-spectrum β-lactamases has changed due to the emergence of metallo-β-lactamase (MBL)-producing strains (Jesudason et al., 2005). They bear a penemic together with the beta-lactam ring inhibiting bacterial cell wall synthesis by binding to and inactivating Penicillin Binding Proteins (PBPs).
Multi-drug resistant (MDR) P. aeruginosa are capable of producing enzymes that can inactivate beta-lactams such as metallo-β-lactamase (MBL) which is responsible for a significant proportion of carbapenem resistance in these bacteria (Moya et al., 2009, Borgianni et al., 2010). These enzymes can hydrolyse all classes of β-lactam drugs and withstand neutralization by β-lactamase inhibitors (Wan Nor Amilah et al., 2012). Imipenem, panipenem, meropenem, biapenem, ertapenem, doripenem and tebipenem belong to the carbapenem family. Each one present different characteristics that influence their way of administration and their usefulness as anti-pseudomonal agents.Carbapenem resistance mechanisms in P. aeruginosa may be classified as enzymatic, mediated by carbapenemases (beta-lactamases hydrolyzing carbapenems among other beta-lactams). Carbapenem resistance, however, develops frequently due to the concomitant presence of more than one mechanism (El Amin et al., 2005; Hammami et al., 2009). Another resistant mechanism of P. aeruginosa to carbapenem is the reduction of outer membrane (OM) permeability through alterations in or decreased production of outer membrane porin D (OprD). This porin allows the cellular entry of carbapenems (Farra et al., 2008).
1.1.4 Metallo-Beta-Lactamase
Metallo-beta-lactamases (MBLs) are enzymes that make bacteria resistant to a broad range of beta-lactam antibiotics one of which includes the cabapenem family (Kumarasamy et al., 2010). They belong to class B of the structural classification of β-lactamases and are able to efficiently hydrolyze all β-lactams with the exception of monobactams (Yan et al., 2006; Gutierrez et al., 2007; Palzkill, 2013). The enzymes require divalent cations, usually zinc, as metal cofactors for enzyme activity and are inhibited by metal chelators such as ethylenediamine tetra acetic acid (EDTA) (Maltezou, 2009). MBLs are encoded either by genes that are part of the bacterial chromosome in some bacteria or by heterologous genes acquired by transfer of mobile genetic elements. Therefore, acquired MBL can be spread among various strains of bacteria such as P. aeruginosa (Cornaglia et al., 2011).
1.2 Statement of the Problem
Scientifically, P. aeruginosa is known to be a notorious organism because it is highly resistant to virtually all antibiotics. Infections to which it is implicated are always difficult to treat. This may be as a result of most virulence factors and mechanisms of resistance in P. aeruginosa. Although P. aeruginosa is ubiquitous, pathogenic and possibly a zoonotic agent, there is a death of information on its isolation from domesticated animals especially asymptomatic cattle and the susceptibility of these isolates from cattle to different antibacterial agents. Though cattle are not carriers of P. aeruginosa, they are often infected by them (mastitis being the most common infection). The presence of multidrug resistant P. aeruginosa poses a threat not only to the cattle but also to the cattle herders, beef retailers, beef handlers and beef consumers.
Consequently, this study will determine
- The possibility of isolating pathogenic pseudomonas from the nasal cavity of healthy cattle as against the expected Staphylococcus aureus which are usually considered natural microflora of the nasal cavity.
- Whether the different antibiotics readily available would be effective in inhibiting the growth of aeruginosa strainsisolated from the nasal cavity of cows or not for effective therapy in infections.
- The multi-drug resistant profile of aeruginosa isolated from cattle
- If the isolates are capable of producing metallo-beta-lactamase which is a resistance mechanism and to what extent or degree.
1.3 Objective of the Study
The general objective of this study is to educate the public on the presence of P. aeruginosa if isolated and to evaluate the increasing prevalence of multi-drug and carbapenem resistant P. aeruginosa isolated from cattle in Kara market, Ogun State, Nigeria. The specific objectives are to:
- isolate aeruginosa from the nasal cavity of male and female cattle;
- identify aeruginosa from the nasal cavity of male and female cattle;
- determine whether all strains of aeruginosa are pigment producing or non-pigment producing P. aeruginosa ;
- determine the antibiogram of the isolates and compare the antibiotic resistant pattern between the pigment producing aeruginosa and non-pigment producing P. aeruginosa;
- determine metallobeta-lactamase production in the isolated aeruginosa from nasal cavity of asymptomatic cattle.
1.4 Research Questions
- Can aeruginosa be isolated from the nasal cavity of healthy cattle?
- How is aeruginosa identified when isolated from the nasal cavity of healthy cattle?
- Are non-pigmented Pseudomonas truly aeruginosa
- How can the isolated pigment producing aeruginosa bedifferentiated from the non-pigment producing P. aeruginosa?
- How are the multiple antibiotic resistance index of the isolate calculated using the antibiogram of the isolate?
- How is the test for metallo-beta-lactamase carried out?
1.5 Significance of the Study
This study would possibly indicate cattle as a nasal carrier of Pseudomonas aeruginosa and indicate the susceptibility profile of the isolates. It would also, educate the general public on the danger of negligence of multidrug resistant P. aeruginosa in cattle and its effect in eating undercooked beef as well as providing a more recent data on the increasing prevalence of resistance in multidrug resistant P. aeruginosa.
1.6 Justification for the Study
This study will most importantly provide baseline information and a more recent epidemiological data on the increasing prevalence of multi-drug resistant P. aeruginosa resulting from cattle.
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