TABLE OF CONTENTS
Title page i
Approval page ii
Dedication iii
Acknowledgement iv
List of tables v
List of figures vi
Abstract xi
CHAPTER ONE 1
Introduction 1
Classification of ESBL 2
Diversity of the types of ESBL 2
Global epidemiology of ESBL 5
Phenotypic identification of ESBL 10
Description of ESBL detection tests 11
CHAPTER TWO 15
Literature review 15
Antibiotics resistance 15
Antibiotics resistance mechanism 16
Genetics of antibiotics resistance 17
Treatment 19
Prevention and control 20
Aim and objective 20
CHAPTER THREE 21
Materials 21
Sample collection 21
Culture media preparation 21
Identification of organism 23
Antibiotics susceptibility test 25
Double disc synergy test 26
CHAPTER FOUR 27
Results 27
CHAPTER FIVE 33
Discussion 33
Conclusion 34
References 35
LIST OF FIGURES Pg
Figure 1: phenotypic confirmation of ESBL production using DDST 12
Figure 2: DDST test 32
LIST OF TABLES Pg
Table 1: morphology of isolates 27
Table 2: frequency of probable organisms 28
Table 3: IMViC test result and coagulase test result 29
Table 4: antibiotics sensitivity test result 30
Table 5: double disc synergy test result 31
ABSTRACT
Extended-spectrum beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam antibiotics, including penicillin, cephalosporin, and the aztreonams. The aim of this present study is to phenotypically identify and establish the presence of ESBL-producing organism among students in the university community. Within the University community of Godfrey Okoye University, Enugu, early morning urine samples of midstream-catch were collected into sterile bottles from sixty (60) students between ages 18 and 25years from the 2ndMay to 31st May. Thirty (30) male students and thirty (30) female students were sampled. Eighteen (18) isolates were identified after the following biochemical test were carried out: Gram staining, IMViC test (Indole test, methyl red test, Vogesproskauer test and citrate utilization test), and coagulase test. Twelve (12) isolates were from female students and six (6) isolates were from male students. The organisms identified were: Streptococcus spp, Corynebacteriumspp, Staphylococcus spp, and Escherichia coli. All theisolates were Gram positive except for one which was Gram negative. The double disc synergy test (DDST) was also carried out to phenotypically confirm the presence of ESBL producing organisms. All isolates were sensitive to the test drugs in the antimicrobial susceptibility test but there was no obvious DDST zones of inhibition. The result of the study suggests the absence of ESBL producing organisms among the students involved in this study.
CHAPTER ONE
1.1 Introduction
Extended-spectrum beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam antibiotics, including penicillin, cephalosporin, and aztreonams (Bush and Jacoby, 2010).
Extended-spectrum Beta(β)-lactamases (ESBLs) are a group which are mostly plasmid-mediated, diverse, complex and rapidly evolving enzymes that are posing a major therapeutic challenge today in the treatment of hospitalized and community-based patients. Infections due to ESBL producers range from uncomplicated urinary tract infections (UTI) to life-threatening sepsis. These enzymes share the ability to hydrolyze third-generation cephalosporin and aztreonam and yet, are inhibited by clavulanic acid. In addition, ESBL-producing organisms exhibit co-resistance to many other classes of antibiotics, resulting in limitation of therapeutic option. Because of inoculum effect and substrate specificity, their detection is also a major challenge (Deepthiet al 2010).
Numerous studies have barbed towards high incidence rate of UTI associated with Escherichia coli (E. coli) and antibiotic resistance. The emergence of Multi Drug Resistant (MDR) variant of E. colihas been accounted. MDR is defined as resistance to at least two antibiotics of different classes including aminoglycosides, chloramphenicol, tetracycline and/or erythromycin. MDR in many bacteria is due to the action of multi-drug efflux pumps and by the accumulation on Resistance (R) plasmids or transposons of genes with each coding for resistance to a specific agent. Nowadays, in UTIs, ESBL -expressing Gram-Negative Bacilli (ESBL-GNB) generally cause community-acquired infections. The resistance of Gram-negative bacteria is typically owed to plasmid mediated enzymes of ESBL. ESBL producing bacteria are typically associated with multi-drug resistance (MDR) and antibacterial choice is often complicated by multi-drug resistance (Prakash and Yadav, 2017).
1.2 CLASSIFICATION OF ESBL
There are two major classification systems for β-lactamases:
- Molecular classification is based on the amino acid sequence and divides β-lactamases Ambler classes into A (serine penicillinases), C (cephalosporinases), and D (oxa-cillinases) enzymes which utilize serine for β-lactam hydrolysis and class B metalloenzymes which require divalent zinc ions for substrate hydrolysis (Bush and Jacoby, 2010).
- Functional classification scheme was initially proposed by Bush in 1989 and then expanded in 1995. It takes into account substrate and inhibitor profiles in an attempt to group the enzymes in ways that can be correlated with their phenotype in clinical isolates (Bush and Jacoby, 2010).
- DIVERSITYOF THE TYPES OF ESBL
- TEM beta-lactamases
The first plasmid-mediated beta-lactamase in gram-negative bacteria was discovered in Greece in the 1960s. It was named TEM after the patient from whom it was isolated (Temoniera). Although TEM-type beta-lactamases are most often found in Escherichia coli and Klebsiellapneumoniae, they are also found in other species of Gram-negative bacteria with increasing frequency (Clark et al., 1990).
- SHV beta-lactamases
Sulfhydryl variable, (SHV) shares 68 percent of its amino acids with TEM and has a similar overall structure. The SHV beta-lactamase is most commonly found in Klebsiellapneumoniae and is responsible for up to 20% of the plasmid-mediated ampicillin resistance in this species. ESBLs in this family also have amino acid changes around the active site. More than 60 SHV varieties are known (Chow et al., 2010).
- CTX-M beta-lactamases
Cefotaximase Munich (CTX-M), these enzymes were named for their greater activity against cefotaxime than other oxyimino-beta-lactam substrates (e.g., ceftazidime, ceftriaxone, or cefepime). Rather than arising by mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on the chromosome of Kluyvera species, a group of rarely pathogenic commensal organisms. These enzymes are not very closely related to TEM or SHV beta-lactamases in that they show only approximately 40% identity with these two commonly isolated beta-lactamases. More than 80 CTX-M enzymes are currently known. Despite their name, a few are more active on ceftazidime than cefotaxime. They have mainly been found in strains of Salmonella entericaserovartyphimurium and E. coli, but have also been described in other species of Enterobacteriaceae(Chow et al., 2010).
- OXA beta-lactamases
The OXA-type β-lactamases are so named because of their oxacillin-hydrolyzing abilities. OXA beta-lactamases were long recognized as a less common but also plasmid-mediated beta-lactamase variety that could hydrolyze oxacillin and related anti-staphylococcal penicillin. These beta-lactamases differ from the TEM and SHV enzymes in that they belong to molecular class D and functional group 2d. The OXA-type beta-lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli, Klebsiellapneumoniae, and other Enterobacteriaceae, the OXA-type ESBLs have been found mainly in Pneumoniaeaeruginosa. The OXA beta-lactamase family was originally created as a phenotypic rather than a genotypic group for a few beta-lactamases that had a specific hydrolysis profile. Therefore, there is as little as 20% sequence homology among some of the members of this family. However, recent additions to this family show some degree of homology to one or more of the existing members of the OXA beta-lactamase family. Some confer resistance predominantly to ceftazidime (Chow et al., 2010).
- PER type
The PER-type ESBLs share only around 25–27% homology with known TEM- and SHV-type ESBLs. PER-1 β-lactamase efficiently hydrolyzes penicillin and cephalosporin and is susceptible to clavulanic acid inhibition. PER was first detected in Pseudomonas aeruginosa, and later in Salmonella entericaserovarTyphimurium and Acinetobacter isolates as well. In Turkey, as many as 46% of nosocomial isolates of Acinetobacter spp. and 11% of P. aeruginosa were found to produce PER, which shares 86% homology to PER-1, has been detected in S. entericaserovarTyphimurium, E. coli, K. pneumoniae, Proteus mirabilis, and Vibrio cholerae (Danish et al 2015).
- GES type
GES was initially described in a K. pneumoniae isolate from a neonatal patient just transferred to France from French Guiana. GES has hydrolytic activity against penicillin and extended-spectrum cephalosporin, but not against cephamycin or carbapenem, and is inhibited by β-lactamase inhibitors. These enzymatic properties resemble those of other class A ESBLs; thus, GES was recognized as a member of ESBLs (Danish et al 2015).
- VEB-1, BES-1, and other ESBL type
Other unusual enzymes having ESBL have also been described (e.g. BES, CME, VE-B, PER, SFO, and GES). These novel enzymes are found infrequently (Danish et al 2015).
- GLOBAL EPIDEMIOLOGY OF ESBL
Antimicrobial resistance has been declared a global threat to public health, as a massive increase in this problem has been observed in different parts of the world (Kang and Song 2013). The reported frequency of MDRs is increasing, putting strain on the public health organizations that are attempting to control this issue in many countries. The alarming increase in the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has serious consequences for treatment outcomes (Pitout 2010). E. coli and Klebsiella species are important pathogens isolated from community-acquired and nosocomial-acquired infections, and have been studied extensively. The ESBL enzymes produced by these bacteria make them resistant to the first-choice antibiotic therapies that are commonly used. ESBL-positive strains are associated with a delay in the commencement of suitable antibiotic therapy, which consequently lengthens hospital stay and raises hospital costs. Failure of antibiotic therapy is responsible for higher mortality rates in patients infected with these bacteria. MDRs are posing a treatment challenge, and are emerging as a major cause of morbidity and mortality worldwide. Unfortunately, proper surveillance and documentation of such pathogens is very limited, especially in developing countries like Nigeria (Hayat et al, 2018).
The epidemiology of health-care associated infections has been characterized by the emergence of gram-negative multi drug resistant organisms, including ESBL-producing Enterobacteriaceae during the past decade. While nosocomial transmission was initially considered by their principal cause of spread, earlier report points to the importance of the food-chain as a continuous source of dissemination (Kluytmanset al 2013). In addition to a growing body of literature regarding the detection of ESBL- producing Enterobacteriaceae in retail meat and food worldwide, food has been reported as a vector for transmission of ESBL- producing Klebsiellapneumoniae in a hospital outbreak (Calboet al 2011). This leads to the conclusion that control teams should consider extending their surveillance towards food as it is a vector of ESBL.
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