MOLECULAR EPIDEMIOLOGY OF EXTENDED-SPECTRUM BETA-LACTAMASE (ESBL) PRODUCING ENTEROBACTERICEAE FROM THREE TERTIARY HOSPITALS IN SOUTHERN GHANA

ABSTRACT
Antimicrobial resistance is fast becoming a global concern in both healthcare settings and in the community due to the rapid emergence of multi drug resistant organisms. High proportions of enterobacteria have developed resistance to the commonly prescribed antimicrobial drugs. Comprehensive data upon which to advocate control interventions are scanty. Hence this study determined the Molecular Epidemiology of ESBL producing enterobactericeae from three Tertiary Hospitals in Southern Ghana. A total of 167 non repetitive isolates consisting of 51 E. coli strains, 43 K. pneumoniae, 16 P. mirabilis, 21 P. aeruginosa ,12 A. baumannii and 24 other unspecified organisms were collected and tested for their antimicrobial susceptibility, ESBL production by double synergy method and the ESBL genotypes were determined by PCR.
Major beta-lactams to which resistance was found in this study included Ampicillin (94.7%), Cefuroxime (81.6%), Cefamandole (71.9%), Ceftriaxone (69.4 %,), Augmentin (66.7%), Cefpodoxime (78.1%), Cefotaxime (78.9%). All these beta-lactams registered more than 50% resistance. ESBL percent prevalence were; 6.7% for Acinetobacter baumannii followed by 8.3% Proteus mirabilis, 15.0% Pseudomonas aeruginosa, 18.3% Klebsiella pneumoniae and 40% for E. coli. Other isolates recorded 11.7%. ESBL genotypes (TEM, SHV and CTX-M) were found in 118 out of 167 ESBLs phenotypically identified. The overall prevalence of ESBL detected was (71.51 %). The high prevalence of ESBL calls for immediate intervention strategies to prevent further spread. Training of laboratory personnel on phenotypic testing of ESBLs in addition to training clinical staff and prescribers on ESBL issues are advocated.

CHAPTER ONE
INTRODUCTION
Chapter one captures the introduction and background of Extended Spectrum Beta-lactamase producing Enterobacteria, including their definitions, types, biological activities, their mechanism of drug resistance and their fate in the environment. The problem statement gives an insight into how ESBL contributes to drug resistance in Ghana. The problem statement further indicates the need for surveillance and continues monitoring which will serve as a basis for the disease control and monitoring. The justification section under this chapter elucidates some problems caused by ESBL in Ghana and globally. The chapter also raises some specific objectives and research questions to be answered in this study.

Bacterial resistance to antibiotics is increasing globally in both healthcare settings and in the community and this remains a global health threat due to the rapid emergence and spread of multi drug resistant organisms. In recent times, susceptible bacteria pathogens such as members of the enterobacteriaceae family are spontaneously developing resistance to these first-line choice drugs used for treatment of severe infections. This is an issue of due public health concern (Sangare et al., 2017).

Background
The members of the Enterobacteriaceae are a large family of Gram-negative bacteria that include many harmless symbionts and many of the more familiar pathogens, such as Salmonella typhi, Escherichia coli, Yersinia pestis, Klebsiella spp, and Shigella spp. Other disease-causing bacteria in this family include Serratia, E. cloacae, Proteus mirabilis, and Citrobacter freundii, usually resident in the gastrointestinal tract. This group of organisms are responsible for common illnesses and cause various hospital acquired infections like gastrointestinal, urinary tract and pyogenic infections (Giddi et al., 2017). Options for treatment of infections due to enterobacteriaceae include beta-lactam antibiotics (Penicillin; Cephalosporins; Carbapenems; and the Monobactam, Aztreonam) or those beta-lactam antibiotics combined with beta-lactamase inhibitors, quinolones, TMP-SMX, Aminoglycosides, and Tigecycline (Richards et al., 2000). Enterobacteriaceae produce many different beta-lactamase enzymes with some having the capability to hydrolyze only Penicillins and 1st, 2nd and 3rd generation Cephalosporins. Overuse of drugs have been identified as a major cause of resistance to β-lactams among Gram-negative bacteria globally, a basis of the emergence of Beta-lactamases (Shaikh et al., 2015) and has been increasingly detected in resource limited regions such as Africa.

Beta-lactamases are hydrolytic enzymes which cleave the beta-lactam ring by hydrolyzing the amide bond of the beta-lactam ring and are the primary mechanism of conferring bacterial resistance to beta-lactam antibiotics especially in Gram-negative bacilli (Dhillon & Clark, 2012; Chaudhary & Aggarwal, 2004). The genes that code for the beta-lactamase associated resistance can be carried on bacterial chromosomes, an inherent resistant property of the organism or may be plasmid-mediated with the potential to move between bacterial populations, a clear implications regarding spread of infection (Shoorashetty et al., 2011). Beta-lactamases in present times have been detected in numerous countries and are known to hydrolyze the extended-Spectrum Cephalosporins and the monobactams (Thenmozhi et al., 2014) hence these new beta-lactamases were coined extended-spectrum – lactamases (ESBLs) (Sirot et al., 1987). ESBLs are therefore a group of beta-lactamases rapidly evolving which share the ability to hydrolyze third-generation cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone ) and monobactams (eg. aztreonam ) but do not affect cephamycins (e.g., cefoxitin) or carbapenems (e.g., meropenem or imipenem) and can hydrolyze all penicillins yet are inhibited by clavulanic acid (Livermore, 2012).

Beta-lactamases are classified by two different schemes; according to structural homology (Ambler’s classification) (Ambler, 1980) and their functional property (Bush’s and Jacoby’s classification) (Bush et al., 1995). ESBLs belong to the Ambler molecular class A as well as the Bush-Jacoby functional group 2be (Bush & Jacoby, 2010). These enzymes have been identified in organisms in different geographical areas particularly in Enterobacteriaceae and are significantly detected in various E. coli strains. Major antibiotics to which resistance has been detected include the ampicillin, tetracycline and cotrimoxazole which are capsules or tablets. Also many ESBL producers have become multi-resistant to non-beta lactam antibiotics, comprising fluoroquinolones and aminoglycosides, sulfonamides which most often is encoded by the gene concealed by the same plasmids that determine the ESBL type (Hijazi et al., 2016; Sangare et al., 2017). The main ESBL-producing organisms isolated globally remain Klebsiella pneumonia and Escherichia coli (Giddi et al., 2017) but has also been identified in several other members of the enterobacteriaceae family and in certain non-fermenters. The acquisition and spread of the genes that code for beta-lactamase enzymes among gram-negative bacterial species together with opportunistic bacteria has led to widespread resistance to many beta-lactam agents, which is becoming an increasingly significant burden on human health (Iredell J. et al., 2016).

Over the years, emergence of resistance to beta-lactam antibiotics has increased exponentially. This began even before the first beta- lactam, penicillin was developed and was firstly identified in Escherichia coli prior to its release for medical use (Abraham et al.,1940). The introduction of the third-generation cephalosporins into clinical practice due to the increased prevalence of ESBLs in the early 1980s, signified a major breakthrough in the fight against beta-lactamase-mediated bacterial resistance to antibiotics (Paterson & Bonomo, 2005). These have broad spectrum activity and typically are effective against most beta-lactamase-producing organisms and had the major advantage of lessened nephrotoxic effects compared to aminoglycosides and polymyxins.(Paterson & Bonomo, 2005; Hijazi et al., 2016)

ESBLs possess strong and ubiquitous selection pressure and have seemingly been accompanied by a shift from "natural" resistance, such as inducible chromosomal enzymes, membrane impermeability, and drug efflux, to the modern paradigm of mobile gene pools that largely determine the epidemiology of modern antibiotic resistance. An in-depth account on this resistance mechanisms to each class of antibiotics by ESBLs have been reported (Doddaiah & Anjaneya, 2014). Also, modulation of the phenotype by host bacteria makes gene transmission less obvious. This in a way explains why tracking and control of ESBLs resistance has been particularly problematic in the enterobacteriaceae (Iredell J et al., 2016). Due to the selective pressure employed by beta-lactam producing bacteria, some soil organisms found in the environment currently exhibit drug resistance (Ghuysen, 1991). Gram-negative bacteria mostly possess naturally occurring chromosomally mediated beta-lactamases but ESBLs are mostly plasmid related and these ESBL gene-encoded plasmids can be transmitted beyond bacterial species (Shibasaki et al., 2016). Plasmids coding for ESBLs may also carry additional beta-lactamase genes as well as genes conferring resistance to other antimicrobial classes (Carattoli, 2009). This can limit the chemotherapeutic options for ESBL-producing pathogens and facilitate the intra and interspecies dissemination of ESBLs (Bush & Fisher, 2011;Zahar et al., 2009).

The first report of plasmid-encoded beta-lactamases which is able to hydrolyze the extended-spectrum cephalosporins was discovered in 1965 in Escherichia coli isolated from a patient named Temoniera in Greece hence designated TEM and published in 1983 (Datta & Kontomichalou, 1965). The presence of TEM 1 on various plasmids and its association with a transposon aided the spread to other bacteria within a few years after its isolation and is now located in different species of the family Enterobacteriaceae globally (Bonnet, 2004). Subsequently, other beta-lactamases were soon discovered which were closely related to TEM-1 and TEM-2. These also possess the ability to confer resistance to the extended-spectrum cephalosporins (Thenmozhi et al., 2014). Another common plasmid mediated β-lactamase SHV-1(named after the Sulfhydryl-variable active site), has been identified in Klebsiella spp and Escherichia coli (Kilebe et al., 1985). This gene encoding the beta–lactamase showed a mutation of a single nucleotide likened to the gene encoding TEM-1. In the early 1980s, a Klebsiella ozaenae isolate from Germany passed a beta-lactamase SHV-2 which efficiently hydrolyzed cefotaxime and to a lesser extent ceftazidime (Kilebe et al.,1985). Recently another type of ESBL (CTX-M) has been described which preferentially hydrolyze cefotaxime over ceftazidime and also hydrolyze cefepime with high efficiency (Bonnet,2004;Bush,2014). The majority of isolated ESBL-producing Enterobactericeae from studies has been established to have multiple genes (blaTEM, blaSHV, and blaCTX-M), where CTX-M are the predominant and CTX-M-9 and CTXM-15 are the most widespread ESBL types ( Paterson & Bonomo, 2005). It has been found that most ESBLs were derivatives of TEM-1 type, TEM-2 type and SHV-1 type of beta-lactamase. And these are composed of one or several point code gene mutations that alter the amino acid configuration around the active site of these beta-lactamases with CTX-M-type produced by Proteus Mirabilis also emerging in recent times (Thenmozhi et al., 2014; Song et al., 2011). This extends the spectrum of beta-lactam antibiotics susceptible to hydrolysis by these enzymes.

An increasing number of ESBLs not of TEM or SHV lineage have recently been described. There are more than 1,600 known beta-lactamases, a list that is rapidly expanding with TEM, SHV, and CTX-M-type mostly (Bush, 2014). In recent times over 100 clinical strains of CTX-M encoding genes have been located on plasmid. These commonly vary in size from 7kb-260kb with majority of these plasmids being IncFII plasmids, either alone or in association with Inc FIA and FIB (Carattoli et al., 2008).Plasmids encoding blaCTX-M-15 are found mainly in Enterobacteriaceae and named recently as plasmids of resistance responsible for outbreaks due to their capacity to acquire and transfer genes of resistance among bacteria (Hijazi et al., 2016).

Intestinal colonization by ESBL producing isolates may thus represent a reservoir for ESBLs in the community not detected in clinical isolates (Fernandez-Reyes et al., 2014). In recent times, clinical impact of ESBL-producing pathogens on morbidity and mortality in infectious diseases in adults, as well as their economic burden has been documented to be on the increase and that gram negative organisms are the most common cause of serious bacterial infection in young infants (Lukac et al., 2015).

The ESBL prevalence varies across Africa. This emerging threat has been pointed out in numerous studies within communities in Africa. In North Africa, it was detected to be 16.4–77.8%, the highest and least in South Africa (8.8–13.1%). In East Africa, studies report a prevalence ranging from 37.4 to 62.8% (Kittinger et al., 2016). Sub-Saharan Africa reports considerably high intestinal ESBL carriage rates between 10% and 45%. ESBL within population and in animals is on the increase due to overuse of antimicrobials in veterinary medicine (Kittinger et al., 2016). In Ghana, it was reported from the largest tertiary care hospital (Korle-Bu Hospital, Accra) that 50% of the Klebsiella pneumoniae and 29% of the Escherichia coli bloodstream isolates were ESBL producers. However, this study did not distinguish between hospital or community acquired strains and genotyping for these isolates were not performed (Obeng-Nkrumah et al., 2013). Feglo and Opoku established that there is high prevalence of AmpC- and ESBL- producing P. aeruginosa and P. mirabilis strains circulating in the Komfo Anokye Teaching Hospital and in the community with higher antimicrobial resistance than the non AmpC and ESBL strains (2014). Also recently, a study conducted at Komfo Anokye Teaching hospital with three other tertiary hospitals in the Northern belt indicates the prevalence of ESBL production to be 57.8% among the isolates; a significantly high level (Adu, 2016). This means that more than half of the Enterobacteria isolates tested produced ESBL. This exponential global spread of ESBLs conferring resistance to the majority of beta-lactam antibiotics, including third-generation cephalosporins, constitutes a major public health threat in both health care and community settings (Friedrich et al., 2016, European Centre for Disease Prevention and Control, 2015). Implementing infectious disease management globally especially in developing countries is also challenging due to intestinal colonization and globalization.

Options for treatment of these infections are generally limited, and given that fewer antibiotics are approved for use in children, the problem is critically important to address. This brings to the fore the significance of the phenotypic and genotypic detection of ESBLs among Enterobacteriaceae species; an important contraption for epidemiological purposes as well as for limiting the spread of resistance mechanisms. This study therefore aims at addressing the emergence of ESBL-producing isolates in some tertiary hospitals of the southern belt in Ghana and comparing the molecular epidemiology between these institutions for informed decision on healthcare and community based infection prevention.

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Item Type: Ghanaian Postgraduate Material  |  Attribute: 116 pages  |  Chapters: 1-5
Format: MS Word  |  Price: GH110 ($20)  |  Delivery: Within 30Mins.
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