PHENOTYPIC DETECTION OF EXTENDED SPECTRUM BETA LACTAMASES PRODUCING ORGANISM
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.
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.
In Africa, the prevalence of ESBL in Enterobacteriaceae has been researched at local levels in various countries, but there is no summarizing research on how prevalent ESBL is on the continent, what type of genes are involved, and where research is missing (Victor, 2014).
In patients treated in African hospitals, the prevalence of ESBL-producing Enterobacteriaceae has been shown to vary between countries and the type of specimen studied. There is a trend of higher prevalence of ESBL in stool samples than in other specimens. There is also a trend of increasing prevalence over time. This is noticeable in the Tunisian setting, where a large amount of studies are available. In two hospitals studied (study periods: 1999–2005 and 2010), ESBLs have increased from 11.7 to 77.8% among K. pneumoniae. (Aouniet al, 2010). In other settings, the trend is not noticeable among the few studies available. In the studied countries in Africa, the prevalence is widely different: in Algeria, it was between 16.4 and 31.4% in mainly urine samples (Barguiguaet al, 2012) and even 99% among Salmonella enterica in stool samples (Bentchoualaet al, 2011) 19 and 42.9%, respectively, in urine and stool samples in Egypt (Domanyet al, 2012); 32.6% among stool samples in Guinea-Bissau (Giskeet al, 2012); 11.7–77.8% in mainly urine, blood, and stool samples from Tunisia (Kechridet al, 2011); 62.8% in stool and blood samples from Ethiopia (Asratet al, 2011); 38.3% in urine samples from Rwanda (Bayinganaet al, 2011); 55.3 and 82.8% in stool samples from Cameroon (Assoumouet al, 2013); 10.3–27.5% in mainly urine and stool samples from Nigeria (Aibinuet al, 2012); and 8.8–13.1% in urine, nasopharyngeal, and wound samples from South Africa (Dubeet al, 2009).
220.127.116.11 Northern Africa
In Algerian hospitals, ESBLs existed in 16.4–31.4% of the samples. Class A ESBLs were most common, but plasmid-encoded AmpC (pAmpC) was also present (Canicaet al, 2011).
In Egypt, ESBLs were found in 11–42.9% of samples in both hospitals and communities; the genes involved were class A ESBLs (Eletrebyet al, 2009).
In Guinea-Bissau and Libya, class A and D ESBLs and a carbapenemase were found in 32.6 and 16%, respectively, in rectal or stool samples (Giskeet al, 2012).
In Morocco, class A and D ESBLs, pAmpC, and carbapenemases were found in hospital settings (Carattoliet al, 2012). In the community setting, class A and D ESBLs were found in between 1.3 and 7.5% of acquired urine samples (Amarouchet al, 2011).
In Tunisia, class A and D ESBLs, pAmpC, and carbapenemases were present, and the prevalence ranged from 11.7 to 77.8% in hospitals and was 0.7 and 7.3% in two communities (Kechridet al, 2011).
18.104.22.168 Eastern Africa
In Ethiopia and Kenya, 62.8 and 37.4%, respectively, of hospital and community samples were ESBLs (Asratet al, 2011). Class A ESBLs and pAmpC were present in the Kenyan sample (Butayeet al, 2012). In samples taken from Kenya and Malawi, class A and D ESBLs were found (Boyle et al, 2011).
In Rwanda, ESBLs were found in 38.3% of hospital urine samples and in 5.9% of community urine samples (Bayinganaet al, 2011).
In Tanzania, class A ESBLs were found in various samples from hospital settings (Chakrabortyet al, 2011).
- Central Africa
In Cameroon, class A and D ESBLs were found in 55.3 and 82.8% of hospital stool samples and in 17.2% of community stool samples (Assoumouet al, 2013).
In the Central African Republic, ESBLs were found in 11.3% of community urine samples (Bercionet al, 2009).
22.214.171.124 Southern Africa
In South Africa, class A and D ESBLs and pAmpC were present, and the prevalence ranged from 8.8 to 13.1% in hospitals and was 0.3 and 4.7% in two communities (Chunderikaet al, 2008).
126.96.36.199 Western Africa
In Ghana and Mali, class A ESBLs were found in 49.4 and 63.4–96%, respectively, in hospital and community samples (Bougoudogoet al, 2009).
In Niger, 40% of hospital samples carried class A ESBLs or pAmpC (Andremontet al, 2011).
In Senegal, class A and D ESBLs were found in 10% of community stool samples. (Andremontet al, 2009)
188.8.131.52NIGERIA (West Africa)
In Nigeria, class A and D ESBLs and pAmpC were found in hospital settings, and the prevalence ranged from 10.3 to 27.5% (Aibinuet al, 2012). In a mixed sample from a hospital and a community, the prevalence was 11.7%. (Afunwaet al, 2011)
In Nigeria, an ESBL prevalence of 9.25% was recorded in a study conducted to screen for ESBLs production among isolates of Enterobacteriaceae (Aliyuet al, 2010).
In another study conducted in a tertiary health center in Nigeria to determine ESBL prevalence in Escherichia coli and Klebsiella Species; an ESBL prevalence of 2.5% for Escherichia coli and 5% for Klebsiellapneumoniae were recorded (Aboderin and Olowe, 2010).
1.5 PHENOTYPIC IDENTIFICATION OF ESBL
Extended-spectrum β-lactamase (ESBL) detection tests should accurately discriminate between bacteria producing these enzymes and those with other mechanisms of resistance to β-lactams, e.g., broad-spectrum β-lactamases, inhibitor-resistant β-lactamases and cephalosporinase overproduction. Several phenotypic detection tests, based on the synergy between a third-generation cephalosporin and clavulanate, have been designed: the double-disk synergy test (DDST), ESBL E-tests, and the combination disk method. These tests often need to be refined in order for them to detect an ESBL in some bacterial strains, such as those that also overproduce a cephalosporinase. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The use of cefepime, a fourth-generation cephalosporin that is less rapidly inactivated by cephalosporinase than by ESBL, improves the detection of synergy with clavulanate when there is simultaneous stable hyperproduction of a cephalosporinase; alternatively, the cephalosporinase can be inactivated by performing phenotypic tests on a cloxacillin-containing agar. Some β-lactamases can hydrolyze both third-generation cephalosporins and carbapenems, such as the metallo-β-lactamases, which are not inhibited by clavulanate, but rather by Ethylenediaminetetraacetic acid (EDTA). The production of an ESBL masked by a metallo-β-lactamase can be detected by means of double inhibition by EDTA and clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly inhibited by clavulanate and not inhibited by EDTA, their detection is difficult in the routine laboratory (Danish et al 2015).
1.6 DESCRIPTION OF THE ESBL DETECTION TESTS
1.6.1 DOUBLE-DISK SYNERGY TEST
The first test specifically designed to detect ESBL production in Enterobacteriaceae was the double disk synergy test (DDST) (Jarlier et al, 1988). It was initially designed to differentiate between cefotaxime-resistant strains, that is, those overproducing cephalosporinase, and those producing ESBLs. The test is performed on agar with a 30-μg disk of cefotaxime (and/or ceftriaxone and/or ceftazidime and/or aztreonam) and a disk of amoxicillin–clavulanate (containing 10 μg of clavulanate) positioned at a distance of 30 mm (center to center). The test is considered as positive when a decreased susceptibility to cefotaxime is combined with a clear-cut enhancement of the inhibition zone of cefotaxime in front of the clavulanate-containing disk, often resulting in a characteristic shape-zone referred to as ‘champagne-cork’ or ‘keyhole’. The DDST was first used in epidemiological studies to assess the spread of ESBL-producing Enterobacteriaceae in French hospitals (Brossieuret al, 2008). It has been shown to work well with a wide range of Enterobacteriaceaespecies and ESBL types, and it is generally regarded as a reliable method for the detection of ESBLs, although it is sometimes necessary to adjust the disk spacing. It is important to note that reducing the distance between the clavulanate-containing disk and the third-generation cephalosporin disk (e.g., to 20 mm) significantly improves the test sensitivity (Brossieuret al, 2008).
FIG 1: CAZ……………………AMC……………………CTX
Phenotypic confirmation of ESBL production using DDST(Afunwa, 2018).
The image in FIG 1 shows an increased zones of inhibition indicating the presence of ESBL producing organism.
1.6.2 ESBL Etests
ESBL Etests have been developed in order to quantify the synergy between extended-spectrum cephalosporins and clavulanate. The Etests called CT/CTL, TZ/TZL and PM/PML are two-sided strips containing gradients of cefotaxime (CT), or ceftazidime (TZ) or cefepime (PM), either alone (at one end of the strip), or combined with clavulanate 4 mg/L (on the other end). The ESBL test is considered as positive when the MIC value of the tested drug is reduced by more than three doubling dilution steps (MIC ratio ≥8) in the presence of clavulanate. The test is also considered as positive when there is either: (a) a rounded zone (phantom zone) just below the lowest concentration of CTL, TZL or PML gradients, or (b) a deformation of the CT, TZ or PM inhibition ellipse at the tapering end. The presence of a phantom zone or an ellipse deformation indicates ESBL production. Interpreting results of the ESBL Etest strips is delicate and requires training(Brossieuret al, 2008).
1.6.3 COMBINATION DISK METHOD
Several manufacturers have developed ESBL detection tests based on the combination disk method. The principle of this method is to measure the inhibition zone around a disk of cephalosporin and around a disk of the same cephalosporin plus clavulanate. Depending on the disk type, a difference of ≥5 mm between the two diameters (i.e., corresponding to a two-fold dilution), or a zone expansion of 50% are considered as indicating ESBL production. The test is easy to perform and its interpretation is straightforward. Sensitivity and specificity for this method were first reported to be 96% and 100%, respectively. Evaluation of the performance of the Oxoidcefpodoxime 10 ng ± 1 μgclavulanate combination disks to distinguish ESBL producers from AmpC overproducers and Klebsiellaoxytoca isolates overexpressing K1 enzyme was done. The presence of clavulanate enlarged the zone of inhibition by ≥5 mm for all 180 ESBL-producing organisms, and by <1 mm for AmpC overproducers and K. oxytoca isolates overexpressing K1 enzyme (Brossieuret al, 2008).
1.6.4 AUTOMATED METHOD
The VITEK 2 ESBL test is based on the simultaneous assessment of the antibacterial activity of cefepime, cefotaxime and ceftazidime, measured either alone or in the presence of clavulanate. This test relies on card wells containing 1.0 mg/L of cefepime, or 0.5 mg/L of cefotaxime or ceftazidime, either alone or associated with 10 or 4 mg/L of clavulanate, respectively. After inoculation, cards are introduced into the VITEK 2 machine, and for each antibiotic tested, turbidity is measured at regular intervals. The proportional reduction of growth in wells containing a cephalosporin combined with clavulanate is then compared with that achieved by the cephalosporin alone and is interpreted as ESBL-positive or – negative through a computerized expert system (Advanced Expert System). (Brossieuret al, 2008). The automated Phoenix ESBL test (Becton Dickinson, Sparks, MD, USA) also relies on the growth response to selected expanded-spectrum cephalosporins. This test is composed of five wells, each containing a cephalosporin alone or in combination with clavulanic acid (cefpodoxime, ceftazidime, and ceftazidime with clavulanic acid, cefotaxime with clavulanic acid and ceftriaxone with clavulanic acid). In this system, the results are also interpreted through a computerized system. (Brossieuret al, 2008)
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