Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin

Extended-spectrum β-lactamases (ESBLs) continue to be a major challenge in clinical setups worldwide, conferring resistance to the expanded-spectrum cephalosporins. The present study focused on the prevalence of ESBL-producing E. coli clinical isolates among patients diagnosed of Wound and Urinary Tract Infections attending Federal Teaching Hospital Abakaliki. A total of one hundred and ninety two clinical isolates of E. coli was studied for their susceptibility patterns to cephalosporin antibiotics and detection of ESBL producers was carried out by double disc synergy test (DDST) and Brilliance ESBL Agar. Of the 192 isolates tested for their antibiogram, 19(9.9%), 41(21.4%), 132(68.7%); 48(25.0%) and 144(75.0%) isolates were from wound, high virginal swab, urine, male and female respectively. The isolates showed higher susceptibility to cefepime (a 4th-generation cephalosporin) with percentage susceptibility of 78.9, 85.4 and 73.5 to the isolates from wound, HVS and urine respectively. Higher resistance was recorded among the 3-generation cephalosporins which include Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Among the resistant isolates of E. coli, 20 isolates were phenotypically confirmed ESBL producers by the DDST and Brilliance ESBL Agar methods. Ten (10) ESBL producing E. coli was confirmed using DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. Brilliance ESBL Agar was found to be better than DDST in the detection of ESBLs. Continuous monitoring of drug resistance and regulating the use of cephalosporin drugs in our hospitals is vital for proper infectious disease management and treatment.


Introduction
Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria are emerging pathogens. Clinicians, microbiologists, infection control practitioners, and hospital epidemiologists are concerned about ESBL-producing bacteria because of the increasing incidence of such infections, the limitations of effective antimicrobial drug therapy, and adverse patient outcomes (Lautenbach et al., 2001;Paterson, 2006). The rapidity of the development and spread of resistance is a complex process that is influenced by selective pressure, pre-existence of resistance genes and poor use of infection control measures in our hospitals. Extended spectrum cephalosporins (including the third generation cephalosporins) gained widespread clinical use in the early 1980s and were developed because of the increasing prevalence of ampicillinhydrolyzing β-lactamases such as TEM-1, TEM-2 and SHV-1 enzymes in Enterobacteriaceae, notably in Escherichia coli (Mark and Paul, 2003). The production of beta-lactamase enzymes by Gramnegative bacteria is one of the most important mechanisms of resistance to beta-lactam antibiotics.
ESBLs therefore, are acquired plasmid-mediated beta-lactamases that hydrolysis broader spectrum beta-lactam antibiotics including the oxyimino-3 rdgeneration cephalosporins and the penicillins (Paterson and Bonomo, 2005). Some data supported that foreign travel as well as household and community exposure may be important risk factors leading to colonization of the human intestinal tract by ESBL-producing E. coli (Laupland et al., 2008;Valverde et al., 2008). Person-to-person transmission and acquisition from a common source, probably related to food and poor hygiene clearly contribute to ESBL dissemination within families or community (Rodriguez-Bano et al., 2008). Similarly, other studies have reported a high prevalence of ESBLproducing bacteria in certain communities. The high prevalence of these bacteria in healthy carriers may be an important source of community-acquired infections, including urinary tract infections (UTIs). However, whether these ESBL-producing bacteria can be causative infectious agents in the carrier has not yet been confirmed (Niki et al., 2011 Bacteriology: All the clinical samples of wound swabs and urine samples were inoculated on MacConkey agar (MAC), Chocolate agar (CA) and Cystein Lactose Electrolyte Deficient (CLED) medium (Oxoid, UK), and incubated at 37°C for 18-24 hrs. Suspected bacterial colonies were subcultured onto freshly prepared MAC, CLED and CA for the isolation of pure cultures of the bacteria. E. coli isolates were identified based on colony morphology and biochemical reactions using standard microbiological conventional identification techniques (Cheesbrough, 2010;Stephan and William, 2006).

Screening for ESBL production:
Bacterial isolates that showed decreased susceptibility (intermediate by CLSI criteria) to any of the third generation cephalosporins including ceftazidime and cefotaxime were suspected to produce ESBL and further tested for ESBL production phenotypically. According to CLSI guidelines, strains showing zone of inhibition of ≤ 22 mm for ceftazidime, ≤ 27 mm for cefotaxime, and ≤ 25 mm for ceftriaxone were selected for ESBL confirmatory test.

Phenotypic determination of ESBL using Double Disc Synergy Test (DDST):
The E. coli isolated colonies were inoculated in peptone water at 37°C for 2-6 h. The turbidity was adjusted to 0.5 McFarland standards and lawn culture was made on Mueller-Hinton agar using sterile swab stick. Amoxicillin plus Clavulanic acid disk (20 µg and 10 µg, respectively) was placed in the centre of plate. A disc of cefotaxime (30 μg) and ceftazidime (30 μg), were placed at a distance of 15 mm away from the centrally placed disc (Amoxicillin plus Clavulanic acid disk). The plate was incubated at 37°C overnight. An increase of ≥ 5 mm in the inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxycillin-clavulanic acid versus its zone when tested alone confirms ESBL production phenotypically (Pituot et al., 2004).  Table 2. The results showed that cefepime are most effective drugs against the E. coli isolates. Higher levels of resistance by the E. coli isolates were recorded for ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam. Phenotypic characterization of ESBLs-producing E. coli isolated from patient attending FETHA was shown in Table 3. Among the resistant isolates of E. coli, 15 were ESBL producers detected by DDST and Brilliance ESBL Agar. Ten ESBL producing E. coli was confirmed using DDST whereas 15 ESBL producing E. coli was confirmed using Brilliance ESBL Agar. Figure 1 shows a keyhole effect produced by E. coli isolate expressing ESBL using DDST method. This appearance is the characteristic for ESBL-producing bacteria due to the synergistic effect produced between amoxicillin-clavulanic acid (a betalactamase inhibitor) and the third generation cephalosporins such as ceftazidime and cefotaxime.

Discussions
Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM and SHV-type beta-lactamases by mutations that alter the amino acid configuration around the active site of these earlier β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. ESBLs are often located on plasmids that are transferable from strain to strain and between bacterial species. However, it can still be chromosomal mediated. There is no doubt that ESBL-producing organisms are of enormous clinical and microbiological significance. Such bacteria are associated with severe infections such as bacteraemia, intra-abdominal infection, wound infection, urinary tract infections (particularly in the community setting), and respiratory tract infections (Pituot et al., 2004). A total of one hundred and ninety two (192) isolates of E. coli was studied for their susceptibility patterns to cephalosporin antibiotics and detection of ESBL producers by double disc synergy test (DDST) and Brilliance ESBL Agar. Of the 192 isolates tested for their antibiogram, 19 (9.9%), 41 (21.4%), and 132 (68.7%) isolates were from wound, high vaginal swab, and urine samples respectively while 48 (25.0%) and 144 (75.0%) isolates were from male and female patients respectively. The E. coli isolates showed higher susceptibility to 4 th -generation cephalosporins (cefepime). This is akin with previous works in which cefepime was reported as the drug of choice for the treatment of bacterial infections caused by ESBL-producing organisms (Lye et al., 2008;Walsh et al., 2005 andPaterson, et al., 2005). Higher resistance of the E. coli isolates were also recorded among the 3 rd -generation cephalosporins which includes Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Higher levels of resistance of ESBL-producing E. coli isolates as obtainable in this study have also been previously reported (Bradford, 2001 ESBL producing E. coli was confirmed using the DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. The false negative results in ESBL detection phenotypically observed using DDST is as a result of co-production of other related enzymes such as metallo-beta-lactamases (Walsh et al., 2005), plasmid-mediated and chromosomal inducible AmpC beta-lactamase in the test bacteria other than ESBLs (Jacoby et al., 2005). However, the brilliant result observed using Brilliance ESBL Agar is as a result of formulation with antibiotic and chromogenic mixture which inhibits every other enzymes present and allows the production of ESBL enzyme only to grow and produce colour pigments. In this present study, Brilliance ESBL Agar was found to be better than DDST in the phenotypic detection of ESBLs. Continuous monitoring of drug resistance, proper susceptibility studies and regulating the use of cephalosporin drugs in our hospitals is important in the containment of the emergence and spread of ESBL-producing bacteria in the hospital environment.