Detection and antimicrobial susceptibility of some gram negative bacteria producing carbapenemases and extended spectrum β-lactamases

International Journal of Microbiology and Immunology Research Vol.2(6), pp. 064-069, November, 2013 Available online at http://www.apexjournal.org ISSN 2315-8743 2013 Apex Journal International Full Length Research Paper Detection and antimicrobial susceptibility of some gram negative bacteria producing carbapenemases and extended spectrum β-lactamases Ejikeugwu Chika 1 *, Ugwu Malachy 1, Iroha Ifeanyichukwu 2, Gugu Thaddeus 1, Duru Carissa 1, Eze Peter 1, Esimone Charles 1 1 Department of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe University, P.M.B 5025, Awka, Nigeria. 2 Department of Applied Microbiology, Ebonyi State University, P.M.B 053, Abakaliki, Nigeria. Accepted 23 October, 2013 The objective of this research was to determine phenotypically the occurrence of carbapenemase enzymes and extended spectrum β-lactamase (ESBL) producing Gram negative bacteria from some clinically important bacteria. Ninety nine clinical isolates comprising of Escherichia coli (n=40), Klebsiella pneumoniae (39) and Pseudomonas aeruginosa (n=20) were used for this study. Susceptibility testing for all isolates was performed by Kirby-Bauer disk diffusion method. Imipenem and meropenem was the most effective antibiotic while sulphamethoxazole showed least antibacterial activity. ESBL was detected in 7.5 % E. coli and 33.3 % K. pneumoniae. None of the P. aeruginosa isolates in our study showed ESBL production. Carbapenemase production was detected in 12.5 % E. coli, 7.7 % K. pneumoniae and 15 % P. aeruginosa isolates. Plasmid curing experiment showed that the ESBL phenotypes in our study were both plasmid-borne and chromosomally-mediated. Five (5) out of the eight (8) positive ESBL isolates were successfully transconjugated. Our study reveals the prevalence of ESBL and carbapenemase phenotypes in this environment, and these were resistant to some conventional antibiotics. Awareness, proper infection control measures, and prompt and accurate detection of ESBLs and carbapenemases from clinically important microbes are required for optimal care of infected patients and affected population. Key words: Carbapenemases, ESBLs, Gram negative bacteria, Antimicrobial Resistance INTRODUCTION The existence of new β-lactamases including carbapenemases and extended spectrum enzymes amongst clinically important pathogens is an important mechanism by which bacteria develop resistance to available antibiotics. Their increasing prevalence is of global concern as they are known to make the treatment of bacterial related infections difficult (Jacoby et al., 2005). This puts the efficacy of available antibiotics into risk, thus worsening patient s health condition. Extended *Corresponding author: ejikeugwu_chika@yahoo.com, Tel: +2348097684562 spectrum β-lactamases (ESBLs) are plasmid-mediated β-lactamases capable of hydrolyzing β-lactam antibiotics including 3 rd -generation cephalosporins and monobactams (SCIEH, 2004; Abhilash et al., 2010), but are yet inhibited by clavulanic acid, a β-lactamase inhibitor (Bonnet, 2004). ESBLs arise by mutations in genes for common plasmid-mediated β-lactamases (especially TEM and SHV enzymes) that alter the amino acid configuration of the enzyme near its active site to increase the affinity and hydrolytic ability of the β- lactamases for oxyimino cephalosporins (Jacoby et al., 1996). Their prevalence has also been noted worldwide in the community and hospital environments (Jacoby et al., 2005; Abhilash et al., 2010; Bonnet, 2004; Spanu et

Ejikeugwu et al 065 al., 2001; Iroha et al., 2008). Carbapenemases (e.g. metallo-β-lactamase) are β-lactamases that hydrolyze and confer resistance to carbapenems and other β- lactam antibiotics (SCIEH, 2004, Walsh et al., 2005; Franklin et al., 2006). Carbapenemases are mostly found in non-lactose fermenters such as Pseudomonads and Acinetobacter species; and to a less extent in lactosefermenting Enterobacteriaceae (Walsh et al., 2005), and they have spread across the world (Libisch et al., 2006; Aibinu et al., 2007). ESBLs and carbapenemases are chiefly produced by the Enterobacteriaceae and Pseudomonads respectively, and they confer on these bacterial pathogens the exceptional ability of having a broad substrate profile in terms of the number and classes of antibiotics that they are resistant to. Pathogenic microorganisms producing ESBLs and carbapenemases have serious clinical implications as they are usually associated with high rate of morbidity, mortality, increased length of hospital stay and high treatment costs (Thompson, 2010; Spanu et al., 2002; Bradford, 2001; Bashir et al., 2011). In the present study, we sought to investigate presumptively, the production of carbapenemases and ESBLs from clinically important Gram negative bacteria from a tertiary hospital in Enugu, Nigeria. MATERIALS AND METHODS Bacterial Isolates Ninety nine (99) bacterial isolates comprising of Escherichia coli (n=40), Klebsiella pneumoniae (n=39), and Pseudomonas aeruginosa (n=20) were isolated over a seven month period (May-November, 2011). All bacterial isolates was subcultured onto fresh growth medium, purified and confirmed by conventional microbiological tests (Cheesbrough, 2000). Susceptibility studies The antibiograms were tested by the Kirby-Bauer disk diffusion method after overnight incubation at 37 C on Mueller-Hinton agar plates as recommended by the clinical laboratory standard institute, CLSI (15). The antibiotics tested included (drug concentrations in µg): sulphamethoxazole-trimethoprim (25), ciprofloxacin (5), ofloxacin (5), ceftazidime (30), cefotaxime (30), amoxicillin-clavulanic acid (20/10), and gentamicin (10) [Oxoid, UK]. Escherichia coli ATCC 25922, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 standard organisms were used as the control strains for susceptibility testing (CLSI, 2009). Double Disk Synergy Test (DDST) ESBL production was confirmed phenotypically in the E. coli, K. pneumoniae and P. aeruginosa clinical isolates that showed reduced susceptibility to any of the screening cephalosporins as per the CLSI guidelines by double disk synergy test method as previously described (Aibinu et al., 2007; Bradford, 2001; CLSI, 2009). ESBL production was confirmed when there is an increase of 5mm in inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxycillin-clavulanic acid versus its zone when tested alone. Carbapenemase detection The Modified Hodges Test (MHT) was performed according to a previously described method (Aibinu et al., 2007; Thompson, 2010; Bashir et al., 2011). A standard strain of E. coli ATCC 25922 adjusted to 0.5 McFarland turbidity standard was inoculated on a Mueller-Hinton (MH) agar (Oxoid, UK) plate, and the plate was allowed to dry for 10 mins. One imipenem disk (10µg) (Oxoid, UK) was applied aseptically at the center of the inoculated MH agar plate, and a suspension of the test bacterium was heavily streaked from the edge of the imipenem disk (10µg) to the edge of the MH agar plate. After incubation (at 37 C for 18-24hrs), the MH agar plates were observed for Cloverleaf effect (symbol that is typical of carbapenemase production) at the intersection of the test bacterium and the E. coli ATCC 25922 standard organism, within the inhibition zone of the imipenem disk (10µg). The presence of growth of the test bacterium towards the imipenem disk (10µg) is considered and interpreted as a positive result for MBL production phenotypically (Bashir et al., 2011). Plasmid curing Plasmid curing experiment was undertaken for all ESBL positive isolates to determine the location (plasmid or chromosomal) of the drug resistance determinants, and this was performed according to a previously described method (Iroha et al., 2008). Conjugation studies Conjugation studies was undertaken to determine the transferability of resistance plasmids from ESBL positive organisms to non-esbl producing bacteria. A previously described method was used to undertake this experiment (Iroha et al., 2008). RESULTS The clinical samples from which these isolates were isolated from included: urine (56), sputum (19), ear swab

066 Int. J. Microbiol. Immunol. Res. Table 1. Distribution of the clinical isolates by hospital location/sites. Clinical isolates GOPD CAS SOP GYNAE MOP A/E ENT MMW CHER CHOP EYE WARD FMW TOTAL E. coli 28 1 4 1 0 4 0 0 0 1 0 1 40 K. pneumoniae 29 0 1 1 0 3 1 1 2 0 0 1 39 P. aeruginosa 7 0 1 0 1 3 5 1 0 0 1 1 20 Total 64 1 6 2 1 10 6 2 2 1 1 3 99 Key: GOPD-General out-patient department, CAS-Casualty, SOP-Surgical out-patient, GYNAE-Gynecology ward, MOP-Medical out-patient, A/E- Accident & emergency, ENT-Ear, nose & throat, MMW-Male medical ward, CHER-Children emergency, CHOP-Children out-patient, FMW-Female medical ward 120 E.coli K. pneumoniae P. aeruginosa 100 % Susceptible 80 60 40 20 0 CTX CAZ SXT CN OFX CIP IPM MEM E.coli 45 47.5 2.5 45 25 35 95 92.5 K. pneumoniae 25.6 25.6 12.8 46.2 41 30.8 87.2 92.3 P. aeruginosa 25 50 0 55 30 45 100 100 Figure 1. Percentage Susceptibility of the E. coli (n=40), K. pneumoniae (n=39) and P. aeruginosa (20) Clinical Isolates. (7), pleural aspirate (1), conjunctival swab (1), wound swab (14), and High Vaginal swab, HVS (1). The frequency of isolation of the Gram negative bacilli (E. coli, K. pneumoniae, and P. aeruginosa) employed in this study from various hospital sites/location is illustrated in Table 1. There was a high percentage of isolates from general out-going patients department (GOPD) when compared to isolates from other hospital sites. The reason for this high level of isolates from the GOPD might be in part to the high influx of patients to the GOPD. The result of susceptibility studies is shown in Figure 1, and the percentage susceptibility to tested agents are: CTX (E. coli 45%, K. pneumoniae 25.6%, P. aeruginosa 25%), CAZ (E. coli 47.5%, K. pneumoniae

Ejikeugwu et al 067 Table 2. Frequency of ESBL and Carbapenemase producers. Clinical isolates No of isolates ESBL positive n (%) Carbapenemase positive n (%) E. coli 40 3 (7.5) 5(12.5) K. pneumoniae 39 13 (33.3) 3(7.7) P. aeruginosa 20 0 (0) 3(15) Total 99 16 (16.2) 11(11.1) Table 3. Plasmid Curing Analysis of ESBL Positive isolates with acridine orange (0.1mg/ml). Clinical isolate Pre-curing (n) Cured (Plasmid-borne) n (%) Not cured (chromosomal) n (%) E. coli 3 2(12.5) 1(6.3) K. pneumoniae 13 5(31.3) 8(50) Total 16 7(43.8) 9(56.3) Key: n=number Table 4. Antibiotic susceptibility of ESBL and Carbapenemase phenotypes to some selected non-β-lactam antibiotics. Antibiotics (µg) Carbapenemase ESBL phenotypes (n=16) phenotypes (n=11) No. % No. % Gentamicin (10) 3 18.8 8 72.7 Ofloxacin (5) 4 25 5 45.5 Sulphamethoxazole- Trimethoprim (25) 1 6.3 0 0 Ciprofloxacin (5) 4 25 7 63.6 25.6%, P. aeruginosa 50%), SXT (E. coli 2.5%, K. pneumoniae 12.8%, P. aeruginosa 0%), CN (E. coli 45%, K. pneumoniae 46.2%, P. aeruginosa 55%), OFX (E. coli 25%, K. pneumoniae 41%, P. aeruginosa 30%), CIP (E. coli 35%, K. pneumoniae 30.8%, P. aeruginosa 45%), IPM (E. coli 95%, K. pneumoniae 87.2%, P. aeruginosa 100%) and MEM (E. coli 92.5%, K. pneumoniae 92.3%, P. aeruginosa 100%). The production of extended spectrum β-lactamase (ESBL) and carbapenemases was phenotypically detected by the double disk synergy test (DDST) and modified Hodges test (MHT) methods respectively, and the results are as shown in Table 2. Plasmid curing experiments was carried out on all the ESBL positive bacteria (n=16) using acridine orange (0.1mg/ml), and the result is illustrated in Table 3. Table 4 shows the antimicrobial susceptibility of ESBL and Carbapenemase phenotypes to some selected non-βlactam antibiotics. DISCUSSION One of the major problems facing the health sector today is the rapid emergence and spread in the resistance of multidrug resistant (MDR) pathogenic bacteria to readily available antibiotics (especially the beta-lactams). This growing resistance of pathogens to antibiotics is a challenge to medical health practitioners when it comes to treating and managing most infections caused by MDR organisms (Jacoby et al., 2005; Walsh et al., 2005). All test isolates showed varied rates of susceptibility to the test antibiotics (Figure 1). The carbapenems (imipenem and meropenem) were the most effective agent with substantial antibacterial activity. This was followed by gentamicin, ofloxacin, ceftazidime and cefotaxime. Sulphamethoxazole-trimethoprim was the least active agent tested (Figure 1). Overall, the ESBL production in our study was 7.5 % and 33.3 % in E. coli and K. pneumoniae isolates respectively. Notably, none of the P. aeruginosa isolates in our study showed ESBL production (Table 2). The prevalence of ESBL production of 16.2 % among the tested Enterobacteriaceae in our study (33.3 % K. pneumoniae and 7.5 % E. coli) is consistent with data from Singapore (44 % Klebsiella, 16.1 % E. coli), Pakistan (58.7 % K. pneumoniae) and Kano in Northern Nigeria where ESBL production was 66.7 % in Enterobacteriaceae (Chlebicki et al., 2004; Chakraborty et al., 2010; Yusha u et al., 2010). However, our results of ESBL production of 7.5 % E. coli and 33.3 % K. pneumoniae was in contrast to a similar work done

068 Int. J. Microbiol. Immunol. Res. in Saudi Arabia where ESBL production was 4.4 % and 95.6 % in K. pneumoniae and E. coli respectively (Kader et al., 2009). The unanticipated non-production of ESBL in P. aeruginosa isolates in this study is in contrast to a similar work carried out in Pakistan and Nigeria where ESBL was detected in P. aeruginosa isolates from blood specimens (Mumtaz et al., 2008; Aibinu et al., 2007). A possible reason for this development in our study (i.e. the non-production of ESBLs by P. aeruginosa isolates) could be due to the non-transferability of ESBL genes (or resistance plasmids) from ESBL-producing Enterobacteriaceae to non-enteric bacteria (in this case P. aeruginosa) in this environment. Nevertheless, it is still important to screen clinically important bacteria that are multiply resistant for possible production of ESBLs in order to forestall any outbreak due to their infections. Carbapenemase enzyme production was detected in 12.5 %, 7.7% and 15 % of E. coli, K. pneumoniae and P. aeruginosa isolates respectively (Table 2). This observed prevalence of carbapenemase production in our study is high, and in contrasts to the results obtained in India (Chakraborty et al., 2010). However, our result of carbapenemase production corresponds to those obtainable in Kashmir (Bashir et al., 2011). Carbapenemase production according to reports is found to be more prevalent in P. aeruginosa isolates than the Enterobacteriaceae (Walsh et al., 2005; Chakraborty et al., 2010; Franklin et al., 2006). Our study nevertheless, has presumptively shown that Enterobacteriaceae and P. aeruginosa isolates producing carbapenemase enzymes exist in southeastern Nigeria. The ESBL phenotypes in our study were both plasmid-mediated and chromosomally-mediated. Particularly, 2 (12.5 %) out of the 3 ESBL positive E. coli isolates lost their plasmids while 5 (31.3 %) of the 13 ESBL positive K. pneumoniae isolates lost their plasmid following curing by acridine orange (at a sub-inhibitory concentration of 0.1 mg/ml). Bacteria Population containing plasmids when subjected to Curing agents such as acridine orange will become more and more dominated by plasmid- free cells with time (Iroha et al., 2008). Bacterial plasmids play a very important role in the spread of antibiotic resistance traits (such as ESBLs) amongst bacterial population through various means of genetic transfers including conjugation, transformation and transduction (Jacoby et al., 2005; Thompson, 2010). In our study, the antibiogram of our ESBL and carbapenemase phenotypes as illustrated in Table 4 shows that sulphamethoxazole-trimethoprim had no antibacterial activity on the tested bacteria while gentamicin, ofloxacin and ciprofloxacin were moderately active. Having in mind the antibiotic susceptibility patterns of ESBL- and carbapenemase- producing organisms is useful in choosing the right antibiotic therapy in the face of an infection. Our conjugation experiment show some evidence of transfer of plasmidmediated resistance in some of the transconjugated isolates. It was noted from our study that more than half of the recipient strain was successfully transconjugated as the transconjugants were observed to be resistant to virtually all the antibiotics (CAZ, CTX, CN, SXT, OFX, CIP, MEM and IPM) tested. Particularly, 5 out of the 8 ESBL positive bacteria isolates transconjugated successfully transferred their antibiotic resistant determinants to the recipient strain (E. coli ATCC 25922). The ability of antibiotic resistant bacteria to successfully transfer their resistant plasmids to susceptible population of bacteria is the single most popular reason (amongst other criteria) by which multidrug resistant traits such as ESBLs can be spread amongst bacterial population in a given hospital environment. From our study, those bacterial isolates that successfully transferred their plasmids have their resistance genes in their plasmids, and those that failed to successfully transfer theirs were inferred to be chromosomally-borne. Conclusion Conclusively, our study has clearly shown that ESBLand carbapenem- producing pathogens occur in Enugu, southeastern Nigeria; and that these pathogens are resistant to some readily available drugs. ESBLs and carbapenemases now exist worldwide, and they hydrolyze and confer resistance to the extended spectrum cephalosporins and the carbapenems respectively. 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