Bacterial identification and ESBL detection
From the study area, a total of 524 Gram-negative bacteria were isolated, of which 366 isolates (69.8%) showed multi-drug resistance (MDR) to different class of antibiotics tested. E. coli was found to be the dominant isolate (n= 135) followed by K. pneumoniae (n=49), Klebsiella oxytoca (n=32), Aeromonas hydrophila (n=18), Enterobacter aerogenes (n=15), Pseudomonas aeruginosa (n=15), Enterobacter cloacae (n=12), Pseudomonas putida (n=12), Citrobacter sp. (n=11), Salmonella sp. (n=9) and Acinetobacter baumannii (n=7) in various samples screened from direct hospital effluents. In comparison, E. coli (n=74), K. pneumoniae (17), E. aerogenes (13), P. aeruginosa (7) and A. baumannii (n=3) were obtained from aquaculture farms downstream of the above sites.
The CLSI recommends screening of E. coli, K. pneumoniae, K. oxytoca, and Proteus mirabilis isolates for ESBL production by the use of cefpodoxime, ceftazidime, aztreonam, cefotaxime, or ceftriaxone, followed by phenotypic confirmation with clavulanate. Only E. coli and K. pneumoniae isolates that showed high resistance to cephalosporin class of antibiotics were subjected to ESBL detection by Double Disc Synergy test (Fig. 1). Of the 209 E. coli isolated from different points, 103 isolates (37.4%) were positive for ESBL production; 76 isolate from direct hospital effluents (27.6%) (H1=23, H2=41, H3=12) and 27 isolates (9.8%) from F3 site. Among the 66 K. pneumoniae isolates, 25 (9%) isolates from hospital points (H1=10, H2=15) and 6 isolates (2.1%) from F3 site were found to be ESBL producers. None of the other bacterial isolates from direct hospital and aquaculture farm samples were positive for ESBL production.
Antimicrobial susceptibility testing and MIC of ESBL isolates
Among the 76 ESBL positive E. coli isolates from direct hospital effluents, all the isolates showed complete resistance to ampicillin, amoxicillin, cefoxitin, cefotetan, cefepime, cefpodoxime, amoxycillin/clavulanate, ceftazidime, cefotaxime, ceftriaxone, ciprofloxacin, ofloxacin, nalidixic acid, aztreonam, trimethoprim/sulfamethoxazole, amikacin, gentamicin and imipenem whereas all the strains were susceptible to azithromycin, meropenem and chloramphenicol. In comparison, out of the 49 hospital effluent K. pneumoniae isolates, 25 ESBL positive isolates (H1=10, H2=15) showed complete resistance to ampicillin, amoxicillin, amoxycillin/clavulanate, cefotaxime, cefotetan, cefoxitin, ceftazidime, ceftriaxone, cefpodoxime and cefepime, whereas K. pneumoniae isolates from H2 (n=15) showed resistance to ciprofloxacin, ofloxacin, nalidixic acid, imipenem, meropenem, aztreonam and trimethoprim/sulfamethoxazole. However, they were sensitive to amikacin, gentamicin, azithromycin and chloramphenicol.
In aquaculture farms (F3 site), 27 E. coli and 6 K. pneumoniae isolates showed high resistance to cephalosporin and quinolone class of antibiotics, the farm which was adjacent (800 m) to the hospital discharge site. The pattern of antibiotic resistance of E. coli and K. pneumoniae from F3 was similar to that found in the direct hospital effluent samples. E. coli and K. pneumoniae isolates from the remaining farms were susceptible to all the tested antibiotics. The antibiotic resistance pattern of ESBL positive isolates collected from direct hospital effluents and aquaculture farm (F3) are shown in Fig. 2.
Majority of the ESBL positive isolates from direct hospital effluent and aquaculture samples had MIC values of ˃32-256 µg/ml to each of the third generation cephalosporin tested, thus confirming high resistance. All the ESBL positive isolates exhibited high level resistance to penicillins (˃256 µg/ml). Frequency of antibiotic resistance was high in those isolates of E. coli and K. pneumoniae which had TEM, SHV and CTX-M genes. E. coli isolates carrying CTX-M ESBL confer high level resistance to ceftazidime (MIC range 64-256 µg/ml) and they had co-resistance to quinolone class of antibiotics and showed an elevated level of MIC value ranging from 8-64 µg/ml. However, in case of K. pneumoniae isolates at the concentration of 128 µg/ml of ceftazidime, only twelve isolates showed resistance, while the remaining thirteen isolates had MIC value of ˃64 µg/ml towards ceftazidime. At the concentration of ≥128-256 µg/ml of cefotaxime and ceftriaxone, all the ESBL positive E. coli isolates were resistant. On the other hand, ESBL positive K. pneumoniae isolates had MIC levels of 64-128 µg/ml of cefotaxime and ceftriaxone; while, MIC range of 32-˃256 μg/ml was observed among E. coli and K. pneumoniae isolates for cefepime. In aquaculture farms, specifically ESBL positive E. coli and K. pneumoniae from F3 site showed elevated level of MIC towards ceftazidime, ceftriaxone and cefotaxime (Table 2).
Molecular characterization of ESBL encoding genes
For a reliable epidemiological investigation of antimicrobial resistance, molecular detection and identification of resistance encoding genes would be necessary. blaCTX-M, blaTEM, blaOXA, blaCMY and blaSHV resistance genes were detected in E. coli and K. pneumoniae isolates. For ESBL resistance encoding genes, blaCTX-M was found to be more prevalent (n=38) followed by blaTEM (n=21) and blaSHV (n=18). The plasmid mediated cephalosporin resistance coding genes of blaSHV group was detected in 18 E. coli isolates in combination with blaCTX-M resistance gene. Two SHV genotypes such as blaSHV-1 (n=11) and blaSHV-61 (n=7) were found among E. coli isolates in H2 site. It is noteworthy that the number of β-lactamase genes in E. coli was clearly associated with the frequency of the ESBL producer at each sampling site, specifically at the H2 site. In hospital effluent samples, for blaCTX-M positive isolates, blaCTX-M-15 was the predominant genotype (n=38). For blaTEM, only TEM-95b variant was detected from H1 and H2 E. coli isolates. However, blaOXA-and the most numerous and diverse AmpC gene type blaCMY-6 was detected in six and five E. coli isolates from H2 site respectively. It is interesting to note that in none of the ESBL positive K. pneumoniae isolates, the blaCTX-M, blaTEM, blaOXA and blaCMY genes were detected; however, three types of blaSHV genotypes like blaSHV-266 (n=7), blaSHV-211 (n=7) and blaSHV-148 (n=5) were detected in K. pneumoniae isolates from direct hospital effluent samples. In aquaculture farm (F3 site), ESBL-positive E. coli isolates with the encoding genes blaCMY-6 (n=5), blaCTX-M-15 (n=6) and blaOXA-48 (n=3) and ESBL positive K. pneumoniae isolates with blaSHV-148 (n=6) were detected.
Bacterial plasmid conjugation
The plasmid-mediated ESBL resistance was successfully transferred from E. coli with blaCTX-M-15 gene to azide-resistant E. coli J53 isolate. Plasmid isolated from the trans-conjugants and analyzed by PCR showed the specific amplification of blaCTX-M-15 gene (550 bp) in the trans-conjugants. All the trans-conjugant strains tested showed resistance to extended-spectrum cephalosporins. They were more resistant to cefotaxime than to ceftazidime and were susceptible to imipenem. MIC of trans-conjugants for cefotaxime and ceftazidime were related to those for each wild-type isolate, and showed decreased ciprofloxacin sensitivity (MIC 0.2 to 1 µg/ml).