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Detection and Characterization of b-lactamases and Biofilm-producing Escherichia coli and Klebsiella pneumoniae from Ducks and Duck Environments in West Bengal
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Methods: Around 197 duck and environmental samples were collected from different districts of West Bengal. E. coli and K. pneumoniae were isolated from collected samples as per standard methods followed by serotyping of the E. coli isolates. The detection of ESBL/ACBL producing isolates was done both phenotypically and genotypically by detecting the presence of antibiotic-resistant genes. Virulence property and biofilm-producing ability of these isolates were also studied. Antibiogram of the ESBL and biofilm positive isolates were done using common 12 antibiotics by disc diffusion method.
Result: A total of 134(68.02%) Escherichia coli and 33(16.75%) Klebsiella pneumoniae isolates were recovered and confirmed by PCR. The prevalence of E. coli and K. pneumoniae in cloacal swabs were less in comparison to the environmental samples (72.09% and 20.93%). Approx. 79.85% E. coli and 72.73% K. pneumoniae isolates were found to be the ESBL/ACBL producers in vitro. E. coli and K. pneumoniae isolates possessed all major antibiotic-resistant genes with the blaAmpC (64.18% & 48.48%) being the most prevalent one. Twenty three (17.16%) E. coli isolates were found positive for the virulence genes, of which stx1 (39.13%) was the most common one as detected by multiplex PCR. No virulence gene was found in K. pneumoniae isolates. Ninety one (67.91%) E. coli and 26(78.79%) K. pneumoniae isolates possessed the biofilm-producing genes. All the ESBL and biofilms producing E. coli and K. pneumoniae isolates showed 100% - 95.24% resistance to ampicillin. A high percentage of resistances were also seen against cefotaxime, ticarcillin/ clavulanic acid, ertapenem, ceftazidime, norfloxacin and ciprofloxacin, which may be due to several reasons. Maximum overall sensitivity was detected against chloramphenicol, co-trimoxazole and gentamicin.
Ducks may get affected by a few pathogens like Escherichia coli, Salmonella spp., Klebsiella pneumoniae, Pasteurella multocida, Reimerella anatipestifer and Streptococcus spp., etc. Among these, E. coli and K. pneumoniae have virulence properties, biofilm production and antimicrobial resistance properties, which are a matter of concern. These can infect humans too via contaminated water and food. These gram-negative bacteria can produce β-lactamases (Wickens and Wade, 2005) which creates antimicrobial resistance in them. Most of them are biofilm producers too (Melo et al., 2012). Both can cause a wide variety of diseases causing up to 20-30% of mortality (Khelfa and Morsy, 2015). In this background, samples were collected from ducks and duck environments from different districts of West Bengal, to detect the prevalence of antibiotic-resistant and biofilm-producing E. coli and K. pneumoniae isolates to insight into the depth of the problem.
MATERIALS AND METHODS
A total of 197 samples (154 cloacal swabs and 43 environmental samples [soil 19, water collected from duck environment/sheds 13, duck feed 11]) were collected aseptically with sterilized cotton swabs (HiMedia, India) from ducks and their environments during the year 2020-21. The swab samples were transported under ice cover to the Department of Veterinary Microbiology laboratory, F/VAS, WBUAFS, Kolkata via the shortest route.
Isolation and characterization of E. coli and Klebsiella pneumoniae isolates
The enriched samples were streaked on sterilized MacConkey’s agar plates (HiMedia, India) and overnight incubated at 37°C for the isolation of E. coli. The rose-pink colonies were selected and sub-cultured and incubated on EMB agar plates (HiMedia, India) for detection of E. coli isolates with metallic sheen’. All isolates were preserved in nutrient agar slants for further study.
The Klebsiella selective agar (HiMedia, India) plates were used for Klebsiella pneumoniae isolation. The characteristic magenta-coloured colonies were selected and subsequently preserved on nutrient agar (HiMedia, India) slants. Morphological and biochemical characterizations were performed as per Quinn et al., (1994) and Edward and Ewing (1972).
Molecular characterization of E. coli and Klebsiella pneumoniae isolates
The tentatively confirmed E. coli and Klebsiella spp. isolates were processed for molecular confirmation by PCR as per the methods described by Wang et al., (1996) and Brisse and Verhoef (2001) with some modifications. Then the positive Klebsiella spp. isolates were tested for species-specific PCR for the detection of Klebsiella pneumoniae (Liu et al., 2008).
Serogrouping of E. coli isolates
The positive E. coli samples from ducks were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh, India for serotyping.
Phenotypic detection of ESBL/ACBL production in E. coli and K. pneumoniae isolates
Antibiotic discs containing cefotaxime (30 µg, Hi-Media) and ceftazidime (30 µg, Hi-Media) with and without clavulanate (10 µg, Hi-Media) were used in double-disc diffusion assay (DDSA) for phenotypic confirmation of the presence of ESBLs in all E. coli and Klebsiella pneumoniae isolates. A difference of ³ 5 mm between the zone of inhibition in the presence and absence of clavulanate was considered for phenotypic confirmation of ESBL production (CLSI, 2012). All the E. coli and Klebsiellae spp. isolates were subjected to cefoxitin-cloxacillin double disc synergy (CC-DDS) test for phenotypic confirmation of AmpC β-lactamase production (Tan et al., 2009).
Detection of ESBL and ACBL genes in E. coli and K. pneumoniae isolates
All the positive E. coli and Klebsiella pneumoniae isolates including negative control were subjected to PCR for detection of major antibiotic-resistant genes such as blaTEM, blaSHV, blaCTX-M and blaAmpC (Bert et al., 2002; Colom et al., 2003; Weill et al., 2004 and Féria et al., 2002).
Detection of Virulence genes in E. coli and K. pneumoniae isolates
All positive E. coli isolates were further subjected to multiplex PCR for detection of the Shiga-toxin gene (stx1, stx2) and eaeA, hlyA genes as described by Paton and Paton (1998) with some modifications. Likewise, in K. pneumoniae isolates, PCR detection of the rmpA gene was done (Nahavandinejad and Asadpour, 2017).
Molecular detection of biofilm-producing genes among E. coli and K. pneumoniae isolates
Detection of biofilm genes like csgA, rpoS, rcsA and sdiA was performed in isolated positive E. coli and K. pneumoniae isolates by PCR (Silva et al., 2013; Adamus-bialek et al., 2015).
Antibiotic sensitivity assay
All the ESBL and biofilm-producing E. coli and K. pneumoniae isolates were tested for their sensitivity and resistance pattern to 12 different antibiotics by the disc diffusion method (CLSI, 2012). The antibiotics used were ampicillin (10 µg), chloramphenicol (30 µg), co-trimoxazole (25 µg), tetracycline (30 µg), gentamicin (10 µg), amikacin (30 µg), norfloxacin (5 µg), ciprofloxacin (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), ticarcillin/clavulanic acid (30/10 µg) and ertapenem (30 µg).
RESULTS AND DISCUSSION
All 134 nos. tentatively positive E. coli isolates were having the 16S rRNA gene (585 bp) specific for E. coli (Fig 1). Around 33 isolates were confirmed to be Klebsiella pneumoniae with 130 bp amplified product in PCR (Fig 2). A total of 12 different serogroups were found such as O98, O157, O135, O120, O63, O26, O121, O101, O8, O118, O119 and O22 among which, O120 was the most prevalent one, in serogrouping of E. coli isolates (Banerjee et al., 2019; Wang et al., 2010).
Out of 134, 107 (79.85%) Escherichia coli isolates (Fig 3) and out of 33, 24 (72.73%) Klebsiella pneumoniae isolates (Fig 4) were detected as phenotypical ESBL/ACBL producers based on double-disc synergy assays. During molecular detection of the resistance genes, a total of 36(26.86%) E. coli and 6(18.18%) K. pneumoniae isolates were found to possess the blaCTX-M gene (540bp) with other genes (blaTEM and blaSHV) too (Table 1). The presence of the blaAmpC gene (634 bp) was quite high in both bacteria. Nalband et al., (2020), Ma et al., (2012) also reported much higher prevalence of ESBL genes in E. coli which is almost similar to the present study. The findings of Banerjee et al., (2019), Raza et al., (2017) also match this report and thus stand confirmed.
A total of 23 (17.16%) Escherichia coli isolates were found to be positive for different virulence genes viz. stx1 (180bp), stx2 (255bp), eaeA (384bp) and hlyA (534bp) in the multiplex PCR assay (Table 2, Fig 5). Majumder et al., (2017) and Banerjee and Acharyya (2020) reported approx. 20-24% STEC producing E. coli from cloacal samples of duck which was higher than the present findings.
No Klebsiella pneumoniae isolate was found positive for the rmpA gene (535bp) in this study. Yang et al., (2019) reported a very less (10.6%) prevalence of the rmpA gene in human origin Klebsiella pneumoniae. Animal origin Klebsiella spp. may possess this gene (Younis et al., 2016) but the present variation might be due to variation in geographical variances.
Around 67.91% Escherichia coli and 78.79% Klebsiella pneumoniae isolates were found to possess at least one of these biofilm-producing genes viz. rpoS (120 bp), csgA (178 bp), sdiA (239 bp) and rcsA (306 bp) (Table 3). Approx. 27 E. coli and 13 K. pneumoniae isolates were found to possess more than one biofilm-producing gene in this study which was also supported by Bakhtiari and Javadmakoei (2017) and Banerjee and Acharyya (2020).
This study showed that 30.43% ESBL-E. coli and 47.82% biofilm-producing E. coli to possess virulence genes, but 56 ESBL-E. coli were positive for biofilm production too. Again only 02 isolates were positive for the ESBL, Biofilm producing and virulence genes as revealed in this study. This reveals a significant relationship were also reported by Jegadeesh Kumar et al., (2016) and Fattahi et al., (2015), which confirms these present findings too.
All the ESBL and biofilm-producing E. coli isolates were resistant to ampicillin and approx. 61-86% resistant to norfloxacin, ertapenem, ciprofloxacin, tetracycline and ceftazidime respectively (Table 4). Na et al., (2019) also reported resistance to tetracycline (59.3%) and other drugs of E. coli in their study.
A total of 95.24% of all ESBL and biofilm-producing K. pneumoniae isolates (n=21) were resistant to ampicillin followed by 85.71%, 85.71%, 80.95%, 76.19%, 57.14% and 52.38% resistant to cefotaxime, ticarcillin/ clavulanic acid, ertapenem, ceftazidime, norfloxacin and ciprofloxacin respectively. Sensitivity was detected against chloramphenicol, co-trimoxazole and gentamicin (@61-92%) [Table 5]. Almost similar reports were shown by Nnachi et al., (2015) and Kumar et al., (2022), thus the current result stands confirmed.
Conflict of interest
- Adamus-bialek, W., Kubiak, A. and Czerwonka, G. (2015). Analysis of uropathogenic Escherichia coli biofilm formation under different growth conditions. Acta Biochimica Polonica. 62(4): 765-771.
- Adzitey, F., Liew, C.Y., Aronal, A.P. and Huda, N. (2012). Isolation of Escherichia coli from ducks and duck-related samples. Asian Journal of Animal and Veterinary Advances. 7(4): 351-355.
- Bakhtiari, N.M. and Javadmakoei, S. (2017). Survey on biofilm production and presence of attachment factors in human uropathogenic strains of Escherichia coli. Jundishapur Journal of Microbiology. 10(6): e13108.
- Banerjee, A. and Acharyya, S. (2020). Molecular characterization of STEC isolated from ducks and its relation to ESBL production. Ukrainian Journal of Veterinary and Agricultural Sciences. 2: 24-29.
- Banerjee, A., Bardhan, R., Chowdhury, M., Joardar, S.N., Isore, D.P., Batabyal, K., Dey, S., Sar, T.K., Bandyopadhyay, S. and Samanta, I. (2019). Characterization of beta- lactamase and biofilm producing Enterobacteriaceae isolated from organized and backyard farm ducks. Letters in Applied Microbiology. 69(2): 110-115.
- Bariha, U.N., Mishra, R., Kundu, A.K., Rath, P.K., Mishra, C., Das, S. and Soren, N. (2019). Microbial Etiology of Duck Mortality in Odisha, India. International Journal of Current Microbiology and Applied Sciences. 8(8): 1577-1585.
- Bert, F., Branger, C. and Lambert-Zechovsky, N. (2002). Identification of PSE and OXA â-lactamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism. Journal of Antimicrobial Chemotherapy. 50(1): 11-18.
- Brisse, S. and Verhoef, J. (2001). Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. International Journal of Systematic and Evolutionary Microbiology. 51(3): 915-924.
- Clinical and Laboratory Standards Institute, (2012). Performance standards for antimicrobial susceptibility testing. Twenty second informational supplement update. CLSI document M100-S22 U. Clinical and Laboratory Standards Institute, Wayne, PA. 31: 100-121.
- Colom, K., Pérez, J., Alonso, R., Fernández-Aranguiz, A., Lariño, E. and Cisterna, R. (2003). Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA–1 genes in Enterobacteriaceae. FEMS Microbiology Letters. 223(2): 147-151.
- Edwards, P.R. and Ewing, W.H. (1972). Identification of Enterobacteriaceae. 3rd edition Burgess Publication Company, Minneapolis, Minnesota.
- Fattahi, S., Kafil, H.S., Nahai, M.R., Asgharzadeh, M., Nori, R. and Aghazadeh, M. (2015). Relationship of biofilm formation and different virulence genes in uropathogenic Escherichia coli isolates from Northwest Iran. GMS Hygiene and Infection Control. 10: 2196-5226.
- Féria, C., Ferreira, E., Correia, J.D., Gonçalves, J. and Caniça, M. (2002). Patterns and mechanisms of resistance to â-lactams and â-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal. Journal of Antimicrobial Chemotherapy. 49(1): 77-85.
- Jegadeeshkumar, D., Rajen, K.S., Nirmala, P., Gopinath, L.R. and Prakash, B. (2016). International Journal of current research in biosciences and plant biology. International Journal of Current Research in Biosciences and Plant Biology. 3(10): 174-178.
- Khelfa, D.G. and Morsy, E.A. (2015). Incidence and distribution of some aerobic bacterial agents associated with high chick mortality in some broiler flocks in Egypt. Middle East Journal of Applied Sciences. 5: 383-394.
- Kumar, K., Sharma, N.S., Kaur, P. and Arora, A.K. (2022). Molecular detection of antimicrobial resistance genes and virulence genes in E. coli isolated from sheep and goat faecal samples. Indian Journal of Animal Research. 56: 208-214.
- Liu, Y., Liu, C., Zheng, W., Zhang, X., Yu, J., Gao, Q., Hou, Y and Huang, X. (2008). PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. International Journal of Food Microbiology. 125(3): 230-235.
- Ma, J., Liu, J.H., Lv, L., Zong, Z., Sun, Y., Zheng, H., Chen, Z.L. and Zeng, Z.L. (2012). Characterization of extended-spectrum â-lactamase genes found among Escherichia coli isolates from duck and environmental samples obtained on a duck farm. Applied and Environmental Microbiology. 78(10): 3668-3673.
- Majumder, S., Akter, M.M., Islam, M.M., Hussain, K., Das, S., Hasan, I., Nazir, K.H.M.N.H. and Rahman, M. (2017). Prevalence, isolation and detection of virulent gene in Escherichia coli from duck. Journal of Advances in Medicine and Medical Research. 20(2): 1-8.
- Melo, L., Bott, T.R., Fletcher, M. and Capdeville, B. (2012). Biofilms- science and technology. Springer Science and Business Media. 223: 13.
- Na, S.H., Moon, D.C., Choi, M.J., Oh, S.J., Jung, D.Y., Sung, E.J., Kang, H.Y., Hyun, B.H. and Lim, S.K. (2019). Antimicrobial resistance and molecular characterization of extended- spectrum â-lactamase-producing Escherichia coli isolated from ducks in South Korea. Foodborne Pathogens and Disease. 16(12): 799-806.
- Nahavandinejad, M. and Asadpour, L. (2017). Mucoviscosity determination and detection of magA and rmpA genes in clinical isolates of Klebsiella pneumoniae in Northern Iran. Crescent Journal of Medical and Biological Sciences. 4: 104-107.
- Nalband, S.M., Kolhe, R.P., Deshpande, P.D., Jadhav, S.N., Gandhale, D.G., Muglikar, D.M., Kolhe, S.R., Bhave, S.S., Jagtap, U.V. and Dhandore, C.V. (2020). Characterization of Escherichia coli Isolated from Bovine Subclinical Mastitis for Virulence Genes, Phylogenetic Groups and ESBL Production. Indian Journal of Animal Research. 54: 1265-1271.
- Nnachi, A.U. Egbo, L.U., Ukaegbu, C.O., Okoroafor, I., Igwe, C.C. and Daniel, L.E. (2015). Antimicrobial susceptibility profile and ESBL-production of Klebsiella species isolated from duck cloaca. Scholars Academic Journal of Biosciences. 3(2B): 207-213.
- Paton, A.W. and Paton, J.C. (1998). Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111 and rfbO157. Journal of Clinical Microbiology. 36(2): 598-602.
- Quinn, P.J., Carter, M.E., Markey, B. and Carter, G.R. (1994). Veterinary Clinical Microbiology. Wolfe Publication, London, UK. pp. 254-258.
- Rajput, D.S., Singh, S.P., Sudipta, G. and Nema, R.P. (2014). Duck farming, fascinating option in India. Journal of Veterinary Science and Technology. 5(3): 181.
- Raza, S., Mohsin, M., Madni, W.A., Sarwar, F., Saqib, M. and Aslam, B. (2017). First report of blaCTX-M-15 type ESBL-producing Klebsiella pneumoniae in wild migratory birds in Pakistan. Ecohealth. 14(1): 182-186.
- Silva, V.O., Espeschit, I.F. and Moreira, M.A.S. (2013). Clonal relationship of Escherichia coli biofilm producer isolates obtained from mastitic milk. Canadian Journal of Microbiology. 59(5): 291-293.
- Tan, T.Y., Ng, L.S.Y., He, J., Koh, T.H. and Hsu, L.Y. (2009). Evaluation of screening methods to detect plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Antimicrobial Agents and Chemotherapy. 53(1): 146-149.
- Wang, R.F., Cao, W.W. and Cerniglia, C.E. (1996). PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Applied and Environmental Microbiology. 62(4): 1242-1247.
- Wang, Y., Tang, C., Yu, X., Xia, M. and Yue, H. (2010). Distribution of serotypes and virulence-associated genes in pathogenic Escherichia coli isolated from ducks. Avian Pathology. 39(4): 297-302. doi: 10.1080/03079457.2010.495742.
- Weill, F.X., Lailler, R., Praud, K., Kérouanton, A., Fabre, L., Brisabois, A., Grimont, P.A.D., Cloeckaert, A. (2004). Emergence of extended-spectrum-â-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France. Journal of Clinical Microbiology. 42(12): 5767-5773.
- Wickens, H. and Wade, P. (2005). Understanding antibiotic resistance. Pharmaceutical Journal. 274(7346): 501-504.
- Yang, F., Deng, B., Liao, W., Wang, P., Chen, P. and Wei, J. (2019). High rate of multi-resistant Klebsiella pneumoniae from human and animal origin. Infection and Drug Resistance. 12: 2729-2737.
- Younis, G., Awad, A., El-Gamal, A. and Hosni, R. (2016). Virulence properties and antimicrobial susceptibility profiles of Klebsiella species recovered from clinically diseased broiler chicken. Advances in Animal and Veterinary Sciences. 4(10): 536-542.
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