In the present study a lung sample was collected from autopsied rabbit, which was suspected to have died due to E. coli infection and cultured on MacConkey agar. Itrevealed typical cultural and biochemical characteristics of E. coli. Molecular confirmation of E. coli was carried out using 16s RNA (ECO1) universal eubacterial primers with positive 585 bp amplicon. A total 26 Antibiotic disc used for antibiotic sensitivity test. Out of 26 Antibiotic disc, only two antibiotics (Imipenem and Cefoxitin) sensitive against isolated E. coli. Phenotypic characterization of ESBL by combine disk diffusion method. The difference between zone of inhibition is 20 mm around the combined disk containing clavulanic acid then the corresponding disk with Ceftazidime and cefotaxime is indicate positive for ESBL producing E. coli. Upon genotypic conformation of ESBL, the isolate was found positive for CTX-M1, CTX-M2, and TEM and negative for CTX-M9, SHV and OXA genes.
HIGHLIGHTS
Multi drug resistant ESBL producing E. coli was isolated from rabbit.
Harboring of CTXM-1, CTXM-2 and TEM genes and absence of CTX-M9, SHV and OXA genes were confirmed by PCR.
Emergence of multidrug resistance (MDR) E. coli is a major concern for human and animals due to their rapid spread. MDR is linked with high disease burden and economic consequences on people and nations. Continuous surveillance of antimicrobial resistance in bacteria that cause infections in humans and animals is essential to know the antimicrobial susceptibility dynamics of these organisms over the time. Extended spectrum β-lactamases (ESBLs) are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and cefotetan) or carbapenems (e.g., meropenem or imipenem). Such enzymes have been most commonly detected in E. coli and Klebseillapneumoniae. ESBLs are inhibited by inhibitors such as clavulanic acid, tazobactam and sulbactam. There are more than 200 different types of ESBL enzymes of which TEM, SHV and CTX-M types are more frequent (Rahman et al., 2017). Its ubiquitous presence and wide host range justifies its designation as a sentinel organism for antimicrobial resistance development in different types of animals (Ashbolt et al., 2013). Sufficient concern has though been given towards the emergence and spread of extended spectrum β-lactamase (ESBL) producing E. coli associated with cattle and other farm animals(Bush and Jacoby, 2010; Pfeifer et al., 2010), but there is very few reports about ESBL producing E. coli in laboratory animals like rabbit, rat, mice etc. A steady increase of these strains have been reported worldwide and in this connection, the present communication givesa report of ESBL producing E.coli in rabbit. Best of author’s knowledge this is the first report of presence of ESBL in rabbit from India.
How to cite this article: Patel, S.S., Patel, A.C., Sharma, K.K., Chauhan, H.C., Raval, S.H., Shrimali, M.D., Shinde, R.S., and Pandya, M.M. (2020). Molecular detection of extended spectrum beta lactamase (ESBL) producing multidrug resistant E. coli from rabbit. J. Anim. Res., 10(5): 691–695.
Source of Support: None; Conflict of Interest: None
MATERIALS AND METHODS
In the present study, a lung sample was aseptically collected from necropsied rabbit. Necropsy findings suggested E. coli infection as cause of death. The sample was streaked on MacConkey agar and EMB agar medium and incubated aerobically at 37°C for overnight. The colony morphology was observed and the features of microorganisms were studies using Gram’s staining method. Further isolate was confirmed by biochemical tests like Indole, Methyl red, Voges-Proskaur, Citrate utilization and Oxidase as per process recommended by Edwards and Ewing (1972). A drug sensitivity pattern was studied by disk diffusion method following the method of Bauer et al. (1966). In total 26 different antibiotics commonly used for animal treatments were used in the study (Table 1).
Phenotypic characterization of ESBL was done by combine disk diffusion method (CLSI VET08Ed4E). The difference between zone of inhibition is >5 mm around the combined disk containing clavulanic acid then the corresponding disk with Ceftazidime and cefotaxime is indicate positive for ESBL production.
For molecular characterization genomic DNA was extracted from the freshly grown culture by heat method and the species of the isolate was confirmed by PCR using species-specific 16S rRNA primer ECO-1 (Fratamico et al., 2000). Genotypic evaluation of ESBL production was carried out by detecting CTX-M-1, CTX-M-2, CTX-M-9, TEM, OXA and SHV using gene specific primer (Dallenn et al., 2010; Koovapra et al., 2016). PCR was standardized in a total reaction volume of 25 (J for each gene, containing 12.5 id of 2X PCR master mixture, 10 pmol of forward and reverse primers each (Table 2), 2 id DNA as template and nuclease free water up to 25 id. The reaction was standardized in a thermal cycler (Eppendorf, Germany) as per table 3. The amplified product was electrophoresed in 1.5% agarose gel stained with ethidium bromide (0.5 ug /ml) and image was documented by gel documentation system (Mini BiSBio Imaging System).
RESULTS AND DISCUSSION
The organisms were successfully isolated from the rabbit’s lung tissue showing characteristic lactose fermenting pink coloured colonies and greenish metallic sheen on MacConkey agar and eosin methylene blue (EMB) agar, respectively. On gram’s staining organisms looks like gram negative bacilli, whereas in biochemical tests it shows negative for oxidase test and + + - - pattern for IMViC test. In antibiotic sensitivity test only two antibiotics viz. Imipenem and Cefoxitin were found sensitive against the isolate. In phenotypic characterization of ESBL by combine disk diffusion method difference between zone of inhibition was 20 mm which is indicative of ESBL producing E. coli (Fig. 1).
List of Antibiotic disc used for Antibiotic Sensitivity Test and its result against Isolated E.coli
Details of primers for amplification of ECO1, CTX-M1, CTX-M2, CTX- M9, TEM and SHV genes of E. coli
Steps and conditions of thermal cycling for various PCR
Phenotypic demonstration of ESBL producing Escherichia coli by combine disk diffusion method showing inhibition of ≥5 mm around the combined disk containing antibiotic and clavulanic acid than the corresponding antibiotic disk
Molecular confirmation of E. coli isolates was carried out using 16s RNA universal eubacterial primers (ECO-1) which shows a amplification of target band of 585 bp size which is indicative of E.coli. The isolate was screened for presence of CTX-M1, CTX-M2, CTX-M9, TEM, OXA and SHV genes using PCR and found positive for CTXM-1 (688 bp), CTXM-2 (404 bp) and TEM (800 bp) genes (Fig. 2, A-D); whereas it found negative for CTX-M9, SHV and OXA genes. Brinas et al. (2002) reported E. coli is the commonest bacteria which causes intestinal and extra intestinal infection in different animals and humans with ESBL. Zaho et al. (2018) and Silva et al. (2010) isolate multidrug resistance E. coli from cases of collibacillosis from domestic rabbits and wild rabbits, respectively. But in the present study it was recovered from extra intestinal tissue. In present study, ESBL enzymes and their genes have been demonstrated with both phenotypic and molecular methods, respectively. Though these were not reported in few earlier studies. In china Zaho et al. (2018) reported 50.90 % MDR-ESBL producing E. coli, It also detect 98.20% and 94.50% ^-lactamase genes, blaTEM and blaCTX-M respectively form 55 Isolated E.coli from rabbit, but they had isolated it from fecal sample. Pandor (2019) reported out of 25 ESBL E. coli isolates, 19 (35.85%), 8 (15.09%) and 7 (13.21%) isolates positive for TEM, SHV and CTX-M gene, respectively. Whereas 6 (15.09%) isolates harbored two genes i.e., CTX-M and TEM; TEM and SHV and 3 (5.66%) isolates harbored all the three genes CTX-M, TEM and SHV. This indicate spread of such deleterious genes in laboratory animal population. Similar to Brinas et al. (2002) and Zaho et al. (2018). CTX-M and TEM genes were detected in this work which are said to be commonest among ESBL genes whereas SHV and OXA genes were less prevalent among population, as previously recorded by Brinas et al. (2002).
PCR based confirmation of E. coli (A) and presence or absence of antibiotic resistant genes (B-D)
Plate A - Gene Pilot 100 bp ladder (L1) and positive samples for ECO1gene; Plate B - Gene Pilot 100–2000 bp ladder (L2) and positive samples for CTX1 gene (2) and CTX2 gene (3); Plate C -Gene Pilot 100 bp ladder (L3) and positive samples for CTX1 gene (2) and TEM gene (4); Plate D - Gene Pilot 100 bp ladder (L3) and negative samples for OXA gene (5)
CONCLUSION
It was concluded that the rabbit was died due to ESBL producing multidrug resistant E. coli. The occurrence of MDR strains is a potential threat not only to animal health but also to workers associated with maintenance of lab animals’ colonies.
REFERENCESAshbolt, N.J.Amezquita, A., Backhaus, T., Borriello, P., Brandt, K.K., Collignon, P., Coors, A., Finley, R., Gaze, W.H., Heberer, T., Lawrence, J.R., Larsson, D.G., McEwen, S.A., Ryan, J.J., Schonfeld, J., Silley, P., Snape, J.R., Van den Eede, C. and Topp, E.2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ. Health Perspect., 121: 993–1001.Bauer, A.W., Kirly, W.M.M., Sherris, J.C. and Turck, M.1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Path, 45: 493–496.Brinas, L., Zarazaga, L., Saenz, Y., Ruiz-Larrea and Torres, C.2002. β-Lactamases in ampicillin resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob. Agents Chemother., 46: 3156–3163.Bush, K. and Jacoby, G.A.2010. Updated functional classification of β-lactamases. Antimicrob. Agents Chemother., 54: 969-976.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, VET08, 4th Edition table 7A, pp. 122.Dallenne, C., DaCosta, A., Decre, D., Favier, C. and Arlet, G.2010. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother., 65(3): 490495.Edwards, P.R. and Ewing, W.E.1972. Identification of Enterbacteriaceae, 3rd Ed., Burgess Publishing CO, Minneapolis, pp. 288–291.Fratamico, P.M., Bagi, L.K. and Pepe, T.A.2000. Multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157: H7 in foods and bovine feces. J. FoodProt, 63: 1032–1037.Koovapra, S., Bandyopadhyay, S., Das, G., Bhattacharyya, D., Banerjee, J., Mahanti, A., Samanta, I., Nanda, P.K., Kumar, A., Mukherjee, R., Dimri, U. and Singh, R.K.2016. Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India. Infect. Genet. Evol., 44: 395–402.Pandor, B.R.2019. Isolation and molecular characterization of E. coli from bovine with emphasis on extended spectrum beta-lactamase production. M.V.Sc thesis, SDAU, Sardarkrushinagar.Pfeifer, Y., Cullik, A. and Witte, W.2010. Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int. J. Med. Microbiol., 300(6): 371–379.Rahman, M.A., Rahman, A.K.M.A., Islam, M.A. and Alam, M.M.2017. Antimicrobial resistance of Escherichia coli isolated from milk, beef and chicken meat in Bangladesh. Bangl. J. Vet. Med., 15(2): 141–146.Silva, N., Igrejas, G., Figueiredo, N., Goncalves, A., Radhouani, H., Rodrigues, J. and Poeta, P.2010. Molecular characterization of antimicrobial resistance in Enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus). Sci. Total Environ., 408: 4871-4876.Zhao, X., Yang, J., Ju, Z., Chang, W. and Sun, S.2018. Molecular Characterization of Antimicrobial Resistance in Escherichia coli from Rabbit Farms in Tai'an, China. BioMedRes. Inter., 1: 1–7.