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Molecular Detection of Antimicrobial Resistance Genes and Virulence Genes in E. coli Isolated from Sheep and Goat Faecal Samples
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First Online 28-01-2021|
Methods: During the period 2018-19, a total of 100 faecal samples from goats (n=50) and sheep (n=50) were processed for isolation and identification of E.coli. All the E. coli isolates were tested for resistance against 10 different antibiotics. The prevalence of various antibiotic resistance genes viz bla TEM, tetA and tetB and virulence genes Stx1, Stx2 and hlyA in all E. coli isolates was determined by PCR.
Result: Out of 100 samples, 40 isolates of E.coli (24 from goats and 16 from sheep) were isolated, identified biochemically and by PCR. Majority of the E.coli isolates were found resistant to penicillin (100%) followed by ampicillin (93%), tetracycline, (68.8%), gentamicin (37.4%), ceftriazone (18.8%), amikacin (16.7%), chloramphenicol (4.2%), co- trimoxazole (4.2%) and ofloxacin (4.2%). Out of all the isolates, 12 isolates were positive for blaTEM, 10 for tetB, 19 for tetA, five for Stx1 and hlyA and none carried Stx2 gene.
Resistance and virulence have shared some common evolution strategy. Transfer of these factors between bacteria is through horizontal gene transfer, bioflim producing microorganisms always associate with resistance and virulence, development of efflux pumps, porins, cell wall alterations which repress or express the various genes are other common characters for both resistance and virulence (Burrus and Waldon, 2004; Tsai et al., 2011; Moya et al., 2008). Based on Global Antimicrobial Surveillance System (GLASS), occurrence of antibacterial resistance is reported in about 5,00,000 people among 22 countries. According to GLASS mostly reported resistant bacteria were Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae and Salmonella spp. (WHO, 2015).
India has been described as the world’s AMR capital (Chaudhry and Tomar, 2017). Although, the emergence of new multi-drug-resistant (MDR) species presents new diagnostic and therapeutic challenges, on the other hand, India is still struggling to eliminate dangerous old pathogens such as tuberculosis, malaria and cholera pathogens, which are becoming increasingly drug-resistant(Swaminathan et al., 2017) and India has also framed its National Action Plan (NAP) for AMR (GOI, 2017).
Escherichia coli is a Gram-negative, motile and facultative anaerobe. Though E.coli represents a significant part of the commensal microbiota of the different hosts, but various pathogenic types are also present which can cause severe infections in animals and humans and can produce diseases. Pathogenic E. coli strains fall into two categories: those that cause intestinal pathologies and those that cause extraintestinal pathologies. E. coli consists of a diverse group of bacteria. Pathogenic E. coli strains are categorized into pathotypes. Six pathotypes are associated with diarrhea and collectively are referred to as diarrheagenic E. coli such as Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Diffusely adherent E. coli (DAEC) (Kaper, 2005).
One of the major concern is possible transmission of resistant and virulent E. coli between animals and humans through various pathways, such as direct contact with animals, contact with animal excretions, or via the food chain (Dho-Moulin and Fairbrother, 1999). Animals, humans and the ecosystem like water supplies serve as natural habitats of virulent E. coli strains. Though E. coli is susceptible to almost all relevant antibacterial agents but it has a great capacity to accumulate resistance genes, mostly via horizontal gene transfer. The most problematic mechanisms in E. coli correspond to the acquiring of genes coding for extended-spectrum β-lactamases (broad-spectrum cephalosporins resistance), carbapenemases (resistance to carbapenems), plasmid-mediated quinolone resistance genes (for fluroquinolones), 16S rRNA methylases (pan-resistance to aminoglycosides) and mcr genes (for polymyxins resistance) (deJong et al., 2018; Cattoir and Nordmann, 2009; Michael et al., 2017; Ewers et al., 2012; Doi et al., 2016). More importantly, ESBL/AmpC genes have been widely noticed in the commensal E. coli isolated from faecal samples of different large and small ruminant food producing animals (Dierikx et al., 2013; Haenni et al., 2014).
The information about antimicrobial resistance and virulence in bacteria isolated from small ruminants is not much reported in India. In West Bengal, prevalence of multidrug resistance in shiga-toxigenic E.coli is reported among small ruminants especially in goats (Mahanti et al., 2015 and Wani et al., 2003) reported STEC serogroup O157 from ovine faecal samples in Srinagar. One of the study from Arunachal Pradesh also indicates that ETEC is a major cause for frequent diarrhoeal episodes in lambs (Bandyopadhyay et al., 2011). Therefore, study of AMR spreading pattern in small ruminant population is necessary in other part of India and also occurrence of resistance and virulence in commensal/pathogenic bacteria is necessary to overcome the antimicrobial resistance spreading among small ruminants and also in humans. The objective of this study was to estimate the antimicrobial resistance of E. coli isolated from small ruminants in Punjab and also virulotyping of E.coli isolates studied.
MATERIALS AND METHODS
A total of 100 faecal samples [50 samples from healthy (n=33) and diarrhoeic (n=17) sheep whereas 50 samples from healthy (n=26) and diarrhoeic (n= 24) goats] were collected from farms in and around Ludhiana, Punjab. The faecal samples were collected in the year 2018 to 2019. Faecal samples were collected directly from the rectum of individual animals and transported to the laboratory in ice box. The faecal samples brought to the laboratory were inoculated on BHI, MLA and EMB (Himedia) agar. The inoculated plates were incubated at 37°C for 16-24 hours. The suspected colonies after incubation were subjected to Gram’s staining for identification and various biochemical tests for confirmation.
Genomic DNA extraction
The DNA of E. coli isolates was extracted using NucleoSpin® Microbial DNA kit (Macherey Nagel) as per manufacturer’s instructions. Purity and concentration of the extracted genomic DNA was estimated by using a Nanodrop (Thermo Scientific, USA). The optical density (OD) at 260 nm and 280 nm of the sample was measured by using nuclease free water as blank and the ratio of 260/280 OD was calculated. A ratio of 1.6 to 1.8 was considered satisfactory. DNA was stored at -80°C until further analysis by PCR.
Polymerase chain reaction (PCR) for detection of E. coli
Confirmation of E. coli isolates was done by PCR primers as per Riffon et al., (2001) (Table 1). Amplification reaction mixture (for one reaction) was prepared in a volume of 25 μl consisting of 12.5 μl mastermix (Promega), forward primer 20pmol (1 μl), reverse primer 20pmol (1 μl), 3 μl of Template DNA and 7.5 μl of nuclease free water. PCR cycling conditions consisted of an initial denaturation at 94°C for 5 minutes followed by 30 cycles each of denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds and extension at 72°C for 1 minute. This was followed by a final extension at 72°C for 10 minutes and amplified products were analysed with Agarose gel electrophoresis.
Antimicrobial susceptibility test
All the isolates obtained were tested for sensitivity to various antibiotics as per the disc diffusion method of Bauer et al., (1966). Ten different antibiotics used were chloramphenicol (30 mcg), Ampicillin/sulbactum (10/10 mcg), penicillin (1U), tetracycline (30 mcg), amoxyclav (30 mcg), cotrimoxazole (25 mcg), gentamicin (10 mcg), ofloxacin (5 mcg), ceftriazone (30 mcg) and amikacin (30 mcg). Diameter of Zone of inhibition was measured and compared with standard reference values in order to classify the isolates as sensitive, intermediate resistance or resistant to a particular antibiotic (Clinical Laboratory Standards Institute, 2016).
Molecular detection of antibiotic resistance and virulence genes of E. coli
All the E. coli isolates were tested for the presence of blaTEM, tetA and tetB antibiotic resistance genes and virulence genes Stx1, Stx2 and hlyA. PCR was done for detection of these genes by using primers, as shown in Table 1. PCR protocol for detection of genes followed as 25 μl reaction mixture was made consisting of 12.5 μl mastermix (Promega), forward primer (20 pmol/μl) 1 μl, reverse primer (20 pmol/μl) 1 μl, 3 μl of Template DNA and 7.5 μl nuclease free water. PCR was performed in a thermocycler (Veriti, ABI, USA) with the following conditions; an initial denaturation at 95°C for 4 minutes followed by 35 cycles each of denaturation at 95°C for 60 seconds, annealing at 64°C for 60 seconds and extension at 72°C for 1 minute. This was followed by a final extension at 72°C for 7 minutes and amplified products were analysed by electrophoresis at 80V for 1 hr in 1.0% agarose gel in 1x TBE buffer containing ethidium bromide and visualized under Alpha imager gel documention system (Alpha innotech) and photographed.
RESULTS AND DISCUSSION
On BHI, colonies were spherical, creamy mucoid in nature; on MLA lactose fermenting pink colour colonies were present and on EMB, colonies showed green metallic sheen. Pink colour coccobacilli were seen in Gram’s staining and the E. coli cultures were confirmed biochemicaly viz. Catalase test (+ve) Oxidase (-ve) and IMViC test, [Indole (+ve), methyl red (+ve), Voges Proskauer (-ve) and citrate (-ve)]. A total of 40 E. coli isolates isolated from sheep and goats faecal samples were screened for antibiogram against 10 antibiotic agents. Majority of isolates were resistant to penicillins (100%). In goats, resistance to other antimicrobial agents in decreasing order was ampicillin (91.7%), gentamicin (37.5%) followed by amikacin (16.7%), ceftriaxone and tetracycline (12.5%), chloramphenicol, cotrimoxazole and ofloxacin (4.2%) and amoxicillin (0%). In sheep, resistance to antimicrobial agents in decreasing order was ampicillin (93.3%), tetracycline (68.8%), gentamicin (37.5%), amoxycillin, chloramphenicol, clotrimoxazole and ofloxacin (31.3%), ceftriaxone (18.8%) and amikacin (12.5%) (Fig 1). The details of each isolate and resistance percentage is given in the Table 2. We have carried out phenotypic and genotypic detection of antibiotic resistance and virulence genes in E. coli, isolated from sheep and goat faecal samples. The colony characteristics of the isolated E. coli in different media resemble the study observation of Ali et al., (1998). All the E. coli isolates were found to be positive for catalase, methyl-red positive and indole positive but negative for VP test, which supports the findings of Beutin et al., (1993). And also the isolated E. coli organisms fermented dextrose, maltose, lactose, sucrose and mannitol with the production of both acid and gas. Results of MR, Indole test of the E. coli isolates were positive. In Gram’s staining, the morphology of the isolated bacteria exhibited pink, small rod shape, gram negative bacilli which was supported by several authors Buxton and Fraser, (1977); Freeman, (1985).
Antimicrobial agents play a crucial role in human and animal life around the world Hudson et al., (2017). Moreover, overuse of antimicrobial agents in food-producing animals has always resulted in the development of antimicrobial-resistant bacteria and antibiotic-resistant genes in human and animal pathogens. β-Lactam antibiotics such as ampicillin, amikacin, cephalosporins can be the most effective therapeutic medicines for the treatment of bacterial infections as when combined show protection and high potency against gram-negative bacteria, such as members of enterobacteriaceae family, including E. coli. Nevertheless, gentamicin, tetracycline, ampicillin, enrofloxacin, chloramphenicol and penicillin are highly prescribed by veterinarians. In our study we found that majority of E. coli isolates were penicillin resistance. There is one similar study done by Momtaz et al., (2013), they describe that majority of their E. coli isolates isolated from meat samples of small and large ruminants were penicillin resistant and some were resistant to other antibiotic like tetracycline, streptomycin and gentamicin.
Molecular confirmation of E.coli was carried out by PCR using primers as per Riffon et al., (2001). A 232 bp amplification band was obtained for E.coli isolates (Fig 2).
Detection of resistance genes and virulence genes
Molecular detection of antibiotic resistance genes was performed in all E. coli isolates. Virulotyping was also done with Stx1, Stx2 and hlyA genes for all E. coli isolates. Out of 40 isolates, 12 isolates were positive for bla TEM gene, 19 for tetA and 10 for tetB gene (Fig 3, 4, 5). In a study conducted by Colom et al., (2003), blaTEM was detected in 45 out of 51 amoxycillin-clavulinic acid resistant isolates. In another study, blaTEM was found in 97% of ampicillin resistant strain of E. coli (Medina et al., 2011). Aslam et al., (2009) also studied same gene in E. coli isolates, they found that the majority of streptomycin-resistant E. coli isolates (76%) were positive for the strA and strB genes together. The bla(CMY), bla(TEM) and bla(SHV) genes were found in 12%, 56% and 4%, of ampicillin-resistant E. coli isolates respectively. Boerlin et al., (2005) carried out studies on antimicrobial resistant and virulence genes of E. coli isolates from swine in Ontorio and in that study they found 98% of E. coli isolates were having tet A gene. Aslam et al., (2009) also studied same genes in E. coli isolates and they foundthat about 50% of tetracycline-resistant E. coli isolates were positive for tet(A) (14%), tet(B) (15%), or tet(C) (21%) genes or both tet(B) and tet(C) genes together (3%). The occurrence of antibiotic resistance genes found in our study was not that much higher as compare to the above studies. This might be due to lower use of antimicrobial agents by the Punjab farmers or the local veterinarians prescribing less antibiotics to them, different geographic zone, their customs and even use of high quality animal nutrition.
In our study we detected Stx1, Stx2 and hlyA virulence genes from E. coli isolates. Among that five isolates were found positive for the presence of Stx1 gene, none of the isolates carried Stx2 gene and five isolates were found to be positive for the presence of hlyA gene (Fig 6,7). These results are in accordance with those of Khan et al., (2002). They analysed 63 strains of E. coli and by using PCR they detected virulence gene Stx1 (36.5%), Stx2 (19%) and hlyA (45.5%). Wani et al., (2004) investigated the presence or absence of shiga toxin producing Escherichia coli (STEC) in avian species. They collected faecal samples from 500 chicken and 25 free flying pigeon and screened for the presence of E. coli. Out of 525 samples processed, a total of 426 (chicken 401, pigeons 25) E. coli strains were isolated. All isolates were subjected to multiplex polymerase chain reaction (mPCR) for the detection of Stx1, Stx2, eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of Stx1, Stx2 or their variants and saa genes. The strains of these harbouring stx2 virulence genes have been implicated in human infections with haemolytic colitis and haemorrhagic uremic syndrome and strain carrying stx1 may trigger diarrhoea in immunocompromised individuals (Farrokh et al., 2013). Some of our E. coli isolates from sheep and goats have two virulence genes, so these are more pathogenic. One of the similar study was done by Jajarmi et al., (2017) and they also found pathogenic strains of E. coli that harbour more than two virulence genes like stx1, stx2, hlyA and eaeA. In our study, phenotypic and genotypic characters varies, this might be due to presence of some other genes related to that particular antibiotic, like instead of blaTEM gene may be blaSHV could be present or due to cross resitance and coresistance. The potential explanation for absence of virulence gene could be that the isolates might have lost the gene during subculture process (Karch et al., 1992). Momtaz et al., (2013) also studied resistant genes like blaSHV, tetA, tetB etc. and virulence genes like Stx1, Stx2, ehly etc. and they found that the incidence of blaSHV was maximum while the incidences of cmlA and cat1 antibiotic resistance genes were minimum and virulence genes like Stx1, Stx2, eaeA and ehly were prevalent in the STEC strains isolated from ruminants meat samples. Since some of the isolates were carrying more than two resistance genes and virulence genes, so they are considered as potentially pathogenic and absence of these genes in some isolates in our study should not be underestimated.
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