Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 9 (september 2021) : 1049-1056

Transferable blaCTX-M Carrying Multidrug Resistant Escherichia coli from Pig Population of North Eastern Region of India

Rajkumari Mandakini1, T.K. Dutta1,*, P. Roychoudhury1, P.K. Subudhi1, I. Samanta2, S. Bandopaddhay3, G. Das1, A.K. Samanta1
1Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
2Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, KB Sarani, Kolkata-700 037, West Bengal, India.
3ICAR-Indian Veterinary Research Institute, Eastern Regional Station, 37, Belgachhia Road, Kolkata-700 037, West Bengal, India.
Cite article:- Mandakini Rajkumari, Dutta T.K., Roychoudhury P., Subudhi P.K., Samanta I., Bandopaddhay S., Das G., Samanta A.K. (2021). Transferable blaCTX-M Carrying Multidrug Resistant Escherichia coli from Pig Population of North Eastern Region of India . Indian Journal of Animal Research. 55(9): 1049-1056. doi: 10.18805/IJAR.B-4162.

ABSTRACT

Background: We investigated the occurrence of blaCTX-M carrying extended spectrum beta lactamase (ESBL) producing Escherichia coli in pigs from 8 North-eastern states of India with special emphasis on the transferability of ESBL gene from resistant E. coli strains to the susceptible Salmonella strains by in vitro and in vivo.

Methods: Fecal samples (n=790) were collected from pigs reared under organized and unorganized farming set up of entire North-eastern region of India. All the samples were processed for isolation and identification of E. coli. All the isolates were subjected to antimicrobial sensitivity assay by disc diffusion method followed by determination of ESBLs producing ability by double disc synergy test (DDST). All the ESBLs producing isolates were screened for blaCTX-M gene by PCR using specific primers. The representative blaCTX-M gene positive isolates were used as donor to determine the ability to transfer of resistance gene in Salmonella by in vitro and in vivo assays with and without antibiotic selection pressure.  

Result: A total of 2,291 E. coli was isolated, of which 1113 and 1178 were from organized and unorganized farms, respectively. Majority of the isolates were multi-drug resistant with highest resistance against amoxicillin (84.81%) followed by cefalexin (77.17%), sulphafurazole (56.79%), piperacillin (46.40%), tetracycline (38.29%) and cefexime (35.66%). Isolates from unorganized farms showed higher resistance than the isolates recovered from organized farms. A total of 654 (28.55%) isolates were confirmed as ESBL producers by double disc synergy test (DDST) method, of which 65 (2.84%) isolates were positive for blaCTX-M gene. Genotypically, isolates with specific amino acids substitution revealed variation in their antibiotic susceptibility by phenotypic method. blaCTX-M gene could be successfully transferred horizontally from E. coli (donor) to Salmonella (recipient) by in vitro (3.6±2.07x10-8 to 4.4±2.88x10-8 transconjugate per donor) and in vivo method. By in vivo method in pig model, the frequency of transfer was higher under the antibiotic selection pressure (6.6±3.05x10-5 to 7.2±1.92x10-5 trans-conjugants per donor) than without antibiotic pressure (5.6±2.3x10-4 to 6.8±3.35x10-4 trans-conjugants per donor).

INTRODUCTION

The incidence of drug resistance is increasing at an alarming rate and posing serious problems in the treatment of infectious diseases caused by multi-drug resistant (MDR) bacteria (Lewis et al., 2007; Collignon and McEwen, 2019). Antimicrobial resistance genes emerge either by being mobilized from obscure strains or by evolving from obscure ancestral genes, which may be situated on chromosomes, plasmids, integrons or on transposons. In large scale poultry and livestock production facilities, antimicrobial agents may be used for therapeutic and prophylactic purposes or as growth promoters at sub-therapeutic levels. It is well known that such use of antimicrobial agents puts selective pressure on commensals and pathogenic bacteria contributing to clonal expansion of MDR (Holmes et al., 2016; Baker et al., 2018).
       
E. coli is an important gastrointestinal flora known to be capable of accepting and transferring plasmids, which under stress readily transfers it to other species and is, therefore, considered an important reservoir of transferable antibiotic resistance (Lim et al., 2007; Salinas et al., 2019). The frequency of resistance in E. coli to β-lactam is increasing worldwide (Kallen et al., 2010). Among the worldwide array of antibiotics, β-lactams are the most widely used agents (Kong et al., 2010). The most common cause of resistance to β-lactam antibiotics is the production of β-lactamases. The prevalence of ESBLs in India has now  reached epidemic proportions ranging from 62 to 100% and majority of them are in E. coli and Klebsiella pneumoniae isolates. Apart from blaTEM and blaSHV types, isolates from India additionally produce CTX-M enzymes (Mathai et al., 2009), in which CTX-M-15 appeared to be the predominant ESBLs in Northern India (Manoharan et al., 2011). Earlier studies conducted in North Eastern India confirmed the presence of ESBLs (blaCTX-M and blaSHV) in E. coli from pigs of Mizoram (Lalzampuia et al., 2013). ESBL-producing E. coli possessing blaCTX-M and blaSHV associated with human diarrhoea has been also reported from this region (Dutta et al., 2013). The present study was undertaken to conduct an extensive investigation among the pig population of entire North Eastern states of India to record the blaCTX-M carrying E. coli and to establish their potential of spreading the traits horizontally.

MATERIALS AND METHODS

Sample collection
 
A total of 790 fresh fecal samples were collected from pigs of 8 North-eastern states of India including Arunachal Pradesh (n=100), Assam (n=103), Manipur (n=103), Meghalaya (n=96), Mizoram (n=98), Nagaland (n=82), Sikkim (n=94) and Tripura (n=104). Samples were collected from different farms of each states including Arunachal Pradesh (n=12), Assam (n=13), Manipur (n=11), Meghalaya (n=10), Mizoram (n=11), Nagaland (n=9), Sikkim (n=9) and Tripura (n=14). Samples were collected randomly from pigs maintained under organized (n=388) and unorganized (n=402) farming system irrespective of age, sex and with or without history of diarrhea. All the samples were collected using a sterilized adsorbent cotton swab. However, for collection of samples from distant locations, a sterilized swab dipped in brain heart infusion broth (HiMedia, Mumbai) was used as transport medium and transported to the laboratory under cold chain for further processing.
 
Isolation and identification of Escherichia coli
 
Individual samples were directly inoculated on MacConkey’s Agar (Hi-Media, Mumbai) plates and incubated at 37°C overnight. From each plate five lactose fermenting pure colonies were randomly selected and sub-cultured on eosin methylene blue (EMB) agar (Hi Media, Mumbai) plates and incubated overnight at 37°C. Colonies with characteristic metallic sheen were further studied for their morphological characteristics for identification of E. coli. All the isolates were further confirmed by BD Phoenix™ automated bacterial identification system. All the isolates were stored as pure culture in semisolid agar at 4°C as well as in Luria Bertani (LB) broth (HiMedia, Mumbai) containing 25% glycerol (v/v) at -80°C for further use.
 
Antibiotic susceptibility test and confirmatory test for ESBLs
 
Antimicrobial susceptibility test was done on Mueller-Hinton agar (HiMedia, Mumbai) plate as per the recommendation of Clinical Laboratory Standard Institute (CLSI, 2014) using the following commercially available antibiotic discs: amoxicillin (30 µg), ampicillin (10 µg), aztreonam (30 µg), cefalexin (30 µg), cefexime (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), Co-trimoxazole (1.25/23.75 µg), gentamicin (10 µg), imipenem (10 µg), nalidixic acid (30 µg), piperacillin (100 µg), streptomycin (10 µg), sulphafurazole/sulfisoxazole (300 µg), tetracycline (30 µg) and trimethoprim (30 µg). Isolates exhibited inhibition zone of ≤ 22 mm for ceftazidine (30 µg), ≤ 25 mm for ceftriazone (30 µg) and ≤ 27 mm for cefotaxime (30 µg) were identified as potential ESBLs producer. Confirmatory test for ESBLs production was carried out in Mueller Hinton agar plate using cefotaxime (30 µg), amoxicillin (30 µg) and ceftazidime (30 µg) alone as well as cefotaxime/clavulanate (30/10 µg), amoxicillin/clavulanate (30/10 µg) and ceftazidime/clavulanate (30/10 µg) combination as per the recommendation of CLSI (2014).
 
PCR detection of blaCTX-M gene in potential ESBLs producing isolates
 
blaCTX-M gene was detected by PCR assay using specific oligonucleotide primers (F: 5'-CAATGTGCAGCACCAGTAA-3' and R: 5'-CGCGATATCGTTGGTGGTG-3') (Perez and Hanson, 2002). The repeatability of the assay was checked by repeating the PCR for three times.
 
Cloning and nucleotide sequencing
 
Cloning of the PCR amplicons for the resistance genes was performed using InsT/A cloneTM PCR product cloning kit (MBI Fermentas). DH5α E. coli culture was used for transformation of the plasmid using TransformAid bacterial transformation kit as per the instruction of the manufacturer. Subsequently, the clone was confirmed by PCR. PCR amplicons were cloned in to pTZ57R/T vector using InsT/A cloneTM PCR product cloning kit (MBI Fermentas) and sequenced by the DNA sequencing facility, University of Delhi, South Campus, New Delhi, India.
 
In vitro horizontal gene transfer (HGT)
 
E. coli isolates with the antibiotic resistance pattern of ampicillin, amoxicillin, aztreonam, cefexime, cefotaxime, ceftazidime, ceftriaxone and cephalexin were studied for presence of target resistance gene (blaCTX-M). The isolates that were found to be positive for blaCTX-M gene in its plasmids were selected as donor strain. Salmonella isolate (obtained from the Department of Veterinary Microbiology, CVSc and AH, CAU, Aizawl) was used as recipient strain, which was sensitive to ampicillin, amoxicillin, aztreonam, cefexime, cephalexin, ceftazidime, ceftriaxone and cefotaxime and was not carrying blaCTX-M gene in their plasmid as confirmed by PCR analysis.
       
In vitro mating study was carried out by broth mating (Schmitt et al., 2007), filter paper mating (Sun et al., 2010) and plate mating (Gniadkowski, 2001) methods. Selected transconjugants (Salmonella) were further characterized for their antimicrobial susceptibility, ESBL phenotype, plasmid profiling and the presence of blaCTX-M gene by PCR.

In vivo horizontal gene transfer (HGT)
 
In vivo mating experiment was performed in pig model. The mating experiment was performed with and without any antibiotic pressure. The animal experiments were approved by The Institutional Animal Ethics Committee, College of Veterinary Science and Animal Husbandry, Central Agriculture University, Aizawl, Mizoram, India (No. CVSC/CAU/IAEC/11-12/P1) and were performed by skilled personnel.
       
Twelve crossbred (Zovawk and Large White Yorkshire) pigs were used for colonization and antimicrobial resistance transfer studies. Six animals each were used for treatment with antibiotic pressure and treatment without antibiotic pressure. All the animals were maintained in separate locations with ad-libitum feed and water.
       
Prior to inoculation of the donor or recipient strains, fecal samples from each pig were tested for the presence of indigenous bacteria with similar resistance. The recipient strain (1010 cfu/pig) was administered to each pig by oral drench. Donor and recipient strains (109 cfu/pig and 1010 cfu/pig) were administered on different days in order to decrease the chance of the occurrence of bacterial conjugation in the mouth of the animals. Pigs were inoculated with recipient (days 0, 2, 4, and 6) and donor (days 1, 3 and 5) stocks by oral drench. As the recipient stock included Salmonella enterica in high numbers, pigs were monitored for clinical signs consistent with salmonellosis, including diarrhea, inappetance and dehydration. Cefotaxime @ 25 mg/kg BW as divided doses was administered intramuscularly to the pigs assigned to the treatment with antibiotic pressure on 0 day and subsequently every 24 h for 5 doses. Thus, pigs had received both donor and recipient strains prior to administration of the first dose of cefotaxime. Fecal samples were collected from each pig on day 2, 4, 6, 8 and 10 post inoculations. The samples were processed for detection of donor, recipient and transconjugates on selective medium. Selected transconjugants (Salmonella) were further characterized for their antimicrobial susceptibility, ESBL phenotype, plasmid profiling and the presence of blaCTX-M gene by PCR.

RESULTS AND DISCUSSION

Isolation of E. coli and antimicrobial susceptibility
 
A total of 2,291 E. coli were isolated from 790 fecal samples of pigs, of which 1113 and 1178 were from organized and unorganized farms, respectively (Table 1). All the isolates exhibited resistance to at least 3 antimicrobial classes. Amoxycillin (84.81%) showed highest level of resistance followed by cefalexin (77.17%), sulphafurazole (56.79%), piperacillin (46.40%), tetracycline (38.29%), cefexime (35.66%), co-trimoxazole (27.94%), trimethoprime (26.32%), ampicillin (26.09%), ceftazidime (22.87%), nalidixic acid (21.82%), aztreonam (18.37%), gentamicin (16.27%), streptomycin (9.12%), cefotaxime (8.25%), ceftriaxone (7.51%), ciprofloxacin (6.11%), and imipenem (0.22%) (Table 2). Of the 1113 isolates obtained from organized farms, imipenem (99.82%) was found to be the most sensitive antibiotic followed by amoxicillin (81.94%), sulphafurazole (48.70%), piperacillin (42.95%), tetracycline (39.64%), cefixime (36.03%), ceftazidime (27.94%), ampicillin (27.13%), nalidixic acid (22.46%), trimethoprim (21.38%), aztreonam (20.58%), co-trimoxazole (21.20%), gentamicin (14.93%), streptomycin (11.41%), ceftriaxone (8.18%), cefotaxime (6.92%), ciprofloxacin (4.85%) and imipenem (0.18%). Similarly, of the 1178 isolates obtained from unorganized farms, amoxicillin (87.52%) exhibited the highest level of resistance followed by cefalexin (71.05%), sulphafurazole (64.43%), piperacillin (49.66%), tetracycline (37.01%), cefexime (35.31%), co-trimoxazole (34.30%), trimethoprime (30.98%), ampicillin (25.09%), nalidixic acid (21.22%), ceftazidime (18.08%), gentamicin (17.57%), aztreonam (16.30%), cefotaxime (9.51%), ciprofloxacin (7.30%), streptomycin (6.96%), ceftriaxone (6.88%) and imipenem (0.25%).
 

Table 1: Antibiotic resistance profile of E. coli isolated from fecal samples of pig from organized and unorganized farms of 8 NER states of India


 

Table 2: Antimicrobial resistance profile of E. coli isolated from fecal samples of pig from organized and unorganized farms of the 8 states of North East India.


       
Amoxicillin is a widely used antibiotic against E. coli infection in man and animals (Xia et al., 2012). However, because of indiscriminate use, misuse and abuse of such antibiotic, bacteria developed the highest level of resistance, which makes the drug a completely irrelevant option against E. coli. As mentioned in Table 1, amoxicillin (84.81%) exhibited highest level of resistance, which is also in corroboration of the observation by Nijsten et al., (1996), who reported prevalence of E. coli isolates from pigs in the south of Netherlands with 92-100% resistance against amoxicillin.
       
The resistance pattern of E. coli isolates against all the antimicrobial agents are variable within the isolates obtained from different farms of the different states of NER. In this study, the resistance pattern of E. coli isolates against all the four third generation cephalosporins exhibited variable resistance (Table 1). National Antimicrobial Resistance and monitoring System (NARMS) report showed that resistance to ceftriaxone ranged from 6.3% to 13.5% among E. coli isolated from chickens during 2000-2008. In contrast to our result, Sasirekha et al., (2010) and Singh and Goyal (2003) reported that 84%, 75% and 85% E. coli were resistant to cefotaxime, ceftriaxone and ceftazidime, respectively. On the other hand, Rosengren et al., (2008) reported no resistant isolates against ceftriaxone and less than 1% resistant to cefoxitin and ceftiofur. Gebreyes et al., (2004) also mentioned that resistant strains are more commonly isolated from the pigs in the production system that used antimicrobials, including beta-lactams and drugs of the tetracycline class more often than from pigs in another type of large production system. With the availability of third and fourth generation of cephalosporins and without any restriction upon their use in veterinary and human health practice, a high level of resistance against them are increasingly evidenced (Myeyer, 2010; Moghnieh et al., 2019). Although, some antimicrobial agents are not in use for veterinary practices in India, but a high level of resistance against them is recorded in the fecal E. coli of swine. The persistence of resistance might result from the process of co-selection by other antimicrobials being used in pigs (Binch et al., 2008).
       
Interestingly, imipenem under-performed against E. coli isolates as compared to high susceptibility in other studies (Shams et al., 2018). Imipenem is not in use for veterinary practices in any of the North eastrern region (NER) states of India. Detection of low level of resistant E. coli in pig intestine is an indication of environmental transfer of such organism from human to animals. The reverse activity of zoonotic transfer of such organisms from animals to man may worsen the situation of treatment option in human practice. Decreased susceptibility to imipenem is a matter of great concern for treating infections caused by gram negative bacteria and indicates the urgent need for improved infection control strategies.
       
Most of the ESBL producing organisms were found to be co-resistant to flouroquinolones, aminoglycosides and co-trimoxazole, which corroborates with the study done by Jabeen et al., (2005) and Denholm et al., (2009). Perez et al., (2007) also reported the ESBLs producing enteric bacteria resistant to other group of antibiotics including aminoglycosides, tetracycline, sulfonamides, trimethoprim and chloramphenicol. Development of co-resistance against other antibiotics along with β-lactam antibiotics by the ESBLs producing organisms generally appeared in the large plasmids, where most of the resistance genes may co-exist. Extended-spectrum cephalosporins are important therapeutic agents in veterinary and human medicine and are often used as first line agents for invasive Gram-negative infections. The prevalence of third generation cephalosporin resistance in E. coli in pigs in this study suggests that these agents may be rapidly losing their efficacy as treatment option. Additionally, high rates of co-resistance to other classes of antimicrobial agents are further aggravating the situation. Over 10% of the isolates, which are resistant to third generation cephalosporins are also co-resistant to tetracycline, sulfonamides, trimethoprim, gentamycin, streptomycin, ciprofloxacin and aztreonam.
 
Confirmatory test for ESBLs
 
Of the 1,094 (47.75%) E. coli isolates found to be positive for suspected ESBL by antimicrobial sensitivity test, 654 (28.55%) were confirmed as ESBL producers by DDST screening method, of which 121 (41.44%), 60 (22.30%), 44 (14.86%), 108 (34.39%), 87 (33.72%), 73 (29.80%), 55 (23.61%) and 106 (27.60%) were from Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura, respectively (Table 3). Several studies from India and abroad have also reported the prevalence of ESBLs producers varying from 6.6% to 91% from time to time (Jain et al., 2003; Wattal et al., 2005; Bhattacherjee et al., 2008; Basavaraj et al., 2011). The wide variation in the prevalence of ESBLs producing organisms are probably due to the variation in the risk factors as well as the extent of antibiotics used. The relatively high rate of resistance to β-lactam antibiotics in primary screening test in comparison to the confirmatory DDST method may be because of different factors, viz., decreased affinity of the target penicillin binding proteins (PBPs), decreased permeability of the drug into the target cell or the antibiotic is attributed only to the production of β-lactamase enzymes (Sanders and Sanders, 1992; Al-Chharak et al., 2011; Jacoby et al., 2011).
 

Table 3: Detail results of in vitro and in vivo experimental horizontal gene transfer (HGT) between the selected E. coli (donors) and Salmonella spp. (recipient).


 
Genotypic detection of beta-lactamase gene blaCTX-M
 
Out of the 654 DDST positive E. coli isolates, 65 (2.84%) isolates were found to be positive for blaCTX-M gene. Amongst all the E. coli isolates from entire 8 NER states, a total of 10 (3.42%), 4 (1.49%), 4 (1.35%), 5 (1.59%), 13 (5.04%), 7 (2.85%), 6 (2.57%) and 16 (4.16%) isolates were found positive for blaCTX-M gene in Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura, respectively. Globally, the prevalence of CTX-M producing E. coli is varied between 0.8% - 3.4% (Adeleke et al., 2012; Senthilkumar, 2012). In some Danish farms, the production of CTX-M-1 β-lactamase in E. coli strains recovered from feces of pigs has been associated with ceftiofur use (Jørgensenet_al2007). On the other hand, significant decreases in the carriage prevalence of CTX-M-producing E. coli and fecal counts of CTX-resistant coliforms were detected during the pig production cycle (Hansen et al., 2013). The variation of results might be due to lower expression of CTX-M gene. blaCTX-M gene spread throughout the community, mostly through the transmission of plasmids, and some studies have also reported that animals may serve as a possible source for the dissemination of ESBL-encoding genes to humans (Dutta et al., 2013).
 
Cloning and sequencing
 
Nucleotide blast search of sequence of blaCTX-M (KF650780, KJ863555 and KJ863556) showed high degree of homology (99-100%) with the sequence reported for blaCTX-M-15. A phylogenetic and bootstrap analysis for the blaCTX-M gene showed minor nucleotide differences among the sequences. In silico translation showed non-synonymous amino acid sequence. There were three amino acid differences within the sequence fragment. There was substitution of ‘G’ (glycine: non-polar hydrophobic aa) by ‘D’ (Aspartic acid: charged amino acid) in isolates PE:34/ii and PE:137/iv. Then ‘R’ (Arginine) is changed with ‘H’ (histidine), both positively charged hydrophilic amino acid in isolate No. PE:34/ii and PE:137/iv and substitution of ‘N’ (aspargine) by ‘H’ in in isolate No. PE:34/ii. Antibiotic profile of those isolates (PE:34/ii and PE:137/iv) revealed wider zone of inhibition against cefotaxime and ceftriazone, which indicated that our isolates were similar to blaCTX-M-15 isolates reported from other sources and any differences in the activities towards various β-lactam antibiotics may be due to point mutations at selected places and the differences in the amino acid sequence leading to extended spectrum activity.
 
Horizontal gene transfer
 
The trans-conjugates were detected only by broth mating method, where the target gene was detected by PCR from the trans-conjugates but was absent in recipient strain. The frequency of transfer from donor to the recipient was between 3.6±2.07´10-8 to 4.4±2.88×10-8 trans-conjugants per donor for blaCTX-M gene. The pigs were tested prior to inoculation of the donor and/or recipient strains and no E. coli and/or Salmonella were isolated with the resistance characteristics of the donor, recipient or putative trans-conjugants strains. Both donor and recipient strains were detected within 24 hours from each pig after oral feeding. Recovered recipient organisms were tested for antibiotic susceptibility testing and produced similar pattern, which indicated the colonization of recipient strains in the pig intestinal tract. No pigs showed signs of clinical salmonellosis during the experiments.
       
Trans-conjugates were obtained from all the pigs and were resistant to cefotaxime and carrying the plasmids with target genes. Trans-conjugates were detected from the pigs with antibiotic treatment group from the 6th day post inoculation, whereas, it could be detected from the 8th day post inoculation from the group without any antibiotic pressure. The detection of trans-conjugates from both the groups could be done up to 10 days post inoculation. The target gene was detected from the trans-conjugates obtained from the plate, whereas it was absent in recipient strain, as detected by PCR assay. In case of in vivo trans-conjugation study without any antibiotic selection pressure, all the isolates positive for blaCTX-M gene could transfer their resistance trait to the recipient strain. But unlike the in vitro study, the frequency of transfer from donor to the recipient was between 5.6±2.3×10-4 to 6.8±3.35×10-4 trans-conjugants per donor for blaCTX-M gene. On the other hand, the frequency of transfer from all the donors to recipient was increased 6.6±3.05×10-5 to 7.2±1.92×10-5 trans-conjugants per donor under the antibiotic selection pressure. The frequency of transfer of resistance plasmids from donor to recipient may vary depending upon the copy number of the conjugative plasmids in the donor strain. In addition, the interspecies transfer of conjugative plasmids is less efficient than intra-species transfer. Failure or poor rate of transfer of resistance plasmid from donor to recipient by plate mating method is also reported by other workers (Faure et al., 2009).
       
Similar kind of experimental data on horizontal gene transfer with variable results are also published by several workers (Amavisit et al., 2003; Schjorring et al., 2008; Faure et al., 2009) using either gnotobiotic mice model or large animal model or even conventional colonization model to mimic the intestinal environment of human being. Using the animal model containing normal bacteria, flora always gives more realistic results than any in vitro or gnotobiotic study. The normal bacterial flora barrier and the present immune system give the used animal model advantages in mimicking the human gastrointestinal tract. Transfer of resistance gene under in vivo condition can’t be calculated from the extrapolation of in vitro experiments. The indigenous flora can act as a reservoir and transfer resistance genes to pathogenic bacteria that might lead to infections with limited treatment possibilities. Transfer of any microbial resistance genes is threat, but transfer to ESBL resistance genes is in a category of its own, which might result in the limitation of treatment and in worst cases treatment failure (Pitout and Laupland, 2008; Schjorring et al., 2008; Dhillon and Clark, 2012; Sing et al., 2017).
       
It may be stated that both clonal spread and transfer of transposable genetic elements might contribute to the proliferation of multidrug resistant ESBLs producing bacteria in the environment. Mobile drug resistance genes are capable of crossing bacterial species and are likely to accelerate dissemination of drug resistance between animals and humans through animal proteins or contact. It is, therefore, important to monitor the spread of extended spectrum cephalosporin resistant bacteria. Further studies of the basic mechanism for the evolution and dissemination of resistance as well as improved tools for the risk assessment of the spread of resistant organisms might enable us to control the global spread of antimicrobial resistance under the future influence of globalization.

ACKNOWLEDGEMENT

We are thankful to the Dean, College of Veterinary Sciences and Animal Husbandry, DBT project on ADMaC (No. DBT-NER/LIVS/11/2012) and DBT project on Institutional Biotech Hub (No. BT/B1/12/042/2007) for providing all the facilities to conduct the present work.

COMPETING INTEREST

Authors have declared that no competing interests exist.

REFERENCES


  1. Adeleke, O.E., Onyenwe, N.E., Mbata, T.L. (2012). Detection of blaCTX-M gene on resistant Salmonella enterica from a hospital in Southeast Nigeria. Internationall. Journal of Biochemistry and Biotechnology. 1(5): 162-166.

  2. Al-Charrakh, A.H., Yousif, S.Y., Al-Janabi, H.S. (2011). Occurence and detection of extended spectrum β-lactamases in Klebsiella isolates in Hilla,. Iraq, African Journal of Biotechnology. 10(4): 657-665.

  3. Amavisit, P., Lightfoot, D., Browning, G.F., Markham, P.F. (2003). Variation between Pathogenic Serovars within Salmonella Pathogenicity Islands. Journal of Bacteriology. 185(12): 3624-3635.

  4. Baker, S., Thomson, N., Weill, F.X., Holt, K.E. (2018). Genomic insights into the emergence and spread of antimicrobial-resistant bacterial pathogens. Science. 360(6390): 733-738.

  5. Basavaraj, M.C., Jyothi, P., Basavaraj, V.P. (2011). The prevalence of ESBL among Enterobacteriaceae in a Tertiary Care Hospital of North Karnataka, Indian Journal of Clinical Diagnostics. Research. 5(3): 470-475.

  6. Bhattacharjee, A., Sen, M.R., Prakash, P., Gaur, A., Anupurba, S. (2008). Increased prevalence of extended-spectrum β- lactamase producers in neonatal septicaemic cases at a tertiary referral hospital. Indian Journal of Medical. Microbiology. 26(4): 356-360.

  7. Binch, C.T.T., Heuer, H., Kaupenjohann, M., Smalla, K. (2008). Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. FEMS Microbiology and Ecology. 66: 25-37.

  8. Clinical and Laboratory Standards Institute CLSI. (2014). Performance standards for antimicrobial susceptibility testing. 28th ed. CLSI supplement M100. Wayne, PA, U.S.A.

  9. Collignon, P., McEwen, S. (2019). One health-its importance in helping to better control antimicrobial resistance. Tropical. Medicine and Infectious. Diseases. 4: 22.

  10. Denholm, J.T., Huysmans, M., Spelman, D. (2009). Community acquisition of ESBL-producing Escherichia coli: a growing concern. Medical Journal of Australia. 190(1): 45-46.

  11. Dhillon, R.H. and Clark, J. (2012). ESBLs: A Clear and Present Danger? Critical. Care Research and Practice. 625170.

  12. Dutta, T.K., Warjri, I., Roychoudhury, P., Lalzampuia, H., Samanta, I., Joardar, S.N., Bandyopadhyay, S., Chandra, R. (2013). Extended- Spectrum β- Lactamase producing Escherichia coli isolate possessing the Shiga Toxin Gene (stx1) belonging to the O64 serogroup associated with human disease in India. Journal of Clinical Microbiology. 51(6): 2008-2009.

  13. Faure, S., Guyomard, A.P., Delmas, J.M., Laurentie, M. (2009). Impact of therapeutic treatment with β-lactum on transfer of the blaCTX-M-9 resistance gene from Salmonella enterica serovar Virchow to Escherichia coli in gnotobiotic rats. Applied and Environmental. Microbiology. 75: 5523-5528.

  14. Gebreyes, W.A., Thakur, S., Davies, P.R., Funk, J.A., Altier, C. (2004). Trends in antimicrobial resistance phage types and integrons in Salmonella serotypes from pigs. Journal of Antimicrobial Chemotherapy. 53: 997-1003.

  15. Gniadkowski, M. (2001). Evolution and epidemiology of extended- spectrum β- lactamases (ESBLs) and ESBL-producing microorganisms. Clinical. Microbiology and Infection. 7: 597-608.

  16. Hansen, K.H., Damborg, P., Andreasen, M., Nielsen, S.S., Guardabassi, L. (2013). Carriage and fecal counts of cefotaxime M-producing Escherichia coli in pigs: a longitudinal study. Applied. Environmental. Microbiology. 79(3): 794-798.

  17. Holmes, A.H., Moore, L.S.P., Sundsfjord, A., Steinbakk, M., Regmi, S., Karkey, A., Guerin, P.J., Piddock, L.J. (2016). Understanding the mMechanisms and Ddrivers of Aantimicrobial Rresistance. Lancet. 387: 176-187.

  18. Jabeen, K., Zafar, A., Hasan, R. (2005). Frequency and sensitivity pattern of extended spectrum beta-lactamase producing isolates in a tertiary care hospital laboratory of Pakistan. Journal of Pakistan Medical. Association. 55: 436-439.

  19. Jacoby, G.A., Muniz-Price, L.S. (2005). The new β-lactamase. New England Journal of Medicine. 352: 380-391.

  20. Jain, A., Roy, I., Gupta, M.K., Kumar, M., Agarwal, S.K. (2003). Prevalence of extended spectrum beta-lactamase producing Gram negative bacteria in septicaemic neonates in a tertiary care hospital. Journal of Medical. Microbiology. 52: 421-425.

  21. Jørgensen, C.J., Cavaco, L.M., Hasman, H., Emborg, H.D., Guardabassi, L. (2007). Occurrence of CTX-M-1-producing Escherichia coli in pigs treated with ceftiofur. Journal of Antimicrobial. Chemotherapy. 59: 1040-1042.

  22. Kallen, R., Skurnik, D., Pier, G.B., Andremont, A., Szmigielska, A., Krzemien, G., Roszkowska-Blaim, M., Craig, J.C., Williams, G.J., Simpson, J.M. (2010). Antibiotic prophylaxis and recurrent urinary tract infection in children. New England Journal of. Medicine. 362: 555-556.

  23. Kong, K.F., Schneper, L., Mathee, K. (2010). Beta-lactam Antibiotics: From Antibiosis to Resistance and Bacteriology. APMIS. 118(1): 1-36.

  24. Lalzampuia, H., Dutta, T.K., Warjri, I., Chandra, R. (2013). PCR based detection of extended spectrum beta-lactamases (blaCTX-M and blaTEM) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae isolated from pigs in North Eastern India (Mizoram). Indian Journal of Microbiology. 53(3): 291-296.

  25. Lewis, J.S., Herrera, M., Wickes, B., Patterson, J.E., Jorgensen, J.H. (2007). First report of the emergence of CTX-M-type extended-spectrum β-lactamase (ESBLs) as the predominant ESBL isolated in a U.S. health care system. Antimicrobial. Agents and Chemotherapy. 51: 4015-4021.

  26. Lim, S.K., Lee, H.S., Nam, H.M., Cho, Y.S., Kim, J.M., Song, S.W., Park, Y.H., Jung, S.C. (2007). Antimicrobial resistance observed in Escherichia coli strains isolated from fecal samples of cattle and pigs in Korea during 2003-2004. International. Journal of Food Microbiology. 116: 283-286.

  27. Manoharan, A., Premalatha, K., Chatterjee, S., Mathai, D. (2011). Correlation of TEM, SHV and CTX-M extended-spectrum β-lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility. Indian Journal of Medical. Microbiology. 29(2): 161-164.

  28. Mathai, D., Manoharan, A., Vasanthan, G. (2009). Epidemiology and implications of ESBL. Critcal. Care Update. 14: 152-162.

  29. Meyer, E. (2010). Dramatic increase of third-generation cephalosporin-resistant E. coli in German intensive care units: secular trends in antibiotic drug use and bacterial resistance, 2001 to 2008. Critical. Care Update. 14: R113.

  30. Moghnieh, R., Araj, G.F., Awad, L., Daoud, Z., Mokhbat, J.E., Jisr, T., Abdallah, D., Azar, N., Irani-Hakimeh, N., Balkis, M.M., Youssef, M., Karayakoupoglou, G., Hamze, M., Matar, M, Atoui, R., Abboud, E., Feghali, R., Yared, N., Husni, R. (2019). A compilation of antimicrobial susceptibility data from a network of 13 Lebanese hospitals reflecting the national situation during 2015-2016. Antimicrobial. Resistanc and Infection. Control. 8: 41-49.

  31. Nijsten, R., London, N., van den Bogaard, A., Stobberingh, E. (1996). Antibiotic resistance among Escherichia coli isolated from faecal samples of pig farmers and pigs. Journal of Antimicroial. Chemotherapy. 37 (6):1131-1140.

  32. Perez, F.J., Endimiani, A., Hujer, K.M., Bonomo, R.A. (2007). The continuing challenge of ESBLs. Current Opinion in Pharmacology. 7(5): 459-46.

  33. Pérez, J.F. and Hanson, N.D. (2002). Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. Journal of Clinical. Microbiology. 40(6): 2153-2162.

  34. Pitout, J.D.D., Laupland, K.B. (2008). Extended-spectrum beta-lactamase producing Enterobacteriaceae: An emerging public health concern. Lancet Infectious. Diseases. 8: 159-166.

  35. Rosengren, L.B., Waldner, C.L., Reid-Smith, R.J., Checkley, S.L., McFall, M.E., Raji´c, A. (2008). Antimicrobial resistance of fecal Escherichia coli isolated from grow-finish pigs in 20 herds in Alberta and Saskatchewan. Canadian Journal of Veterinary. Research. 72:160-167.

  36. Salinas, L., Cárdenas, P., Johnson, T.J., Vasco, K., Graham, J., Trueba, G. (2019). Diverse commensal Escherichia coli clones and plasmids disseminate antimicrobial resistance genes in domestic animals and children in a semirural community in Ecuador. mSphere. 4: e00316-19.

  37. Sanders, C.G., Sanders, W.E. (1992). β-lactam resistance in Gram negative bacteria: global trends and clinical impact. Clinical. Infectious. Diseases. 15: 824-839.

  38. Sasirekha, B., Manasa, R., Ramya, P., Sneha, R. (2010). Frequency and antimicrobial sensitivity pattern of extended spectrum β-lactamases producing E. coli and Klebsiella pneumoniae iIsolated in a Tertiary Care Hospital, Al Ameen. Journal of Medical. Sciencs. 3(4): 265-271.

  39. Schjorring, S., Struve, C., Krogfelt, K.A. (2008). Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine. Journal of Antimicrobial. Chemotherapy. 62: 1086-1093.

  40. Schmitt, J., Jacobs, E., Schmidt, H. (2007). Molecular characterization of extended-spectrum β-lactamases in Enterobacteriaceae from patients of two hospitals in Saxony, Germany. Journal of Medical. Microbiology. 56: 241-249.

  41. Senthilkumaran, K. (2012). Isolation and detection of antimicrobial susceptibility pattern and ESBL production of Escherichia coli in children with gastroenteritis. Internationall. Journal of Pharmacology. Bioscience. 3(4): 32-34.

  42. Shams, S, Hashemi, A, Esmkhani, M, Kermani, S, Shams, E, Piccirillo, A. (2018). Imipenem resistance in clinical Escherichia coli from Qom, Iran. BMC Research Notes. 11(1):314-321.

  43. Singh, A.S., Lekshmi, M., Prakasan, S., Nayak, B.B., Kumar, S. (2017). Multiple Antibiotic Resistant, Extended Spectrum beta-Lactamase (ESBL)-Producing Enterobacter in Fresh Seafood. Microorganisms. (3): 3-9.

  44. Singh, N.P., Goyal, R. (2003). Changing trends in bacteriology of burns in the burns unit, Delhi, India. Burns. 29(2): 129-132.

  45. Sun, Y., Zeng, Z., Chen, S., Ma, J., He, L., Liu, Y., Deng, Y., Lei, T., Zhao, J., Liu, J.H. (2010). High prevalence of CTX-M extended spectrum β-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. Clinical. Microbiology. Infection. 16: 1475-1481.

  46. Wattal, C., Sharma, A., Oberoi, J.K., Datta, S., Prasad, K.J., Raveendra, R. (2005). ESBL– An emerging threat to antimicrobial therapy. Microbiology Newsletter. 10(1): 1-8.

  47. Xia, L.N., Zhao, H.Q., Su, Y., Guo, Q.Y., Chen, T.T., Gao, P. (2012). Resistance survey of Escherichia coli isolates to antibiotics from hoggery in Xinjiang. Xinjiang Agricultural. Science. 12: 27-29.
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