Detection of extended-spectrum β-lactamases (blaCTX-M-1 and blaTEM) in Escherichia coli, Salmonella spp., and Klebsiella pneumoniae isolated from poultry in North Eastern India

Aim: The present study was conducted to record the association of extended spectrum β-lactamases (ESBLs) producing enteric bacteria with diarrhea of poultry birds in Mizoram, India. Materials and Methods: Fecal samples were collected from poultry birds with the history of diarrhea from different parts of Mizoram. Samples were processed for isolation and identification of Escherichia coli, Salmonella, and Klebsiella pneumoniae. All the isolates were subjected to antibiotic sensitivity assays. Phenotypically, ESBLs production ability was determined by double discs synergy test (DDST) method. ESBLs producing isolates were subjected to polymerase chain reaction (PCR) for detection of ESBLs genes. Plasmids were cured by acridine orange. Transfer of resistance from donor to recipient strains was done by in vitro horizontal method. Results: A total of 134 enteric bacteria was isolated, of which 102 (76.12%), 21 (15.67%) and 11 (8.21%) were E. coli, Salmonella spp. and K. pneumoniae, respectively. By DDST 7 (5.22%) isolates (6 E. coli and 1 K. pneumoniae) were ESBLs producer. PCR analysis confirmed 5 (3.73%) (4 E. coli and 1 K. pneumoniae) isolates harboured blaCTX-M-1 gene and/or blaTEM gene. All the isolates were carrying plasmids ranging between 0.9 kb and ~30 kb. Of the 4 isolates positive for blaCTX-M-1 and/or blaTEM, 2 (1.84%) were confirmed for blaCTX-M-1 gene in their plasmid. No blaTEM gene was detected from plasmid. The resistance plasmid could not be transferred to the recipient by in vitro horizontal gene transfer method. Conclusion: ESBLs producing enteric bacteria are circulating in poultry in North Eastern Region of India. As poultry is one of the most common food animals in this region, these organisms may enter in human population through them.


Introduction
The rapid emergence of extended-spectrum β-lactamases (ESBLs) in the food-producing animals has been recorded and published worldwide [1][2][3][4][5]. It is important to note its impact on the treatment and therapeutic strategy of serious infections [6][7][8][9][10]. Food animals, including poultry are one of the most important sources of development of multi-drug resistant (MDR) bacteria because of continuous use of antibiotics as feed additives and growth promoting factors in a sub-therapeutic level [11][12][13]. This practice may lead to selection of a resistant population in the native microbiota of the animal and the local environment due to shedding through feces. The MDR bacteria may re-enter the human and animal populations through various routes including natural water, irrigation water, drinking water, vegetables, and foods.
Till date, no authentic data are published regarding the association of ESBLs producing enteric bacteria in poultry in North Eastern Region (NER) of India. Hence, the present study is conducted to detect and characterize the ESBLs producing enteric bacteria in poultry in NER of India.

Ethical approval
The work has been carried out after obtaining the approval from the Institutional Animal Ethical Committee, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram.

Bacterial isolates
A total of 51 fecal samples from poultry birds of either sex, irrespective of the age was collected from various districts of Mizoram between September 2011 and March 2012. The birds were reared under different housing system and belonged to organized as well as unorganized farms. Samples were collected using sterilized dry absorbent cotton swab. However, for collection of samples from distant locations, a sterilized swab dipped in nutrient broth was used as a transport medium. Each swab containing the sample was then inserted into separate sterilized test tube, plugged with non-absorbent cotton and carried to the laboratory under cold chain for further processing. For the isolation of Escherichia coli and Klebsiella pneumoniae, the collected fecal samples were inoculated on MacConkey's agar and single colonies were selected and confirmed by standard bacteriological technique. For the isolation of Salmonella spp., samples were first enriched in selenite F broth and streaked on Salmonella Shigella Agar plate. Pure colonies were then selected and identified as per standard bacteriological technique [14].

Phenotypic detection of ESBLs
All the isolates were subjected to in vitro antibiotic sensitivity test by disc diffusion method against commonly used antibiotics as per the recommendation of Clinical Laboratory Standard Institute [15]. The antibiotics used for the experiment were ceftriaxone, cefotaxime, cefixime, cefazolin, cephalexin, ampicillin, erythromycin, chlortetracycline, streptomycin, enrofloxacin, oxytetracycline, and lincomycin. The isolates conferring resistance to the extended spectrum cephalosporin group of antibiotics were selected for confirmation of ESBLs production, which was done by placing cefotaxime and cefotaxime/clavulanate discs on the inoculated Muller Hinton Agar plate at a distance of 30 cm apart. It was incubated overnight, and the increase zone size of more than 5 mm was considered as positive for ESBLs production.

Characterization of ESBLs producing isolates
Bacterial lysate was prepared from all the isolates found to be positive for ESBLs production phenotypically, and were tested for the presence of bla CTX-M-1 and bla TEM genes by polymerase chain reaction (PCR) assay using specific primers (Table-1). PCR was carried out in a 0.2 ml thin-wall PCR tubes using the bacterial lysate as template DNA with a final volume of 25 μl containing 1× buffer, 1.5 mM MgCl 2 , 200 pM of each oligonucleotide primers, 200 μM of each dNTPS, 1 U of Taq polymerase and 4.0 μl DNA lysate. PCR was carried out in a thermal cycler and the cycling condition for bla CTX-M-1 was: Initial denaturation at 94°C for 7 min followed by 30 cycles of amplification with denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s, ending with a final extension at 72°C for 5 min. For bla TEM gene, the annealing temperature was 53°C.
Multiplex PCR was carried out using the same composition of PCR reaction mixture mentioned above. However, the annealing temperature was set to 54°C.

Extraction of plasmid and genomic DNA
Plasmid DNA was extracted as per the method described by Sambrook and Russel [16] and the chromosomal DNA was extracted as per the method of Nazik et al. [17] from the isolates harboring the ESBLs genes. PCR was performed using the plasmid and chromosomal DNA separately following the above-mentioned settings to find out the location of the target genes.

Curing of plasmid
All the isolates, carrying bla CTX-M-1 and/or bla TEM genes in their plasmid were subjected to curing using acridine orange as curing agent following the method described by Silhavy et al. [18] with suitable modifications. In brief, 0.2 ml of overnight culture was inoculated in 5 ml LB broth containing different concentrations (2.5, 1.25, 1.0, 0.7, 0.5, 0.25, and 0.1 mg/ml) of acridine orange. Positive control contained only cells without acridine orange, while negative control contained only acridine orange without cells. All the tubes were incubated (in dark) at 37°C overnight. Next day tubes containing the highest concentration of acridine orange showing growth were selected, and loop-full was streaked on MacConkey's agar plates and incubated overnight.

Horizontal gene transfer
The ability of transfer of antibiotic resistance genes within Enterobacteriaceae group of bacteria was recorded by in vitro conjugation study. E. coli isolates harboring the ESBLs gene were used as donor and Salmonella enteritidis (ATCC 13076), which was made resistant to nalidixic acid was used as recipient strain. The recipient strain was sensitive to cefazolin, cephalexin, ceftriaxone and cefotaxime and was not carrying bla CTX-M-1 and/or bla TEM genes in its plasmid as confirmed by PCR analysis. In vitro mating experiments were performed by broth mating [19], filter paper mating [20] and plate mating [21,22]. Transconjugants were selected on Mac Conkey's agar containing ceftriaxone (50 μg/ml) and nalidixic acid (100 μg/ml). Donor and recipient strains were grown separately in antibiotic-free medium as well as antibiotic medium as control. Selected transconjugants were further characterized for their antimicrobial susceptibility, ESBLs phenotype and presence of bla CTX-M-1 and/or bla TEM genes by PCR.

Sample collection and bacterial isolates
In the present study, a total of 51 fecal samples was collected from various districts of Mizoram, of which 30 (58.82%) were collected from organized farms and 21 (41.18%) were from local backyard farms.

Genotypic characterization of β-lactamase genes
Of the 7 phenotypically positive isolates screened for the presence of bla genes by PCR using bacterial lysate as template DNA, 5 (3.73%) were found to be positive for bla CTX-M-1 gene (950 bp) and/or bla TEM gene (1080 bp). Four isolates (3 E. coli and 1 K. pneumoniae) were positive for bla CTX-M-1 gene, of which 3 were having an additional bla TEM gene. Only 1 E. coli isolate was found to be positive for bla TEM gene alone ( Figure-1 and Table-3).

Plasmid profiling and curing
Agarose gel electrophoresis of the extracted plasmids showed that all the isolates were carrying plasmids ranging between 0.9 kb and ~30 kb (Figure-2). Of 7 isolates positive for bla CTX-M-1 and / or bla TEM genes, 2 isolates were confirmed to harbor the bla CTX-M-1 gene in their plasmid. Only one bla TEM gene was detected in plasmid.
Using acridine orange (1.25-1.5 mg/ml), the E. coli isolates were successfully cured. Confirmation of curing was done by disc diffusion assay, where the organism showed 100% sensitivity against all the antibiotics; plasmid extraction could not trace any plasmids and by PCR assay, no ESBLs genes could be detected.

In vitro horizontal gene transfer
The resistance trait from any of the isolates could not be transferred to the recipient host. The transconjugants remained sensitive to cefixime, cefazolin, cephalexin, ceftriaxone, cefotaxime, enrofloxacin, oxytetracycline, streptomycin, ampicillin, chlortetracycline, erythromycin, and lincomycin. Transconjugants were also found negative for bla CTX-M-1 and bla TEM genes by PCR assay.

Discussion
The present study revealed that bla CTX-M-1 is the most abundant ESBLs type in this region, with E. coli being the major ESBLs producer, which is in accordance with the reports of other investigators from other places [1,3,23]. Furthermore, the presence of more than one bla genes is often reported worldwide [1,23].
Prevalence of CTX-M genes along with TEM gene has been often reported by various workers [5,27,28]. The prevalence of CTX-M may be due to wide use of third generation cephalosporins especially ceftriaxone and cefotaxime or may be associated with high mobilization of the encoding genes [29]. Barlow et al. [30] reported that the bla CTX-M genes have been mobilized to plasmid almost 10 times more frequently than other Class A β-lactamases. The predominance of CTX-M type of ESBLs gene may be an indication of this being common in North East Region of India. Muzaheed et al. [31] also reported high prevalence of CTX-M genes in K. pneumoniae and E. coli from Southern India.
Of the 5 ESBLs positive isolates, 4 isolates were from organized farm. Only 1 positive isolate (AE45) was recorded from the sample collected from local backyard farm. The predominance of ESBLs positive isolates from organized farms may be due to the frequent and routine use of third-generation antibiotics. The prevalence of ESBLs in the farm animals is also reported by other workers from various parts of the world [1][2][3].
Most of the ESBLs producing organisms under this study were also found to be co-resistant to fluoroquinolones, aminoglycosides as well as co-trimoxazole, which is in accordance with the study done by Denholm et al. [32] and Randall et al. [33]. Perez et al. [9] also reported similar results, where the ESBLs producing enteric bacteria were also 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 resistant genes may co-exist.
In the present study, the resistant plasmids could be successfully cured by acridine orange. Although curing provides only the preliminary evidence that genetic traits are of extra-chromosomal nature, loss of growth on antibiotic containing plates shows that the MDR genes may be plasmid-borne. The resistance determining traits are often transposable, which exist in both plasmid and chromosomal locations (flip-flop mechanism) [34]. It is, however, important to note that all antibiotic resistance genes are not plasmid-mediated [35]. The copies of the plasmid lying closer to the membranes are readily eliminated by chemical agents, while those lying closer to the nucleus may escape the curing effect, thereby; one may observe partial curing [36].
During conjugation study, neither of the plasmids carrying any one of the target gene could be transferred horizontally to the recipient isolate. Similarly, low transconjugation success was also reported by other workers in Switzerland and Germany [19,37].
Franiczek et al. [38] also reported that none of the 4 E. coli isolates could transfer their resistance gene to other recipient strains. Yuan et al. [39] reported that plasmids are transferred under the influence of environmental condition (in vitro vs. in vivo). In vitro experiment showed transfer of the plasmids ranging from 108 to 157 kb, while in vivo conjugation experiment showed a transfer of smaller sized plasmids. Failure of conjugation in the present study may be because of the small size plasmids carried by the donors. Involvement of insertion sequence ISEcp1 in the mobility of bla CTX-M has also been confirmed by studies of plasmids [40,41]. Hypothetically, the plasmid may be of different incompatibility group other than IncI1 incompatibility group because most of the conjugative plasmids generally belonged to IncI1 incompatibility group [42,43].

Conclusion
ESBLs producing E. coli, Salmonella spp., and K. pneumoniae, were recorded from poultry birds in Mizoram. PCR analysis confirmed 3.73% isolates harboring bla CTX-M-1 gene and/or bla TEM gene. bla CTX-M-1 gene was detected in the plasmid. Transmission of such organisms to humans via the food chain cannot be dismissed. More strict veterinary antibiotic policies are needed in order to prevent emergence and dissemination of these strains among animals and humans.

Authors' Contributions
HL collected the samples, extracted nucleic acids, detected virulence genes and ESBLs genes. TKD designed the program, analyzed the data, prepared the manuscript. IW isolated the organisms, conducted antibiotic assays. RC monitored the program, prepared and edited the manuscript. All authors read and approved the final manuscript.

Isolates
Zone of inhibition (in mm)