1.Introduction

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Extended-spectrum β-lactamases (ESBLs) are bacterial enzymes that catalyse the hydrolysis of ß-lactam antibiotics, including third generation cephalosporins (e.g. ceftazidime or cefotaxime) and monobactams (aztreonam) [1]. ESBLs represent the most common mechanism of antibacterial resistance in Gram-negative bacilli. With regard to their primary sequence homology [2] and their substrate profiles [3], ESBLs are categorized into classes and groups, whereby the majority belong to Ambler class A and to the Bush group 2be. The classical plasmid-mediated TEM- and SHV-ESBLs, which derived from point mutations in the structural genes of their precursors TEM-1, TEM-2, and SHV-1, were originally detected in the early 1990s in human clinical isolates associated with nosocomial infections [4]. Today, the most important ESBLs belong to the CTX-M family of enzymes which are cefotaximases that originated through mobilization of chromosomal blaCTX-M genes from environmental Kluyvera spp. into mobile genetic elements such as transposons and plasmids [5]. The CTX-M enzymes are classified according to their amino acid similarities into the five major groups CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 [6]. In recent years the rapid and global spread of CTX-M enzymes throughout the human population, food animals, wildlife and the environment has been recognized as a major threat to human and animal health [5]. Food producing animals may serve as a reservoir for ESBL producing Escherichia coli (E. coli), which can then be transmitted to humans either through direct contact or through meat contaminated during the slaughtering process. This represents a risk to human health, as E. coli is not only an important opportunistic pathogen for humans, but may also colonize the human gut as a commensal and transfer resistance genes to pathogenic bacteria [7]. In the EU, CTX-M-1 is the most frequently reported ESBL subtype in E. coli originating from food producing animals (poultry, cattle and pigs) and food [8]. By contrast, in Asia, CTX-M-2, CTX-M-14 and CTX-M-15 are found in cattle and pigs [9].

Bhutan is a small developing country located in the eastern Himalayas. There is a scarcity of data regarding the occurrence of antibiotic resistant bacteria among the human population as well as among animals. In 2013, a study investigating human clinical isolates originating from urinary tract infections revealed that 31.8% of the E. coli isolates were resistant to cefotaxime [10]. A very recent report confirmed the presence of CTX-M-15 in a collection of multidrug resistant human clinical isolates in Bhutan [11]. No data has been available so far to document the occurrence of ESBL producing E. coli in food producing animals in Bhutan. The aim of this study was to evaluate the presence of ESBL producing E. coli in suckling pigs and weaners on three pig breeding farms in Bhutan, and to characterize the isolated strains by (i) antibiotic susceptibility testing, (ii) identification of the extended-spectrum ß-lactamase genes (bla_ESBL), (iii) classification of the isolates into phylogenetic groups and multilocus sequence types (MLST).

2. Materials And Methods

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2.1. Collection of Samples

The investigation involved three governmental pig-breeding farms (farm 1: Yusipang; farm 2:

Gelephu; and farm 3: Lingmethang, respectively) in Bhutan. A total of 83 faecal samples were collected (farm 1: n = 11; farm 2: n=34; and farm 3: n=38). The samples were sent to the National Centre for Animal Health, Thimphu, Bhutan for the isolation of E. coli.

2.2. Microbiological Procedures

E. coli was isolated according to standard procedures on MacConkey agar. From each sample, one suspected colony with typical E. coli morphology was selected for further analysis. Identification of isolates was performed using in-house classical biochemical methods at the National Centre for Animal Health, Bhutan. Thereafter, all E. coli isolates were sent to the Institute for Food Safety and - Hygiene, Zürich, Switzerland for further characterization.

The detection of ESBL producers was performed using chromogenic Brilliance ESBL agar plates (Oxoid, Hampshire, UK). Plates were incubated at 37°C for 24 h under aerobic conditions. Colonies were picked from the selective plates and subcultured on Mueller Hinton agar plates (Becton Dickinson, Allschwil, Switzerland) at 37°C for 24 h. Isolates were subjected to susceptibility testing against 13 antimicrobial agents by the disc diffusion method according to CLSI protocols and evaluated according to CLSI criteria [12]. The panel included ampicillin (AM), amoxicillin-clavulanic acid (AMC), cephalothin (CF), cefotaxime (CTX), nalidixic acid (NA), ciprofloxacin (CIP), gentamicin (GM), kanamycin (K), streptomycin (S), sulfamethoxazole (SMZ), trimethoprim (TMP) tetracycline (TE), and chloramphenicol (C) (Becton Dickinson, Heidelberg, Germany).

Multidrug resistance (MDR) was defined as such for isolates that were resistant to three or more classes of antimicrobials, counting ß-lactams as one class.

2.3. Molecular Biological Analysis of ß-Lactamase Genes

Isolates identified as potential ESBL producers were selected for further analysis. DNA was extracted by a standard heat lysis protocol and analysed by using the polymerase chain reaction (PCR) technique for the presence of bla genes. Synthesis of primers and DNA custom sequencing was carried out by Microsynth (Balgach, Switzerland). Purification of amplicons was done using a PCR purification kit (Qiagen Courtaboeuf, France).

Screening for blaCTX-M alleles belonging to CTX-M groups 1, 2, 8, 9, and 25 was performed as described by Woodford et al. [13]. Amplicons for sequencing individual open reading frames were generated using primers described by Geser et al. [14] Screening for blaTEM and blaSHV was carried out using primers described previously [15].

2.4. Phylogenetic Classification of E. coli Isolates

DNA from isolates was subjected to triplex PCR targeting the chuA gene, the yjaA gene and an unspecified DNA fragment termed TspE4.C2, as described by Clermont et al. [16]. Each isolate was assigned to one of the four phylogenetic groups designated A, B1, B2 or D. Group A and B1 typically contain commensal E. coli strains while groups B2 and D consist of virulent extra-intestinal strains [17].

2.5. Multilocus Sequence Typing

For multilocus sequence typing of the isolates, internal fragments of the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were amplified by PCR, as described by Wirth et al. [18]. Sequencing of the amplification products was performed by Microsynth (Balgach Switzerland). Sequences were imported into the E. coli MLST database website (http://mlst.ucc.ie/mlst/dbs/Ecoli) to determine MLST types.

2.6. Plasmid Incompatibility Typing

Plasmids were assigned to incompatibility groups on the basis of the presence of specific replicon sequences identified by PCR [19].

3. Results and Discussion

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Of the 83 E. coli isolates investigated in this study, two (2.4%) were ESBL producers. PCR amplification and sequencing of the bla_ESBL genes revealed blaCTX-M-15 in both isolates. In addition, both isolates harboured additionally the ß-lactamase blaTEM-1 and both were MDR (Table 1). This is the first report of the detection of blaCTX -M-15 harbouring E. coli in food producing animals in Bhutan. While none of the isolates belonged to extra-intestinal pathogenic phylogenetic groups B2 or D, B1 was identified in both strains. The detection of phylogenetic group B1 suggests that CTX-M-15 producers may have become established among commensal E. coli in the intestinal flora of pigs in Bhutan. As described in previous studies, this may be a consequence of the use of ß-lactam antibiotics in the swine industry [20,21]. The isolates contained plasmids which were assigned to plasmid incompatibility groups IncY((strainB2 L43 (S) and colE (strainB3 GS8 (S)). Strain B2 L43 (S) from farm 3 belonged to ST4173 and strain B3 GS8 (S) from farm 2 to ST156. Of particular interest is the description of CTX-M-15 producing E. coli belonging to ST156, which belongs to the clonal complex (CC) 156. This clone has been detected globally as a CTX-M-15 producer in sea gulls [22], water fowl and river water in Bangladesh [23], as well as in broilers and turkeys on farms in the United Kingdom and Italy [24,25]. The detection of this clone in pigs provides further evidence for its dissemination among animals.

Table 1

Compared to other countries, ESBL producing E. coli appear to be rare in human clinical isolates and in animal isolates in Bhutan. However, an increase of ESBLs, including CTX-M-15 in bacteria is to be expected in future. In order to limit the dissemination of multidrug resistant bacteria, enhanced surveillance, transmission control strategies and antimicrobial stewardship in human and veterinary medicine are of major importance.

4.Conclusion

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The results of the present study show that ESBL producing E. coli have emerged among breeding pigs in Bhutan. In addition, the isolates were found to be MDR. Monitoring for ESBL producing bacteria should be continued in humans and animals in order to protect public health.

5.Acknowledgements

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We would like to thank the National Piggery Research and Development Centre, Gelephu; Regional Pig Breeding Centre, Yusipang; and Regional Pig and Poultry Breeding Centre, Lingmethang under the Ministry of Agriculture and Forests, Thimphu, Bhutan for the access to the farms.