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Pig farm environment as a source of beta-lactamase or AmpC-producing Klebsiella pneumoniae and Escherichia coli

Annals of Microbiology volume 68, pages 781–791 (2018)Cite this article

Abstract

The present study was undertaken to detect the occurrence of beta-lactamase-/AmpC-producing Klebsiella and Escherichia coli in healthy pigs, feed, drinking water, and pen floor or surface soil. The study also intended to detect the clonal relationship between the environmental and porcine isolates to confirm the route of transmission. Rectal swabs and environmental samples were collected from apparently healthy pigs kept in organized or backyard farms in India. The pigs had no history of antibiotic intake. Production of phenotypical beta-lactamase, associated genes, and class I integron gene was detected in E. coli and Klebsiella isolates. The phylogenetic relationship among the isolates was established on the basis of Random amplification of polymorphic DNA banding pattern. Beta-lactamase-producing Klebsiella were isolated from healthy pigs (20.0%), pen floor swabs/surface soil swabs (14.0%), and drinking water (100%). Escherichia coli isolated from healthy pigs (14.4%), pen floor/surface soil (8.0%), and drinking water (33.3%) were detected as beta-lactamase producers. Majority of beta-lactamase-producing isolates possessed blaCTX-M-9. Further, 35 (81%) Klebsiella and all the E. coli isolates were detected as AmpC beta-lactamase ACBL producers and possessed blaAmpC. Sixteen beta-lactamase-producing Klebsiella (37.20%) and 13 E. coli (86.67%) possessed class I integron. Few resistant isolates from environmental sources (surface soil swab and drinking water) and the studied pigs were detected within the same cluster of the dendrogram representing their similarities. The study indicated about the possible role of contaminated environment as a source of beta-lactamase/AmpC-producing Klebsiella and E. coli in pigs.

Introduction

Environment plays a significant role in emergence and spread of antibiotic resistance determinants. The environmental “resistome” is created with exposure of environmental bacteria to antibiotics, shedding of resistant bacteria into environment from human, livestock or other resources, and transfer of resistance genes from donor to recipient bacteria (Wright 2010). Anthropogenic activities since industrialization released bulk amount of antibiotics into the environment through wastewater effluents, farm animal manures, treatment of crop diseases, and aquaculture (Czekalski et al. 2012). The environmental bacteria although carried genes for antibiotic resistance even before the “antibiotic era,” such that evolution of class A β-lactamase was detected 2.4 billion years ago with emergence of active on Cefotaxime, first isolated in Munich CTX-M progenitors around 200–300 million years ago (D’Costa et al. 2011).

Rapid dissemination of antibiotic resistance mostly depends on horizontal gene transfer between competent bacterial population, i.e., either from fecal bacteria to environmental isolates or vice versa (Baquero et al. 2008). Extended spectrum beta-lactamases (ESBLs) are well documented resistance determinants acquired through horizontal gene transfer (Carattoli 2013). ESBLs belong to clinically important class A β-lactamase enzymes, found in Enterobacteriaceae, frequently in Klebsiella pneumoniae and Escherichia coli. It can confer resistance to a variety of β-lactam antibiotics, including penicillins, 2nd-, 3rd-, and 4th-generation cephalosporins and monobactams (e.g., aztreonam), but usually not the carbapenems or the cephamycins (e.g., cefoxitin) (EFSA Panel on Biological Hazards 2011). Moreover, AmpC β-lactamase-producing organisms can produce resistance against cephalosporins, penicillins, cephamycins, monobactams, and also against β-lactamase inhibitors such as clavulanic acid. There are three classical ESBLs, i.e., TEM (except TEM-1), sulphydryl variable SHV (except SHV-1 and 2), and CTX-M (EFSA Panel on Biological Hazards 2011). Among them, CTX-M is observed as the most prevalent type in clinically infected humans throughout the world (Carattoli 2013). The origin of CTX-Ms was detected in Kluyvera species, an environmental bacterium, present abundantly in water, soil, sewage, hospital sinks, and animal originated food items (Forsberg et al. 2012). Four major groups of CTX-M enzymes (1, 2, 8, and 9) identified in clinical isolates were originated from different species of Kluyvera (Humeniuk et al. 2002). AmpC β-lactamases were initially detected as intrinsic cephalosporinase found in the chromosome of gram-negative bacteria (“chromosomal”) as well as acquired by plasmids, known as “plasmidic” AmpCs (Liebana et al. 2012).

In human, ESBL or AmpC-producing Klebsiella pneumoniae or E. coli causes nosocomial or community acquired infections associated with increased morbidity and mortality, prolonged hospital stays, and subsequent economic burden (Schwaber and Carmeli 2007). Role of environment was evidenced earlier in transmission of human infection with antimicrobial resistant bacteria such as methicillin-resistant Staphylococcus aureus (Hower et al. 2013). Similarly, transmission of ESBL-producing Klebsiella or E. coli in a community is also dependent on environmental “resistome,” in which livestock farm plays a significant role (Gao et al. 2015; von Salviati et al. 2015). Earlier works documented the spread of ESBL-producing organisms from farms to the community through air, water, and crops irrigated with contaminated water and flies (Søraas et al. 2013; Njage and Buys 2015; Solà-Ginés et al. 2015).

There is always scarcity of data for identification of environmental risk factors associated with transmission of ESBL-/AmpC-producing Enterobacteriaceae in livestock farms such as pig farms. A few reports are available where the studies confirmed emission of ESBL-producing bacteria from the pig farms to the surrounding environment but excluded the possibility of transmission from the environment (Gao et al. 2015; von Salviati et al. 2015). The present study was undertaken to detect the occurrence of ESBL-/AmpC-producing Klebsiella and E. coli in healthy pigs kept in organized or backyard farms, their feed, drinking water, and pen floor swabs or surface soil swabs from roaming arena. The study was also intended to reveal the occurrence of class I integron genes in Klebsiella and E. coli isolates along with their clonal relationship. The study was conducted with the samples from different agro-climatic zones of West Bengal, a major pig rearing state in India. The pigs are reared due to high consumption of pork observed among the northeast inhabitants and Chinese people living in the state (Tiwari and Arora 2005).

Materials and methods

Collection of samples

The rectal swabs were collected from apparently healthy pigs of either sex kept in farms (organized or backyard) at three different agro-climatic zones of West Bengal, India during 2017. The organized farms were selected on the basis of production level, whereas the backyard farms were selected on the basis of convenient location and willingness of the farmers to participate in the study. The rectal swabs were collected from the pigs selected randomly from the farms. The pigs of organized farms were 1–2 years old with no history of antibiotic intake with feed. The indigenous pigs belonged to 5–6 months age, and all the studied backyard farms had no history of costly antibiotic intake (cephalosporins) either with feed or with during therapy. Occasionally the pigs were treated with tetracycline or gentamicin by local veterinarian or paraveterinarians. The organized piggeries kept the pigs in brick-made pens with concrete floors and asbestos shed. The backyard pigs were kept in houses during night only made of bamboo and jute-stick with earthen floors. The organized piggery and backyard farm offered the feed and water in cement-casted and earthen mangers, respectively. Properly treated water was not used in both the types of farms. The pigs kept in backyard farms roamed in a fenced place during daytime.

The rectal swabs (n = 120) and pen floor swabs/swabs from surface soils of roaming arena (backyard pigs) (n = 48) were collected with the help of sterile cotton swab sticks (HiMedia, India) in sterile vials containing transport medium (HiMedia, India) as per the standard guideline (OIE 2002). Drinking water (n = 10) and feed samples (n = 6) were collected randomly from the studied pig farms into sterile vials containing transport medium (Table S1, supporting information). Feed and drinking water samples were collected in replicate in consecutive days from three different agro-climatic zones. All the samples collected were brought into the laboratory maintaining the cold chain for further examination. The study was approved by Institutional Biosafety Committee.

Isolation and Identification of Klebsiella spp. and E. coli from collected samples

The samples collected from the pigs and environment were inoculated into Klebsiella selective agar (HiMedia, India) and incubated at 37 °C for overnight. Next day, characteristic colonies (more than one colony per sample) were picked and streaked into nutrient agar (HiMedia, India) slant for further morphological and biochemical confirmation as per the standard methods (Quinn et al. 1994).

Similarly, for isolation of E. coli, the samples collected in transport medium were inoculated into MacConkey’s agar (HiMedia, India) and incubated at 37 °C for overnight. Next day, rose pink colonies were transferred into EMB agar (HiMedia, India) and again incubated overnight at 37 °C. The colonies were observed after incubation and single colony was streaked into nutrient agar (HiMedia, India) slants for further morphological and biochemical confirmation (Quinn et al. 1994).

PCR-based confirmation of Klebsiella spp. and E. coli

For PCR-based confirmation of Klebsiella spp. and E. coli isolates, DNA was extracted from all the morphological and biochemically confirmed Klebsiella spp. and E. coli isolates. For confirmation of Klebsiella spp. and E. coli, all the suspected samples including positive (ATCC 27736, ATCC 4157, HiMedia, India) and negative controls (nuclease free water) were subjected to PCR (Table 1). All the PCR positive Klebsiella spp. isolates including positive (ATCC 27736, HiMedia, India) and negative control (nuclease free water) were further tested for the identification of Klebsiella pneumoniae by specific PCR (Table 1). The amplified products were visualized by gel documentation system (UVP, UK) after electrophoresis in 2% (w/v) agarose (SRL, India) gel containing ethidium bromide (0.5 μg ml−1) (SRL, India).

Table 1 Oligonucleotide sequences and PCR conditions used in the study
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Double disc diffusion test

PCR confirmed Klebsiella spp. and E. coli isolates were subjected to screening for extended spectrum beta-lactamase production by antibiotic sensitivity test containing cefotaxime (30 μg, HiMedia, India) and ceftazidime (30 μg, HiMedia, India) antibiotic discs with or without clavulanate (10 μg, HiMedia, India). A difference of ≥ 5 mm between the zone diameters of either of the cephalosporin discs and their respective cephalosporin/clavulanate discs was considered to be phenotypically positive for ESBL production (CLSI 2014). Further, cefoxitin-cloxacillin double disc synergy (CC-DDS) was performed with all Klebsiella spp. and E. coli isolates for phenotypic confirmation of ACBL production (Tan et al. 2009).

Detection of beta-lactamase genes (bla CTX-M, bla TEM, bla SHV) in Klebsiella spp. and E. coli

All the phenotypically ESBL-producing Klebsiella spp. and E. coli isolates including controls were subjected to PCR for detection of blaCTX-M, blaTEM, and blaSHV genes using the primers and the cycle conditions as described in Table 1. The positive controls used in the study were provided by Department of Veterinary Microbiology, CAU, Aizawl, India. The PCR products were sequenced from commercially available sources (Xcelris Genomics, India). The sequence homology searches were conducted using the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST), and selected sequences were submitted into DDBJ.

Detection of chromosomal bla ampC and plasmid-mediated ampC β-lactamase gene (bla CMY) in Klebsiella spp. and E. coli

All the phenotypically ACBL-producing Klebsiella spp. and E. coli isolates including controls were subjected to PCR for detection of blaampC and blaCMY genes using the primers and the cycle conditions as described earlier (Table 1). The positive controls used in the study were provided by Department of Veterinary Microbiology, CAU, Aizawl, India. The PCR products were sequenced from commercially available sources (Xcelris Genomics, India). The sequence homology searches were conducted using the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST), and selected sequence was submitted into DDBJ.

Detection of class I integron gene in Klebsiella spp. and E. coli isolates

All the beta-lactamase-producing Klebsiella spp. and E. coli isolates were subjected to PCR for detection of class I integron as described earlier (Table 1).

Serotyping of ESBL-producing E. coli isolates

All the beta-lactamase-producing E. coli isolates were sent to Central Research Institute, Kasuli, HP, India for serotyping and storage in the repository.

Antimicrobial sensitivity of beta-lactamase-producing Klebsiella spp. and E. coli isolates

All the beta-lactamase-producing Klebsiella spp. and E. coli isolates were further tested for their sensitivity/resistance to different common antibiotics such as amikacin, gentamicin, tetracycline, chloramphenicol, levofloxacin, amoxicillin/clavulanic acid, ceftriaxone, piperacillin/tazobactam, imipenem, cotrimoxazole, ertapenem, cefpodoxime, cefoperazone, cefepime, and ceftriaxone/tazobactam by disc diffusion method (CLSI 2014).

Clonal relationship of beta-lactamase-producing Klebsiella spp. and E. coli

The molecular typing of all the beta-lactamase-producing Klebsiella spp. and E. coli isolates were done by relative afferent pupillary defect (RAPD)-PCR (Table 1). All the images taken by the gel documentation system were analyzed by using the Doc-itLs image analysis software supplied with the system as per manufacturer’s instruction (UVP, UK). By comparing the difference in the RAPD-PCR banding pattern, phylogenetic relationship among the isolates was established. An unrooted phylogenetic tree was made by using neighbor joining method.

Statistical analysis

Occurrence of beta-lactamase-producing Klebsiella spp. and E. coli in different agro-climatic zones from which the samples were collected was compared by chi-square test using SPSS software version 17.0 (SPSS Inc.).

Results

In total, 227 Klebsiella spp. were isolated and identified by biochemical tests and PCR from healthy pigs (91/120, 75.8%), pen floor/surface soil (29/48, 60.4%), drinking water (1/6, 16.6%), and feed (1/6, 16.6%) samples. Each of the 227 Klebsiella spp. isolates showed typical biochemical tests results and generated a PCR amplicon of approximately 441 bp. Further, 113 (113/227, 49.7%) Klebsiella spp. isolates were identified as Klebsiella pneumoniae by specific PCR with characteristic amplicon.

Similarly, 114 E. coli were isolated and identified by biochemical tests and PCR from healthy pigs (83/120, 69.1%), pen floor/surface soil (25/48, 52.0%), drinking water (3/6, 50.0%), and feed (3/6, 50.0%) samples. Each of the 114 E. coli isolates showed typical biochemical tests results characteristics of E. coli and generated a PCR amplicon of approximately 585 bp.

In total, 43 Klebsiella spp. (43/227, 18.9%) and 15 E. coli strains (15/114, 13.1%) isolated from different samples were phenotypically detected as ESBL producers in double disc synergy test. Klebsiella spp. isolated from healthy pigs (35/175, 20.0%), pen floor swabs/surface soil swabs (7/50, 14.0%), drinking water (1/1, 100%), and feeds (0/1, 0%), respectively were detected as phenotypical ESBL producers (Table S1, supporting information). Escherichia coli isolated from healthy pigs (12/83, 14.4%), pen floor swabs/surface soil swabs (2/25, 8.0%), drinking water (1/3, 33.3%), and feeds (0/3, 0%), respectively were detected as phenotypical ESBL producers. Isolation rate varies significantly between different agro-climatic zones (p < 0.05; Table S1, supporting information). Among them, 27 Klebsiella spp. isolates (27/43, 62.79%) and six E. coli isolates (6/15, 40%) were phenotypically confirmed as CTX-M producers with cefotaxime and cefotaxime/clavulanate double disc. Majority of the beta-lactamase-producing E. coli isolates belonged to O88, O149, and O22 serogroups.

Out of 43 beta-lactamase-producing Klebsiella spp. and 15 beta-lactamase-producing E. coli isolates, 35 Klebsiella spp. (35/43, 81.3%) and all the E. coli isolates (15/15, 100%) were detected as ACBL producers with cefoxitin-cloxacillin double disc synergy.

All the CTX-M-producing Klebsiella spp. (n = 27) and E. coli isolates (n = 6) possessed blaCTX-M in PCR. Further, 19 Klebsiella spp. (19/43, 44.18%) and two E. coli isolates (2/15, 13.3%) were found positive for blaSHV gene in PCR, whereas 10 Klebsiella spp. (10/43, 23.25%) and 10 E. coli (10/15, 66.7%) isolates were detected to possess the studied blaTEM gene in PCR (Tables 2 and 3). The sequences of the PCR products were compared and found 98% cognate with blaCTX-M-9, blaSHV-12 and blaTEM-1 in BLAST search. The sequences were published by DDBJ with accession numbers LC420321 (blaCTX-M-9), LC421936 (blaSHV-12), and LC421935 (blaTEM-1).

Table 2 Genotype profile of ESBL- and ACBL-producing Klebsiella spp. isolated from pigs and farm environment in West Bengal, India
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Table 3 Genotype profile of ESBL- and ACBL-producing E. coli isolated from pigs and farm environment in West Bengal, India
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All the ACBL-producing Klebsiella spp. and E. coli isolates possessed blaAmpC in PCR. No plasmid mediated blaCMY was found in any of the ACBL-producing Klebsiella spp. and E. coli isolates (Tables 2 and 3). The sequences of the PCR products were compared and found 99% cognate with blaAmpC in BLAST search. The sequence was published by DDBJ with accession number LC421937 (blaAmpC).

Sixteen beta-lactamase-producing Klebsiella spp. (16/43, 37.20%) and 13 beta-lactamase-producing E. coli isolates (13/15, 86.67%) were detected as positive for class I integron gene in PCR with desired product size of approximate 481 bp (Tables 2 and 3).

Phenotypical resistance of beta-lactamase-producing Klebsiella spp. was observed most frequently against cefotaxime (88.4%), cefepime (74.5%), cefpodoxime (88.4%), ceftazidime (79.1%), amoxicillin/clavulanic acid (83.7%), cefoxitin (65.2%), and ceftriaxone (60.5%). Majority of beta-lactamase-producing E. coli isolates were resistant to amoxicillin/clavulanic acid (80%), ceftazidime (86.8%), cefpodoxime (73.4%), cefotaxime (60%), and ceftriaxone (53.4%). Majority of the ESBL-producing Klebsiella and E. coli isolates were detected as sensitive to ertapenem, imipenem, amikacin, colistin, chloramphenicol, and levofloxacin.

In RAPD-PCR of beta-lactamase-producing Klebsiella spp., amplified fragments ranging from 112 to 3464 bp were detected (calculated by Doc-itLs image analysis software, UVP, UK). The phylogenetic analysis of ESBL-producing Klebsiella spp. generated a dendrogram where the strains isolated from environment and animals in same agro-climatic zone (K1, K2, K4; KP-1, KP-2, KP-3, KP-6, KP-9, KP-10, K3; K8, K13, K17; K14, K20) were detected in same cluster (Fig. 1).

Fig. 1
figure 1

Phylogenetic analysis of ESBL/AmpC-producing Klebsiella spp. strains isolated from healthy pigs and environment in West Bengal (India). The neighbor-joining method was used to summarize the similarity of RAPD-PCR profiles of ESBL/AmpC-producing Klebsiella spp. strains in a dendrogram

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Similarly, in RAPD-PCR of beta-lactamase-producing E. coli, amplified fragments ranging from 171 to 3965 bp were detected (calculated by Doc-itLs image analysis software, UVP, UK). The phylogenetic analysis of ESBL-producing E. coli generated a dendrogram, where the strains isolated from environment and animals in same agro-climatic zone (EC-18, EC-24; EC-6, EC-9; EC-13, EC-19) were detected in same cluster (Fig. 2).

Fig. 2
figure 2

Phylogenetic analysis of ESBL/AmpC/Shiga toxin-producing E. coli strains isolated from healthy pigs and environment in West Bengal (India). The neighbor-joining method was used to summarize the similarity of RAPD-PCR profiles of ESBL/AmpC/Shiga toxin-producing E. coli strains in a dendrogram. Figure 2 was constructed with both ESBL-and Shiga toxin-producing E. coli isolates, although the data of STEC was not included in the manuscript

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Discussion

The present study detected Klebsiella spp. (n = 227) and E. coli (n = 114) in healthy pigs (75.8 and 69.1%, respectively) and farm environment in West Bengal, India. Earlier study in Thailand and India (West Bengal and Mizoram) detected lower occurrence of Klebsiella spp. (7%) and E. coli (28–48%) in healthy pigs (Lalzampuia et al. 2013; Boonyasiri et al. 2014; Samanta et al. 2015). Use of selective medium and more than one colony was the probable reason for higher isolation rate of Klebsiella spp.

Moderate occurrence of beta-lactamase-producing Klebsiella spp. (20.0%) and E. coli strains (14.4%) was observed in the studied healthy pig population. Similar occurrence of ESBL-producing Klebsiella spp. (19.2%) and E. coli (15.3%) in swine was reported from Republic of Korea and Switzerland, respectively (Rayamajhi et al. 2008; Geser et al. 2012). Earlier study in northeastern India (Mizoram) although could not detect any ESBL-producing Klebsiella spp. from pigs (Lalzampuia et al. 2013). Lower occurrence of ESBL-producing E. coli in healthy pigs was noted in Portugal (5.7%) (Machado et al. 2008), Hong Kong and Japan (2–3%) (Duan et al. 2006; Hiroi et al. 2012), and in our earlier study in West Bengal (6%) (Samanta et al. 2015). Presence of beta-lactamase-producing bacteria in healthy pigs in the present study with higher occurrence rate than European and Asian countries, even higher than our own study conducted 2 years ago in the same state, indicates either generation or transmission of beta-lactamase genes from the environmental resistome with an increasing trend. Mostly, the studied pigs did not receive higher generation cephalosporins and synthetic analog of thiamphenicol (florphenicol) through their feed for growth promotion or therapeutic purposes which are acknowledged risk factors for shedding of ESBL-producing bacteria (Paterson and Bonomo 2005; Meunier et al. 2010). Reduction of cephalosporin intake was recommended as a control measure for ESBL-producing E. coli in slaughter pigs (Hammerum et al. 2014).

The feed, drinking water, and pen floor/surface soil swabs from roaming arena were evaluated to detect the role of farm environment in transmission of beta-lactamase-producing bacteria. Beta-lactamase-producing Klebsiella spp. and E. coli were detected in drinking water (33.3–100%) and pen floor/surface soil swabs (8–14%). The water bodies present in the globe (e.g., rivers) acts as a source of ESBL-producing bacteria except the water present at an altitude of more than 1000 m, indicating the anthropogenic activities as responsible factor for the contamination (Zurfluh et al. 2013). In developing countries, even the drinking water is not an exception. Municipal drinking water was detected as a carrier of NDM-1 beta-lactamase in India (Walsh et al. 2011). Water samples adjacent to pig or duck farms in China were reported earlier as a source of ESBL-producing E. coli (Ma et al. 2012; Hu et al. 2013). Cultivated soil was also reported earlier as a reservoir of ESBL-producing E. coli in France, where application of manure, sewage sludge, or irrigation with wastewater was assumed as a source of infection (Hartmann et al. 2012). Sewage sludge was identified as a potential reservoir of ESBL-producing E. coli in Europe (Spain and Austria) (Mesa et al. 2006; Reinthaler et al. 2010). The surface soils, collected from different locations in India, irrigated with wastewater, showed the presence of AmpC-β-lactamase (Malik et al. 2007). Isolation of beta-lactamase-/AmpC-producing bacteria varied significantly between different agro-climatic zones in the present study which indicated differences in the environmental contamination level.

Majority of beta-lactamase-producing Klebsiella spp. and E. coli were phenotypically CTX-M producers and all of them possessed blaCTX-M. Nucleotide sequencing of representative PCR products revealed the presence of blaCTX-M-9, blaSHV-12, and blaTEM-1. The studies with ESBL characterization in Enterobacteriaceae revealed the global emergence of CTX-M in the last decade replacing TEM and SHVs. Among four main groups of CTX-M associated with clinical infection, CTX-M-9 and CTX-M-1 were detected as most prevalent throughout the world especially in Asia including swine feces and aquatic environment (Ewers et al. 2012). In China, maximum numbers of ESBL-producing E. coli strains isolated from pigs and local water bodies possessed CTX-M-9 alone or in combination with CTX-M-1 (Hu et al. 2013). Similarly, SHV-12 was reported from swine feces in Portugal (Machado et al. 2008) and pig slurry in Spain (Escudero et al. 2010). TEM-1 was prevalent in ducks and water samples collected from ponds adjacent to the duck farm in China (Ma et al. 2012).

All the ACBL-producing Klebsiella spp. and E. coli isolates possessed blaAmpC, not plasmid mediated blaCMY-2. Although, CMY-2 variant was detected as more prevalent than AmpC in United States (EFSA Panel on Biological Hazards 2011). In Asian countries such as in China and Japan, ESBL-producing bacteria isolated from pigs and aquatic environment did not possess AmpC gene (Hu et al. 2013; Norizuki et al. 2018). In India, our earlier study also revealed occurrence of AmpC in E. coli strains isolated from poultry and cattle with mastitis (Kar et al. 2015).

Transmission of ACBL-/ESBL-producing organisms is regulated by mobile genetic elements specially class 1 integrons. Sometimes class 1 integrons detected from animals and human ACBL-/ESBL-producing isolates belonged to homologous in nature which confirms their role in transmission (EFSA Panel on Biological Hazards 2011). The present study revealed higher occurrence of class 1 integron in beta-lactamase-producing Klebsiella spp. (16/43, 37.20%) and E. coli (13/15, 86.67%) isolates depicting their high transmission possibility.

Beta-lactamase-/AmpC-producing bacteria isolated from food animals including pigs possessed co-resistance against other common veterinary drugs such as aminoglycosides, sulfonamides, trimethoprim, fluoroquinolones, and tetracyclines (Deng et al. 2011; Liebana et al. 2012). Beta-lactamase-producing Klebsiella or E. coli isolates did not show cross-resistance against non-β-lactams such as gentamicin, tetracycline, levofloxacin, and cotrimoxazole, even though the studied pigs were infrequently treated with tetracycline or gentamicin. It seems that possession of antimicrobial resistance genes or phenotypes in the studied pigs was not correlated with antibiotic intake.

The clonal relationship of beta-lactamase-/AmpC-producing Klebsiella spp. and E. coli placed the isolates from environmental sources (surface soil swab and drinking water) and the studied pigs within the same cluster of the dendrogram. Presence of the strains within the same cluster of a dendrogram indicates their similarity. For example, K1 (pigs), K2 (pigs), and K4 (pen floor swab) Klebsiella isolates were in the same cluster indicating pen floor as a source of infection (Fig. 1; Table 2). Similarly, EC18 (soil surface) and EC 24 (pig) E. coli isolates were detected within same cluster indicating soil as a source of infection (Fog 2; Table 3). Earlier PFGE-based study showed limited similarities and indicated the cross-transmission possibility of ESBL-producing E. coli from pigs to environmental water (Hu et al. 2013).

The study indicated about the possible role of contaminated environment as a source of beta-lactamase-/AmpC-producing Klebsiella spp. and E. coli in healthy pigs. Possible explanation for the presence of beta-lactamase-/AmpC-producing bacteria in the studied environment (water and soil) is either persistence of bacteria which was excreted from earlier herd especially in organized farms. Horizontal transfer of ESBL genes through plasmid or other mobile genetic elements is another option which is possible in soil and water (Trevors and Oddie 1986).

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Acknowledgements

The authors provide sincere thanks to honorable Vice Chancellor, West Bengal University of Animal and Fishery Sciences for the infrastructure and facilities. We also acknowledge the Director, Central Research Institute, Kasuli, HP, India for serogrouping of E. coli isolates.

Funding

The study was partially funded by Department of Biotechnology, Government of India (grant no.: BT/PR16149/NER/95/85/2015) and West Bengal State Council for Science and Technology (grant no.: 929(Sanc.)/ST/P/S and T/ 1G-25/2016).

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Authors and Affiliations

  1. Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata, West Bengal, 700037, India

    Arindam Samanta, Achintya Mahanti, Souvick Chatterjee, Siddhartha Narayan Joardar & Indranil Samanta

  2. Eastern Regional Station, ICAR-Indian Veterinary Research Institute, Kolkata, West Bengal, India

    Samiran Bandyopadhyay

  3. Department of Veterinary Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata, West Bengal, 700037, India

    Tapas Kumar Sar

  4. Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata, West Bengal, 700037, India

    Guru Prasad Mandal

  5. Department of Veterinary Microbiology, Central Agricultural University, Aizawl, Mizoram, India

    Tapan Kumar Dutta

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  1. Arindam Samanta
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  2. Achintya Mahanti
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  6. Tapas Kumar Sar
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  9. Indranil Samanta
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Correspondence to Indranil Samanta.

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Samanta, A., Mahanti, A., Chatterjee, S. et al. Pig farm environment as a source of beta-lactamase or AmpC-producing Klebsiella pneumoniae and Escherichia coli. Ann Microbiol 68, 781–791 (2018). https://doi.org/10.1007/s13213-018-1387-2

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  • DOI: https://doi.org/10.1007/s13213-018-1387-2

Keywords

Annals of Microbiology

ISSN: 1869-2044

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