Phenotypic and molecular characterization of β-lactamase and plasmid-mediated quinolone resistance genes in Klebsiella oxytoca isolated from slaughtered pigs in Thailand

Background and Aim: Over recent years, antimicrobial-resistant Klebsiella species in humans, animals, food animals, food products, and agricultural environments have been the center of attention due to its role in the evolution of antimicrobial resistance. The emergence of resistance to fluoroquinolones and cephalosporins of third and higher generations in Klebsiella oxytoca has not received much attention in animal husbandry compared to that in Klebsiella pneumoniae. Reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand. Materials and Methods: Microbiological examination was conducted on 384 Klebsiella spp. isolates recovered from slaughtered pigs in ten provinces of Thailand. Seventy-two K. oxytoca isolates (18.75%) were examined for antimicrobial-resistant genes (β-lactamase [blaTEM, blaCTX-M, and blaSHV]) and fluoroquinolone-resistant genes (qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB, aac(6’)-Ib-cr, and qepA). Results: The most common genotype was blaCTX-M (58/72, 80.55%), followed by blaTEM with blaCTX-M (7/72, 9.72%) and blaTEM (6/72, 8.33%). The most common blaCTX-M group was blaCTX-M-1 (19/58, 32.76%), followed by blaCTX-M-9 (1/58, 1.72%). Plasmid-mediated quinolone resistance genes were identified in 13 (18.05%) isolates: qnrS (16.70%) and qnrB (1.4%). All 13 isolates had qnrS transferable to an Escherichia coli recipient, whereas qnrB was not detected in any transconjugants. Either blaCTX-M or blaTEM harbored by one K. oxytoca strain was transferable to an E. coli recipient. Analysis of antimicrobial susceptibility revealed that more than 90% of the blaCTX-M-carrying K. oxytoca isolates were susceptible to chloramphenicol, trimethoprim, ceftazidime, cefepime, cefotaxime, amoxicillin-clavulanic acid, piperacillin–tazobactam, and fosfomycin. All K. oxytoca isolates (13) harboring qnr were susceptible to carbapenem and ceftriaxone; however, 43 (74.13%) of the K. oxytoca isolates harboring blaCTX-M exhibited extended-spectrum β-lactamase activity. Most of the K. oxytoca isolates from pigs were highly resistant to ampicillin, azithromycin, and gentamicin. Conclusion: To prevent further transmission of Klebsiella spp. Between food animals and humans, strict control of antibiotic use in clinical and livestock settings is necessary along with routine disinfection of the livestock environment and efforts to increase awareness of antimicrobial resistance transmission.


Introduction
Extended-spectrum β-lactamase (ESBL) was first isolated from Klebsiella species and later from Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and other Gram-negative bacilli [1,2]. ESBL can confer resistance to a variety of β-lactam antibiotics, including penicillins and broad-spectrum cephalosporins with an oxyimino side chain, such as cefotaxime, ceftriaxone, and ceftazidime, but not often to carbapenems or cephamycins, such as cefoxitin [3]. ESBL-producing Gram-negative bacilli have rapidly spread and become a serious threat to human health worldwide, resulting in significant morbidity and mortality [4]. Various types of β-lactamase genes (including ESBL genes), such as bla CTX-M , bla SHV , and bla TEM , have been reported in K. oxytoca strains obtained from patients and healthy persons [5]. Within the bla CTX-M family, bla CTX-M-2 -carrying K. oxytoca strains have been identified in milk samples obtained from cows with mastitis in Japan [6]. In a study Available at www.veterinaryworld.org/Vol.15/February-2022/10.pdf conducted in Italy, bla CTX-M-9 , bla SHV-12 , and bla DHA-1 genes were detected in K. oxytoca isolates [7].
Plasmid-mediated resistance to quinolones (PMQR) genes, including qnrA, qnrB, qnrS, qnrC, and qnrD genes, has been demonstrated to play a significant role in quinolone resistance by protecting DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones [8,9]. Aminoglycoside acetyltransferase (aac(6')-Ib-cr) gene has been shown to enzymatically modify fluoroquinolones [9]. The production of efflux pumps is enhanced by qepA, acrAB, and oqxAB genes, which also contribute to quinolone and fluoroquinolone resistance [10]. Klebsiella oxytoca isolated from cats and dogs in Italy were positive for qnr genes (qnrA1 and qnrB4), and one K. oxytoca isolate was positive for the aac(6')-Ib-cr gene [7].
The spread of K. ocytoca strains carrying β-lactamase and PMQR genes may be a threat to the "One Health" program. In Thailand, where antibiotics are used without restriction in the healthcare, livestock, and food production sectors, research on the epidemiology of antibiotic-resistant bacteria in food animals and its associated public health implications has been conducted in common bacteria, such as E. coli, Klebsiella pneumoniae, and Salmonella spp.
However, reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand.

Ethical approval
Ethical review and approval were not required for this study as the samples were collected from slaughtered pigs at the slaughterhouses as per standard collection procedure.

Study period and location
The study was conducted from October 2014 to September 2015. The sampling locations were the slaughterhouses in 10 provinces nationwide: Bangkok, Nakhon Pathom, Lop Buri, Chiang Mai, Lampang, Chon Buri, Roi-Et, and Khon Kaen. Surat Thani, and Songkhla ( Figure-1).

Bacterial isolates and identification
We followed the procedures for isolation and identification of Klebsiella spp. as described by Phetburom et al. [11]. We recovered 384 isolates of Klebsiella spp. from slaughtered pigs. Four slaughterhouses were randomly selected from each province, and 50 carcass surface swab samples were randomly collected from each slaughterhouse, which results in a sample size of 2000 swab samples. Using a single swab for each carcass, 400 cm 2 of one side of the carcass was swabbed. The swab samples were immediately stored on ice in zip-lock bags and transported to the microbiological laboratory. Isolation and identification of Klebsiella spp. were performed using 10-fold serial dilutions of swab samples achieved using buffered peptone water (BPW; pH 7.2). Each diluted BPW sample was spread onto MacConkey agar (Difco Laboratories, Detroit, Mich. USA ) and incubated at 37°C for 24 h. The presumptive identification of Klebsiella species was confirmed using conventional biochemical tests, including motility, Voges-Proskauer, ornithine decarboxylation, lysine decarboxylation, malonate, and O-nitrophenyl-β-D-galactopyranoside [12]. In total, 384 Klebsiella isolates were recovered and all isolates were stored at −80°C in a laboratory freezer until use in this study. The presumptive Klebsiella species for up to five colonies were cultured on MacConkey agar (Difco Laboratories) and their DNA was prepared using ZymoBIOMICS™ DNA Miniprep Kits (Zymo Research Corp., CA, USA) according to the manufacturer's protocols. Polymerase chain reaction (PCR) was performed using a thermal cycler to identify K. oxytoca, as described elsewhere [13]. The PCR program included initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and a final cycle of amplification at 72°C for 7 min.

Detection of β-lactamase genes
Isolates carrying β-lactamase genes (bla TEM , bla C-TX-M , and bla SHV ) were detected through multiplex PCR using a thermal cycler with a PCR program that included initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and amplification at 72°C for 7 min [14]. The bla CTX-M -harboring K. oxytoca was classified into CTX-M groups (bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-5 , bla CTX-M-8 , bla CTX-M-9 , and bla CTX-M-25 groups) using multiplex PCR [15]. PCR amplification was performed using a thermal cycler with a PCR program of denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 25 s, annealing at 52°C for 40 s, extension at 72°C for 50 s, and amplification at 72°C for 6 min.

Detection of ESBL production based on combination disk test
The production of ESBL was tested for in 58 bla CTX-M -harboring K. oxytoca isolates using the combined disk method and separate commercial disks containing cefotaxime (30 µg) and ceftazidime (30 µg) with or without clavulanic acid (10 µg) [17]. An increase in zone size ≥5 mm for cefotaxime and ceftazidime with or without clavulanic acid was considered to indicate ESBL production [17].

Detection of ESBL Nordmann-Dortet-Poirel (NDP) assay
The bla CTX-M -harboring K. oxytoca isolates were tested for extended-spectrum β-lactamase activity using the ESBL NDP assay [18]. One calibrated inoculated loop (10 µL) of the tested strain was briefly suspended in 100-µL B-PER II ® Bacterial Protein Extraction Reagent (Thermo Scientific, USA) buffer and centrifuged at room temperature for 5 min. The supernatant (30 µm) was mixed with 100 µL of a 1-mL solution of pH 7.8 phenol red solution with or without 6 mg of purified cefotaxime sodium salt (Tokyo Chemical Industry Co., Ltd, Japan), incubated at 37°C for 2 h, and observed for color change. Extendedspectrum β-lactamase-producing strains were identified as they broke down cefotaxime into acidic products, changing the color of the phenol red indicator to yellow.

Conjugation assay
Conjugation assays were performed using all isolates of K. oxytoca harboring bla CTX-M and qnrS as donors and streptomycin-resistant E. coli UB1637 as recipients, as described elsewhere [19]. Mueller-Hinton agar plates supplemented with 3200 µg/mL of streptomycin were used to select transconjugants. Transconjugants were confirmed as E. coli using PCR [20], and PCR was used to determine the presence of ESBL and PMQR genes.

Transferability of β-lactamase and PMQR genes
The 61 K. oxytoca isolates carrying bla CTX-M , qnr, or both were subjected to conjugation assay using streptomycin-resistant E. coli UB1637 as the recipient. Not all K. oxytoca donors carrying only bla CTX-M (n=50) transferred resistance to recipient E. coli cells. On the contrary, K. oxytoca isolates carrying only qnrS (n=8) and those harboring either qnrS with bla CTX-M or qnrS with bla TEM (n=4) successfully transferred resistance to recipient E. coli (Table-2). Among these transconjugants, qnrS, qnrS with bla CTX-M , and qnrS with bla TEM were successfully transferred, whereas qnrB was not detected in any of the transconjugants in this study (Table-2).

Discussion
Our study revealed that the majority of β-lactamase-carrying K. oxytoca isolates possessed bla CTX-M , especially bla CTX-M-1 . In the current study, we did not detect bla SHV . The dissemination of bla CTX-M-2 among K. oxytoca isolates collected from pigs has been reported [21,22], and bla CTX-M-15 and bla TEM-1 were detected in K. oxytoca isolates collected from six provinces in China [23]. K. oxytoca isolates harboring bla CTX-M were highly susceptible to cefotaxime, ceftazidime, and cefepime (93.1-98.3%) and 100% susceptible to ceftriaxone. Combination disk assay revealed that they were non-ESBL producers; however, ESBL NDP assay demonstrated that 43 (74.1%) of these isolates exhibited β-lactamase activity on cefotaxime. Another study observed that K. oxytoca showed ostensibly positive resistance to cefotaxime and cefepime and rare resistance to ceftazidime, with only borderline resistance to these cephalosporins (MIC 2-8 mg/L), suggesting that there was hyperproduction of K1 (KOXY) chromosomal β-lactamase rather than ESBL production [24]. The positive result for ESBL NDP assay but the negative result for combination disk assay seen in our study may suggest the presence of K1 β-lactamase in this organism.
In the current study, qnrS and qnrB-harboring K. oxytoca strains were widely distributed in the isolates and were resistant to ciprofloxacin (66.67%), levofloxacin (88.9), or both (55.56%). These results contrast with the findings of Hossain et al. [25] that K. oxytoca strains harboring qnrA, qnrA with qnrB, and qnrB with qnrS were highly susceptible to ciprofloxacin (100%) and ofloxacin (100%). In addition, qnrB and qnrA were the most prevalent genes (37.50%) in K. oxytoca strains isolated from pet turtles in Korea [26]. In this study, all isolates demonstrated capacity to transfer qnrS to an E. coli recipient. Literak et al. [27] found that qnrS genes were carried by plasmids of the N, X1, and X2 incompatibility groups and were transferable by conjugation to E. coli and/or Salmonella spp. This could support  This study found that some qnrS, qnrS with bla CTX-M , and qnrS with bla TEM genes were transferred to E. coli recipients. However, most of the bla CTX-M in K. oxytoca donors were not transferred to recipient E. coli cells. On the contrary, Zhang et al. [23] reported that the bla CTX-M and bla TEM genes were successfully transferred from K. oxytoca to E. coli 600. This difference may be because bla CTX-M genes may be located on chromosomes or nonconjugative plasmids. Thus, the characterizations to understand the origin, evolution, and spread of antimicrobial resistance genes are required for further investigation using whole-genome analysis.
Conclusion bla CTX-M-1 and qnrS were the predominant resistance genes in K. oxytoca strains isolated from slaughtered pigs in Thailand. Further study and ongoing efforts are necessary to develop a complete understanding of the epidemiology, risk factors, transmission dynamics, and public health implications associated with food animals as reservoirs of antimicrobial-resistant bacteria.

Authors' Contributions
PB and AK: Contributed to conceptualization and design of the study. NP: Performed the laboratory work. PB, PC, and RH: Performed validation. SN: Analyzed and interpreted the data. PB: Prepared and wrote the original draft. PB and AK: Reviewed and edited the manuscript. All authors read and approved the final manuscript.
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