Epidemiological study of antimicrobial-resistant bacteria in healthy free-ranging bantengs (Bos javanicus) and domestic cattle

Background and Aim: Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand. Materials and Methods: Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types. Results: All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79. Conclusion: Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.


Introduction
The global human population has been rapidly increasing [1], necessitating the expansion of land uses for anthropogenic activities, such as agriculture to provide sufficient food resources.This expansion has caused habitat loss for wild animals, creating new wildlife-human-livestock (WHL) interfaces [2].These interfaces have raised various public health concerns [3,4], including the potential transmission of antimicrobial-resistant microorganisms (ARMs) between humans, livestock, and wildlife [3].The emergence of ARMs in wild species poses several challenges and warrants the identification (ID) of potential sources and the mechanisms behind such transmissions.Inappropriate drug use has significantly contributed to the emergence of ARMs [3], particularly in non-human applications (e.g., livestock and companion animals).This is because antibiotics are heavily Copyright: Konputtar, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.used as precautionary measures for high-risk animals and as growth promoters for healthy livestock, often mixed with their feed [5].These applications illustrate violations of rational drug use proposed by the World Health Organization to prevent ARM emergence [6].Thus, ARMs are likely to emerge in livestock and potentially spread to wildlife and ecosystems [7].
The emergence of ARMs has complicated the treatment of infectious diseases, often leading to treatment delays and/or failures.Beta-lactam antibiotics have been extensively prescribed in livestock due to their broad-spectrum activities [8].These antibiotics primarily disrupt cell wall synthesis, resulting in bacterial death [9].However, ARMs can produce beta-lactamase enzymes that hydrolyze the beta-lactam ring of these antibiotics [9], essentially degrading their pharmacological effects.The emergence of ARMs is primarily due to genetic mutation and selection [10]; however, genetic materials that encode antimicrobial-resistant genes (ARGs) can also be acquired from other microorganisms through horizontal gene transfer (HGT) [11].The HGT mechanism is crucial because environments contaminated with ARM and non-pathogenic bacteria can serve as ARG reservoirs, facilitating the indirect transmission of these genes, even without the coexistence of the organisms at the WHL interfaces.Escherichia coli is a common flora that inhabits the gastrointestinal tracts of healthy organisms, with some strains found to exhibit pathogenicity and toxigenesis [12].The previous study by Rupp and Fey [13] have shown that E. coli plasmid harbors genes encoding ampicillin-hydrolyzing beta-lactamases (e.g., TEM-1, TEM-2, and SHV-1) and cephalosporins-hydrolyzing extended-spectrum beta-lactamases (ESBLs).In this study, we focused on ESBL-producing E. coli strains crucial for the development of drug resistance [13].Extended-spectrum beta-lactamase genes confer resistance to third-generation cephalosporins, which have been extensively used in veterinary practices for their broad-spectrum effects [8].With over 150 documented ESBLs, they are differentiated from other ampicillin-hydrolyzing beta-lactamases by their ability to hydrolyze oximino-cephalosporins [14].Most E. coli strains can tolerate extreme environmental changes and survive adverse conditions for months [15].Thus, non-pathogenic E. coli strains are excellent reservoirs of ESBLs before these genes are transmitted to susceptible species.
This study aimed to identify the types of ESBLs present in ESBL-producing E. coli strains isolated from a free-ranging population of bantengs (Bos javanicus) in the Lam Pao non-hunting area (Kalasin, Thailand) and domestic cattle in neighboring human communities.The banteng, listed as "endangered" on the red list of threatened species of the International Union for Conservation of Nature since 2014, is estimated to have only 4000-8000 individuals left in their natural habitats [16].Identifying ESBL profiles in wild bantengs and domestic animals would address the spillover of ARMs between wild and domestic animals, facilitating further studies aimed at preventing this issue at other WHL interfaces.

Ethical approval
This study was approved by the Institutional Animal Care and Use Committee, Mahasarakham University (IACUC-MSU), Thailand (Approval number IACUC-MSU-32/2021).Works involving legally protected animals and areas were approved by the Department of Natural Parks, Wildlife, and Plant Conservation, Thailand (MNRE 0907.4/439).

Study period and location
The study was conducted from August 2021 to October 2022.The Lam Pao non-hunting area was established in 1988 with an area of approximately 338 km 2 in Kalasin Province, North-east Thailand.Of the total area, three square kilometers have been inhabited by a population of about 100 free-ranging bantengs as well as other wild species such as Siamese hares (Lepus peguensis), various species of squirrels, Asian palm civets (Paradoxurus hermaphroditus), and migratory and non-migratory bird species [17].This area is known for the local communities as Suan Sa-On (GPS coordinates 16.61345, 103.45910).The north, south, and west boundaries of Suan Sa-On are surrounded by the Pao River, and the east is adjacent to the land and human communities.

Sample collection
Fecal samples from the bantengs were collected from six locations (Figure -1) within the non-hunting area in August 2021.The locations were selected based on the preferred grazing areas of the bantengs, which were identified using observational records.Fecal samples from domestic cattle were collected from nearby farms within a 3-km radius of the non-hunting area that had records of antibiotic use.To avoid contamination, approximately 20 g of fresh feces from the middle portions that did not come into contact with the environment (i.e., air and soils) were sampled.Each sample was packaged individually in a sealed plastic bag and labeled with a unique sample ID number (Supplementary data).Overall, 45 banteng and 15 domestic cattle samples were collected.All samples were then preserved in ice-cooled containers (~4°C) and promptly transferred to the microbiology laboratory at the Faculty of Veterinary Sciences, Mahasarakham University, where they were refrigerated until further processing.

Identification of E. coli
Escherichia coli strains were identified based on colony characteristics (i.e., pink, flat, smooth, and round) and biochemical tests.Colonies that showed characteristics consistent with E. coli on MacConkey agar (Himedia ® , Mumbai, India) were considered potential colonies and verified using triple sugar iron (TSI) [18] and motile indole lysine (MIL) tests [19].Three colonies (hereafter referred to as isolates) were selected from each sample that tested positive for both TSI and MIL, and these isolates were cultured on nutrient agar (Himedia ® ) and incubated at 37°C for 24 h.An isolate was collected using an inoculated loop and mixed with 400 µL of distilled water in a microcentrifuge tube.Next, the tube containing the isolate was boiled in 100°C water for 10 min and left to cool at room temperature (28-30°C).Centrifugation was conducted at 18,000× g and 4°C for 10 min.The supernatant (approximately 300 µL for each sample) was collected and kept at −20°C as a DNA template for polymerase chain reaction (PCR) analysis.
The PCR was performed for each sample using 6.25 µL of OnePCR ® Ultra solution (GeneDirex, USA), 0.05 µL of the ECO-1 primer, 0.05 µL of the ECO-2 primer (Table -1 for 2 s.Polymerase chain reaction products were visualized using 1.5% agar rose gel electrophoresis in a 1× Tris-acetate-EDTA (TAE) buffer, compared to a 100 base pairs (bp) DNA ladder (VC 100 bp DNA ladder, Vivantis, Malaysia) and positive controls.Samples displaying a band of 585 bp were considered positive.

Bacterial culture and ID of cefotaxime (CTX)-resistant E. coli
To identify CTX-resistant E. coli, each sample was cultured on a Petri dish containing MacConkey agar (Himedia) supplemented with 2 µg/mL of CTX.The samples were then incubated at 37°C for 24 h.Only the E. coli-like colonies were selected for further confirmation, while the rest were preserved in tryptone soya broth with 10% glycerol at −20°C.The TSI [18] and MIL [19] tests were conducted, and samples positive for both tests were considered CTXresistant E. coli.

Identification of ESBL-producing CTX-resistant E. coli using the double-disk synergy test
The samples categorized as CTX-resistant E. coli were cultured in individual Petri dishes containing nutrient agar (Himedia).Three growth colonies were selected from each sample (three isolates per sample) and individually diluted in 0.85% NaCl to achieve a turbidity equivalent to 0.5 McFarland standard.A sterile cotton swab was immersed in the solution and then spread on Mueller-Hinton agar (MHA; Himedia).On an MHA Petri dish, an amoxicillin/clavulanate (20/10 µg/disk, respectively) disk (Oxoid, UK) and a CTX (30 µg/disk) disk (Oxoid) were placed at the center with a distance of 2.5 cm between them.Incubation was conducted at 37°C for 16-24 h, and the increase in the inhibition zone between both disks was considered an indicator of ESBL production [20].

Disk diffusion test to determine resistant patterns
A drug susceptibility test was conducted for samples confirmed to be ESBL-producing.Each sample was cultured on nutrient agar (Himedia) and incubated at 37°C for 24 h.A colony was selected and diluted in 0.85% NaCl to achieve a turbidity equivalent to 0.5 McFarland standard.A sterile cotton swab was immersed in the solution and spread on a dish containing MHA (Himedia).Disks containing the following antibiotics were used for the drug susceptibility test: meropenem (MER) (10 µg), gentamicin (GEN; 10 µg), tetracycline (TET; 30 µg), ciprofloxacin (CIP; 5 µg), nalidixic acid (NAL; 30 µg), chloramphenicol (CHL; 30 µg), and sulfamethoxazole/trimethoprim (SXT; 23.75/1.25 µg).These antibiotics were selected from drugs commonly used in the area.To determine resistant patterns, the results were interpreted following the zone diameter interpretive criteria recommended by the Clinical and Laboratory Standards Institute [21].

Identification of ESBL gene groups using the PCR technique
DNA extraction was conducted on the isolates identified as ESBL-producing and CTX-resistant, which served as DNA templates for subsequent PCR procedures.Extended-spectrum beta-lactamase genes were identified using the multiplex PCR technique.To identify CTX-M genes for each isolate, a PCR reaction mixture of 13 µL was prepared, which contained the following: 6.25 µL of 2X ViRed Taq Mastermix ® (Vivantis); 0.05 µL of the CTXmGr1_F, CTXGr1_R, CTXmGr2_F, CTXmGr2_R, CTXmGr9_F, CTXmGr9_R, CTXmGr8_F, CTXmGr25_F, and CTXmGr8/25_R primers (Table-1); 1.0 µL of DNA templates; and 5.30 µL of nuclease-free water.The PCR (Proflex ® , Applied Biosystem, Bangkok) protocol followed these steps: (1) Initial denaturation at 94°C for 2 min, (2) denaturation at 98°C for 10 s, (3) annealing at 55°C for 30 s, ( 4) an extension at 72°C for 30 s, (5) steps 2-4 repeated for 25 cycles, and (6) a final extension at 72°C for 7 min.The PCR products were visualized using 1.5% of agar rose gel electrophoresis in a 1× TAE buffer.Extended-spectrum beta-lactamase gene groups were identified based on the sizes of PCR products relative to a 100 bp DNA ladder (VC 100 bp DNA ladder ® , Vivantis) and positive controls of different groups.

Identification of ESBL genes using DNA sequencing
To identify ESBL genes, sample preparation for DNA sequencing was conducted for each isolate using 10 µL of 2X ViRed Taq Mastermix (Vivantis); 0.05 µL of the SeqCTX-M-1_for, SeqCTX-M-1_rev, SeqCTX-M-9_for, and SeqCTX-M-9_rev primers (Table-1); 2.0 µL of DNA templates; and 7.90 µL of nuclease-free water.The same amplification protocol employed to identify ESBL gene groups using the PCR technique was followed.For quality control, 2.0 µL of PCR products were subjected to 2% agar rose gel electrophoresis (i.e., validation of E. coli based on amplicon sizes and confirmation of DNA materials).The PCR product from each isolate that showed a well-defined electrophoretic band with an appropriate size was selected to represent each resistant pattern.These PCR products were submitted for DNA sequencing (Solgent Co. Ltd., South Korea) and were examined for ESBL genes using the basic local alignment search tool [22] available on the National Center for Biotechnology Information website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Identification of E. coli
Based on the TSI and MIL tests, as well as the PCR analyses, all 135 isolates from the 45 banteng samples and 45 isolates from the 15 domestic cattle samples were confirmed as E. coli (Supplementary data).
The double-disk synergy test confirmed ESBL production in all 15 CTX-resistant banteng samples.Similarly, the only CTX-resistant sample among the domestic cattle was confirmed to be ESBL-producing.These results accounted for 33.3% and 6.7% of the total banteng and domestic cattle samples, respectively (Table -2).

Identification of ESBL gene groups using the PCR technique
Forty-five banteng isolates were obtained from the 15 CTX-resistant and ESBL-producing samples.Among these isolates, 84.5% (38) belonged to the CTX-M1 group, while 11.1% (5) and 4.4% (2) were categorized under the CTX-M9 and SHV groups.All three isolates (100%) obtained from one domestic cattle sample were classified under the CTX-M1 group (Supplementary data).

Identification of ESBL genes using DNA sequencing
Thirty-two banteng isolates and three domestic cattle isolates were selected for sequencing based on their well-defined electrophoretic bands with appropriate sizes.Cefotaxime-M15 was detected in 20 banteng isolates and one domestic cattle isolate, while CTX-M55 was observed in six banteng isolates and one domestic cattle isolate.Furthermore, CTX-M14 was observed in five banteng isolates, and CTX-M79 was observed in one banteng and one domestic cattle isolate (Supplementary data).

Discussion
To the best of our knowledge, this study is the first to identify ESBL-producing E. coli among the wild banteng population of Thailand.This population has never been treated with antibiotics, suggesting the potential sharing of ARMs between bantengs and domestic cattle.The existence of these organisms in other wild species has been reported worldwide [7,23,24].The first publication discussing the spread of cephalosporin-resistant bacteria in wild animals (e.g., wild boars, Sus scrofa) was published in 2004 [25].Subsequently, the number of related studies steadily increased, with a notable acceleration between 2016 and 2020, during which approximately 60% of these studies were published [23].The CTX-M1 group identified in this study exhibited the highest prevalence among free-ranging bantengs, consistent with previous studies by Homeier-Bachmann et al. [24], Torres et al. [26], Formenti et al. [27], Sasaki et al. [28] and Niumsup et al. [29] on other wild ungulates and healthy individuals in rural Thailand regions.The prevalence of CTX-M1 and CTX-M9 observed in this study is consistent with the findings reported in other studies involving domesticated animals [30,31] and humans [28,29] in South-east Asia.In addition, most ESBL genes identified through DNA sequencing were shared between the bantengs and domestic cattle.Only one gene (i.e., CTX-M14) observed in the bantengs was absent in domestic cattle.This consistency across studies and the shared genes between these animal groups highlight the potential for ARG transmission among humans, wildlife, and domestic animals.However, regarding the number of gene counts, the banteng samples exhibited more ESBL genes than the domestic cattle samples.Homeier-Bachmann et al. [24] reported a contrary finding, with fewer ESBL genes observed in wild animals than in domestic species.The dissimilarity in our study is likely due to the differing sample sizes between the wild banteng (45 samples) and domestic cattle (15 samples) populations, as a smaller sample size limits the detection of less common ESBLs.However, regardless of sample size, our results suggest that the bantengs did not solely acquire ESBL genes from the domestic cattle.Instead, they suggest the existence of other ESBL sources [32].The relationships between various factors and the prevalence of ESBLs in wildlife have been reported in various studies.These factors include high cattle densities [33], the presence of wastewater treatment plants [34], and the existence of scavenger species [35].Potentially, the bantengs in our study might act as reservoirs hosting ARMs from multiple origins, implying that the ARMs in the population were not solely introduced by the domestic cattle.The results are concerning because this banteng population inhabits an area that serves as an important source of freshwater, nourishing the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Conclusion
The presence of ARMs in the wild banteng population poses a threat to public health and wildlife conservation.The results of our study highlight the need for further studies to identify contamination sources and establish surveillance programs for monitoring ARM in wild animal populations.

Figure- 1 :
Figure-1: Map of Suan Sa-On non-hunting area (Courtesy of Geo-Informatics Center for Thailand).The six sampling sites of bantengs samples are shown as red asterisks.Note that the area is surrounded by local agricultural communities and the Pao River.

Table - 3
: Resistant patterns revealed from the disk diffusion test of the CTX-resistant ESBL-producing Escherichia coli.

Table - 2
: Percentages of samples that were deemed as CTX-resistant Escherichia coli.