The emergence of multi-drug resistant and virulence gene carrying Escherichia coli strains in the dairy environment: a rising threat to the environment, animal, and public health

Escherichia coli is a common inhabitant of the intestinal microbiota and is responsible for udder infection in dairy cattle and gastro-urinary tract infections in humans. We isolated E. coli strains from a dairy farm environment in Xinjiang, China, and investigated their epidemiological characteristics, phenotypic and genotypic resistance to antimicrobials, virulence-associated genes, and phylogenetic relationship. A total of 209 samples were collected from different sources (feces, slurry, water, milk, soil) and cultured on differential and selective agar media (MAC and EMB). The presumptive identification was done by the VITEK2 system and confirmed by 16S rRNA gene amplification by PCR. Antimicrobial susceptibility testing was done by micro-dilution assay, and genomic characterization was done by simple and multiplex polymerase chain reaction (PCR). A total of 338 E. coli strains were identified from 141/209 (67.5%) of the samples. Most of the E. coli strains were resistant to sulfamethoxazole/trimethoprim (62.43%), followed by cefotaxime (44.08%), ampicillin (33.73%), ciprofloxacin (31.36%), tetracycline (28.99%), and a lesser extent to florfenicol (7.99%), gentamicin (4.44%), amikacin (1.77%), and fosfomycin (1.18%). All of the strains were susceptible to meropenem, tigecycline, and colistin sulfate. Among the resistant strains, 44.4% were identified as multi-drug resistant (MDR) showing resistance to at least one antibiotic from ≥3 classes of antibiotics. Eighteen out of 20 antibiotic-resistance genes (ARGs) were detected with sul2 (67.3%), blaTEM (56.3%), gyrA (73.6%), tet(B) (70.4%), aph(3)-I (85.7%), floR (44.4%), and fosA3 (100%, 1/1) being the predominant genes among different classes of antibiotics. Among the virulence-associated genes (VAGs), ompA was the most prevalent (86.69%) followed by ibeB (85.0%), traT (84.91%), ompT (73.96%), fyuA (23.1%), iroN (23.1%), and irp2 gene (21.9%). Most of the E. coli strains were classified under phylogenetic group B1 (75.45%), followed by A (18.34%), C (2.96%), D (1.18%), E (1.18%), and F (0.30%). The present study identified MDR E. coli strains carrying widely distributed ARGs and VAGs from the dairy environment. The findings suggested that the dairy farm environment may serve as a source of mastitis-causing pathogens in animals and horizontal transfer of antibiotic resistance and virulence genes carrying bacterial strains to humans via contaminated milk and meat, surface water and agricultural crops.


Introduction
Escherichia coli (E. coli) is an opportunistic and common inhabitant of the intestinal microbiota of animals as well as humans (He et al., 2019). Moreover, E. coli is also the most common organism responsible for causing udder infection in animals (Cheng et al., 2019) and bloodstream infections in humans (Jara et al., 2021). The use of antibiotics to prevent disease and promote the health of growing animals remains an integral part of livestock farming. It has been 50 years since antibiotic-supplemented feeds were first approved for livestock to improve overall health and increase the productivity of animals (Afema et al., 2018). However, the emergence and spread of pathogens resistance to multiple antibiotics has become a growing problem for veterinary medicine and public health (Murray et al., 2022). It was estimated that antimicrobial resistance results in $55 billion annual economic loss in USA (Dadgostar, 2019). The China also rank high in consumption of antibiotics for food producing animals especially in dairy sector.
Transmission of antimicrobial resistance (AMR) may occur by multiple ways, but contact with human and animal feces is the most common pathway . Dairy cattle act as a potential source of spread of antibiotic-resistant and zoonotic bacterial strains, especially Shiga-toxin producing E. coli (STEC) through the GRAPHICAL ABSTRACT Frontiers in Microbiology 03 frontiersin.org contamination of the farm environment and food products such as milk and meat, and direct contact with animals (Amézquita-López et al., 2018;Sobur et al., 2019). Animal farming, especially intensive livestock farming, plays a major role in AMR transmission between humans, animals, and the environment (Manyi-Loh et al., 2018). Due to the widespread use of antimicrobials in livestock production, livestock manure is considered a hotspot for the spread and transmission of AMR genes. Genetically diverse E. coli strains exist in animal manures, and their ability to survive in various ecological niches (Beattie et al., 2020). E. coli strains carrying bla CTX-M and bla CMY genes confer resistance to β-lactam antibiotics are frequently found in animal manure (Cookson et al., 2022). Therefore, animal manure is thought to be harming to animals via udder infections by environmental pathogens such as E. coli, humans via contaminated food products, and environment by using manure as fertilizer in soil or waste water (Sarowska et al., 2019). This increases the potential of antibiotic resistance genes (ARGs) to integrate into human intestinal microbiota by horizontal gene transfer mechanism (Lima et al., 2020). A better understanding of the transmission and spread of AMR, especially in areas with intensive livestock production, is important to understand. Therefore, the present study investigated the prevalence of E. coli in the dairy farm environment and their drug resistance characteristics. We also investigated the diversity of virulence associated genes (VAGs) responsible for pathogenicity and their distribution within phylogenetic groups.

Sample sources and collection strategy
A total of 209 environment samples were collected from 2017-2019 from a large dairy farm (herd size = 25,000 animals) in Xinjiang province, China. The environmental samples included were fecal samples (n = 50), manure slurry from a storage tank (n = 36), raw milk (n = 90), water samples from the residential area (n = 9), soil samples (n = 12), and crop field soil (n = 12) based on random sampling technique ( Figure 1). A 50 g of manure sample was collected from the animal living area and storage tank from five different sites using a five-point mixed sampling method (Sharp et al., 2012) and stored in sterile zipper bags. Raw milk samples (10 mL) were collected and transferred to sterile falcon tubes according to the guidelines of the National Mastitis Council (Hogan and Smith, 1992). The water samples (50 mL) were collected in sterile water bottles from the residential area by randomly selecting three different water outlets. The blank and crop field soil samples were collected from different sites on farm and fodder growing fields, respectively. All the collected samples were kept at 4°C and transferred to the laboratory within 24 h for further processing.

Isolation and identification of Escherichia coli
The 25 g of fecal, manure, and soil samples were first mixed in 225 mL of phosphate-buffered saline (PBS) to solubilize them. After mixing, 1 mL of the liquid was transferred to a 10 mL LB broth tube for bacterial enrichment by incubation at 37°C with continuous mixing at 160 rpm. From each tube, 100 μL of the enrichment culture was sub-cultured on MacConkey (MAC) agar under prior mentioned incubation conditions. However, the water and milk samples were swabbed directly onto MacConkey agar and incubated at 37°C overnight. Based on colony shape and color, large, smooth, and pink colonies were picked and further streaked onto Eosin Methylene Blue (EMB) agar and incubated overnight at 37°C. The appearance of a metallic green sheen with dark center colonies on EMB agar was indicative of E. coli growth (Peng et al., 2022). Further, presumptive identification was done by the VITEK2 system (BioMerieux, France) (Alfinete et al., 2022) and confirmation by 16S rRNA gene amplification by PCR using primers reported previously . The PCR-amplified product was visualized on 1% agarose gel under the GelDoc XR system (Supplementary Figure S1). The confirmed isolates were preserved in 20% glycerol at −80°C for further analysis.

Detection of virulence-associated genes
The 16S rRNA-confirmed E. coli isolates were subjected to the identification of seven VAGs by the previously described method . The genomic DNA was extracted using a DNA extraction kit (Tiangen Biotech Beijing, Co., Ltd.) following the manufacturer's guidelines. The virulence genes were identified by PCR amplification of target gene primers mentioned in Supplementary Table S1. The PCR reaction mixture (25 μL) consisted of 12.5 μL PreTaq Mix (Vazyme Biotech, China), 1 μL forward primer, 1 μL reverse primer, 1 μL genomic DNA, and 9.5 μL of deionized water under the following conditions; prior denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for the 30s, annealing at varying temperatures mentioned in Supplementary Table S1 for 30s, initial extension at 72°C for 30s followed by a final extension at 72°C for 5 min. After amplification, the PCR product was run on 1% agarose gel electrophoresis at 180 V/200 mA followed by ethidium bromide staining for visualization, and images were taken under the GelDoc XR system (Supplementary Figure S2).

Antimicrobial susceptibility testing
The AST was done by broth micro-dilution assay following the EUCAST guidelines. 1 Briefly, the preserved isolates were thawed at room temperature and re-suspended in LH broth by vigorous mixing (120 rpm) at 37°C for 12 h. The loopful enriched broth was streaked on MacConkey agar following the overnight incubation. The bacterial inoculum was prepared by adjusting the cell density at 5 × 10 5 CFU/mL. The 96-well round bottom plate was used for broth dilution assay and 100 μL of Mueller Hinton (MH) broth was added from the 1st well to the 12th well with a micropipette. Next, 50 μL of prepared bacterial inoculum was added from the 1st to 11th well by keeping the 12th well as a negative control. The antibiotics were selected based on medical and veterinary use which includes trimethoprim-sulfamethoxazole (SXT), ampicillin (AMP), cefotaxime (CTX), tetracycline (TET), ciprofloxacin (CIP), gentamicin (GEN), amikacin (AMK), colistin sulfate (CS), florfenicol (FFC), meropenem (MEM), and tigecycline (TIG) were added from 1st to 10th well by keeping 11th well as a positive control. The reference strain  Table S2). The strains showing resistance to at least one antibiotic from ≥3 classes were classified as MDR.

Detection of antibiotic resistance genes
Phenotypically resistant E. coli strains were subjected to the detection of 20 ARGs from eight antibiotic classes (Supplementary Table S3) according to the method described previously (Yu et al., 2020). The bacterial DNA was extracted using a DNA extraction kit (Tiangen Biotech Beijing, Co., Ltd.) and used as a template for PCR amplification of 20 ARGs (listed in Supplementary Table S3). The PCR reaction mixture (25 μL) consisted of 12.5 μL PreTaq Mix (Vazyme Biotech, China), 1 μL of forward and reverse primer each, 1 μL of bacterial DNA, and 9.5 μL of deionized water. The reactions were performed under the following conditions: denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30s, annealing at varying temperatures (see Supplementary Table S3) for 30s, and extension at 72°C for 30s, followed by a final extension at 72°C for 5 min. After amplification, the PCR product was separated on 1% agarose gel at 180 V/200 mA and stained with ethidium bromide for visualization using the GelDoc XR system.

Phylogenetic analysis
The phylogenetic grouping of E. coli strains was carried out by 2 sets of PCR using primers listed in Supplementary Table S4. The quadruple PCR reaction mixture (25 μL) consists of Premix Taq TM 12.5 μL, 1 μL of Distribution of samples collected from different sources.
Frontiers in Microbiology 05 frontiersin.org each forward and reverse primers (chuA, yjaA, tspE4C2), 2 μL of each arpA forward and reverse primers, 1.5 μL DNA template, and 1 μL dd H 2 O. PCR was carried out under the following conditions; pre-denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 5 s followed by annealing at 59°C for 20 s, and extension at 72°C for 5 min. The PCR reaction for group E and C identification consisted of Premix Taq TM 12.5 μL, 0.6 μL of trpBA forward and reverse primers each, 1 μL of each group-specific primer (Supplementary Table S4), 1.5 μL DNA template, and 2.8 μL dd H 2 O. In the PCR reaction solution, trpBA primers were added as an internal control. The PCR amplifications conditions were pre-denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 5 s, annealing at 57°C (group E) or 59°C (group C) for 20 s, and final extension at 72°C for 5 min. After the PCR amplification, the PCR product was run on 1% agarose gel and visualized under the GelDoc XR system (Supplementary Figure S3), and the phylogenetic group was identified by comparing the results with Supplementary Table S4.

Data analysis
The prevalence was calculated using the formula described by Thrusfield (2018).

Prevalence
No of positive isolates Total isolates % .
( ) = × 100 The antimicrobial susceptibility data were analyzed by descriptive statistics using Microsoft Excel. Moreover, the data for various factors such as sampling source and sampling year affecting the prevalence, AMR and virulence rates were analyzed using the Pearson's Chi-Squared test keeping the level of significance, α = 5% Ma et al., 2022). p-value < 0.05 was considered statistically significant and vice versa. The graphical representation of data was done by GraphPad Prism version 8.2.1 and Microsoft Excel.

AMR characteristics of Escherichia coli strains isolated from different sources
Most of the E. coli strains from all samples were resistant to trimethoprim/sulfamethoxazole (SXT) and 100% susceptible to meropenem (MEM), tigecycline (TIG), and colistin sulfate (CS). Moreover, E. coli strains from fecal samples exhibited higher resistance to ampicillin (AMP), ciprofloxacin (CIP), and tetracycline (TET) in 2017 than other sampling years with a significant difference ( Figure 4A). A similar trend was observed for E. coli strains isolated from milk and manure slurry ( Figures 4B,C). Furthermore, E. coli strains isolated from blank and crop field soil in 2017 exhibited 100% resistant to CTX, CIP, TET, and SXT, while none of the strains isolated from 2018 and 2019 was resistant ( Figures 4D,E). In addition, E. coli strains from crop field showed 50% resistance to AMP and florfenicol (FFC).
The correlation between phenotypic resistance and genotypic detection of ARGs was noted variable. For example, no strains showed resistance to colistin sulfate phenotypically but one strain was carrying the ARG upon genotypic analysis. Moreover, the number of strains carrying ARGs was noted higher as compared to phenotypic resistance among sulfonamides, beta-lactams, and aminoglycosides-resistant strains while the inverse was noted among quinolone, tetracycline, and amphenicol-resistant strains. However, the phenotypic and genotypic expression was observed 100% correlated for fosfomycinresistant strains (Table 2).

Discussion
Antimicrobial resistance particularly in the Enterobacteriaceae family possesses a major threat to global public health. The present study isolated E. coli from the dairy environment, which serves as a reservoir of bacterial pathogens and ARGs and a source of spread of ARGs between the bacterial species via horizontal gene transfer and to humans via fecal contamination of drinking water and milk. The isolation rates of E. coli in this study were found similar to the findings of Sobur et al. (2019) who reported 75% prevalence of E. coli from dairy cattle and farm environment. Other studies conducted by Li et al. (2022) (Imre et al., 2022). Beattie et al. (2020) also reported a similar isolation rate of E. coli from dairy manure in the USA. It is also reported that the presence of E. coli in the dairy environment may be the cause of clinical mastitis in dairy cows (Su et al., 2016). The antimicrobial susceptibility results showed higher resistance to SXT, followed by CTX, AMP, CIP, TET, and the least resistance to FFC, GEN, AMK, and FOS. These results are consistent with the findings of Peng et al. (2022) who isolated E. coli strains from pigs that were highly resistant to SXT (80.38%), AMP (92.86%), and TET (96.26%). A similar study conducted by Beattie et al. (2020) reported that E. coli strains from manure isolates showed higher resistance to AMP and CTX. However, a lower resistance rate to AMK and GEN was also noted by Lu et al. (2022) and Liu et al. (2021) respectively. We noted the E. coli strains were susceptible to the "last resort" antimicrobials such as MEM, TIG, and CS, which is consistent with the findings of a study conducted by Hu et al. (2019) in the human setting. Moreover, other studies conducted by Wang et al. (2021), Zou et al. (2021), and Ma et al. (2022) in animal settings also reported 100% susceptibility of MEM and TIG against E. coli strains. We noted no resistance to CS, which is consistent with a previous report that colistin resistance is decreasing in animal and human settings because of the CS ban in China . The percentage of MDR E. coli was noted at 44.4% in the current study, which is comparable with what was previously Frontiers in Microbiology 08 frontiersin.org reported (54.4%) by Su et al. (2016) and lower than what was reported by Yu et al. (2020) in dairy milk. Another study conducted by Salinas et al. (2019) reported higher resistance to SXT, CIP, AMP, CTX, and TET by E. coli isolated from child and domestic animal origin, which is also consistent with current findings. We identified 18 ARGs out of 20 belonging to different classes of antibiotics. The most prevalent ARGs were sul2 (67.3%, sulfonamides), bla TEM (56.3%, beta-lactam), gyrA (73.6%, quinolones), tet(B) (70.4%, tetracycline's), aph(3)-I (85.7%, aminoglycosides), floR (44.4%, amphenicol), and fosA3 (100%, fosfomycin). Previous studies reported AMR in humans is linked to food animals raised for milk and meat purposes because of environmental contamination and drug residues (Bacanlı and Başaran, 2019;Pormohammad et al., 2019;Ma et al., 2021). The use of antimicrobial drugs in food animals also enhances the percentage of MDR bacteria and ARGs in human microbiota (Ma et al., 2022). Moreover, E. coli is also known to serve Frontiers in Microbiology 09 frontiersin.org as donor bacteria for horizontal gene transfer within and between species (Oladeinde et al., 2019). Lima et al. (2020) highlighted the importance of animal manure and manure-substituted agriculture lands as a major source of antibiotic residues, ARGs, and AMR bacteria in the environment posing a potential threat to public health via horizontal gene transfer mechanisms with the help of mobile genetic elements such as plasmids, transposons, and integrons. Qian et al. (2018) detected 109 ARGs from the fresh manure of chicken, cattle, and pigs responsible for AMR to a class of antibiotics widely used in human and animal settings. We investigated multiple VAGs in the isolated E. coli and most of the investigated VAGs (ompA, ibeB, traT, ompT, fyuA, iroN, irp2) belong to ExPEC, which may cause urinary tract infections in humans. VAGs are responsible for the production of virulence factors which play an important role in the pathogenicity of bacteria through multiple mechanisms such as adhesion, invasion, toxin production, and immune evasion (Kudva et al., 2020). Virulence genes investigated in the present study have various functions. ompA encodes for outer membrane protein A, ibeB is an invasion protein gene, traT encodes for complement resistance protein, ompT encodes for outer membrane protease protein, fyuA encodes for yersiniabactin receptor, iroN encodes for aerobactin receptor, and irp2 gene encodes for iron-responsive element binding protein 2. Zhang et al. (2021)  Drug-resistance spectrum of E. coli strains isolated from the dairy environment.  Frontiers in Microbiology 10 frontiersin.org Percentage distribution of ARGs among E. coli strains.  Khalifeh and Obaidat (2022) identified the iroN gene in E. coli strains from milk and fecal origin similar to the present study. In the current study, most of the E. coli strains were classified under phylogenetic group B1, which is consistent with the findings of Raimondi et al. (2019). These results suggest that the occurrence of ARGs and VAGs may vary by antimicrobial use and other unknown factors. This study also suggests regular monitoring of antimicrobial usage on dairy farms and proper manure treatment before disposal be ensured.

Conclusion
The present study identified multi-drug resistant E. coli strains carrying various ARGs and VAGs in the dairy environment, which may pose a potential threat to human, animal, and environmental health. Moreover, all of the E. coli strains were susceptible to meropenem, tigecycline, and colistin sulfate, which may be considered as critical antibiotics for therapeutic purposes in human and animal settings. Given the widespread distribution of AMR in the dairy environment, it is a potential reservoir of transferring ARGs genes to humans via various direct and indirect gene transfer mechanisms. This prudent the use of antibiotics on dairy farms, proper manure treatment, and enhancement of sanitation, especially in milk processing and transportation, are necessary to reduce the risk to food safety, public health, and environmental health.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Author contributions
MS: writing-original draft. ZH and XG: data curation and formal analysis. MT and RH: graphical representation of data. SW and RS: review and editing. XW and HZ: project administration. WP: conceptualization, supervision, project visualization, and funding acquisition. All authors contributed to the article and approved the submitted version.