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Article

Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow’s Milk of Large-Scale Dairy Farms in China

by
Jiaojiao Gao
1,2,†,
Yating Wu
1,†,
Xianlan Ma
1,2,
Xiaowei Xu
1,2,
Aliya Tuerdi
1,2,
Wei Shao
2,
Nan Zheng
3 and
Yankun Zhao
1,2,3,*
1
Ministry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Key Laboratory of Agro-Products Quality and Safety of Xinjiang, Institute of Quality Standards & Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
2
Xinjiang Meat and Milk Herbivore Nutrition Laboratory, College of Animal Science Xinjiang Agriculture University, Urumqi 830052, China
3
Key Laboratory for Quality and Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(2), 454; https://doi.org/10.3390/ijms26020454
Submission received: 2 December 2024 / Revised: 25 December 2024 / Accepted: 3 January 2025 / Published: 8 January 2025
(This article belongs to the Special Issue Molecular Aspects of Bacterial Infection)
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Abstract

:
Escherichia coli is a common cause of mastitis in dairy cows, which results in large economic losses to the livestock industry. The aim of this study was to investigate the prevalence of E. coli in raw milk in China, assess antimicrobial drug susceptibility, and identify key antibiotic resistance genes carried by the isolates. In total, 350 raw milk samples were collected from large-scale farms in 16 provinces and cities in six regions of China to assess the resistance of E. coli isolates to 14 antimicrobial drugs. Among the isolates, nine resistance genes were detected. Of 81 E. coli isolates (23.1%) from 350 raw milk samples, 27 (33.3%) were multidrug resistant. Antimicrobial susceptibility testing showed that the 81 E. coli isolates were resistant to 13 (92.9%) of the 14 antibiotics, but not meropenem. The resistance gene blaTEM was highly distributed among the 27 multidrug-resistant isolates with a detection rate of 92.6%. All isolates carried at least one resistance gene, and 19 patterns of resistance gene combinations with different numbers of genes were identified. The most common gene combinations were the one-gene pattern blaTEM and the three-gene pattern blaTEM-blaPSE-blaOXA. The isolation rate of E. coli in raw milk and the identified resistance genes provide a theoretical basis for the rational use of antibiotics by clinical veterinarians.

1. Introduction

Escherichia coli is a Gram-negative, opportunistic pathogen commonly found in natural environments and a major cause of persistent and recurrent mastitis in dairy cows [1]. E. coli present in raw milk and dairy products can lead to serious foodborne illnesses in humans, including hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, hemorrhagic colitis, and bloody diarrhea [2,3,4]. Mastitis is a common disease in dairy cows. It significantly reduces milk yield and quality if not addressed promptly and effectively, leading to considerable economic losses [5,6]. Mastitis is induced by a variety of factors, including bacteria, fungi, viruses, and other microorganisms and environmental factors, of which E. coli is one of the most frequently isolated pathogens in bovine intramammary infections [7,8]. E. coli is not only associated with subclinical and clinical mastitis in cows, but it can also transmit antimicrobial resistance (AMR) to humans through the consumption of inadequately heated milk or milk products [9,10]. Risk factors associated with E. coli contamination include the type of milk container, mammary gland cleaning practices, and farm hygiene management. Cow’s milk can act as a reservoir for antibiotic-resistant enteropathogenic E. coli, posing health risks to both animals and humans [11]. As a zoonotic pathogen, E. coli is highly transmissible and can rapidly acquire drug resistance, causing clinical diseases across a wide range of hosts, including humans and animals of varying ages [12]. Currently, E. coli ranks among the top three bacterial diseases affecting agriculture globally.
For decades, controlling E. coli infections in livestock has heavily relied on antibiotic therapy with β-lactams, fluoroquinolones, methotrexate-sulfamethoxazole complexes, and tetracyclines [13,14]. These antibiotics are not only employed for treating infections of E. coli-related diseases in veterinary and human medicine but are also used prophylactically and as antimicrobial growth promoters in livestock feed in many countries [15]. However, the increasing reliance on antibiotics has driven the rapid development of AMR, which has become a significant global public health challenge. E. coli has developed resistance to many antibiotics, which is not confined to specific regions but is, rather, a global phenomenon. AMR arises through horizontal transfer between bacteria or through mutations that develop under selective pressure [16]. The rapidly rising resistance of E. coli to multiple antibiotics in human medicine and animal husbandry has been widely documented globally [17]. The inappropriate use of antibiotics in veterinary practice, such as overdosing, improper compounding, or incomplete treatment regimens, exacerbates the development of AMR of E. coli. In addition, constant exposure to different antimicrobial drugs fosters the transfer of resistant plasmids, leading to multidrug-resistant (MDR) E. coli [18]. For instance, Mwasinga et al. [19] found that 51.2% (214/418) of E. coli isolates from raw milk samples were MDR in the Namwala district in Zambia. Similarly, a study by Tripathi et al. [20] showed that 89.8% (575/640) of E. coli isolates from raw milk samples were resistant to more than two antimicrobial agents. The spread of MDR bacteria is a considerable challenge to human health and the livestock industry [21,22].
Given the critical role of E. coli in mastitis, selecting appropriate antibiotics is essential for effective treatment [23]. Many countries, including India [24], the Kwara State in Nigeria [25], Ethiopia [26], the North Sinai Governorate in Egypt [27], and Romania [28], have conducted studies on the genotypes of AMR E. coli in raw milk. While a previous study investigated the distribution of E. coli in raw milk from northern China [29], a nationwide study of all regions is needed to provide a comprehensive understanding of AMR E. coli in China. The presence of pathogenic E. coli in raw milk not only facilitates the transmission of antibiotic-resistance genes to the human gut but also complicates treatment approaches. Monitoring antibiotic-resistance profiles is crucial to devise effective therapeutic strategies. Continuous surveillance of the resistance gene patterns of AMR E. coli will help to assess the risks associated with E. coli contamination of raw milk. Therefore, the aim of this study was to monitor the prevalence and trends of antibiotic resistance in E. coli and provide scientific evidence for developing effective control strategies, promoting the rational use of antibiotics in clinical practice, and protecting public health.

2. Results

2.1. Prevalence of E. coli

Of 81 E. coli isolates (23.1%) from 350 samples, 6 (13.6%) were from 44 samples in Northeast China, 5 (11.6%) from 43 samples in North China, 4 (11.1%) from 36 samples in South China, 14 (33.3%) from 42 samples in East China, 50 (29.2%) from 171 samples in Northwest China, and 2 (14.3%) from 14 samples in Southwest China. There was a significant difference in the E. coli isolation rates among the six regions (p < 0.01).

2.2. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing showed that 81 E. coli isolates from raw milk samples exhibited significant and variable resistance to 14 antimicrobials (Table 1). Notably, phenotypic resistance was observed for 13 (92.9%) of the 14 tested antimicrobials, with the exception of meropenem. Isolates showed the highest resistance to cephalothin (63.0%), with no significant differences observed among the six regions (p > 0.05). The resistance rates of E. coli isolates for other antimicrobials ranged from 0.0% to 30.9%, with no resistance to meropenem. The resistance rates of the E. coli isolates to kanamycin, gentamicin, doxycycline, fosfenicol, doxorubicin, ciprofloxacin, and cotrimoxazole were low at 2.5%, 6.2%, 8.7%, 2.5%, 7.4%, 6.2%, and 5.0%, respectively.
In Northeast China, isolates had the highest rate of resistance to cefotiophene (75.0%), followed by amoxicillin/clavulanic acid and polymyxin E (33.3%), but were sensitive to meropenem, kanamycin, gentamicin, tetracycline, doxycycline, florfenicol, ciprofloxacin, sulfisoxazole, and cotrimoxazole. Isolates from North China had the highest rate of resistance to cefthiophene (60.0%), with sensitivity to amoxicillin/clavulanic acid, meropenem, kanamycin, gentamycin, tetracycline, doxycycline, flucytosine, ciprofloxacin, ciprofloxacin, and cotrimoxazole. Isolates from South China had the highest rate of resistance to cephalothin (50.0%), while all isolates were susceptible to amoxicillin/clavulanic acid, meropenem, kanamycin, gentamicin, tetracycline, doxycycline, florfenicol, polymyxin E, ciprofloxacin, and cotrimoxazole. Isolates from East China had the highest rate of resistance to cefotaxime (71.4%), with sensitivity to meropenem, florfenicol, and polymyxin E. Isolates from Northwest China had the highest rate of resistance to cefthiophene (62.0%) and sensitivity to meropenem. Isolates from Southwest China were resistant only to cefthiophene and sensitive to all other tested antibiotics.

2.3. Multidrug Resistance

Multidrug resistance is defined as resistance to at least three or more antimicrobial classes. Of the 81 E. coli isolates, 59 (72.8%) were resistant to at least one antibiotic and 27 (33.3%) were resistant to more than three. The MIC distribution of the 27 MDR isolates is shown in Figure 1. The MDR isolates were most commonly resistant to ampicillin, amoxicillin/clavulanic acid, cephalosporins, ceftiofur, tetracyclines, and sulfisoxazole.

2.4. Screening Antibiotic Resistance Genes

In total, 9 antibiotic resistance genes were identified among the 27 MDR E. coli isolates, which included β-lactam resistance genes (blaTEM, blaSHV, blaPSE, blaOXA, blaCTX-M), tetracycline resistance genes (tetA and tetB), and sulfonamide resistance genes (sul1 and sul2). The blaTEM β-lactam resistance gene was the most prevalent, with a detection rate of 92.6% among the 27 MDR isolates. The detection frequencies of blaSHV, blaPSE, blaOXA, blaCTX-M, tetA, tetB, sul1, and sul2 were 25.9%, 14.8%, 37.0%, 37.0%, 37.0%, 29.6%, 11.1%, and 25.9%, respectively (Figure 2).
The E. coli isolates exhibited varied resistance phenotypes to different antimicrobial drugs. A comparison of phenotypic resistance and the corresponding resistance genes of E. coli isolates from raw milk samples showed that isolates resistant to β-lactams, tetracyclines, and sulfonamides also harbored the respective resistance genes (blaTEM, blaSHV, blaPSE, blaOXA, blaCTX-M, tetA, tetB, sul1, and sul2) (Table 2 and Figure 2). The resistance rate of the isolates to β-lactam antibiotics was 100% and most of the isolates were able to amplify the corresponding resistance genes. The different combination patterns of resistance genes detected in this study are shown in Table 2. All of the isolates carried at least 1 resistance gene and 19 unique gene combinations were detected. The most frequent patterns involved isolates carrying three genes (n = 7, 25.9%), followed by one gene (n = 6, 22.2%), five genes (n = 5, 18.5%), two genes (n = 4, 14.8%), four genes (n = 3, 11.1%), and six genes (n = 2, 7.4%). The most common gene combinations were the single-gene pattern of blaTEM (n = 5, 18.5%) and the three-gene pattern of blaTEM-blaPSE-blaOXA (n = 3, 11.1%).

3. Discussion

Mastitis is a devastating disease in dairy farms that not only reduces milk production and quality but also increases culling rates among lactating cows. E. coli, a widespread environmental pathogen, is a key contributor to mastitis [30,31]. In addition, E. coli is a zoonotic pathogen that is particularly capable of acquiring AMR, which may have serious implications for animal food safety and human health [32]. At present, treatment of E. coli infections primarily relies on antimicrobial agents [33]. However, the prevalence of drug resistance continues to escalate, particularly MDR bacteria, often linked to the overuse or inappropriate application of antibiotics [34]. The rise in drug resistance affects livestock production and poses a threat to human health, complicating treatment protocols and potentially leading to increased healthcare costs and treatment failures. Continuous monitoring and comprehensive research into MDR E. coli are essential for developing effective infection control strategies and promoting responsible antibiotic use. Therefore, in this study, we analyzed the prevalent drug-resistant phenotypes and genotypes of 81 E. coli isolates from 350 raw milk samples collected from healthy cows at large-scale farms in 16 provinces in six regions of China. Our findings enhance the understanding of the prevalence of E. coli in raw milk and associated drug resistance risks, thereby supporting efforts to rationalize antimicrobial use, minimize misuse in animal husbandry, and protect public health.
The results indicate a prevalence of E. coli in raw milk of 23.1% (81/350). This detection rate was lower than in previous reports of 70.4% in Indonesia [35], 70% in Bangladesh [36], 45% in Northern China [37], and 41.2% in Tennessee, USA [38]. In contrast, our findings were higher than the 11.8% incidence of E. coli in raw milk in Kenya [39]. Moreover, our results are similar to those of Awadallah et al. [40] who reported that 22.4% of raw milk samples were positive for E. coli in the Sharkia Governorate area. There were no significant differences in the prevalence of E. coli or the resistance patterns of the isolates depending on the source of the samples. Overall, the high isolation rate of E. coli in the raw milk samples of this study indicates the risk of contamination during the production process, suggesting that the frequent occurrence of mastitis in dairy cows might be caused by irregular operation during milking and poor environmental hygiene conditions.
Antibiotics are commonly administered for the treatment of mastitis, although this practice contributes to the emergence of MDR isolates [41]. In China, approximately 6000 tons of veterinary antibiotics are used annually, mostly as feed additives, including β-lactams and tetracyclines [42]. This widespread use indicates multiple pathways for the development of resistance genes, which can be transferred between pathogens.
In the present study, drug sensitivity testing of 81 E. coli isolates revealed varying levels of resistance to several antimicrobial drugs, except for meropenem. Among the isolates, 63.0% were resistant to cephalothin and all were sensitive to meropenem. These findings are similar to a study by Ibrahim et al. [43] that suggested 97.1% of E. coli isolates were resistant to ampicillin and 71.4% were resistant to compounded amoxicillin, cefotaxime, ceftazidime, and ceftiofur. A study conducted in Bangladesh by Rana et al. [44] revealed that the highest resistance rates were observed for ampicillin and tetracycline (100%), followed by amoxicillin (79.17%), ceftazidime (62.5%), streptomycin (58.53%), and gentamicin (60%). In contrast, the bacteria were most sensitive to vancomycin, ciprofloxacin, and meropenem. A phenotypic study of E. coli isolates from raw milk samples showed that 82.25% of isolates were resistant to ampicillin and 50% to sulfamethoxazole [45]. Antibiotic susceptibility of E. coli is crucial for the selection of appropriate antibiotics for the treatment of mastitis. These studies have pointed out that E. coli exhibits a high degree of resistance to β-lactams, tetracyclines, and sulfisoxazoles, consistent with the results of the present study. This phenomenon may be due to the widespread use of β-lactams, tetracyclines, and sulfisoxazoles against E. coli infections in clinical therapy, suggesting that antibiotics should be used rationally in veterinary clinics.
In recent years, MDR E. coli has become an increasingly serious public health concern worldwide [46]. In the present study, 9 (33.3%) of 27 E. coli isolates exhibited multidrug resistance, consistent with a report by Lan et al. [47] that suggested 34.80% of E. coli isolates exhibited multidrug resistance in some parts of China. In comparison, the multi-resistance rate in the present study was significantly lower than the 84.20% rate reported by Bag et al. [48] in Bangladeshi dairy cows and the 100% rate reported by Eldesoukey et al. [49]. The high prevalence of E. coli infection and the increasing prevalence of multidrug resistance poses a significant risk to public health and food safety. Differences in multidrug resistance rates among regions may be due to differences in the type and amount of antibiotics used on different ranches. Therefore, rational control of antibiotic use is key to preventing the development of multidrug resistance. The emergence of MDR E. coli is related to the irrational use of antimicrobial drugs and the drug-resistance genes carried by E. coli. β-lactamase is the main cause of bacterial resistance to β-lactam antibiotics, which is a bacterial enzyme that can inactivate β-lactam antibiotics through hydrolysis, thus rendering these drugs ineffective [50]. In the present study, all 27 E. coli isolates carried β-lactamase-encoding genes and the detection rates of blaPSE, blaSHV, blaOXA, blaCTX-M, and blaTEM were 14.8%, 25.9%, 37.0%, 37.0%, and 92.6%, respectively. Among the genes, blaTEM had the highest detection rate, which was similar to the blaTEM gene (n = 69, 83.1%) reported by Yu et al. [51], who have shown that treatment of mastitis with cephalosporins increased the proportion of blaTEM in milk samples during the withdrawal period, thus likely contributing to the high detection rate of blaTEM [52]. The blaTEM and blaOXA genes were detected in 10 E. coli isolates, blaTEM and blaCTX-M in 8 isolates, and blaTEM and blaSHV in 6 isolates, suggesting a high detection rate of β-lactamase resistance genes. Thus, E. coli can serve as a reservoir of antibiotic resistance and possible gene transfer to other pathogenic species. In general, tetA and tetB are the most prevalent tetracycline resistance genes in E. coli of animal origin [53]. These genes are components of the small nonconjugative transposons Tn1721 (tetA) [54] and Tn10 (tetB) [55], which are frequently integrated into conjugative and nonconjugative plasmids. Among the 27 MDR isolates in this study, the detection rates of tetA and tetB were 37.0% and 29.6%, respectively, consistent with several previous studies [56,57]. Sulfonamide resistance mediated by the sul gene has become globally prevalent, especially in E. coli in food and companion animals, where sulfonamide antibiotic resistance is severe [58]. In the present study, the detection rates of sul1 and sul2 were 11.1% and 25.9%, respectively, consistent with previously reported data on E. coli isolated from food and animals [59,60]. In total, 19 different resistance gene combination patterns were identified in this study, with 7 (25.9%) E. coli isolates carrying three resistance genes. In this study, the isolates were resistant to β-lactams, tetracyclines, and sulfonamides, consistent with the detected resistance genes (blaTEM, blaSHV, blaPSE, blaOXA, blaCTX-M, tetA, tetB, sul1, and sul2). The resistance rate of the isolates to β-lactams was 100% and most of the isolates were able to amplify the corresponding resistance genes. The presence of these resistance genes is key to the development of drug resistance in E. coli.
In summary, E. coli, the main pathogen causing mastitis in dairy cows, has demonstrated resistance to a wide range of antibiotics. E. coli is known to be a very efficient reservoir of antibiotic-resistance genes and can transfer these genes to other pathogens. Therefore, analyzing E. coli isolates for resistance and related resistance genes is essential for screening suitable antimicrobial drugs.

4. Materials and Methods

4.1. Sample Collection

Overall, 350 raw milk samples were collected from large-scale farms across six regions in China (Northeast, North, South, East, Northwest, and Southwest), which encompassed 16 provinces and municipalities, including autonomous regions (Beijing, Tianjin, Heilongjiang, Inner Mongolia, Gansu, Shaanxi, Hebei, Shandong, Jiangsu, Zhejiang, Fujian, Guangdong, Chongqing, Yunnan, Guizhou, and Xinjiang). In total, 44, 43, 36, 42, 171, and 14 raw milk samples were collected in Northeast, South, East, Northwest, and Southwest China, respectively. The raw milk samples were collected from the top, middle, and bottom of bulk tanks, mixed well, and then transferred into sterile bottles and immediately transported to our laboratory at 4 °C.

4.2. Isolation and Characterization of E. coli

An aliquot of 25 mL from each milk sample was mixed with 225 mL of trypticase soy broth and then incubated at 37 °C for 16 h with shaking to detect E. coli. Following incubation, the samples were incubated on MacConkey agar plates (Beijing Luqiao Technology Co., Ltd., Beijing, China) for 18–24 h at 37 °C. Presumptive E. coli colonies, which appeared small and pink, with smooth, moist surfaces and neat edges, were selected for Gram staining and biochemical identification. Gram-negative E. coli isolates appeared microscopically as red, short rods, positive for methyl red, and negative for Voges–Proskauer and citric acid. Also, the isolates were cultured in 3 mL of trypsinized soy broth at 37 °C for 18–24 h whilst being shaken for DNA extraction, which was conducted with the TIANamp Bacteria DNA Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) in accordance with the manufacturer’s instructions. The extracted genomic DNA samples were stored at −20 °C until further use.

4.3. PCR Amplification

PCR amplification was performed by Beijing Bio-Tech Co., Ltd. (Beijing, China) using 16S rRNA universal primers and specific primers for the phoA gene (Supplementary Table S1). Each 30 μL PCR reaction mixture consisted of 15 μL of 2×Taq PCR Master Mix, 1 μL each of the forward and reverse primers (10 pmol/μL), 2 μL of DNA template, and 11 μL of double-distilled H2O. The PCR amplification program included an initial denaturation step at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 90 s. The PCR amplification products were analyzed by electrophoresis on a 1.0% agarose gel and the amplicons were sequenced by Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). The obtained sequences were assessed against the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/ (accessed on 1 March 2024)) using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 March 2024)).

4.4. Antimicrobial Susceptibility Patterns

Antimicrobial susceptibility testing for 14 antimicrobial agents was conducted using the broth dilution method as recommended by the American Committee for Clinical and Laboratory Standardization (CLSI, 2024). The bacterial suspensions were adjusted to a turbidity of 0.5 McFarland units using a turbidimeter and inoculated into 96-well plates containing different concentrations of the following antimicrobial agents: ampicillin (0.25–128 mg/L), amoxicillin/clavulanic acid (0.25/0.12–128/64 mg/L), cephalothin (0.25–128 mg/L), ceftiofur (0.25–128 mg/L), meropenem (0.25–128 mg/L), kanamycin (0.25–128 mg/L), gentamicin (0.25–128 mg/L), tetracycline (0.25–128 mg/L), doxycycline (0.25–128 mg/L), florfenicol (0.25–128 mg/L), polymyxin E (0.25–128 mg/L), ciprofloxacin (0.25–128 mg/L), sulfisoxazole (2–1024 mg/L), and sulfamethoxazole (0.12/2.4–64/1216 mg/L) (YNK (Tianjin) Biotechnology Co., Ltd., Tianjin, China). The plates were incubated at 35 °C for 18–20 h. The minimum inhibitory concentration (MIC), defined as the lowest concentration of an antimicrobial agent that inhibited visible bacterial growth, was recorded. The interpretation of the results was based on the guidelines of CLSI (2024) (Supplementary Table S2).

4.5. Detection of Drug Resistance Genes

Five genes related to β-lactamase resistance (blaTEM, blaSHV, blaPSE, blaOXA, and blaCTX-M), two related to tetracycline resistance (tetA and tetB), and two related to sulfisoxazole resistance (sul1 and sul2) of the E. coli isolates were detected by multiplex PCR (Supplementary Table S1). The PCR amplification conditions included an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at different temperatures for 30 s, extension at 72 °C for 45 s, and extension at 72 °C for 10 min. E. coli strain ATCC 25922 served as a positive control in each run. Supplementary material provides specific details of sample numbers (Supplementary Tables S3 and S4).

4.6. Statistical Analysis

All data were recorded and organized with Excel 2019 software (Microsoft Corporation, Redmond, WA, USA). Statistical comparisons between groups, including prevalence rates, AMR phenotypes, and the distribution of resistance genes, were performed using one-way analysis of variance and the least significant difference multiple comparisons with IBM SPSS Statistics for Windows (version 20.0; IBM Corporation, Armonk, NY, USA). A probability (p) value < 0.05 was considered statistically significant.

5. Conclusions

In this study, E. coli isolated from raw milk produced in China was characterized for the first time. The results showed that the prevalence of E. coli in raw milk in China was as high as 23.1% and the multidrug resistance rate was 33.3%. Therefore, the in-depth monitoring of the prevalence of E. coli and antibiotic resistance is particularly important for the development of effective preventive and control measures, and to promote the rational clinical use of antibiotics. Further studies are warranted to assess the mechanism employed by E. coli to acquire, transfer, and transmit multidrug resistance genes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26020454/s1.

Author Contributions

Sample collection, isolation and identification of bacteria, conduction of experiments, generation of graphs, and writing of the manuscript, J.G., Y.W. and X.M.; Data analysis and summarization, X.X.; RNA extraction and PCR amplification, A.T.; Project supervision and revision of the manuscript, W.S. and N.Z.; Funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant number, 32060797), Tianshan Talent Training Program (grant number, 2023TSYCCY0034), Project of Fund for Stable Support to Agricultural Sci-Tech Renovation (grant number, xjnkywdzc-2024003-51) and Xinjiang Outstanding Youth Fund (grant number, 2024D01E12).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available in Supplementary Materials Tables S1–S4.

Conflicts of Interest

The authors declare no conflicts of interest.

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Ijms 26 00454 g001
Figure 1. Patterns of multidrug resistance for Raw Bovine Milk-Derived E. coli based on resistance classifications as susceptible (S), resistant (R), or intermediate (I).
Figure 1. Patterns of multidrug resistance for Raw Bovine Milk-Derived E. coli based on resistance classifications as susceptible (S), resistant (R), or intermediate (I).
Ijms 26 00454 g001
Ijms 26 00454 g002
Figure 2. Detection rate of drug resistance genes blaTEM (92.6%), blaSHV (25.9%), blaPSE (14.8%), blaOXA (37.0%), blaCTX-M (37.0%), tetA (37.0%), tetB (29.6%), sul1 (11.1%), and sul2 (25.9%). The resistance genes of blaTEM, blaSHV, blaPSE, blaOXA, and blaCTX-M belong to beta-lactams; the tetA and tetB genes belong to tetracyclines; and the sul1 and sul2 genes belong to sulfonamides.
Figure 2. Detection rate of drug resistance genes blaTEM (92.6%), blaSHV (25.9%), blaPSE (14.8%), blaOXA (37.0%), blaCTX-M (37.0%), tetA (37.0%), tetB (29.6%), sul1 (11.1%), and sul2 (25.9%). The resistance genes of blaTEM, blaSHV, blaPSE, blaOXA, and blaCTX-M belong to beta-lactams; the tetA and tetB genes belong to tetracyclines; and the sul1 and sul2 genes belong to sulfonamides.
Ijms 26 00454 g002
Table 1. Antibiotic resistance of isolated E. coli strains.
Table 1. Antibiotic resistance of isolated E. coli strains.
Name of Antibacterial DrugsNo. (%) of Positive Strains
Northeast China (n = 6)North China (n = 5)South China (n = 4)East China (n = 14)Northwest China (n = 50)Southwest China (n = 2)Total
(n = 81)
Ampicillin1 (16.7)2 (40.0)1 (25.0)7 (50.0)14 (28.0)0 (0.0)25 (30.9)
Amoxicillin/Clavulanic acid2 (33.3)0 (0.0)0 (0.0)4 (28.6)3 (6.0)0 (0.0)9 (12.4)
Cephalothin4 (75.0)3 (60.0)2 (50.0)10 (71.4)31 (62.0)1 (50.0)51 (63.0)
Ceftiofur1 (16.7)2 (40.0)1 (25.0)1 (7.1)9 (18.0)0 (0.0)14 (17.3)
Meropenem0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Kanamycin0 (0.0)0 (0.0)0 (0.0)1 (7.1)1 (2.0)0 (0.0)2 (2.5)
Gentamicin0 (0.0)0 (0.0)0 (0.0)3 (21.4)2 (4.0)0 (0.0)5 (6.2)
Tetracycline0 (0.0)0 (0.0)0 (0.0)2 (14.3)9 (18.0)0 (0.0)11 (13.6)
Doxycycline0 (0.0)0 (0.0)0 (0.0)1 (7.1)6 (12.0)0 (0.0)7 (8.7)
Florfenicol0 (0.0)0 (0.0)0 (0.0)0 (0.0)2 (4.0)0 (0.0)2 (2.5)
Polymyxin E2 (33.3)1 (20.0)0 (0.0)0 (0.0)3 (6.0)0 (0.0)6 (7.4)
Ciprofloxacin0 (0.0)0 (0.0)0 (0.0)1 (7.1)4 (8.0)0 (0.0)5 (6.2)
Sulfisoxazole0 (0.0)1 (20.0)1 (25.0)6 (42.9)5 (10.0)0 (0.0)13 (16.1)
Sulfamethoxazole0 (0.0)0 (0.0)0 (0.0)1 (7.1)3 (6.0)0 (0.0)4 (5.0)
Table 2. Main resistance patterns of 27 multidrug-resistant strains of E. coli isolated from raw milk.
Table 2. Main resistance patterns of 27 multidrug-resistant strains of E. coli isolated from raw milk.
No. of Resistance
Genes		Combination Patterns of Resistance GenesNo. of Resistance Gene Combination PatternsNo. of E. coli Isolates (%)
(n = 27)
1blaTEM56 (22.2)
blaCTX-M1
2blaTEM-blaOXA24 (14.8)
blaSHV-blaCTX-M1
blaTEM-blaCTX-M1
3blaTEM-blaPSE-blaOXA37 (25.9)
blaTEM-blaSHV-blaCTX-M2
blaTEM-tetA-tetB1
blaTEM-blaCTX-M-tetA1
4blaTEM-blaSHV-blaPSE-blaOXA13 (11.1)
blaTEM-blaSHV-blaOXA-sul11
blaTEM-tetA-tetB-sul21
5blaTEM-blaCTX-M-tetA-sul1-sul215 (18.5)
blaTEM-blaSHV-tetA-tetB-sul21
blaTEM-blaOXA-tetA-tetB-sul21
blaTEM-blaOXA-blaCTX-M-tetA-tetB1
blaTEM-tetA-tetB-sul1-sul21
6blaTEM-blaOXA-blaCTX-M-tetA-tetB-sul212 (7.4)
blaTEM-blaSHV-blaCTX-M-tetA-tetB-sul21
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Gao, J.; Wu, Y.; Ma, X.; Xu, X.; Tuerdi, A.; Shao, W.; Zheng, N.; Zhao, Y. Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow’s Milk of Large-Scale Dairy Farms in China. Int. J. Mol. Sci. 2025, 26, 454. https://doi.org/10.3390/ijms26020454

AMA Style

Gao J, Wu Y, Ma X, Xu X, Tuerdi A, Shao W, Zheng N, Zhao Y. Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow’s Milk of Large-Scale Dairy Farms in China. International Journal of Molecular Sciences. 2025; 26(2):454. https://doi.org/10.3390/ijms26020454

Chicago/Turabian Style

Gao, Jiaojiao, Yating Wu, Xianlan Ma, Xiaowei Xu, Aliya Tuerdi, Wei Shao, Nan Zheng, and Yankun Zhao. 2025. "Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow’s Milk of Large-Scale Dairy Farms in China" International Journal of Molecular Sciences 26, no. 2: 454. https://doi.org/10.3390/ijms26020454

APA Style

Gao, J., Wu, Y., Ma, X., Xu, X., Tuerdi, A., Shao, W., Zheng, N., & Zhao, Y. (2025). Prevalent and Drug-Resistant Phenotypes and Genotypes of Escherichia coli Isolated from Healthy Cow’s Milk of Large-Scale Dairy Farms in China. International Journal of Molecular Sciences, 26(2), 454. https://doi.org/10.3390/ijms26020454

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