Study on drug resistance, biofilm formation, phylogenetic and virulence gene analysis of Escherichia coli from diarrheic lambs

Abstract

Background

This study comprehensively characterized E. coli from diarrheal lambs in Aksu, China. We evaluated growth kinetics, phylogenetic groups, virulence and resistance genes, antimicrobial susceptibility, biofilm formation, and pathogenicity in mice to determine their potential risks.

Methods

Fresh fecal samples from diarrheic lambs were collected for bacterial isolation. E. coli was identified via specific PCR and 16 S rRNA sequencing. A microbial growth analyzer was used to determine growth curves, and motility medium was used to detect active motility. Biofilm formation was assessed by crystal violet staining. The Kirby-Bauer disk diffusion method was used to test the antimicrobial resistance of the strains, while PCR was performed to identify phylogenetic groups, virulence genes, and resistance genes. Pathogenicity was confirmed via a murine infection model.

Results

A total of 28 E. coli strains were isolated from 21 diarrheal lambs. Growth kinetic analysis revealed that all the strains entered the logarithmic growth phase after approximately 5 h of cultivation. Among the strains, 53.6% exhibited active motility. Phylogenetic classification revealed a predominance of Group B1 (53.6%), followed by Group D (35.7%) and Group A (10.7%). Thirteen virulence genes and nine resistance genes were detected. The murine infection model demonstrated that 39.2% of the strains tested were pathogenic, with significant pathological lesions observed in the liver, lungs, spleen, kidneys, and small intestine of infected mice. Additionally, 64.3% of the strains were multidrug resistant (MDR), and the detection rate of extended-spectrum β-lactamase (ESBL)-producing strains was 53.6%. blaCTX-M4 was the key determinant mediating E. coli resistance to β-lactam antibiotics. Notably, complete phenotype‒genotype concordance was observed for blaNDM-mediated imipenem resistance and cmlA-mediated chloramphenicol resistance. A statistically significant correlation was found between biofilm formation capacity and resistance patterns: strains with stronger biofilm formation were more likely to be MDR- and ESBL-positive.

Conclusion

E. coli from diarrheal lambs poses significant risks to sheep farming and may represent a zoonotic reservoir. These findings highlight the need for effective measures to control E. coli infections on sheep farms.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-026-05296-z.

Introduction

Escherichia coli (E. coli) is a common gram-negative, facultative anaerobic bacterium that typically inhabits the intestinal tracts of humans and other warm-blooded animals [1]. While most strains are harmless commensals, certain pathogenic variants can cause severe intestinal and extraintestinal infections, posing a significant global public health challenge [2]. Of particular concern is the emergence and spread of antimicrobial resistance (AMR) in E. coli, especially multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing strains, which have substantially limited treatment options [3, 4]. The circulation of resistant bacteria and their resistance genes among humans, animals, and the environment underscores the urgency of adopting a One Health approach to mitigate the AMR crisis [5].

Pathogenic E. coli is a major causative agent of lamb diarrhea, which is highly prevalent in livestock production. Diarrheagenic E. coli pathotypes—including enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and enterohemorrhagic (EHEC) E. coli—are classified on the basis of specific virulence genes [6]. For example, Shiga toxin is a defining marker of EHEC, whereas enterotoxin is central to ETEC [1]. In addition to these core genes directly associated with toxin production, various accessory genes—such as those encoding adhesins (e.g., fimH, eae), outer membrane proteins (e.g., ompA, ompC), and iron acquisition systems (e.g., irp2, ireA)—also play important roles in the pathogenicity of E. coli by facilitating colonization, host invasion, and nutrient acquisition [7, 8]. A key concept is the distinction between these two gene categories: core genes are pathotype-defining, whereas accessory genes increase virulence without being pathotype-specific [8]. Furthermore, phylogenetic group analysis provides an essential perspective for understanding the evolutionary history and pathogenic potential of E. coli. The species is classified into several major phylogroups—primarily A, B1, B2, and D—along with more recently identified lineages [9, 10]. Studies have shown that commensal strains frequently belong to phylogroups A and B1, whereas strains associated with extraintestinal infections often fall within phylogroups B2 and D. Additionally, it is evident that E. coli within commensal phylogroups can harbor certain virulence genes, underscoring the intricate relationship between phylogenetic background and pathogenic potential [11].

In veterinary practice, the increasing use of antimicrobials to control bacterial lamb diarrhea has led to a continuous expansion of E. coli AMR profiles and the widespread occurrence of MDR strains [12]. Bacterial resistance is mediated by diverse molecular mechanisms, including enzymatic drug inactivation by β-lactamases, target site mutations, enhanced efflux pump activity (e.g., AcrAB-TolC), and plasmid-mediated horizontal transfer of resistance genes [13–15]. Furthermore, biofilm formation serves as a physical resistance strategy. Regulated by quorum-sensing (e.g., luxS) and stress-response (e.g., rpoS) factors, biofilms can significantly increase bacterial tolerance to antimicrobial agents [16, 17].

Therefore, this study aims to systematically characterize E. coli isolates from diarrheal lambs in the Aksu region of Xinjiang, China, by analyzing their antimicrobial resistance profiles, virulence gene carriage, pathogenicity in a murine model, and biofilm-forming capacity. The findings will help assess their potential role as reservoirs of antimicrobial resistance and virulence determinants, providing a scientific basis for understanding the indirect risks they may pose to public health through the food chain or environmental contamination.

Materials and methods

Sample collection

Rectal swabs and fecal samples (n = 21) were collected from diarrheic lambs in three intensive livestock production facilities located in Aksu, Xinjiang, China, during two distinct epidemiological periods: spring 2023 and winter 2024.

Experimental animals

Six-week-old Kunming strain-bearing mice were obtained from the Experimental Animal Center of Xinjiang Medical University. The mice were used for pathogenicity assays under controlled laboratory conditions (Approval Letter for Ethics Review by Biology Ethics Committee of Tarim University, Approval Number: 2023009).

Bacterial isolation and PCR identification

Following initial enrichment in tryptic soy broth (TSB) (Haibo Biotech, Qingdao, Cat. No. HB4114, China), the samples were subjected to selective purification. For the isolation of presumptive E. coli, samples were first streaked onto MacConkey Agar (MAC) (Haibo Biotech, Qingdao, Cat. No. HB6239-9, China). Pink colonies, which are indicative of lactose fermentation, were then subcultured onto Eosin Methylene Blue (EMB) agar (Haibo Biotech, Qingdao, Cat. No HB0107, China). The colonies that exhibited a characteristic metallic sheen on EMB agar were selected as presumptive E. coli for final purification and confirmation and were subjected to Gram staining (Coolaber Technology, Beijing, Cat. No SL7040, China) (which confirmed them as gram-negative bacilli) as well as microscopic characterization. To ensure a diverse collection, multiple colonies with distinct morphologies were purposefully selected from each animal, and all were characterized independently, with the distribution detailed in Supplementary Table S1. Genomic DNA was extracted via the Bacterial Genomic DNA Extraction Kit (Tiangen Biotech, Beijing, Cat. No. DP302, China). Amplification was performed via E. coli-specific phoA gene primers and universal bacterial 16 S rRNA gene primers, with sequences and detailed information provided in Supplementary Table 2. The 16 S rRNA gene amplicons were subjected to Sanger sequencing by Shanghai Sangon Biotech Co., Ltd. The assembled sequences were analyzed via BLAST alignment against the NCBI database, and a phylogenetic tree was constructed via MEGA software (version 11.0).

Determination of growth curves and motility of isolates

The growth curve of the isolated E. coli was determined via a high-throughput real-time microbial growth analyzer. The test strains were inoculated into TSB (Haibo Biotech, Qingdao, Cat. No. HB4114 China) and incubated overnight at 37 °C with shaking (180 rpm/min). The cultures were subsequently transferred to fresh TSB at a 1:200 volumetric ratio (v/v) and loaded into the analyzer (1 mL total volume). Continuous shaking (800 rpm/min) at 37 °C was maintained for 24 h to monitor growth dynamics.

Bacterial motility was assessed via the semisolid agar plate method [18]. Strains were cultured in TSB at 37 °C (180 rpm/min) until they reached the logarithmic growth phase. Aliquots (2 µL) of bacterial suspensions were inoculated vertically onto agar surfaces. After complete liquid absorption, the plates were incubated upright at 37 °C for 24 h, after which motility halo diameters were measured.

Population typing

Phylogenetic grouping of the E. coli isolates was performed according to the PCR Clermont typing scheme [9]. Amplification reactions were conducted under standardized cycling conditions, with PCR products resolved by agarose gel electrophoresis to determine allelic profiles for hierarchical classification.

Studies on bacterial pathogenicity

Detection of virulence genes

Polymerase chain reaction (PCR) (2X EasyTaq PCR SuperMix: TransGEN Biotech, Beijing, Cat. No. AS111-13, China) was used to detect 20 virulence-associated genes in E. coli isolates, which were categorized into four functional groups on the basis of their roles: adhesin-associated genes (crl, fimH, papC, sfaS, K88, eae, malX, and papC); toxin-producing genes (stx1, stx2, hlyA, hlyE, astA, and estA); iron acquisition system genes (ireA, irp2, and iucD); outer membrane protein genes (ompA and ompC); and invasion-associated genes (invE) [19–22]. The amplified products were resolved by 1% agarose gel electrophoresis to confirm the amplification efficacy of the target genes; the primer sequences and amplification parameters are provided in Table S2.

Pathogenicity testing in mice

The mice were randomly divided into 28 test groups and 1 control group, with 9 mice in each group. E. coli strains were cultured on EMB agar plates, followed by expansion in TSB under standardized conditions (37 °C, 180 rpm, 12 h). The bacterial suspensions were pelleted via centrifugation (5,000 rpm, 5 min), washed twice with phosphate-buffered saline (PBS) (Servicebio Biotech, Wuhan, Cat No. G4202-100ML, China), and adjusted to the target concentrations (1 × 10⁹, 1 × 10⁸, and 1 × 10⁷ CFU/mL). The experimental groups received intraperitoneal injections of 0.5 mL of bacterial suspensions, whereas the control group received 0.5 mL of sterile saline. Over a 10-day observation period, the mice were monitored for clinical signs of systemic infection, including lethargy, piloerection, anorexia, diarrhea, and mortality. Strains that induced any of the aforementioned clinical signs were defined as pathogenic.

Necropsy and pathological examination

The mice that died from infection were subjected to necropsy to assess organ lesions. Additionally, affected tissues were fixed in 4% formaldehyde (Servicebio Biotech, Wuhan, Cat No. G1101-100ML, China), embedded in paraffin, sectioned, and stained for histopathological examination under a microscope.

Studies on antimicrobial resistance

Antimicrobial susceptibility testing and detection of multidrug-resistant bacteria

Antimicrobial susceptibility testing was performed via the Kirby-Bauer disk diffusion method in strict accordance with the Clinical and Laboratory Standards Institute (CLSI, 2022 edition) guidelines [23], with susceptibility interpreted according to the criteria outlined in Table 1. E. coli ATCC 25,922 was used as the quality control strain. A total of 18 antimicrobial agents (Thermo Fisher Scientific, Cat. No. HP0053A, USA) were evaluated in this study, including β-lactams (ampicillin, amoxicillin, imipenem, cefoxitin, ceftazidime, cefotaxime, and ceftriaxone); aminoglycosides (gentamicin, amikacin, kanamycin, and neomycin); phenols (chloramphenicol and florfenicol); tetracyclines (tetracycline and doxycycline); quinolones (ciprofloxacin and norfloxacin); and polymyxins (polymyxin B). MDR isolates were defined in strict accordance with CLSI standards and internationally recognized criteria: isolates that exhibit resistance to at least three different classes of those mentioned above seven antimicrobial categories were classified as MDR strains [24].

Table 1.

Criteria for determining the dosage and drug resistance of tablets

Antibiotic name	Drug content (µg/ tablet)	Criteria for determining the diameter of the antibacterial zone/mm
		R	I	S
AMX	10	≤ 13	14–16	≥ 17
AMP	10	≤ 13	14–16	≥ 17
CTX	30	≤ 22	23–25	≥ 26
CAZ	30	≤ 17	18–20	≥ 21
CRO	30	≤ 19	20–22	≥ 23
FOX	30	≤ 14	15–17	≥ 18
IPM	10	≤ 19	20–22	≥ 23
FON	30	≤ 12	13–16	≥ 17
GM	10	≤ 12	13–15	≥ 16
AK	30	≤ 14	15–16	≥ 17
N	30	≤ 12	13–14	≥ 15
KAN	30	≤ 13	14–17	≥ 18
TE	30	≤ 11	12–14	≥ 15
DOX	30	≤ 10	11–13	≥ 14
CIP	5	≤ 21	22–25	≥ 26
NOR	10	≤ 12	13–16	≥ 17
C	30	≤ 12	13–17	≥ 18

Detection of resistance genes

Polymerase chain reaction (PCR) was used to detect 14antibiotic resistance genes belonging to 6 ategories in E. coli isolates, specifically β-lactam resistance genes (blaTEM, blaCTX-M4, blaNDM and blaSHV); aminoglycoside resistance genes (aadA1 and aacC2); tetracycline resistance genes (tetA and tetM); phenicol resistance genes (cmlA); quinolone resistance determinants (parC, oqxA, oqxB and oqxAB); and polymyxin resistance determinants (mcr-1) [19, 20, 25–27]. The PCR conditions and specific primer information are provided in Table S3.

Detection of extended-spectrum β-lactamase-producing E. coli.

The detection of ESBL-producing E. coli was performed through phenotypic screening and confirmatory assays in strict adherence to Clinical and Laboratory Standards Institute (CLSI) guidelines via the standard disk diffusion method. Two antimicrobial disk pairs—ceftazidime (30 µg) versus ceftazidime/clavulanic acid (30/10 µg) and cefotaxime (30 µg) versus cefotaxime/clavulanic acid (30/10 µg)—were employed [28].

Measurement of biofilm formation ability

The biofilm formation capacity of E. coli was semiquantitatively assessed via a standardized 96-well microtiter plate assay. The bacterial strains were initially inoculated into tryptic soy broth (TSB) and incubated overnight at 37 °C under aerobic shaking conditions (180 rpm). Following subculture in fresh TSB to mid-log-phase turbidity, the suspension was diluted 1:100 in sterile TSB, and 200 µL aliquots were dispensed into 96-well polystyrene plates. After 36–48 h of static incubation at 37 °C, nonadherent cells were removed by three sequential washes with phosphate-buffered saline (PBS). Adherent biofilms were air-dried, heat-fixed at 56 °C for 15 min, and stained with 1% (w/v) crystal violet for 5 min. Excess stain was eliminated through two PBS rinses. Following decolorization with 200 µL of anhydrous ethanol per well, the absorbance at 570 nm was measured by Enzyme-linked Immunosorbent Assay Reader. The critical OD_570C values (OD_570C: the mean of the blank control wells) were used to determine the results: OD₅₇₀ ≤ OD_570C was considered to have no ability to form membrane, OD_570C < OD₅₇₀ ≤ 2OD_570C was considered to have weak ability to form membrane, and 2OD_570C < OD₅₇₀ ≤ 4OD_570C was considered to have moderate ability to form membrane, OD₅₇₀ > 4OD_570C is the strong ability of film formation [29].

Results

Isolation and identification of E. coli.

Following selective culture from 21 diarrheic lambs in Aksu, 28 E. coli strains were isolated and identified. The identification was based on characteristic colony morphology on EMB agar, gram-negative staining, and amplification of the species-specific phoA gene (720 bp). The distribution of isolates among the lamb hosts and representative identification data are provided in Supplementary Table S1 and Supplementary Figure. S1, respectively.

Through homology analysis of the 16 S RNA gene, it was found that the 16 S rRNA gene sequences of 28 isolated strains presented 99.83–100% similarity with the 16 S rRNA sequences of E. coli in the GenBank database, further confirming that these isolates are indeed E. coli. Phylogenetic tree analysis indicated that the isolates were most closely related to E. coli of Indian origin (Fig. 1).

Fig. 1.

Phylogenetic tree based on the 16S rRNA gene sequence. Note: E1--E28 represent the 16S rRNA gene sequence phylogenetic tree of the isolated strains in this study

Analysis of growth curves and motility

Growth kinetics analysis of all the E. coli isolates revealed conserved growth phase transition profiles: initiation of the logarithmic phase at 5 h, attainment of the stationary phase by 10 h, and progression to the decline phase after 16 h (Fig. 2). Motility assays across the 28 isolates revealed strain-dependent variance in radial migration capacity on semisolid media, with 14 strains exhibiting robust motility phenotypes (Figure. S2).

Fig. 2.

Growth curve of Escherichia coli. The growth dynamics of the E. coli strain were monitored by measuring the optical density at 830 nm (OD830) over time in hours (h). The data show that the bacteria entered the logarithmic phase at approximately 5 h and reached the stationary phase around 10 h

Population typing

On the basis of the established typing criteria, among the 28 E. coli strains isolated in this study, 3 belonged to phylogroup A, 15 to phylogroup B1, and 10 to phylogroup D, as illustrated in Fig. 4 and Figure. S2.

Fig. 4.

Comprehensive characterization of Escherichia coli isolates. Distribution of pathotypes, phylogenetic groups, antimicrobial resistance phenotypes, virulence genes, and resistance genes in E. coli isolates, along with correlation analysis between resistance genotypes and phenotypes. athotypes are defined as follows: EPEC, enteropathogenic E. coli; EAEC, enteroaggregative E. coli; ETEC, enterotoxigenic E. coli; EIEC, enteroinvasive E. coli. Antimicrobial agent abbreviations: AMP, ampicillin; AMX, amoxicillin; IPM, imipenem; FOX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; GM, gentamicin; AK, amikacin; KAN, kanamycin; N, neomycin; C, chloramphenicol; TE, tetracycline; DOX, doxycycline; FON, florfenicol; PB, polymyxin B

Detection of virulence genes

The results of the PCR screening for virulence-associated genes are presented in Fig. 4 and Figure. S3. The detection rates were as follows: ompC (100%), crl (96.4%), fimH (96.4%), hlyE (85.7%), astA (39.3%), papC (25.0%), malX (25.0%), invE (17.9%), K88 (14.3%), eae (10.7%), hlyA (10.7%), irp2 (10.7%), and ireA (7.1%). On the basis of these virulence gene profiles, the E. coli isolates were classified into four pathotypes: EPEC, EAEC, EPEC + ETEC and EAEC + EIEC, as illustrated in Fig. 4.

Pathogenicity testing in mice

Murine infection models revealed that 39% of the tested strains exhibited pathogenicity. Within 16–24 h post-challenge, the mice in the experimental groups displayed clinical signs, including lethargy, anorexia, and mortality. The survival status of the mice in each experimental group at different time points is presented in Fig. 3A. Necropsy of the deceased mice revealed varying degrees of enlargement and congestion in the liver, spleen, and kidneys, along with localized enlargement of the small intestine (Fig. 3B) and thinning of the intestinal mucosa. As shown in Fig. 3C, histopathological examination revealed the following observations: a significant reduction in the number of lymphocytes in the white pulp of the spleen and a marked increase in the number of red blood cells in the red pulp; disruption of the hepatic lobule structure in the liver, accompanied by edema and degeneration of hepatocytes; a large number of red blood cells in the alveolar spaces, along with hyperplasia and inflammatory cell infiltration in the pulmonary interstitium; edema and degeneration of renal tubular epithelial cells, with extensive red blood cell accumulation and inflammatory cell infiltration in the renal interstitium; and damage to the intestinal mucosal layer, severe destruction of villous structures, and degeneration and necrosis of epithelial cells.

Fig. 3.

Pathogenicity experiment ofEscherichia coli. A Survival curve of mice. B Pathological examination of mice. C Histopathological observation of mice. Note: The black arrows indicate red blood cells and inflammatory exudate; the red arrows indicate detachment of the small intestinal villi

Antimicrobial susceptibility testing and detection of multidrug-resistant bacteria

The antimicrobial susceptibility results for the 28 E. coli isolates are shown in Fig. 4. The isolates exhibited extensive drug resistance. The degree of resistance to nine antibiotics, including ceftriaxone, cefotaxime, norfloxacin, ciprofloxacin, gentamicin, amoxicillin, ampicillin, tetracycline, and doxycycline, was high (50%-71.4%). Moderate resistance (14.3%-46.4%) to ceftazidime, florfenicol, chloramphenicol, amikacin, and kanamycin was detected. In contrast, resistance to imipenem, cefoxitin, and polymyxin B was low (3.6%-7.1%), and notably, all the isolates were susceptible to neomycin. Furthermore, 64.3% (18/28) of the isolates were classified as MDR.

ESBL-producing E. coli identification

Phenotypic confirmation via the use of ceftazidime/clavulanic acid and cefotaxime/clavulanic acid discs identified 53.6% (15/28) of the isolates as ESBL producers.

Detection rates of resistance genes of E. coli in lambs with diarrhea

As shown in Fig. 4, analysis of 28 E. coli isolates (E1-E28) revealed the prevalence of different resistance genes: the β-lactamase genes blaTEM (100%), blaCTX-M4 (64.3%), blaSHV (21.4%), and blaNDM (7.1%); the aminoglycoside resistance gene aadA1 (57.1%); the tetracycline resistance gene tetA (82.1%); the phenicol resistance gene cmlA (25.0%); and the quinolone resistance genes parC (96.4%) and oqxA (14.3%). The concordance between resistance genotypes and phenotypes varied across all E. coli isolates. For β-lactam resistance genes, the correlation between blaCTX-M4 carriage and the resistance phenotypes of related drugs ranged from 11.1% to 100% in all E. coli isolates, which was significantly greater than that of other β-lactam resistance genes. The concordance between the tetA carriage (a tetracycline resistance gene) and resistant phenotypes was consistently 82.6%. For the aminoglycoside resistance gene aadA1, the correlation between carriage and resistance phenotypes was 50.0%-81.2%. The concordance between parC carriage (a quinolone resistance gene) and resistance phenotypes was 53.6–57.1%. Notably, the carbapenem resistance gene blaNDM and the phenolic resistance gene cmlA showed 100% concordance with their corresponding resistance phenotypes.

Association between biofilm-forming capacity and antimicrobial resistance in E. coli.

Biofilm quantification assays across all 28 E. coli isolates (Fig. 5A and B) revealed varying formation capacities, which were stratified into three categories: strong, moderate, and weak producers. Among strong biofilm formers, MDR and ESBL-producing strains constituted 80% and 60%, respectively. Moderate formers presented lower prevalence rates (MDR: 62%, ESBL: 38%), whereas weak formers presented 40% co-occurrence for both resistance traits (Fig. 5C). This gradient pattern suggests a quantifiable association between biofilm production intensity and antibiotic resistance development in E. coli populations, although mechanistic causality requires further validation.

Fig. 5.

Biofilm-forming capacity and its association with antimicrobial resistance. A Phenotypic assay of biofilm formation strength determined by crystal violet staining. Examples are classified as follows: +++, strong; ++, moderate; +, weak. B Quantitative determination of biofilm formation was performed by measuring the optical density at 570 nm (OD570). The dashed lines represent the thresholds for categorizing biofilm formation strength. C Association between biofilm formation capacity and the prevalence of multidrug resistance (MDR) and extended-spectrum β-lactamase (ESBL) production. Strains with stronger biofilm formation had a greater prevalence of MDR and ESBL phenotypes

Discussion

Phylogenetic analysis assigned the E. coli isolates in this study to phylogroups A (10.7%), B1 (53.6%), and D (35.7%), with no B2 strains detected. Previous studies reported that E. coli isolates from cattle and sheep in Xinjiang (2015–2019) were predominantly assigned to phylogroups A and B1 [19]. Similarly, E. coli from diarrheic lambs in Northwest China were classified into phylogroup B1, whereas poultry-derived isolates from Central China were identified as phylogroups A (50.8%), B1 (1.7%), B2 (17.6%), and D (9.9%) [30, 31]. Among the E. coli isolates from diarrheic calves in Inner Mongolia, phylogroups A, B1, B2, and D accounted for 19.0%, 33.3%, 4.8%, and 42.9%, respectively [32]. Studies on E. coli from diarrheic calves in Iran and Brazil further indicated a predominance of phylogroups A and B1, respectively [33, 34]. Notably, the dominance of phylogroup B1 in Aksu lambs differs from that in some of these reports. Although phylogroups B2 and D are generally associated with greater pathogenicity [35], the relatively high prevalence of phylogroup D (35.71%) and the absence of B2 strains in our collection suggest that phylogroup D may represent a more prevalent pathogenic lineage than B2 in this region. This finding warrants increased attention, given its potential epidemiological and clinical significance.

As a significant zoonotic pathogen, the virulence of E. coli is predominantly mediated by its accessory virulence gene repertoire. This study confirmed this finding. In this study, the E. coli isolates were found to carry a variety of virulence-associated genes, which is consistent with previous domestic and international reports [36, 37]. Among the 13 virulence genes detected, most were general virulence factors or accessory genes that increase bacterial colonization and survival in the host but do not confer human-specific pathogenicity. On the basis of established pathotype classification criteria, some isolates were categorized as ETEC, EAEC, EPEC + ETEC, or EAEC + EIEC hybrid strains according to their virulence gene profiles. Notably, however, key virulence determinants such as K88 exhibit strict host adaptability: K88 is predominantly associated with porcine ETEC and is absent in human-adapted ETEC pathotypes [38]. Although genes such as eae, hlyA, and invE are also present in some human-pathogenic E. coli strains, their expression patterns and pathogenic mechanisms remain host-specific. Furthermore, the absence of human-specific toxin genes—such as stx1, stx2, and estA—in the isolates further supports a low risk of direct infection in humans [39].

In the murine infection model, 39% of the tested strains exhibited pathogenicity. Histopathological analysis revealed that the pathological changes were not confined to the intestinal tract but disseminated systemically, resulting in generalized tissue damage—a manifestation consistent with the pathogenesis of septicemia. Notably, the coordinated expression of virulence factors is essential for the pathogenicity of pathogenic E. coli [40]. In pathogenicity assays, all strains carrying both the eae and hlyA genes caused mortality in mice. Histopathological examination revealed significant alterations and shedding of intestinal villus morphology, as well as disrupted cellular distribution and structural integrity in the intestinal tissue of infected mice. Previous studies have shown that the eae gene mediates the intimate adherence of bacteria to intestinal epithelial cells, leading to the effacement of microvilli and disruption of the intestinal barrier, whereas the hlyA gene induces pore-forming cytotoxic activity and inflammatory cascades [41]. Additionally, strains harboring irp2 and astA genes also caused mortality in mice. The irp2 gene enhances iron acquisition under host nutritional immunity pressure, thereby promoting bacterial survival and proliferation, whereas astA disrupts ion transport in intestinal epithelial cells, exacerbating fluid loss and tissue damage [42]. As one of the molecular markers for EIEC [21], the invE gene was detected in the virulence gene profile of the isolates in this study. Strains carrying both invE and astA were also lethal to mice, suggesting a potential synergistic role of these genes in pathogenicity.

With the intensive farming of animals, to reduce the economic losses caused by various bacterial diseases, antibacterial drugs are used in large quantities, resulting in the continuous emergence of drug-resistant strains [12]. Antimicrobial susceptibility testing revealed a high prevalence of resistance to multiple drug classes. Notably, resistance rates to β-lactams ranged from 3.6% (cefoxitin) to 71.4% (ampicillin), while substantial resistance was also observed for tetracyclines (60.7–71.4%) and quinolones (53.6–57.1%). Among the aminoglycosides, resistance varied from 39.2% (amikacin) to 60.7% (gentamicin), with all the isolates remaining susceptible to neomycin. Lower resistance rates were detected for chloramphenicol (25.0%) and polymyxin B (3.6%). This broad-spectrum resistance profile underscores the high prevalence of MDR strains in our collection. Antimicrobial resistance profiles of E. coli isolates from various regions reveal a notable spectrum of severity. A study of 116 E. coli strains from cattle and sheep in Xinjiang (2015–2019) reported resistance rates ranging from 31.9% to 81.9% for all antimicrobials tested except imipenem (5.17%), with all isolates exhibiting MDR [19]. Similarly, avian-derived E. coli from central and eastern China presented high resistance to ampicillin, tetracycline, chloramphenicol, cefotaxime, and gentamicin, with MDR rates reaching 86.0% and 100%, respectively [43, 44]. These findings indicate that antimicrobial resistance levels in E. coli from certain parts of China are generally greater than those reported in the Aksu region. In contrast, sheep-derived strains from India presented lower resistance rates, between 20% and 25%, for tetracycline, cefotaxime, and amikacin [45]. Moreover, E. coli from diarrheic calves in Slovakia presented resistance rates of 22–76% to ceftazidime, gentamicin, ciprofloxacin, tetracycline, and ampicillin, with 71% of the isolates being MDR [46]. In contrast, cattle-derived E. coli from Canada presented resistance rates ranging from 4.2% to 41% against ceftriaxone, ampicillin, chloramphenicol, and tetracycline, with an associated MDR prevalence of 37% [24]. Moreover, studies from the United States reported significantly lower multidrug resistance rates in livestock-associated E. coli, with sheep-derived isolates showing a prevalence of only 8.2% [47]. This clear resistance gradient situates E. coli isolates from Aksu lambs at a relatively high level of antimicrobial resistance severity within the global One Health context. The extensive use of β-lactam antimicrobials in veterinary practice has contributed to a growing prevalence of ESBL-producing E. coli [48, 49]. In the present study, the detection rate of ESBL-producing E. coli was 53.57%. Although data on ESBL-producing E. coli from sheep in Xinjiang remain limited, a study conducted in northern Xinjiang reported an ESBL detection rate of 19% among cattle-derived E. coli [50]. Internationally, the prevalence of ESBL-producing E. coli in sheep has been reported to be 28.8% in South Africa [51] and between 40% and 49% in isolates from Jordan, Turkey, Canada, and Pakistan [52–54]. These comparisons indicate that the detection rate of ESBL-producing E. coli from diarrheic lambs in the Aksu region is relatively high, which is associated with the widespread multidrug resistance observed in local E. coli isolates.

Molecular characterization of antimicrobial resistance genes revealed a wide distribution of β-lactam resistance determinants, with detection rates ranging from 7.1% (blaNDM) to 100% (blaTEM). Notably, the blaCTX-M4 gene (64.3%) presented strong phenotypic concordance (72.2%-100%) with resistance to ampicillin, amoxicillin, and cefotaxime, indicating that blaCTX-M4 serves as the dominant driver of β-lactam resistance in E. coli from the Aksu region. This pattern differs somewhat from the correlations between the phenotype and genotype of β-lactam resistance reported in some previous studies [55]. The tetracycline resistance gene tetA was detected in 82.1% of the isolates and showed 82.6% concordance with the corresponding resistance phenotype. Consistent with this, studies have identified tetA as the primary gene mediating tetracycline resistance in sheep-derived E. coli from Iran [56]. Among the aminoglycoside resistance genes, aadA1 was present in 57.1% of the isolates and was strongly correlated (87.6%-93.8%) with resistance to gentamicin and kanamycin. Similarly, aadA1 has been reported as a key determinant of aminoglycoside resistance in cattle-derived E. coli from Canada [57]. For quinolones, the carriage rate of the parC gene was very high (96.4%). Mutations in the parC gene of E. coli constitute a key mechanism underlying resistance to quinolone antibiotics [58], suggesting that the high quinolone resistance observed in this region may be attributed to mutations in parC. Notably, perfect phenotype-genotype concordance (100%) was observed for blaNDM and cmlA in relation to imipenem and chloramphenicol resistance, respectively. Numerous studies have confirmed that blaNDM is a critical determinant of carbapenem resistance in E. coli [59].

Previous studies have confirmed an association between bacterial antimicrobial resistance and biofilm-forming capacity [60]. Notably, all E. coli strains isolated in this study exhibited quantifiable biofilm-forming capacity with varying intensities. Between 2015 and 2019, 79.5% of 132 sheep-derived E. coli strains isolated from the Xinjiang region were found to possess biofilm-forming ability [61]. One study reported that the biofilm formation rate of animal-derived E. coli isolates from North China was 86.14% [31]. Furthermore, studies from Tunisia reported biofilm-positivity rates of up to 73.1% in E. coli strains isolated from diarrheic calves and diseased chickens [62]; corresponding research in Egypt documented a biofilm formation rate of 66.3% in animal-derived E. coli isolates [63]. Comparative analysis revealed that the biofilm formation rate of E. coli strains from Aksu, Xinjiang, was significantly greater than that reported in several international studies. These cumulative pieces of evidence suggest that contemporary E. coli strains across diverse geographical regions retain substantial biofilm-forming potential. In general, E. coli exhibits robust biofilm-forming capabilities. Our findings demonstrate clear stratification: among the strong biofilm-forming strains, 80% were MDR, and 60% produced ESBL; intermediate biofilm-forming strains presented 62% MDR and 38% ESBL production, whereas weak biofilm-forming strains presented 40% prevalence of both resistance phenotypes. This biofilm-mediated adaptive strategy confers increased tolerance to environmental stressors and directly potentiates the development of antibiotic resistance. These findings collectively suggest a potential correlation between biofilm-forming capacity and antibiotic resistance in livestock-derived E. coli strains from select regions of Aksu.

Conclusion

This study is the first to systematically characterize E. coli strains isolated from diarrheic lambs in the Aksu region of Xinjiang. Our study revealed that the isolates carry various virulence genes and exhibit a certain degree of pathogenicity. Multiple drug-resistant strains and ESBL-producing strains were detected at high rates, with some resistance genes showing a high degree of concordance with resistant phenotypes. Moreover, there is a certain correlation between biofilm-forming capacity and resistant phenotypes. This study reveals the zoonotic risk of E. coli in this region, providing a reference for the prevention and control of lamb diarrhea and the management of antimicrobial agents.

Authors’ contributions

L.X. Shi and M.J. Xu conducted part of the experiments and completed the writing; S.J. Tang and H.R. Chen collected the experimental samples; Q.L. Cai, D. Xu and H.L. Qin conducted part of the experiments; B.Q. Long, W.Q. Tian and B. Liu performed software analysis; H.A, L.J. Yu and Y.H. Han conducted data analysis; X.P. Zhang and J. Chen designed the experimental protocol; Y.W. Li provided project support, designed the experimental protocol, and completed the manuscript review.

Funding

This study was supported by the Innovation Team for Rapid Diagnosis, Prevention and Control Technology of Mycoplasmal Pneumonia in Sheep, Southern Xinjiang (2023ZD099), and the College Student Innovation and Entrepreneurship Project of Tarim University (202410757006).

Data availability

Data availability：The data presented in this study have been deposited in GenBank, including 28 16 S ribosomal RNA gene sequences with accession numbers ranging from PX567714.1 to PX567741.1. All these sequences are now publicly searchable in the database. The data supporting this study’s findings are available upon request from the corresponding author.

Declarations

Ethics approval and consent to participate

The animal studies were approved by the Animal Management and Ethics Committee of Tarim University (approval no. 2023009). The studies were conducted in accordance with local legislation and institutional requirements.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.