Co-colonization of blaCTX-M and mcr-1 in Avian Pathogenic Escherichia coli in Southern Xinjiang: Current Status and Antimicrobial Resistance Characteristics

Co-colonization of blaCTX-M and mcr-1 in Avian Pathogenic Escherichia coli in Southern Xinjiang: Current Status and Antimicrobial Resistance Characteristics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-colonization of blaCTX-M and mcr-1 in Avian Pathogenic Escherichia coli in Southern Xinjiang: Current Status and Antimicrobial Resistance Characteristics Bin Yang, Guofeng Xing, Wenxuan Ma, Meng Qi, Bo Jing, Jing Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8591920/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Extended-spectrum β-lactamases (ESBLs) constitute one of the principal mechanisms underlying multidrug resistance (MDR) in avian pathogenic Escherichia coli (APEC). To clarify the current epidemiological status and potential transmission risks associated with the co-occurrence of ESBL and mcr-1 genes in southern Xinjiang, 133 liver samples from diseased chickens were collected, from which 104 E. coli strains were isolated; 100 of these isolates (96.15%, 100/104) were confirmed as APEC. Double-disc diffusion testing with cefotaxime-clavulanate (CTL)/cefotaxime (CTX) and ceftazidime-clavulanic acid (CAL)/ceftazidime (CAZ) showed that 65 strains (65.00%, 65/100) were ESBL producers. Among these, five isolates (7.69%, 5/65) were resistant to colistin and carried the mcr-1 gene. All five isolates exhibited MDR phenotypes and were assigned to phylogenetic groups A (n = 1), B1 (n = 3), and D (n = 1). Whole-genome sequencing (WGS) revealed that the five mcr-1 -positive APEC strains belonged to four sequence types (STs): ST6792 (n = 1), ST1196 (n = 2), ST155 (n = 1), and ST162 (n = 1), and harbored three b laCTX-M subtypes ( blaCTX-M-55 [n = 3], blaCTX-M-64 [n = 1], and blaCTX-M-65 [n = 1]). These data suggest that MDR APEC strains co-harboring mcr-1 and ESBL genes particularly those classified as phylogenetic group D (ST162) and ST155 may serve as important reservoirs and potential vehicles for antimicrobial resistance dissemination. APEC ESBL MDR blaCTX-M mcr-1 Drug Resistance Analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Avian pathogenic Escherichia coli (APEC) is a major subtype of extraintestinal pathogenic Escherichia coli (ExPEC) that causes multisystemic infections in poultry, including respiratory disease, septicemia, and perihepatitis, leading to substantial economic losses in the global poultry industry (Kathayat et al. 2021 ; Nawaz et al. 2024 ). The consequences of APEC infection include an approximately 2.7% reduction in feed conversion efficiency, an approximate 20% decline in egg production (Ebrahimi-Nik et al. 2018 ; Ghunaim et al. 2014 ), and mortality rates that may reach 20% (Miao et al. 2023 ). Comparative studies have revealed close genetic and phenotypic relatedness between APEC and human uropathogenic E . coli (UPEC) and neonatal meningitis-associated E. coli (NMEC), including shared serotypes, virulence genes, and molecular typing profiles (Tivendale et al. 2010 ). Among these, sequence types (STs) ST95 and ST131, which are frequently detected in human clinical isolates and associated with multiple virulence determinants, have been designated by the World Health Organization as “priority serotypes of zoonotic pathogens” (Kathayat et al. 2021 ; Usman et al. 2023 ). Although antimicrobial agents are widely used for disease control in poultry, their extensive application has driven the emergence and dissemination of antimicrobial resistance, with multidrug-resistant (MDR) strains now representing a significant challenge to poultry health. The widespread occurrence of extended-spectrum β-lactamase (ESBL)-producing E . coli , mainly carrying b laCTX-M gene, together with the plasmid-mediated colistin resistance gene mcr-1 , has led to resistance to both β-lactam antibiotics and colistin. These genes are frequently located on mobile genetic elements such as plasmids, transposons, and integrons, facilitating their horizontal transfer within and between bacterial populations. In particular, blaCTX-M is commonly associated with IncF and IncI1 type plasmids, whereas mcr - 1 is predominantly linked to IncX4 and IncHI2 type plasmids. The high transferability of these plasmids accelerates the spread of resistance genes across bacterial strains and species (Li et al. 2022 ; Wu et al. 2018 ). Functionally, ESBLs encoded by blaCTX-M hydrolyze third-generation cephalosporins, wereas mcr-1 mediates the modification of lipid A in the bacterial outer membrane, reducing colistin binding affinity and thereby conferring resistance (Dhaouadi et al. 2020 ; Wang et al. 2024 ). The Xinjiang Uyghur Autonomous Region (hereinafter referred to as Xinjiang) is an important poultry production area in China. The present study aimed to determine the prevalence of ESBL-producing APEC isolates obtained from diseased chickens on farms in southern Xinjiang and to identify isolates simultaneously carrying mcr-1 gene. Whole-genome sequencing (WGS) was used to characterize antimicrobial resistance gene profiles, plasmid types, STs, and phylogenetic relationships, thereby providing insight into their pathogenic potential and possible transmission pathways. 2. Materials and Methods 2.1 Sample collection Samples were collected between November 2023 and November 2024 from six locations sampling sites in Aksu City (Aksu 1—2) and Alaer City (Alaer 1—4) in southern Xinjiang, China, including two large-scale layer farms, one medium-scale broiler farm, and three family operated backyard farms. A total of 133 liver samples were obtained from chickens that had died after exhibiting clinical signs consistent with colibacillosis. Of these, 93 samples originated from large-scale layer farms, five from the medium-scale broiler farms, and 35 from family farms. 2.2 Isolation and Identification of APEC All procedures were conducted under aseptic conditions. The surface of each organ sample was first disinfected, and deep tissue was aseptically collected and inoculated into tryptic soy broth (TSB; Haibo Biotech, Qingdao, China). Cultures were incubated at 37°C with shaking at 200 rpm for 12–16 hours to promote bacterial growth. Following incubation, cultures were streaked onto Eosin Methylene Blue (EMB) agar (Haibo Biotech, Qingdao, China) and incubated at 37°C for an additional 12–16 hours. Colonies displaying typical E. coli morphology were selected and purified. Genomic DNA was extracted using the OMEGA DNA Extraction Kit (OMEGA Biotek Inc., USA). Isolates were confirmed as E. coli by PCR amplification of the phoA gene using E. coli- specific primers as described by Hu et al. (2011). Pathogenicity classification followed the method of Kim et al. (2020). Isolates carrying two or more of the virulence genes hlyF , iutA , iroN , ompT , and iss genes were classified as APEC, whereas isolates lacking this virulence gene profile designated non-pathogenic E. coli (NPEC). Each PCR reaction was performed in a total volume of 25 μL, containing of 12.5 μL of 2 × EasyTaq® PCR SuperMix, 0.5 μL of each primer, 10.5 μL of double-distilled water, and 1 μL of DNA template. Primer sequences used for amplification are listed in Supplementary Table 1. 2.3 Screening for ESBL-APEC Strains Revived APEC cultures were obtained by incubating bacterial isolates in tryptic soy broth (TSB) at 37°C with shaking overnight. Screening for ESBL production was conducted using the double-disc diffusion assay according to the standard operating procedures and interpretive criteria described by Ratti et al. (2023). Antibiotic discs containing cefotaxime (CTX) and cefotaxime-clavulanic acid (CTL), as well as ceftazidime (CAZ) and ceftazidime-clavulanic acid (CAL) (Liofilchem, Italy), were used for testing. E. coli ATCC 25922 served as the quality control strain. An isolate was classified as an ESBL-producer if the inhibition zone diameter for around a disc containing clavulanic acid was ≥5 mm larger than that of the corresponding antibiotic disc without clavulanic acid. 2.4 ESBL-APEC Molecular Typing Phylogenetic classification of ESBL producing E. coli isolates was performed using the method described by Clermont et al. (2000). Primer sequences are provided in Supplementary Table 1. Amplification of the chuA , yjaA , and TspE4. C2 as used to assign each isolate to a phylogenetic group. The PCR products were analyzed by 1% agarose gel electrophoresis to verify the amplification patterns. Phylogenetic grouping criteria were as follows: group A ( chuA- / TspE4. C2- ); group B1 ( chuA- / TspE4. C2 +); group B2 ( chuA +/ yjaA +); and group D ( chuA +/ yjaA -). 2.5 Drug sensitivity testing Antimicrobial susceptibility testing of the isolates was performed using the Kirby-Bauer (K-B) disk diffusion method and the broth microdilution method in accordance with guidelines from the standards of the Clinical and Laboratory Standards Institute (CLSI, 2023) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2020). Escherichia coli ATCC 25922 was used as the quality control strain. A total of 14 antimicrobial agents representing multiple antibiotic classes were tested, representing multiple antibiotic classes. The β-lactams included penicillin (P, 10 IU/disc), ampicillin (AMP, 10 μg/disc), cefoxitin (FOX, 30 μg/disc), cefazolin (KZ, 30 μg/disc), and ceftiofur (FUR, 30 μg/disc). The carbapenem tested was imipenem (IMI, 10 μg/disc). The aminoglycosides included kanamycin (K, 30 μg/disc), gentamicin (CN, 10 μg/disc), and streptomycin (S, 10 μg/disc). The tetracycline group comprised doxycycline (DXT, 30 μg/disc) and tetracycline (TE, 30 μg/disc). Additional agents included the phenicol chloramphenicol (C, 30 μg/disc), the rifamycin rifampicin (RD, 5 μg/disc), the sulfonamide combination trimethoprim-sulfamethoxazole (SXT, 25 μg/disc), and polymyxin B sulfate (tested viabrothmicrodilution over a concentration range of 0.08–160 μg/mL). All susceptibility tests were performed in triplicate to ensure reproducibility. Isolates resistant to three or more antibiotic classes were classified as MDR strains, according to the criteria proposed by Magiorakos et al. (2012). 2.6 Detection of Antibiotic Resistance Genes and Integrons Antibiotic resistance genes associated with APEC were detected using PCR assays designed based on previously published studies. Primer sequences are listed in Supplementary Table 1. The targeted genes included those conferring resistance to β-lactams ( blaTEM , blaCTX - M, and blaSHV ); carbapenems ( blaOXA -48, blaVIM , blaNDM , blaKPC , and blaGES ); tetracyclines ( tetA , tetM , and tetB ); aminoglycosides ( aph and ant ( 3’)-Ia ); sulfonamides ( sul1 and sul2 ); chloramphenicol ( clma and floR ); colistin ( mcr-1 and mcr-2 ); and integron integrase genes ( Int1 , Int2 , and Int3 ). 2.7 Biofilm Formation Capability Testing Biofilm formation capacity was assessed according to the method described by Wu et al. (2024). The cutoff optical density (ODc) was defined as the mean OD of the negative control plus three times its standard deviation. Based on this threshold, isolates were categorized as follows: isolates with OD ≤ ODc were considered non-biofilm formers; those with ODc < OD ≤ 2×ODc were classified as weak biofilm formers; those with 2×ODc < OD < 4×ODc were classified as moderate biofilm formers; and those with OD＞4×ODc were classified as strong biofilm formers. All the experiments were performed in triplicate. 2.8 Whole-genome sequencing analysis Genomic DNA from strains co‑harboring blaCTX‑M and mcr‑1 was extracted using the OMEGA DNA Extraction Kit and submitted to Shenggong Biotechnology (Shanghai) Co., Ltd. for Illumina‑based WGS. Assembled genomes were uploaded to the SerotypeFinder database at the Centre for Genomic Epidemiology (CGE) , the BIGSDB database platform, and the PlasmidFinder database to determine serotypes, MLST, and plasmid content, respectively (Carattoli et al., 2014; Joensen et al., 2015; Jolley & Maiden, 2010). Antimicrobial resistance genes were identified using ABRicate with alignment thresholds set at ≥95% nucleotide identity and ≥95% coverage. Complete genome sequences of a APEC isolates from other regions co-carrying blaCTX‑M and mcr‑1 were downloaded from the NCBI Genome database and analyzed using identical bioinformatic pipelines. Single-‑nucleotide polymorphism (SNP) analysis was performed by mapping all APEC genomes to reference strain EC‑92 using Snippy, and a phylogenetic treewas inferred with FastTree (Price et al., 2009). The resulting tree was visualized and annotated using with iTOL (Letunic & Bork, 2021). 2.9 Statistical analysis All statistical analyses were performed using GraphPad Prism 10 software. Fisher’s exact test and Student’s t-test were used to evaluate intergroup differences.. Statistical significance was defined as P < 0.05 (*), and highly significant differences were defined as P < 0.01 (**). 3. Results 3.1 Isolation of APEC A total of 104 E. coli strains were isolated based on colony morphology and molecular identification. According to the APEC identification criteria, 100 isolates were confirmed as APEC. Thus, the overall APEC isolation rate from all collected liver samples was 75.19% (100/133). 3.2 Identification of ESBL Producing APEC In total, 65 APEC isolates were identified as ESBL producers via the double-disc diffusion method, corresponding to a detection rate of 65.00%(65/100). Of these, 35 isolates originated from Alaer City and 30 from Aksu City. No statistically significant difference in detection rates was observed between the two regions (P > 0.05). Further analysis revealed a marked association between ESBL-APEC prevalence and farming practices: high proportions were observed in intensive farms with 75.00% (27/36) in Aksu 1, 76.74% (33/43) in Alaer 1, and 75.00% (3/4) in a medium-scale farm in Aksu 2, whereas substantially lower rates were found in backyard/free-range households, with 28.57% (2/7) in Alaer 2, and 0% in Alaer 3 and Alaer 4 (Table 1 ). Table 1 Distribution of ESBL-APEC isolates by region Region Sampling location Breeding model Sample size APEC Detection Rate(%) ESBL -APEC Detection Rate(%) Aksu Aksu 1 large-scale layer farms 48 75.00(36/48) 75.00%(27/36) Aksu 2 medium-scale broiler farms 5 80.00(4/5) 75.00%(3/4) Subtotal 53 75.47(40/53) 75.00%(30/40) Alar Alar 1 large-scale farm 45 95.56(43/45) 76.74%(33/43) Alar 2 family farms 11 63.63(7/11) 28.57%(2/7) Alar 3 family farms 19 36.84(7/19) 0 Alar 4 family farms 5 60.00(3/5) 0 Subtotal 80 75.00(60/80) 58.33%(35/60) Total 133 75.19(100/133) 65.00%(65/100) 3.3 Phylogenetic Typing of ESBL-APEC Phylogenetic analysis of the 65 ESBL-APEC isolates showed that group B1 was the most prevalent, accounting for 35.38% (23/65) of isolates. Group B2 represented 29.23% (19/65), while groups A and D accounted for 16.92% (11/65) and 18.46% (12/65), respectively. 3.4 Antimicrobial Susceptibility of ESBL-APEC Isolates Antimicrobial susceptibility testing indicated that all 65 APEC isolates exhibited pronounced MDR. Complete resistance (100%) was observed to ampicillin, penicillin, cefazolin, and rifampicin. High resistance rates were also recorded for chloramphenicol (98.46%), ceftiofur (92.31%), and tetracycline (89.23%). Resistance to aminoglycosides, tetracyclines, and sulfonamides ranged from 36.92% to 78.46%. In contrast, all isolates were fully susceptible to imipenem (100%), and high susceptibility was also observed to cefoxitin (95.38%) and polymyxin B sulfate (92.21%) (Fig. 1 ). Regional comparison showed that isolates from Alaer City exhibited significantly higher resistance rates to aminoglycosides (kanamycin and gentamicin), tetracyclines (tetracycline and doxycycline), and sulfonamides (trimethoprim-sulfamethoxazole) than those from Aksu City (P < 0.05; Fig. 2 ). According to the CLSI ( 2023 ) criteria and the MDR definition described in the Methods section (resistance to ≥ 3 antibiotic classes), all 65 ESBL-APEC isolates were classified as MDR strains. Among them, 58.46% (38/65) exhibited resistance to ≥ 10 antibiotic classes (Fig. 3 ). The maximum number of antibiotics resisted by a single isolate was 13. The mean number of antibiotics resisted by isolates from Alaer City (10.71) was significantly higher than that of isolates from Aksu City (8.73; P < 0.01). Twenty-eight distinct resistance patterns were identified among all isolates. The predominant pattern in observed in isolates from Alaer City was AMP + P + KZ + FUR + K + CN + S + DXT + TE + C + RD + SXT, whereas the most frequent pattern in isolates from Aksu City was AMP + P + KZ + FUR + S + TE + C + RD. 3.5 Detection of Antimicrobial Resistance Genes and Integrons Among the 65 ESBL-APEC isolates, 12 antimicrobial resistance genes were detected, often including several occurring in combination. Detection rates exceeded 90.00% for tetA (98.46%, 64/65), blaCTX-M (95.38%, 62/65), blaTEM (93.85%, 61/65), and ant(3’)-Ia (92.31%, 60/65). Detection rates for the remaining genes ( blaSHV , mcr-1 , floR , and sul1 ) ranged from 1.54% to 86.15%. Eight genes: blaIMP , blaVIM , blaOXA - 48 , blaNDM , blaGES , blaKPC , mcr-2 , and tetB were not detected in any isolate. Individual isolates harbored up to nine resistance genes, and 87.69% (57/65) carried six or more (Table 2 ). Integron analysis revealed Int1 in 64 isolates(98.46%), whereas Int2 and Int3 were not detected. Table 2 Detection of ESBL-APEC Resistance Genes and Integrons Antimicrobial Class Resistance Gene No. of Positive Isolates Prevalence (%) β-Lactams blaTEM 61 93.83 blaCTX-M 62 95.38 blaSHV 7 10.77 blaIMP 0 0 blaVIM 0 0 blaOXA-48 0 0 blaNDM 0 0 blaGES 0 0 blaKPC 0 0 Polymyxins mcr-1 5 7.69 mcr-2 0 0 Tetracyclines tetA 64 98.46 tetB 0 0 tetM 1 1.54 Aminoglycosides ant(3'')-Ia 60 92.31 aph(3') 40 61.54 Phenicols floR 56 86.15 cmla 35 53.85 Sulfonamide sul1 27 41.54 sul2 30 46.15 Integrons int1 64 98.46 int2 0 0 int3 0 0 3.6 Biofilm Formation Capability The biofilm-forming ability of the 65 ESBL-producing APEC isolates was assessed using the crystal violet microtiter plate assay. Sterile LB medium served as the negative control, yielding a mean OD value of 0.30 (SD = 0.01). The cutoff OD was therefore calculated as 0.30 + 3 × 0.01 = 0.33. All isolates exhibited OD values ranging from 0.66 to 2.28, exceeding ODc and confirming their capacity to form biofilms. Based on OD classification, 14 isolates were strong biofilm formers, 39 were moderate biofilm formers, and 12 were weak biofilm formers. 3.7 Serotype and MLST WGS of five ESBL-APEC strains co-harboring blaCTX-M and mcr - 1 identified four distinct STs. Isolates from Alaer City belonged to ST6792 (n = 1), ST1196 (n = 2), and ST155 (n = 1), whereas the isolate from Aksu City belonged to ST162 (n = 1). Serotype prediction revealed four profiles: H27-O81 (n = 1), H28-O116 (n = 2), H23-O134 (n = 1), and H9-O9 (n = 1). 3.8 Prediction of Antimicrobial Resistance Genes and Plasmid Types The five ESBL producing E. coli isolates were found to harbored multiple β-lactamase genes, including blaCTX - M ( blaCTX - M-55 , blaCTX-M-64 , and blaCTX - M-65 ), blaEC ( blaEC - 15 and blaEC-18 ), and blaTEM ( blaTEM-1 ), together with additional resistance determinants. Plassmid replicon typing identified ten plasmid types across these isolates: Col440I (1/5), IncI2 (4/5), IncX1 (1/5), p0111 (2/5), IncFIB (4/5), IncI1 (1/5), IncHI2A (1/5), IncHI2 (1/5), and IncFIC (FII) (1/5). 3.9 Phylogenetic Analysis Based on Core SNPs Five ESBL producing APEC isolates carrying both the blaCTX - M and mcr-1 , together with 39 chicken-derived APEC genomes with the same resistance genes retrieved from the NCBI, were included in the analysis, totaling 44 strains. Core SNP analysis revealed that these isolates were distributed across 28 distinct MLST lineages, including ST48, ST93, ST155, and ST162. The predominant O serotypes were O9 (13.64%), O1 (9.09%), and O8 (6.82%), whereas the dominant H serotypes were H10 (13.64%), H28 (11.36%), and H4 (9.09%). Plasmid replicon analysis showed IncFIB as the most prevalent type (86.36%, 38/44), followed by IncFIC(FII) (43.18%) and IncI2 (40.91%). In contrast, Col family plasmids were detected at relatively low frequencies (2.27–18.18%). The most common blaCTX - M subtypes were blaCTX-M-55 (27.27%), blaCTX-M-65 (20.45%), and blaCTX-M-14 (18.18%). All strains carried mcr-1 . Additionally, the APEC strains harbored multiple resistance genes, including aadA2 , aph ( 3” )- Ib , tetA , fosA3 , and sul2 . The 44 APEC isolates originated from several countries, including China, Laos, and Turkey, and were recovered from diverse sources such as poultry carcasses (food chain), feces, and slaughterhouse wastewater (environmental samples), suggesting potential dissemination via poultry products or farming environments. These observations indicate a risk of transregional transmission and support the need for continued surveillance and comprehensive assessment of zoonotic transmission risks within a One Health framework. SNP-based genomic phylogenetic analysis revealed that the ESBL - producing APEC isolates from this study clustered closely with strains reported from Henan and Shandong Provinces in China and from Turkey (Fig. 4 ). Detailed information on each strain—including source, geographic origin, MLST type, serotype, plasmid replicons, and resistance genes—is provided in Supplementary Tables 2 and 3. 4. Discussion This study isolated 100 APEC strains from 133 liver samples collected from diseased chickens at poultry farms in Aksu Prefecture, southern Xinjiang, corresponding to an isolation rate of 75.19% (100/133). Among these, 65 strains were ESBL-APEC (65.00%). These findings indicate that APEC, including ESBL-producing strains, is common in the poultry populations sampled in this study. In comparison, Chenouf et al.(202?) isolated 211 APEC strains (85.08%) from 248 liver samples collected from poultry slaughterhouses and a veterinary clinic in northeastern Algeria, but the detection rate of ESBL-APEC was only 8.06% (17/211). Jhandai et al. (2025) repored an APEC isolation rate of 90.91% (50/55) across 55 farms in Haryana, India, with ESBL-positive strains accounting for 20.00% (10/50) . Saeed et al. (2023) isolated 164 APEC strains (51.25%) from 320 cloacal swab samples in Jhang District, Punjab Province, Pakistan, among which 45.13% (74/164) were ESBL-positive. It is important to note that these studies differed in sample type (e.g., liver from dead poultry, cloacal swabs, or slaughterhouse samples), sources (clinical cases, slaughterhouses, or markets), sampling strategies (random or convenience sampling), geographic and management contexts, and detection methods. These factors can substantially influence detection rates. Therefore, cross-study variations are better interpreted as reflecting epidemiological patterns under different ecological contexts and survey designs, rather than serving as abasis for direct comparison or regional ranking. Nevertheless, the relatively high proportion of ESBL-APEC observed here suggests that the study area may be subject to strong selective pressures for resistance or ecological conditions that favor the maintenance of resistant strains.. Further analysis revealed a significant association between ESBL strain detection rates and farming practices (P < 0.01). Detection rates in large-scale farms (Aksu 1, Aksu 2, and Alaer 1) remained consistently high at 75.00%–76.74%, clearly higher than the 0%–28.57% observed in backyard farms (Alaer 2, Alaer 3, and Alaer 4). This trend is consistent with reports from other regions, such as Egypt, where the detection rate of ESBL strains in large-scale poultry farms reached 65.71% (Salem et al., 2023), and the Netherlands, where the isolation rate of ESBL strains in broiler and layer farms reached 73.05% (H et al., 2015). In contrast, detection rates are generally lower in free-range systems, with reports from Nigeria and Vietnam showing 32.31% and 23.30%, respectively (Kwoji et al., 2019; Nakayama et al., 2017). The higher prevalence of ESBL in large-scale farms may be related to factors such as higher stocking densities, more frequent drug interventions, and continuous exposure to environmental microorganisms. In particular, in intensive farming systems, more frequent and systematic antibiotic use may create sustained selection pressure, thereby promoting the maintenance and spread of ESBL-associated resistance genes (Capparelli et al., 2021). This study observed a spatial correlation between intensive farming practices and high ESBL-APEC detection rates, suggesting that such environments may be more conducive to the accumulation and transmission of ESBL-APEC. However, caution is warranted as the study included a limited number of farms, with samples originating from selected poultry farms. Accordingly, the present findings are more appropriately interpreted as an early warning signal of antimicrobial resistance risks in the specific study area, rather than as arobust estimate of ESBL-APEC prevalence across southern Xinjiang or Xinjiang as awhole. Although ESBL detection rates are lower in free-range farming systems, their products predominantly enter local markets and may constitute a latent transmission chain for resistant bacteria, which should be considered in future surveillance programs. According to the criteria established by Clermont et al. (2000), E. coli strains can be classified into phylogenetic groups A, B1, B2, and D. Among the 65 ESBL strains in this study, groups B1 (35.38%) and B2 (29.23%) predominated. This phylogenetic structure differs from that reported in Tunisia (predominantly groups B2 and A) and the Netherlands (predominantly groups B1 and A) (Dhaouadi et al., 2020; Blaak et al., 2015), which may reflect differences in the genetic background of strains, host populations, or selective pressures in the farming environment across regions. Notably, the B2 and D groups, which are closely associated with ExPEC, collectively accounted for more than 50% of the strains in this study. Previous studies have indicated that B2 and D group strains often carry more virulence factors and are associated with severe infections such as human urinary tract infections, bacteremia, and neonatal sepsis (M et al., 2021). Although groups A and B1 are often regarded as commensal groups, increasing evidence suggests that they can also harbor virulence genes and cause opportunistic infections (da et al., 2017; Villavicencio et al., 2025). Therefore, the high proportion of B2/D group strains detected here suggests that their potential public health relevance merits attention, especially when antimicrobial resistance coexists with virulence, which may enhance cross-host transmission and infection success. These observations further underscore that, in monitoring antimicrobial-resistant bacteria from poultry sources, focusing solely on resistance profiles is insufficient for comprehensive risk assessment; phylogenetic background and virulence profiles should also be considered as important complementary indicators. Antimicrobial susceptibility testing showed that the ESBL-APEC strains in this study exhibited a severe MDR phenotype: 100% resistance to ampicillin and penicillin, with resistance rates exceeding 80% for commonly used veterinary drugs such as ceftiofur (92.31%) and tetracycline (89.23%). According to the criteria established by Magiorakos et al. (2012), all ESBL-APEC strains were classified as MDR (100%). This extensive MDR pattern is consistent with observations for avian-derived E. coli from multiple regions worldwide. Chenouf et al. (2025) reported Algerian ESBL-APEC strains resistant to ampicillin, cefotaxime, and tetracycline (100.00%), with high resistance also observed against antibiotics such as nalidixic acid (76.47%) and co-trimoxazole (70.59%). Jhandai et al. (2025) found that Indian ESBL-APEC strains exhibited high resistance to cephalothin, imipenem, and piperacillin (60.00%-100.00%), as well as substantial resistance to common antibiotics such as tetracycline and ciprofloxacin. Saeed et al. (2023) identified ESBL-positive strains in Pakistan that were resistant to multiple antibiotics, including doxycycline and chloramphenicol, with higher MDR indices than non-ESBL strains. Long-term surveillance across several Chinese provinces by Wu et al. (2018) similarly demonstrated persistent and widespread resistance of ESBL-producing strains to doxycycline, nalidixic acid, and co-trimoxazole, alongside rising resistance rates to florfenicol and polymyxin E. Collectively, these data indicate that the ESBL phenotype in avian-derived E. coli is frequently accompanied by MDR. In this context. Collectively, ESBL-APEC isolates from the farms included in this study showed markedly reduced susceptibility to conventional therapeutic agents, which may increase the risk of treatment failure and persistent bacterial shedding, thereby promoting the stable circulation of resistant bacteria within poultry farms. Analysis of antibiotic resistance genes further clarified the genetic basis of the observed resistance phenotypes. In this study, the detection rates of tetA (98.46%), blaCTX-M (95.38%), blaTEM (93.85%), and ant(3')-Ia (92.31%) all exceeded 90%, which was highly consistent with the phenotypic resistance profiles. This suggests that these genes form a core resistance gene pool for ESBL-APEC resistance in the study region. This finding is largely consistent with reports from several other locations worldwide: Algerian strains also showed high frequencies of blaCTX-M (94.12%) and tetA (100.00%) (Chenouf et al., 2025); Egyptian strains predominantly carried blaSHV (34.78%) and blaTEM (59.78%) (Salem et al., 2023); and a Chinese study reported a blaCTX-M carriage rate of 92.67% and a blaTEM detection rate exceeding 86.00% (Wu et al., 2018). Tunisian strains predominantly harbored blaCTX-M-1 (93.33%) and blaTEM (40.00%) genes (Dhaouadi et al., 2020). Taken together, these data indicate that, despite geographic variation in specific subtypes and gene combinations, genes such as blaCTX-M , blaTEM , and tetA are widely prevalent in avian-derived ESBL-producing E. coli , forming a common core antimicrobial resistance gene pool. Notably, the Int1 integron detection rate reached 98.46% in this study, substantially higher than the 47.06% reported in Algeria (Chenouf et al., 2025). Integrons, which are capable of capturing and expressing resistance gene cassettes, act as important vehicles for the horizontal transmission of MDR (Zhang et al., 2018). The high prevalence of Int1 suggests that integron-mediated gene capture and horizontal transfer may be key mechanisms driving the high-frequency accumulation of genes such as tetA and ant(3')-Ia within the poultry production ecosystem investigated here, thereby contributing to MDR phenotypes.. One notable finding of this study is that five ESBL-APEC strains (7.69%) simultaneously harbored both blaCTX-M and mcr-1 , indicating the potential for concurrent resistance to third-generation cephalosporins and polymyxins (Wu et al., 2018; Lima Barbieri et al., 2017). The coexistence of blaCTX-M and mcr-1 has also been reported in Tunisia and eastern China (Dhaouadi et al., 2020; Wang et al., 2024), suggesting that this high-risk combination is emerging in different regions. WGS in the present study identified plasmid types previously associated with resistance gene dissemination, such as IncI2 (4/5, 80.00%) and earlier work has suggested that IncFIC(FII) (1/5, 20.00%), and previous research suggests IncI2 plasmids can co-carry both genes (Yin et al., 2021), and can transfer at frequencies of 5 × 10⁻⁴ to 7.88 × 10⁻² in in vitro conjugation assays (Aslam et al., 2018). However, this study did not experimentally determine whether blaCTX-M and mcr-1 reside on the same mobile genetic element or can be co-transferred, for example via plasmid conjugation assays, plasmid localization, or long-read sequencing. Accordingly, although the coexistence of these genes clearly heightens the resistance risk, the exact mode and efficiency of their transmission require further experimental clarification. Even so, the simultaneous presence of blaCTX-M and mcr-1 may further limit available antimicrobial options and therefore constitutes a resistance combination of particular concern. Additionally, strains harboring both blaCTX-M and mcr-1 genes belonged to multiple ST types, including high-risk lineages such as ST155 and ST162 that have been implicated in human infections worldwide (Szmolka et al., 2023; Thabet et al., 2023), and carried ExPEC associated virulence genes, such as iroN and iss (Jeong et al., 2012). This combination of a high-risk clonal background key resistance genes, and ExPEC-associated virulence factors may enhance cross-host adaptability and pathogenic potential. However, the actual risk of zoonotic transmission needs to be further evaluated using epidemiological investigations and animal models. ST6792 represents the first report of this ST in avian APEC. It carries an IncHI2-type plasmid, which frequently coexists with heavy metal resistance genes (Y et al., 2016), suggesting that heavy metal use in farming environments (e.g., copper sulfate as a feed additive) may promote plasmid stability through co-selection, thereby providing an additional reservoir and dissemination platform for resistance genes. Core SNP phylogenetic analysis showed that the strains in this study clustered genetically with APEC from chickens in Henan and Shandong, China, and from Turkey. The external strains were isolated from farm clinical cases, slaughterhouse carcasses, and environmental wastewater, providing molecular support for a potential transmission pathway from farms to slaughterhouses, the environment, and ultimately human exposure. Future work should include sequential sampling across farm - slaughterhouse - market - environment interfaces within the same region, combined with WGS-based tracing, to assess whether such transmission chains are present locally and to identify critical control points. In summary, this study documented a high detection rate of ESBL-APEC, clear differences associated with farming practices, a predominance of B1/B2 phylogenetic groups, and severe MDR in isolates from diseased and dead poultry from a limited number of chicken farms in Aksu Prefecture, southern Xinjiang. The coexistence of blaCTX-M and mcr-1 in some strains and the high prevalence of Int1 integrons suggest a substantial potential for resistance gene transmission within the local farming ecosystem. Given the limited number of farms included and the regional and site-specific nature of the sampling, the current findings shouldbe regarded as preliminary evidence and an early warning of antimicrobial resistance risks in the region. Future studies should expand the sampling frame to include more farms and a longer study period, and incorporate environmental samples such as feed, water sources, manure, and slaughterhouse or other environmental matrices. Particular attention should be paid to key resistance genes such as blaCTX-M and mcr-1 and to high-risk clones such as ST155 and ST162. Combining plasmid localization and conjugative transfer experiments to evaluate transmission potential would provide more robust evidence for managing resistance risks management at the livestock production level and for strengthening regional public health defenses. 5. Conclusion This study confirms the presence of MDR E. coli strains co-harboring blaCTX-M and mcr-1 among APEC isolates from chickens in southern Xinjiang. These strains carry diverse integrons and plasmids, supporting genetic diversity, potential cross-regional dissemination, and possible zoonotic transmission risks, and highlighting the need for continued surveillance under a One Health framework. Declarations Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions: Jing Wu ( [email protected] ) and Jing Li ( [email protected] ) conceived and designed this study and wrote the manuscript. Bin Yang ( [email protected] ) , Guofeng Xing ( [email protected] ), Wenxuan Ma ( [email protected] ), Meng Qi ( [email protected] ), Bo Jing ( [email protected] ) performed the experiments and collected and analyzed the data. Bin Yang and Jing Li reviewed and edited the manuscript. Ethics declaration: All chickens that died from disease were sourced from commercial farms and family farms, and the collection of pathological specimens was conducted with the consent of the farm operators and household owners approval from an institutional animal ethics committee was not required. References Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U., Salamat, M. K. F., & Baloch, Z (2018) Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance 11: 1645–1658. https://doi.org/10.2147/idr.S173867 Blaak, H., van Hoek, A. H., Hamidjaja, R. A., van der Plaats, R. 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AMP: ampicillin; P: penicillin; FOX: cefoxitin; KZ: cefazolin; FUR: ceftiofur; IMI: imipenem; K: kanamycin; CN: gentamicin; S: streptomycin; DXT: doxycycline; TE: tetracycline; C: chloramphenicol; RD: rifampicin; PB: polymyxin B sulfate; SXT: trimethoprim-sulfamethoxazole.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/4ebc65ae201a7236aa1c5802.png"},{"id":101236966,"identity":"17dc3cef-2645-4904-8911-6e8a19fe495c","added_by":"auto","created_at":"2026-01-27 14:50:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":965491,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial resistance differences between the Aksu and Alar regions.\u003c/p\u003e\n\u003cp\u003eNote: Green represents the Aksu region; blue represents Alar city. K: kanamycin; CN: gentamicin; DXT: doxycycline; TE: tetracycline; SXT: trimethoprim-sulfamethoxazole. *: P \u0026lt; 0.05; **: P \u0026lt; 0.01; ns: No significant difference.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/86cd5e4e31df5e671bce4585.png"},{"id":101236967,"identity":"a879734b-25d7-49ea-b068-c343a75824a9","added_by":"auto","created_at":"2026-01-27 14:50:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":158748,"visible":true,"origin":"","legend":"\u003cp\u003eMultidrug resistance patterns of ESBL-APEC.\u003c/p\u003e\n\u003cp\u003eNote: Bar charts represent the number of APEC strains with different resistance profiles. The green bars indicate data from Alar city, whereas the blue bars represent data from Aksu Prefecture. Each column in the bubble chart corresponds to a distinct resistance profile: blue circles: β-lactams; magenta circles: carbapenems; red circles: aminoglycosides; green circles: tetracyclines; orange circles: phenicols antibiotics; purple circles: rifamycin antibiotics; magenta circles (magenta circles represent only the resistance patterns of resistant strains; not all ESBL-APEC strains are resistant to polymyxin B): polymyxins; vertical circles: sulfonamides; gray circles: negative.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/19a448abf0742995d8abd636.png"},{"id":101236968,"identity":"5996690b-8758-4cd2-82ae-354319c6f0d2","added_by":"auto","created_at":"2026-01-27 14:50:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":569286,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood Phylogenetic Tree Based on SNPs\u003c/p\u003e\n\u003cp\u003eNote: Circular maximum likelihood phylogenetic tree constructed based on single nucleotide polymorphisms (SNPs). The central radial structure depicts evolutionary relationships among samples. Layers from inner to outer are: Innermost layer: Polymorphic sites differences among 44 APEC strains; Serotype layer: serotype distribution differences among 44 APEC strains; Location layer: sampling locations for all APEC strains; Sample layer: Sample types from which APEC was isolated; Plasmid layer: Plasmid carriage status, with dark green indicating carriage and light green indicating absence; Gene layer: ESBLs gene carriage status, with dark blue indicating carriage and light blue indicating absence.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/d6802e236d0e8c38da8594f1.png"},{"id":102748567,"identity":"cf432516-5596-4ac9-aef1-e07a7550d3fb","added_by":"auto","created_at":"2026-02-16 09:11:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2682820,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/c76aa634-296f-4125-b129-549aaa1d7a48.pdf"},{"id":101236965,"identity":"2876fce1-e03e-43b2-9ebe-11e773f76248","added_by":"auto","created_at":"2026-01-27 14:50:19","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":39376,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8591920/v1/61193452b1840f6e0349557e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Co-colonization of blaCTX-M and mcr-1 in Avian Pathogenic Escherichia coli in Southern Xinjiang: Current Status and Antimicrobial Resistance Characteristics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAvian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC) is a major subtype of extraintestinal pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ExPEC) that causes multisystemic infections in poultry, including respiratory disease, septicemia, and perihepatitis, leading to substantial economic losses in the global poultry industry (Kathayat et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nawaz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The consequences of APEC infection include an approximately 2.7% reduction in feed conversion efficiency, an approximate 20% decline in egg production (Ebrahimi-Nik et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghunaim et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and mortality rates that may reach 20% (Miao et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Comparative studies have revealed close genetic and phenotypic relatedness between APEC and human uropathogenic \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e (UPEC) and neonatal meningitis-associated \u003cem\u003eE. coli\u003c/em\u003e (NMEC), including shared serotypes, virulence genes, and molecular typing profiles (Tivendale et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Among these, sequence types (STs) ST95 and ST131, which are frequently detected in human clinical isolates and associated with multiple virulence determinants, have been designated by the World Health Organization as \u0026ldquo;priority serotypes of zoonotic pathogens\u0026rdquo; (Kathayat et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Usman et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough antimicrobial agents are widely used for disease control in poultry, their extensive application has driven the emergence and dissemination of antimicrobial resistance, with multidrug-resistant (MDR) strains now representing a significant challenge to poultry health. The widespread occurrence of extended-spectrum β-lactamase (ESBL)-producing \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, mainly carrying b\u003cem\u003elaCTX-M\u003c/em\u003e gene, together with the plasmid-mediated colistin resistance gene \u003cem\u003emcr-1\u003c/em\u003e, has led to resistance to both β-lactam antibiotics and colistin. These genes are frequently located on mobile genetic elements such as plasmids, transposons, and integrons, facilitating their horizontal transfer within and between bacterial populations. In particular, \u003cem\u003eblaCTX-M\u003c/em\u003e is commonly associated with IncF and IncI1 type plasmids, whereas \u003cem\u003emcr\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e is predominantly linked to IncX4 and IncHI2 type plasmids. The high transferability of these plasmids accelerates the spread of resistance genes across bacterial strains and species (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Functionally, ESBLs encoded by \u003cem\u003eblaCTX-M\u003c/em\u003e hydrolyze third-generation cephalosporins, wereas \u003cem\u003emcr-1\u003c/em\u003e mediates the modification of lipid A in the bacterial outer membrane, reducing colistin binding affinity and thereby conferring resistance (Dhaouadi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Xinjiang Uyghur Autonomous Region (hereinafter referred to as Xinjiang) is an important poultry production area in China. The present study aimed to determine the prevalence of ESBL-producing APEC isolates obtained from diseased chickens on farms in southern Xinjiang and to identify isolates simultaneously carrying \u003cem\u003emcr-1\u003c/em\u003e gene. Whole-genome sequencing (WGS) was used to characterize antimicrobial resistance gene profiles, plasmid types, STs, and phylogenetic relationships, thereby providing insight into their pathogenic potential and possible transmission pathways.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected between November 2023 and November 2024 from six locations sampling sites in Aksu City (Aksu 1\u0026mdash;2) and Alaer City (Alaer 1\u0026mdash;4) in southern Xinjiang, China, including two large-scale layer farms, one medium-scale broiler farm, and three family operated backyard farms. A total of 133 liver samples were obtained from chickens that had died after exhibiting clinical signs consistent with colibacillosis. Of these, 93 samples originated from large-scale layer farms, five from the medium-scale broiler farms, and 35 from family farms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Isolation and Identification of APEC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were conducted under aseptic conditions. The surface of each organ sample was first disinfected, and deep tissue was aseptically collected and inoculated into tryptic soy broth (TSB; Haibo Biotech, Qingdao, China). Cultures were incubated at 37\u0026deg;C with shaking at 200 rpm for 12\u0026ndash;16 hours to promote bacterial growth. Following incubation, cultures were streaked onto Eosin Methylene Blue (EMB) agar (Haibo Biotech, Qingdao, China) and incubated at 37\u0026deg;C for an additional 12\u0026ndash;16 hours. Colonies displaying typical \u003cem\u003eE. coli\u003c/em\u003e morphology were selected and purified.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted using the OMEGA DNA Extraction Kit (OMEGA Biotek Inc., USA). Isolates were confirmed as \u003cem\u003eE. coli\u003c/em\u003e by PCR amplification of the \u003cem\u003ephoA\u003c/em\u003e gene using \u003cem\u003eE. coli-\u003c/em\u003e specific primers\u003cem\u003e\u0026nbsp;\u003c/em\u003eas described by Hu et al. (2011). Pathogenicity classification followed the method of Kim et al. (2020). Isolates carrying two or more of the virulence genes \u003cem\u003ehlyF\u003c/em\u003e, \u003cem\u003eiutA\u003c/em\u003e, \u003cem\u003eiroN\u003c/em\u003e, \u003cem\u003eompT\u003c/em\u003e, and \u003cem\u003eiss\u003c/em\u003e genes were classified as APEC, whereas isolates lacking this virulence gene profile designated non-pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(NPEC).\u003c/p\u003e\n\u003cp\u003eEach PCR reaction was performed in a total volume of 25 \u0026mu;L, containing of 12.5 \u0026mu;L of 2 \u0026times; EasyTaq\u0026reg; PCR SuperMix, 0.5 \u0026mu;L of each primer, 10.5 \u0026mu;L of double-distilled water, and 1 \u0026mu;L of DNA template. Primer sequences used for amplification are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Screening for ESBL-APEC Strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRevived APEC cultures were obtained by incubating bacterial isolates in tryptic soy broth (TSB) at 37\u0026deg;C with shaking overnight. Screening for ESBL production was conducted using the double-disc diffusion assay according to the standard operating procedures and interpretive criteria described by Ratti et al. (2023). Antibiotic discs containing cefotaxime (CTX) and cefotaxime-clavulanic acid (CTL), as well as ceftazidime (CAZ) and ceftazidime-clavulanic acid (CAL) (Liofilchem, Italy), were used for testing. \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;coli\u003c/em\u003e ATCC 25922 served as the quality control strain. An isolate was classified as an ESBL-producer if the inhibition zone diameter for around a disc containing clavulanic acid was \u0026ge;5 mm larger than that of the corresponding antibiotic disc without clavulanic acid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 ESBL-APEC Molecular Typing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic classification of ESBL producing \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eisolates was performed using the method described by Clermont et al. (2000). Primer sequences are provided in Supplementary Table 1. Amplification of the \u003cem\u003echuA\u003c/em\u003e, \u003cem\u003eyjaA\u003c/em\u003e, and \u003cem\u003eTspE4. C2\u003c/em\u003e as used to assign each isolate to a phylogenetic group. The PCR products were analyzed by 1% agarose gel electrophoresis to verify the amplification patterns. Phylogenetic grouping criteria were as follows: group A (\u003cem\u003echuA-\u003c/em\u003e/\u003cem\u003eTspE4. C2-\u003c/em\u003e); group B1 (\u003cem\u003echuA-\u003c/em\u003e/\u003cem\u003eTspE4. C2\u003c/em\u003e+); group B2 (\u003cem\u003echuA\u003c/em\u003e+/\u003cem\u003eyjaA\u003c/em\u003e+); and group D (\u003cem\u003echuA\u003c/em\u003e+/\u003cem\u003eyjaA\u003c/em\u003e-).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Drug sensitivity testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntimicrobial susceptibility testing of the isolates was performed using the Kirby-Bauer (K-B) disk diffusion method and the broth microdilution method in accordance with guidelines from the standards of the Clinical and Laboratory Standards Institute (CLSI, 2023) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2020). \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 was used as the quality control strain.\u003c/p\u003e\n\u003cp\u003eA total of 14 antimicrobial agents representing multiple antibiotic classes were tested, representing multiple antibiotic classes. The \u0026beta;-lactams included penicillin (P, 10 IU/disc), ampicillin (AMP, 10 \u0026mu;g/disc), cefoxitin (FOX, 30 \u0026mu;g/disc), cefazolin (KZ, 30 \u0026mu;g/disc), and ceftiofur (FUR, 30 \u0026mu;g/disc). The carbapenem tested was imipenem (IMI, 10 \u0026mu;g/disc). The aminoglycosides included kanamycin (K, 30 \u0026mu;g/disc), gentamicin (CN, 10 \u0026mu;g/disc), and streptomycin (S, 10 \u0026mu;g/disc). The tetracycline group comprised doxycycline (DXT, 30 \u0026mu;g/disc) and tetracycline (TE, 30 \u0026mu;g/disc). Additional agents included the phenicol chloramphenicol (C, 30 \u0026mu;g/disc), the rifamycin rifampicin (RD, 5 \u0026mu;g/disc), the sulfonamide combination trimethoprim-sulfamethoxazole (SXT, 25 \u0026mu;g/disc), and polymyxin B sulfate (tested viabrothmicrodilution over a concentration range of 0.08\u0026ndash;160 \u0026mu;g/mL). All susceptibility tests were performed in triplicate to ensure reproducibility. Isolates \u0026nbsp; resistant to three or more antibiotic classes were classified as MDR strains, according to the criteria proposed by Magiorakos et al. (2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Detection of Antibiotic Resistance Genes and Integrons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibiotic resistance genes associated with APEC were detected using PCR assays designed based on previously published studies. Primer sequences are listed in Supplementary Table 1. The targeted genes included those conferring resistance to \u0026beta;-lactams (\u003cem\u003eblaTEM\u003c/em\u003e, \u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM,\u003c/em\u003e and \u003cem\u003eblaSHV\u003c/em\u003e); carbapenems (\u003cem\u003eblaOXA\u003c/em\u003e-48, \u003cem\u003eblaVIM\u003c/em\u003e, \u003cem\u003eblaNDM\u003c/em\u003e, \u003cem\u003eblaKPC\u003c/em\u003e, and \u003cem\u003eblaGES\u003c/em\u003e); tetracyclines (\u003cem\u003etetA\u003c/em\u003e, \u003cem\u003etetM\u003c/em\u003e, and \u003cem\u003etetB\u003c/em\u003e); aminoglycosides (\u003cem\u003eaph\u003c/em\u003e and \u003cem\u003eant\u003c/em\u003e(\u003cem\u003e3\u0026rsquo;)-Ia\u003c/em\u003e); sulfonamides (\u003cem\u003esul1\u003c/em\u003e and \u003cem\u003esul2\u003c/em\u003e); chloramphenicol (\u003cem\u003eclma\u003c/em\u003e and \u003cem\u003efloR\u003c/em\u003e); colistin (\u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-2\u003c/em\u003e);\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003eintegron integrase genes (\u003cem\u003eInt1\u003c/em\u003e, \u003cem\u003eInt2\u003c/em\u003e, and \u003cem\u003eInt3\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Biofilm Formation Capability Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiofilm formation capacity was assessed according to the method described by Wu et al. (2024). The cutoff optical density (ODc) was defined as the mean OD of the negative control plus three times its standard deviation. Based on this threshold, isolates were categorized as follows: isolates with OD \u0026le; ODc were considered non-biofilm formers; those with ODc \u0026lt; OD \u0026le; 2\u0026times;ODc were classified as weak biofilm formers; those with 2\u0026times;ODc \u0026lt; OD \u0026lt; 4\u0026times;ODc were classified as moderate biofilm formers; and those with OD＞4\u0026times;ODc were classified as strong biofilm formers. All the experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Whole-genome sequencing analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA from strains co‑harboring \u003cem\u003eblaCTX‑M\u003c/em\u003e and \u003cem\u003emcr‑1\u003c/em\u003e was extracted using the OMEGA DNA Extraction Kit and submitted to Shenggong Biotechnology (Shanghai) Co., Ltd. for Illumina‑based WGS. Assembled genomes were uploaded to the SerotypeFinder database at the Centre for Genomic Epidemiology (CGE) , the BIGSDB database platform, and the PlasmidFinder database to determine serotypes, MLST, and plasmid content, respectively (Carattoli et al., 2014; Joensen et al., 2015; Jolley \u0026amp; Maiden, 2010). Antimicrobial resistance genes were identified using ABRicate with alignment thresholds set at \u0026ge;95% nucleotide identity and \u0026ge;95% coverage. Complete genome sequences of a APEC isolates from other regions co-carrying \u003cem\u003eblaCTX‑M\u003c/em\u003e and \u003cem\u003emcr‑1\u003c/em\u003e were downloaded from the NCBI Genome database and analyzed using identical bioinformatic pipelines. Single-‑nucleotide polymorphism (SNP) analysis was performed by mapping all APEC genomes to reference strain EC‑92 using Snippy, and a phylogenetic treewas inferred with FastTree (Price et al., 2009). The resulting tree was visualized and annotated using with iTOL (Letunic \u0026amp; Bork, 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 10 software. Fisher\u0026rsquo;s exact test and Student\u0026rsquo;s t-test were used to evaluate intergroup differences.. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*), and highly significant differences were defined as\u003cem\u003e\u0026nbsp;P\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01 (**).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation of APEC\u003c/h2\u003e \u003cp\u003eA total of 104 \u003cem\u003eE. coli\u003c/em\u003e strains were isolated based on colony morphology and molecular identification. According to the APEC identification criteria, 100 isolates were confirmed as APEC. Thus, the overall APEC isolation rate from all collected liver samples was 75.19% (100/133).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Identification of ESBL Producing APEC\u003c/h2\u003e \u003cp\u003eIn total, 65 APEC isolates were identified as ESBL producers via the double-disc diffusion method, corresponding to a detection rate of 65.00%(65/100). Of these, 35 isolates originated from Alaer City and 30 from Aksu City. No statistically significant difference in detection rates was observed between the two regions (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Further analysis revealed a marked association between ESBL-APEC prevalence and farming practices: high proportions were observed in intensive farms with 75.00% (27/36) in Aksu 1, 76.74% (33/43) in Alaer 1, and 75.00% (3/4) in a medium-scale farm in Aksu 2, whereas substantially lower rates were found in backyard/free-range households, with 28.57% (2/7) in Alaer 2, and 0% in Alaer 3 and Alaer 4 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of ESBL-APEC isolates by region\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSampling location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBreeding model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAPEC Detection Rate(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eESBL -APEC Detection Rate(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAksu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAksu 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elarge-scale layer farms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.00(36/48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e75.00%(27/36)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAksu 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emedium-scale broiler farms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.00(4/5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e75.00%(3/4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSubtotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.47(40/53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e75.00%(30/40)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAlar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlar 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elarge-scale farm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.56(43/45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e76.74%(33/43)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlar 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efamily farms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.63(7/11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e28.57%(2/7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlar 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efamily farms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.84(7/19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlar 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efamily farms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.00(3/5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSubtotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.00(60/80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e58.33%(35/60)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.19(100/133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e65.00%(65/100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Phylogenetic Typing of ESBL-APEC\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis of the 65 ESBL-APEC isolates showed that group B1 was the most prevalent, accounting for 35.38% (23/65) of isolates. Group B2 represented 29.23% (19/65), while groups A and D accounted for 16.92% (11/65) and 18.46% (12/65), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Antimicrobial Susceptibility of ESBL-APEC Isolates\u003c/h2\u003e \u003cp\u003eAntimicrobial susceptibility testing indicated that all 65 APEC isolates exhibited pronounced MDR. Complete resistance (100%) was observed to ampicillin, penicillin, cefazolin, and rifampicin. High resistance rates were also recorded for chloramphenicol (98.46%), ceftiofur (92.31%), and tetracycline (89.23%). Resistance to aminoglycosides, tetracyclines, and sulfonamides ranged from 36.92% to 78.46%. In contrast, all isolates were fully susceptible to imipenem (100%), and high susceptibility was also observed to cefoxitin (95.38%) and polymyxin B sulfate (92.21%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Regional comparison showed that isolates from Alaer City exhibited significantly higher resistance rates to aminoglycosides (kanamycin and gentamicin), tetracyclines (tetracycline and doxycycline), and sulfonamides (trimethoprim-sulfamethoxazole) than those from Aksu City (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the CLSI (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) criteria and the MDR definition described in the Methods section (resistance to \u0026ge;\u0026thinsp;3 antibiotic classes), all 65 ESBL-APEC isolates were classified as MDR strains. Among them, 58.46% (38/65) exhibited resistance to \u0026ge;\u0026thinsp;10 antibiotic classes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The maximum number of antibiotics resisted by a single isolate was 13. The mean number of antibiotics resisted by isolates from Alaer City (10.71) was significantly higher than that of isolates from Aksu City (8.73; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Twenty-eight distinct resistance patterns were identified among all isolates. The predominant pattern in observed in isolates from Alaer City was AMP\u0026thinsp;+\u0026thinsp;P\u0026thinsp;+\u0026thinsp;KZ\u0026thinsp;+\u0026thinsp;FUR\u0026thinsp;+\u0026thinsp;K\u0026thinsp;+\u0026thinsp;CN\u0026thinsp;+\u0026thinsp;S\u0026thinsp;+\u0026thinsp;DXT\u0026thinsp;+\u0026thinsp;TE\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;RD\u0026thinsp;+\u0026thinsp;SXT, whereas the most frequent pattern in isolates from Aksu City was AMP\u0026thinsp;+\u0026thinsp;P\u0026thinsp;+\u0026thinsp;KZ\u0026thinsp;+\u0026thinsp;FUR\u0026thinsp;+\u0026thinsp;S\u0026thinsp;+\u0026thinsp;TE\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;RD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Detection of Antimicrobial Resistance Genes and Integrons\u003c/h2\u003e \u003cp\u003eAmong the 65 ESBL-APEC isolates, 12 antimicrobial resistance genes were detected, often including several occurring in combination. Detection rates exceeded 90.00% for \u003cem\u003etetA\u003c/em\u003e (98.46%, 64/65), \u003cem\u003eblaCTX-M\u003c/em\u003e (95.38%, 62/65), \u003cem\u003eblaTEM\u003c/em\u003e (93.85%, 61/65), and \u003cem\u003eant(3\u0026rsquo;)-Ia\u003c/em\u003e (92.31%, 60/65). Detection rates for the remaining genes (\u003cem\u003eblaSHV\u003c/em\u003e, \u003cem\u003emcr-1\u003c/em\u003e, \u003cem\u003efloR\u003c/em\u003e, and \u003cem\u003esul1\u003c/em\u003e) ranged from 1.54% to 86.15%. Eight genes: \u003cem\u003eblaIMP\u003c/em\u003e, \u003cem\u003eblaVIM\u003c/em\u003e, \u003cem\u003eblaOXA\u003c/em\u003e-\u003cem\u003e48\u003c/em\u003e, \u003cem\u003eblaNDM\u003c/em\u003e, \u003cem\u003eblaGES\u003c/em\u003e, \u003cem\u003eblaKPC\u003c/em\u003e, \u003cem\u003emcr-2\u003c/em\u003e, and \u003cem\u003etetB\u003c/em\u003e were not detected in any isolate. Individual isolates harbored up to nine resistance genes, and 87.69% (57/65) carried six or more (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Integron analysis revealed Int1 in 64 isolates(98.46%), whereas Int2 and Int3 were not detected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection of ESBL-APEC Resistance Genes and Integrons\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntimicrobial Class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResistance Gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Positive Isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eβ-Lactams\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaTEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaCTX-M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaSHV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaIMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaVIM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaOXA-48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaNDM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaGES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eblaKPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePolymyxins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emcr-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emcr-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTetracyclines\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etetA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etetB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etetM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAminoglycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eant(3'')-Ia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaph(3')\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePhenicols\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efloR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecmla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSulfonamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esul1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esul2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIntegrons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eint1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eint2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eint3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Biofilm Formation Capability\u003c/h2\u003e \u003cp\u003eThe biofilm-forming ability of the 65 ESBL-producing APEC isolates was assessed using the crystal violet microtiter plate assay. Sterile LB medium served as the negative control, yielding a mean OD value of 0.30 (SD\u0026thinsp;=\u0026thinsp;0.01). The cutoff OD was therefore calculated as 0.30\u0026thinsp;+\u0026thinsp;3 \u0026times; 0.01\u0026thinsp;=\u0026thinsp;0.33. All isolates exhibited OD values ranging from 0.66 to 2.28, exceeding ODc and confirming their capacity to form biofilms. Based on OD classification, 14 isolates were strong biofilm formers, 39 were moderate biofilm formers, and 12 were weak biofilm formers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Serotype and MLST\u003c/h2\u003e \u003cp\u003eWGS of five ESBL-APEC strains co-harboring \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e identified four distinct STs. Isolates from Alaer City belonged to ST6792 (n\u0026thinsp;=\u0026thinsp;1), ST1196 (n\u0026thinsp;=\u0026thinsp;2), and ST155 (n\u0026thinsp;=\u0026thinsp;1), whereas the isolate from Aksu City belonged to ST162 (n\u0026thinsp;=\u0026thinsp;1). Serotype prediction revealed four profiles: H27-O81 (n\u0026thinsp;=\u0026thinsp;1), H28-O116 (n\u0026thinsp;=\u0026thinsp;2), H23-O134 (n\u0026thinsp;=\u0026thinsp;1), and H9-O9 (n\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Prediction of Antimicrobial Resistance Genes and Plasmid Types\u003c/h2\u003e \u003cp\u003eThe five ESBL producing E. coli isolates were found to harbored multiple β-lactamase genes, including \u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM\u003c/em\u003e (\u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM-55\u003c/em\u003e, \u003cem\u003eblaCTX-M-64\u003c/em\u003e, and \u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM-65\u003c/em\u003e), \u003cem\u003eblaEC\u003c/em\u003e (\u003cem\u003eblaEC\u003c/em\u003e-\u003cem\u003e15\u003c/em\u003e and \u003cem\u003eblaEC-18\u003c/em\u003e), and \u003cem\u003eblaTEM\u003c/em\u003e (\u003cem\u003eblaTEM-1\u003c/em\u003e), together with additional resistance determinants. Plassmid replicon typing identified ten plasmid types across these isolates: Col440I (1/5), IncI2 (4/5), IncX1 (1/5), p0111 (2/5), IncFIB (4/5), IncI1 (1/5), IncHI2A (1/5), IncHI2 (1/5), and IncFIC (FII) (1/5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Phylogenetic Analysis Based on Core SNPs\u003c/h2\u003e \u003cp\u003eFive ESBL producing APEC isolates carrying both the \u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e, together with 39 chicken-derived APEC genomes with the same resistance genes retrieved from the NCBI, were included in the analysis, totaling 44 strains. Core SNP analysis revealed that these isolates were distributed across 28 distinct MLST lineages, including ST48, ST93, ST155, and ST162. The predominant O serotypes were O9 (13.64%), O1 (9.09%), and O8 (6.82%), whereas the dominant H serotypes were H10 (13.64%), H28 (11.36%), and H4 (9.09%). Plasmid replicon analysis showed IncFIB as the most prevalent type (86.36%, 38/44), followed by IncFIC(FII) (43.18%) and IncI2 (40.91%).\u003c/p\u003e \u003cp\u003eIn contrast, Col family plasmids were detected at relatively low frequencies (2.27\u0026ndash;18.18%). The most common \u003cem\u003eblaCTX\u003c/em\u003e-\u003cem\u003eM\u003c/em\u003e subtypes were \u003cem\u003eblaCTX-M-55\u003c/em\u003e (27.27%), \u003cem\u003eblaCTX-M-65\u003c/em\u003e (20.45%), and \u003cem\u003eblaCTX-M-14\u003c/em\u003e (18.18%). All strains carried \u003cem\u003emcr-1\u003c/em\u003e. Additionally, the APEC strains harbored multiple resistance genes, including \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eaph\u003c/em\u003e(\u003cem\u003e3\u0026rdquo;\u003c/em\u003e)-\u003cem\u003eIb\u003c/em\u003e, \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003efosA3\u003c/em\u003e, and \u003cem\u003esul2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe 44 APEC isolates originated from several countries, including China, Laos, and Turkey, and were recovered from diverse sources such as poultry carcasses (food chain), feces, and slaughterhouse wastewater (environmental samples), suggesting potential dissemination via poultry products or farming environments. These observations indicate a risk of transregional transmission and support the need for continued surveillance and comprehensive assessment of zoonotic transmission risks within a One Health framework.\u003c/p\u003e \u003cp\u003eSNP-based genomic phylogenetic analysis revealed that the ESBL - producing APEC isolates from this study clustered closely with strains reported from Henan and Shandong Provinces in China and from Turkey (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Detailed information on each strain\u0026mdash;including source, geographic origin, MLST type, serotype, plasmid replicons, and resistance genes\u0026mdash;is provided in Supplementary Tables\u0026nbsp;2 and 3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study isolated 100 APEC strains from 133 liver samples collected from diseased chickens at poultry farms in Aksu Prefecture, southern Xinjiang, corresponding to an isolation rate of 75.19% (100/133). Among these, 65 strains were ESBL-APEC (65.00%). These findings indicate that APEC, including ESBL-producing strains, is common in the poultry populations sampled in this study. In comparison, Chenouf et al.(202?) isolated 211 APEC strains (85.08%) from 248 liver samples collected from poultry slaughterhouses and a veterinary clinic in northeastern Algeria, but the detection rate of ESBL-APEC was only 8.06% (17/211). Jhandai et al. (2025) repored an APEC isolation rate of 90.91% (50/55) across 55 farms in Haryana, India, with ESBL-positive strains accounting for 20.00% (10/50) . Saeed et al. (2023) isolated 164 APEC strains (51.25%) from 320 cloacal swab samples in Jhang District, Punjab Province, Pakistan, among which 45.13% (74/164) were ESBL-positive. It is important to note that these studies differed in sample type (e.g., liver from dead poultry, cloacal swabs, or slaughterhouse samples), sources (clinical cases, slaughterhouses, or markets), sampling strategies (random or convenience sampling), geographic and management contexts, and detection methods. These factors can substantially influence detection rates. Therefore, cross-study variations are better interpreted as reflecting epidemiological patterns under different ecological contexts and survey designs, rather than serving as abasis for direct comparison or regional ranking. Nevertheless, the relatively high proportion of ESBL-APEC observed here suggests that the study area may be subject to strong selective pressures for resistance or ecological conditions that favor the maintenance of resistant strains..\u003c/p\u003e\n\u003cp\u003eFurther analysis revealed a significant association between ESBL strain detection rates and farming practices (P \u0026lt; 0.01). Detection rates in large-scale farms (Aksu 1, Aksu 2, and Alaer 1) remained consistently high at 75.00%\u0026ndash;76.74%, clearly higher than the 0%\u0026ndash;28.57% observed in backyard farms (Alaer 2, Alaer 3, and Alaer 4). This trend is consistent with reports from other regions, such as Egypt, where the detection rate of ESBL strains in large-scale poultry farms reached 65.71% (Salem et al., 2023), and the Netherlands, where the isolation rate of ESBL strains in broiler and layer farms reached 73.05% (H et al., 2015). In contrast, detection rates are generally lower in free-range systems, with reports from Nigeria and Vietnam showing 32.31% and 23.30%, respectively (Kwoji et al., 2019; Nakayama et al., 2017). The higher prevalence of ESBL in large-scale farms may be related to factors such as higher stocking densities, more frequent drug interventions, and continuous exposure to environmental microorganisms. In particular, in intensive farming systems, more frequent and systematic antibiotic use may create sustained selection pressure, thereby promoting the maintenance and spread of ESBL-associated resistance genes (Capparelli et al., 2021). This study observed a spatial correlation between intensive farming practices and high ESBL-APEC detection rates, suggesting that such environments may be more conducive to the accumulation and transmission of ESBL-APEC. However, caution is warranted as the study included a limited number of farms, with samples originating from selected poultry farms. Accordingly, the present findings are more appropriately interpreted as an early warning signal of antimicrobial resistance risks in the specific study area, rather than as arobust estimate of ESBL-APEC prevalence across southern Xinjiang or Xinjiang as awhole. Although ESBL detection rates are lower in free-range farming systems, their products predominantly enter local markets and may constitute a latent transmission chain for resistant bacteria, which should be considered in future surveillance programs.\u003c/p\u003e\n\u003cp\u003eAccording to the criteria established by Clermont et al. (2000), \u003cem\u003eE. coli\u003c/em\u003e strains can be classified into phylogenetic groups A, B1, B2, and D. Among the 65 ESBL strains in this study, groups B1 (35.38%) and B2 (29.23%) predominated. This phylogenetic structure differs from that reported in Tunisia (predominantly groups B2 and A) and the Netherlands (predominantly groups B1 and A) (Dhaouadi et al., 2020; Blaak et al., 2015), which may reflect differences in the genetic background of strains, host populations, or selective pressures in the farming environment across regions. Notably, the B2 and D groups, which are closely associated with ExPEC, collectively accounted for more than 50% of the strains in this study. Previous studies have indicated that B2 and D group strains often carry more virulence factors and are associated with severe infections such as human urinary tract infections, bacteremia, and neonatal sepsis (M et al., 2021). Although groups A and B1 are often regarded as commensal groups, increasing evidence suggests that they can also harbor virulence genes and cause opportunistic infections (da et al., 2017; Villavicencio et al., 2025). Therefore, the high proportion of B2/D group strains detected here suggests that their potential public health relevance merits attention, especially when antimicrobial resistance coexists with virulence, which may enhance cross-host transmission and infection success. These observations further underscore that, in monitoring antimicrobial-resistant bacteria from poultry sources, focusing solely on resistance profiles is insufficient for comprehensive risk assessment; phylogenetic background and virulence profiles should also be considered as important complementary indicators.\u003c/p\u003e\n\u003cp\u003eAntimicrobial susceptibility testing showed that the ESBL-APEC strains in this study exhibited a severe MDR phenotype: 100% resistance to ampicillin and penicillin, with resistance rates exceeding 80% for commonly used veterinary drugs such as ceftiofur (92.31%) and tetracycline (89.23%). According to the criteria established by Magiorakos et al. (2012), all ESBL-APEC strains were classified as MDR (100%). This extensive MDR pattern is consistent with observations for avian-derived \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003efrom multiple regions worldwide. Chenouf et al. (2025) reported Algerian ESBL-APEC strains resistant to ampicillin, cefotaxime, and tetracycline (100.00%), with high resistance also observed against antibiotics such as nalidixic acid (76.47%) and co-trimoxazole (70.59%). Jhandai et al. (2025) found that Indian ESBL-APEC strains exhibited high resistance to cephalothin, imipenem, and piperacillin (60.00%-100.00%), as well as substantial resistance to common antibiotics such as tetracycline and ciprofloxacin. Saeed et al. (2023) identified ESBL-positive strains in Pakistan that were resistant to multiple antibiotics, including doxycycline and chloramphenicol, with higher MDR indices than non-ESBL strains. Long-term surveillance across several Chinese provinces by Wu et al. (2018) similarly demonstrated persistent and widespread resistance of ESBL-producing strains to doxycycline, nalidixic acid, and co-trimoxazole, alongside rising resistance rates to florfenicol and polymyxin E. Collectively, these data indicate that the ESBL phenotype in avian-derived \u003cem\u003eE. coli\u003c/em\u003e is frequently accompanied by MDR. In this context. Collectively, ESBL-APEC isolates from the farms included in this study showed markedly reduced susceptibility to conventional therapeutic agents, which may increase the risk of treatment failure and persistent bacterial shedding, thereby promoting the stable circulation of resistant bacteria within poultry farms.\u003c/p\u003e\n\u003cp\u003eAnalysis of antibiotic resistance genes further clarified the genetic basis of the observed resistance phenotypes. In this study, the detection rates of \u003cem\u003etetA\u003c/em\u003e (98.46%), \u003cem\u003eblaCTX-M\u003c/em\u003e (95.38%), \u003cem\u003eblaTEM\u003c/em\u003e (93.85%), and \u003cem\u003eant(3\u0026apos;)-Ia\u003c/em\u003e (92.31%) all exceeded 90%, which was highly consistent with the phenotypic resistance profiles. This suggests that these genes form a core resistance gene pool for ESBL-APEC resistance in the study region. This finding is largely consistent with reports from several other locations worldwide: Algerian strains also showed high frequencies of \u003cem\u003eblaCTX-M\u003c/em\u003e (94.12%) and \u003cem\u003etetA\u003c/em\u003e (100.00%) (Chenouf et al., 2025); Egyptian strains predominantly carried \u003cem\u003eblaSHV\u003c/em\u003e (34.78%) and\u003cem\u003e\u0026nbsp;blaTEM\u003c/em\u003e (59.78%) (Salem et al., 2023); and a Chinese study reported a \u003cem\u003eblaCTX-M\u003c/em\u003e carriage rate of 92.67% and a \u003cem\u003eblaTEM\u003c/em\u003e detection rate exceeding 86.00% (Wu et al., 2018). Tunisian strains predominantly harbored \u003cem\u003eblaCTX-M-1\u003c/em\u003e (93.33%) and \u003cem\u003eblaTEM\u003c/em\u003e (40.00%) genes (Dhaouadi et al., 2020). Taken together, these data indicate that, despite geographic variation in specific subtypes and gene combinations, genes such as \u003cem\u003eblaCTX-M\u003c/em\u003e, \u003cem\u003eblaTEM\u003c/em\u003e, and \u003cem\u003etetA\u003c/em\u003e are widely prevalent in avian-derived ESBL-producing \u003cem\u003eE. coli\u003c/em\u003e, forming a common core antimicrobial resistance gene pool. Notably, the Int1 integron detection rate reached 98.46% in this study, substantially higher than the 47.06% reported in Algeria (Chenouf et al., 2025). Integrons, which are capable of capturing and expressing resistance gene cassettes, act as important vehicles for the horizontal transmission of MDR (Zhang et al., 2018). The high prevalence of Int1 suggests that integron-mediated gene capture and horizontal transfer may be key mechanisms driving the high-frequency accumulation of genes such as \u003cem\u003etetA\u003c/em\u003e and \u003cem\u003eant(3\u0026apos;)-Ia\u003c/em\u003e within the poultry production ecosystem investigated here, thereby contributing to MDR phenotypes..\u003c/p\u003e\n\u003cp\u003eOne notable finding of this study is that five ESBL-APEC strains (7.69%) simultaneously harbored both \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e, indicating the potential for concurrent resistance to third-generation cephalosporins and polymyxins (Wu et al., 2018; Lima Barbieri et al., 2017). The coexistence of \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e has also been reported in Tunisia and eastern China (Dhaouadi et al., 2020; Wang et al., 2024), suggesting that this high-risk combination is emerging in different regions. WGS in the present study identified plasmid types previously associated with resistance gene dissemination, such as IncI2 (4/5, 80.00%) and earlier work has suggested that IncFIC(FII) (1/5, 20.00%), and previous research suggests IncI2 plasmids can co-carry both genes (Yin et al., 2021), and can transfer at frequencies of 5 \u0026times; 10⁻⁴ to 7.88 \u0026times; 10⁻\u0026sup2; in in vitro conjugation assays (Aslam et al., 2018). However, this study did not experimentally determine whether \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e reside on the same mobile genetic element or can be co-transferred, for example via plasmid conjugation assays, plasmid localization, or long-read sequencing. Accordingly, although the coexistence of these genes clearly heightens the resistance risk, the exact mode and efficiency of their transmission require further experimental clarification. Even so, the simultaneous presence of \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e may further limit available antimicrobial options and therefore constitutes a resistance combination of particular concern.\u003c/p\u003e\n\u003cp\u003eAdditionally, strains harboring both \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e genes belonged to multiple ST types, including high-risk lineages such as ST155 and ST162 that have been implicated in human infections worldwide (Szmolka et al., 2023; Thabet et al., 2023), and carried ExPEC associated virulence genes, such as\u003cem\u003e\u0026nbsp;iroN\u0026nbsp;\u003c/em\u003eand \u003cem\u003eiss\u003c/em\u003e (Jeong et al., 2012). This combination of a high-risk clonal background key resistance genes, and ExPEC-associated virulence factors may enhance cross-host adaptability and pathogenic potential. However, the actual risk of zoonotic transmission needs to be further evaluated using epidemiological investigations and animal models. ST6792 represents the first report of this ST in avian APEC. It carries an IncHI2-type plasmid, which frequently coexists with heavy metal resistance genes (Y et al., 2016), suggesting that heavy metal use in farming environments (e.g., copper sulfate as a feed additive) may promote plasmid stability through co-selection, thereby providing an additional reservoir and dissemination platform for resistance genes. Core SNP phylogenetic analysis showed that the strains in this study clustered genetically with APEC from chickens in Henan and Shandong, China, and from Turkey. The external strains were isolated from farm clinical cases, slaughterhouse carcasses, and environmental wastewater, providing molecular support for a potential transmission pathway from farms to slaughterhouses, the environment, and ultimately human exposure. Future work should include sequential sampling across farm - slaughterhouse - market - environment interfaces within the same region, combined with WGS-based tracing, to assess whether such transmission chains are present locally and to identify critical control points.\u003c/p\u003e\n\u003cp\u003eIn summary, this study documented a high detection rate of ESBL-APEC, clear differences associated with farming practices, a predominance of B1/B2 phylogenetic groups, and severe MDR in isolates from diseased and dead poultry from a limited number of chicken farms in Aksu Prefecture, southern Xinjiang. The coexistence of \u003cem\u003eblaCTX-M\u0026nbsp;\u003c/em\u003eand \u003cem\u003emcr-1\u003c/em\u003e in some strains and the high prevalence of Int1 integrons suggest a substantial potential for resistance gene transmission within the local farming ecosystem. Given the limited number of farms included and the regional and site-specific nature of the sampling, the current findings shouldbe regarded as preliminary evidence and an early warning of antimicrobial resistance risks in the region. Future studies should expand the sampling frame to include more farms and a longer study period, and incorporate environmental samples such as feed, water sources, manure, and slaughterhouse or other environmental matrices. Particular attention should be paid to key resistance genes such as \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003eand to high-risk clones such as ST155 and ST162. Combining plasmid localization and conjugative transfer experiments to evaluate transmission potential would provide more robust evidence for managing resistance risks management at the livestock production level and for strengthening regional public health defenses.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study confirms the presence of MDR \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003estrains co-harboring \u003cem\u003eblaCTX-M\u003c/em\u003e and \u003cem\u003emcr-1\u003c/em\u003e among APEC isolates from chickens in southern Xinjiang. These strains carry diverse integrons and plasmids, supporting genetic diversity, potential cross-regional dissemination, and possible zoonotic transmission risks, and highlighting the need for continued surveillance under a One Health framework.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Wu ([email protected]) and Jing Li ([email protected]) conceived and designed this study and wrote the manuscript. Bin Yang ([email protected]) , Guofeng Xing ([email protected]), Wenxuan Ma ([email protected]), Meng Qi ([email protected]), Bo Jing ([email protected]) performed the experiments and collected and analyzed the data. Bin Yang and Jing Li reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chickens that died from disease were sourced from commercial farms and family farms, and the collection of pathological specimens was conducted with the consent of the farm operators and household owners approval from an institutional animal ethics committee was not required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U., Salamat, M. K. F., \u0026amp; Baloch, Z (2018) Antibiotic resistance: a rundown of a global crisis. 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Microbiome 6(1): 130. https://doi.org/10.1186/s40168-018-0516-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"APEC, ESBL, MDR, blaCTX-M, mcr-1, Drug Resistance Analysis","lastPublishedDoi":"10.21203/rs.3.rs-8591920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8591920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtended-spectrum β-lactamases (ESBLs) constitute one of the principal mechanisms underlying multidrug resistance (MDR) in avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC). To clarify the current epidemiological status and potential transmission risks associated with the co-occurrence of ESBL and \u003cem\u003emcr-1\u003c/em\u003e genes in southern Xinjiang, 133 liver samples from diseased chickens were collected, from which 104 \u003cem\u003eE. coli\u003c/em\u003e strains were isolated; 100 of these isolates (96.15%, 100/104) were confirmed as APEC. Double-disc diffusion testing with cefotaxime-clavulanate (CTL)/cefotaxime (CTX) and ceftazidime-clavulanic acid (CAL)/ceftazidime (CAZ) showed that 65 strains (65.00%, 65/100) were ESBL producers. Among these, five isolates (7.69%, 5/65) were resistant to colistin and carried the \u003cem\u003emcr-1\u003c/em\u003e gene. All five isolates exhibited MDR phenotypes and were assigned to phylogenetic groups A (n\u0026thinsp;=\u0026thinsp;1), B1 (n\u0026thinsp;=\u0026thinsp;3), and D (n\u0026thinsp;=\u0026thinsp;1). Whole-genome sequencing (WGS) revealed that the five \u003cem\u003emcr-1\u003c/em\u003e-positive APEC strains belonged to four sequence types (STs): ST6792 (n\u0026thinsp;=\u0026thinsp;1), ST1196 (n\u0026thinsp;=\u0026thinsp;2), ST155 (n\u0026thinsp;=\u0026thinsp;1), and ST162 (n\u0026thinsp;=\u0026thinsp;1), and harbored three b\u003cem\u003elaCTX-M\u003c/em\u003e subtypes (\u003cem\u003eblaCTX-M-55\u003c/em\u003e [n\u0026thinsp;=\u0026thinsp;3], \u003cem\u003eblaCTX-M-64\u003c/em\u003e [n\u0026thinsp;=\u0026thinsp;1], and \u003cem\u003eblaCTX-M-65\u003c/em\u003e [n\u0026thinsp;=\u0026thinsp;1]). These data suggest that MDR APEC strains co-harboring \u003cem\u003emcr-1\u003c/em\u003e and ESBL genes particularly those classified as phylogenetic group D (ST162) and ST155 may serve as important reservoirs and potential vehicles for antimicrobial resistance dissemination.\u003c/p\u003e","manuscriptTitle":"Co-colonization of blaCTX-M and mcr-1 in Avian Pathogenic Escherichia coli in Southern Xinjiang: Current Status and Antimicrobial Resistance Characteristics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 14:50:14","doi":"10.21203/rs.3.rs-8591920/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f907c8df-dd58-11ec-a6e7-12b504df345a","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-14T09:40:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 14:50:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8591920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8591920","identity":"rs-8591920","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}