Phenotypical and genetic virulence and resistance in atypical avian pathogenic Escherichia coli

Phenotypical and genetic virulence and resistance in atypical avian pathogenic Escherichia coli | 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 Phenotypical and genetic virulence and resistance in atypical avian pathogenic Escherichia coli Angélica Liberalino da Silva, Fabrina Sousa Luna, Joel Fonseca Nogueira, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8781221/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Apr, 2026 Read the published version in International Microbiology → Version 1 posted 11 You are reading this latest preprint version Abstract Avian pathogenic Escherichia coli is a threat to poultry production systems, as it is associated with infections, poor production ratings, and economic losses. The spread of multidrug-resistant bacterial strains has aggravated the situation. Therefore, this study aimed to characterize 28 E. coli isolates from poultry farms in Brazil regarding virulence, as well as genetic and phenotypical resistance. Antimicrobial susceptibility test was carried out by the disk diffusion method, while the genomic information of the isolates was determined by whole-genome sequencing. Resistance was identified for all 19 antimicrobials tested, except imipenem. Elevated resistance was observed for rifampicin (96.4%) and oxacillin (67.9%); intermediate resistance for the carbapenems (ertapenem, meropenem, 10.7%), and tetracyclines (doxycycline, 10.7%) was also detected. Genotypic analyses showed that the most common resistance genes were tetA (39.3%) and gyrA (39.3%). Remarkably, the plasmid-mediated gene, MCR-1.1 , associated with colistin resistance, was detected in two isolates. The isolates tested did not carry the typical avian pathogenic E. coli genetic virulence panel; however, they presented great genetic diversity with prominent pathogenic potential connected to mechanisms of adhesion ( fimH , 96.4%), immune evasion ( ompA and ibeB/C , 100%; traT , 92.9%), and iron acquisition ( febB , 100%; iroN , 60.7%). The overlap between genotypic and phenotypic information emphasizes the health risk imposed by such atypical strains and their dispersion capability amongst both animals and humans. Our findings contribute to a better understanding of the diversity of avian pathogenic E. coli and reinforce the importance of combined diagnostic and surveillance strategies in the control of such bacteria. APEC Colistin resistance E. coli MDR Poultry Figures Figure 1 Figure 2 Figure 3 Introduction One of the most important sanitary problems that aviculture currently faces is the occurrence of colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC) that can, in many cases, lead to significant economic and zootechnical losses (Denamur et al. 2021 ; Barros et al. 2025 ). These E. coli strains are a real threat to intensive production systems, as they are capable of causing local and systemic infections, especially in young chicks, which often leads to mortality and low production ratings (Ha et al. 2025 ; Jhandai et al. 2025 ). Moreover, in recent decades, the issue of the spread of multidrug-resistant E. coli has become a matter of particular concern and is considered a major challenge due to its potential to undermine the effectiveness of conventional antimicrobial therapy, making the control of such infections difficult, and increasing the risk of spreading resistance genes not only to other animals but also among humans (Hasan et al., 2022 ; Ha et al., 2025 ). Another concern is the ability of E. coli strains to develop antimicrobial resistance independently, as well as to acquire and exchange virulence and resistance genes through plasmids, transposons, and other mobile genetic elements with other bacteria, thereby promoting the sharing and persistence of such genes (Dawadi et al. 2021 ; Barros et al. 2025 ). Consequently, poultry products may act as reservoirs of MDR APEC and other pathogenic strains, enabling their transmission to humans (Dawadi et al. 2021 ; Ha et al. 2025 ). The present work aims to characterize the genotypical and phenotypical profile of E. coli isolates from poultry farms in Santa Catarina, Brazil, analyzing both in silico and in vitro data, to try to gain a deeper understanding of the mechanisms connected to the occurrence of colibacillosis outbreaks. Material and Methods Bacterial isolates 28 lyophilized E. coli bacterial isolates were donated by a veterinary diagnostic laboratory, Mercolab, having been previously obtained from colibacillosis outbreaks on poultry farms in Santa Catarina, Brazil. The isolates were registered in the National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen, no. A4E44C8). A standard strain, E. coli ATCC 25922, was used as a control for the phenotypic analysis. Phenotypic Characterization Isolation and Identification of E. coli. E. coli isolates were rehydrated and cultivated in MacConkey agar, and further identification was performed through additional biochemical tests (Markey et al. 2013). Confirmed E. coli isolates were stored in Brain Heart Infusion (BHI) broth with 40% glycerol, at -20 °C for subsequent molecular and antimicrobial resistance analyses. Antimicrobial Susceptibility Test Susceptibility to antimicrobials was determined by disk diffusion method, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2023). 19 antibiotics, representing the following classes of antimicrobials, were used: β-lactams (penicillins (ampicillin 10µg, oxacillin 1µg), cephalosporins (cephalexin 30µg, cefotaxime 30µg), and carbapenems (ertapenem 10µg, imipenem 10µg, meropenem 10µg)), aminoglycosides (gentamicin 10µg, amikacin 30µg), fluoroquinolones (ciprofloxacin 5µg, enrofloxacin 5µg), folate pathway inhibitors ((sulfamethoxazole–trimethoprim 30µg), phenicols (chloramphenicol 30µg, florfenicol 30µg), nitrofurans (nitrofurantoin 100µg), quinolones (nalidixic acid 30µg), tetracyclines (tetracycline 30µg, doxycycline 30µg), and rifamycins (rifampicin 5µg). The results were determined by the diameter (in mm) of the inhibition zone diameters and isolates were classified as “sensitive” (S), “intermediate” (I), or “resistant” (R) to the evaluated antimicrobials (CLSI 2023). The multiple antimicrobial resistance index (IRMA) was calculated based on the number of antimicrobial classes to which each isolate was resistant, divided by the total number of antimicrobials tested (n = 19), as adapted from the method described by Krumperman (1983). The class in which at least one antimicrobial exhibited a resistance profile was considered resistant. Results classified as intermediate were not considered for the calculation of the index. Genotypic Characterization Genomic DNA extraction Molecular characterization was performed by genomic DNA extractions through the method adapted from Ausubel et al . (1989) and Wade et al . (2005). For this purpose, the bacteria were cultured on BHI agar for 24 hours at 37 ºC. Using a calibrated 10 μL loop, colonies were added to a sterile microtube containing 300 μL of TE (Tris/HCl-EDTA, pH 8.0). The suspensions were homogenized using a vortex for 2 minutes, and then they were incubated for 10 minutes using a dry bath at 80 ºC to inactivate the samples. After that, 70 µL of 10% sodium dodecyl sulfate was added, and the homogenization was performed using a vortex again. Subsequently, 100 µL of 5M NaCl and 80 µL CTAB/NaCl (Cromoline) were added with stirring for two minutes. The samples remained in a dry bath at 65 ºC for 10 minutes. At this stage, 700 µL of chloroform/isoamyl alcohol in a ratio of 24:1 (Synth) was added, and the samples were homogenized by inversion for subsequent centrifugation at 11,750 x g for 5 minutes. Then, the first phase was transferred to another sterile microtube, adding 450 µL of isopropanol and homogenizing the solution. The samples were kept in contact with ice for 20 minutes, and then they were centrifuged at 11,750 x g for 15 minutes, after it the supernatant was discarded. 500 µL of 70% ethanol solution was added, and then the homogenization and centrifugation at 11,750 x g for 10 minutes were performed. After that, the supernatant was discarded, and the tubes were kept in a dry bath at 37 ºC. At last, 80 µL of water solution was added to elute the DNA, and then the samples were kept in a freezer at -20 °C. Total DNA extraction was performed using the commercial HiYield™ Genomic DNA Mini Kit (Real Genomics™). DNA concentration and integrity were assessed by spectrophotometry (NanoDrop®) and 1% agarose gel electrophoresis. APEC genome sequencing Whole genome sequencing (WGS) of E. coli isolates was performed through the Illumina MiSeq next-generation sequencing (NGS) platform. Libraries for whole-genome sequencing were prepared using Nextera XT DNA Library Prep Kit (Illumina®), in accordance with the standard protocol. DNA fragmentation and adapter addition were performed simultaneously through enzymatic tagging. A dual index was incorporated into each sample for multiplexing during sequencing. Sequencing was performed on the Illumina MiSeq platform using the v3 reagent kit, generating 300 bp (2×300 bp) paired-end reads. A minimum coverage of 30× was defined to ensure sturdiness of the genomes for subsequent bioinformatic analysis. Quality control on the raw FASTQ files was completed using FastQC. And residual adapters were removed with the Trimmomatic tool before de novo genomic assembly and subsequent analytical steps. In silico analysis The genomic sequences of the 28 isolates were obtained by paired-end sequencing and subjected to de novo assembly using Shovill software. Each assembly was executed with four threads, and the generated contigs were organized individually by sample. QUAST (version 5.3.0) was used to evaluated the quality of the assemblies, and the results were merged into a single report by the MultiQC software version 1.32. For the automated functional annotations of the genomes, Prokka software version 1.15.6 was used. Information on the sequencing locus and center was added, and the functions for detecting conserved RNAs and families were enabled. The screening of antimicrobial resistance genes was performed using two distinct and well-established approaches: the complete VFDB database and VirulenceFinder, both executed via ARIBA v.2.14.7, applying strict identity and coverage criteria. Using RGI v.6.0.5 (Resistance Gene Identifier), the annotated protein files (.faa) were processed against the CARD database, in protein mode, with eight threads and automatic cleaning of intermediate files. In parallel, DeepARG v.1.0.2 software was used with the LS model to identify genes in nucleotide sequence (.ffn), applying a minimum probability of 0.8 and alignment identity of at least 30%. Additionally, the ARIBA package was used for simultaneous screening of resistance genes (argannot bank), plasmids (PlasmidFinder), and virulence factors (VFDB and VirulenceFinder), with the banks previously prepared and updated. ARIBA analyses were performed from the raw fastq files, with ten threads per sample. MLST typing was performed using two independent methods: (i) through ARIBA, using Escherichia coli #1 and #2 schemes extracted from PubMLST, and compiling the results with the compileMLST.py script; and (ii) with the mlst command, using the contig files as input for cross-validation of the allelic profiles. Lastly, the results obtained from the gene annotation were converted into a binary matrix, which was then analyzed using the R environment, and analysis was conducted with the pheatmap library (v.1.0). Further information, such as the Multilocus Sequence Typing (MLST) data and the origin of the isolates, was included for the graphical analysis, which allowed for the generation of heatmaps for the visualization of the genetic data obtained from the isolates. Results Phenotypical susceptibility to antibiotics Different levels of resistance were observed for all 19 antimicrobials tested, with the exception of imipenem. Based on the susceptibility profile (Fig. 1), the highest resistance rate was observed for rifampicin (96.42%) and oxacillin (67.85%). In contrast, the lowest resistance levels were recorded for florfenicol (7.14%) and nitrofurantoin (3.57%). The presence of intermediate resistance was more evident among carbapenems (ertapenem 10.71% and meropenem 10.71%) and tetracyclines (doxycycline 10.7% and tetracycline 3.57%). Moreover, it was possible to identify that 100% of the E. coli isolates in this study exhibited resistance to at least one of the antimicrobials tested. In addition, based solely on IRMA values, 75.86% (22/28) of the isolates presented indices above 0.2 and were therefore classified as multidrug-resistant (MDR), as shown in Fig. 2. These isolates demonstrated concomitant resistance to a high number of the antimicrobials tested. High IRMA values indicate extensive diversity of antimicrobial classes to which these isolates are resistant, suggesting strong selective pressure in the environment of origin and strengthening the potential clinical and epidemiological risks presented by these bacteria, as it shows significantly reduced therapeutic options. Genotypic virulence and resistance Several antimicrobial virulence and resistance genes were observed in the isolates analyzed. Due to the large volume of data, the complete results are available in the supplementary material (Table S1). Virulence factors Out of the 28 isolates, nine isolates simultaneously presented the four classic virulence markers of the APEC panel – iutA, hlyF, ompT, and iroN, reinforcing their pathogenic potential as shown in Table 1. In addition, all of them carried genes with functions related to complement system evasion and survival in serum, such as traT, ompA , and ibeB . Table 1 Most relevant virulence genes identified among the 28 E. coli isolated from poultry Functional Category Gene Frequency among isolates (%) APEC panel (core genes) hlyF 71.42% iroN 60.71% ompT 57.14% iutA 42.85% Adhesion fimH 96.42% Iron acquisition febB 100% sitA 71.42% iro 60.71% chuA 42.85% iucA 42.85% Immune evasion ompA 100% ibeB/ibeC 100% traT 92.85% ompT 57.14% Toxins hlyE 85.71% In spite of its importance in pathogenic E. coli strains, such as APEC, the presence of the iss gene was not detected in any of the isolates examined. The absence of this gene in all the analyzes carried out indicates that this gene may not be part of the genomes evaluated. A few isolates (n=3) carried only three out of the five genes traditionally used as molecular indicators of APEC, rendering them beyond the most restrictive diagnostic limits for this group. However, each carried a number of other relevant virulence genes, such as adhesins ( fimH, papC ), siderophores ( iucA-D, fepA-D, chuA ), and immune evasion factors ( traT, ompT ), contributing to the pathogenic potential of these isolates. Resistance factors Similar to what was observed regarding virulence, the presence of numerous resistance genes was also remarkable. All genes detected are showcased in Fig. 3, as well as the intensity of their expression. ESBL genes were present in some isolates, the most frequent being CTX-M-55 (10.7%), CTX-M-2 (7.1%), and CTX-M-8 (3.6%), all of which were a part of the CTX-M operon. TEM gene variants were also observed in 12 isolates out of the 28 isolates (42.9%), and the most frequent one was TEM-1 (28.6%), followed by TEM-244 (10.7%) and less frequently TEM-135 (3.6%). AmpC was present in 4 isolates (14.3%). For aminoglycoside resistance genes, the majority were detected. ANT(3'')-IIa gene was the most prevalent, detected in ten isolates (35.7%). Other genes of significant frequency included APH(3')-Ia (17.9%) and aadA2 (14.3%). Some forms of the acetyltransferase members of the AAC(3) family were also found, although not as commonly. The quinolone resistance genes were also found, namely gyrA (39.2%) and parC (14.3%), qnrS1 (10.34%) and qnrB5 (10.3%). 13 out of 28 isolates (46.4%) contained sulfonamide resistance genes, the most prevalent being sul2 (28.6%) and sul1 (14.3%), while sul3 was present in a single isolate (3.6%). As far as tetracycline resistance genes are concerned, they occurred in 12 out of 28 isolates (42.9%), with the tet(A) gene prevailing in 11 isolates (39.3%), followed by four isolates (14.3%) in which the tet(B) variant occurred, and the tetR regulatory gene in four samples. The dfrA variant responsible for resistance against trimethoprim was encountered in 11 out of 28 isolates (39.3%). The dfrA1 was the most prevalent gene (17.9%). Other variants that were found included dfrA8, dfrA12, dfrA15b , and dfrA17 . The presence of these genes, especially together with the sul genes, enhances the probability of resistance to the TMP-SMX therapeutic regimen. Genes that encode resistance to phenicol were found in five isolates, with cmlA6 and floR each found in two (7.1%) and catI found in one (3.6%). For the MCR-1.1 gene that induces colistin resistance, this was found in two isolates (7.1%). Discussion In the present study, we chose to highlight the genes considered most relevant from an epidemiological, clinical, and public health perspective, based on their frequency, association with multidrug resistance, or known role in pathogenicity. Virulence profile Our genotypic analysis of the isolates exposed a broad range of virulence genes, with profiles ranging from classical lineages compatible with APEC to atypical strains carrying alternative determinants with comparable pathogenic potential. As previously noted, nine of the 28 isolates analyzed presented the four classical markers of the APEC panel ( iutA, hlyF, ompT , and iroN ) defined by Johnson et al. ( 2008 ), and therefore can be considered APEC. Although the five genes that compose the panel act as genetic markers for avian pathogenic E. coli , it’s important to note that the profile of such strains is not solely defined by the presence of these genes. As stated by Kazimierczak et al. ( 2025 ), APEC strains are highly diverse and may present several virulence markers, frequently including iron acquisition, adhesins, invasins, protectins, and toxin genes. In this study, the APEC strains lacked the iss gene, traditionally associated with increased serum resistance (Tivendale et al. 2004 ). Although this may indicate a specific limitation in complement resistance, it appears to be compensated for by the combined action of other evasion genes such as traT, ompA , and ibeB , all associated with complement evasion and serum survival (Ikeda et al. 2021 ). The recurring nature of cvaC , which is involved in the production of microcin (Bhambure et al. 2024 ), may also provide a competitive edge in challenging environments such as the intestinal tract of birds. Adhesion genes, especially those related to type 1 fimbriae ( fimA-fimH ), were also present at a high frequency, indicating the functional or almost complete operon in most strains. The presence of fimbriae-related genes is a common feature in APEC strains, as described in recent studies on colibacillosis by Jalil et al. ( 2023 ), Saidenberg et al. ( 2024 ), Bhambure et al. ( 2024 ), among others. These genes are responsible for the adhesion of the pathogen to the intestinal tract of the host, and their presence may indicate the horizontal acquisition of intestinal adhesion determinants (Foroogh et al. 2021 ), as described by the genomic plasticity of the strains in this study. Immune evasion and tissue invasion ability were further enhanced by the presence of high numbers of traT, ompA , and ibeB/ibeC genes. Yet, the number of these genes is vastly different among E. coli populations and geographical areas, as observed in studies performed in China (Lu et al. 2022 ) and South Korea (Ha et al. 2025 ). The concurrency of these genes indicates an elevated potential for extracellular survival, systemic infection (Ikeda et al. 2021 ), and colonization of protected sites (Hasan et al. 2022 ). Iron acquisition, a critical process for bacterial survival and a cofactor for various cellular processes (Zhang et al. 2025 ), was indicated by the presence of a wide and strong array of siderophore-related genes, including iucA-D, chuA, sitABCD, iroBDE, febB-G , and iroN . This array may contribute to host colonization and rapid bacterial growth (Spiga et al. 2023 ). In recent studies on avian E. coli , iroN and iutA are the most common iron acquisition-related genes, as part of the APEC panel, as reported by Meng et al. ( 2024 ) and Jhandai et al. ( 2025 ). However, as previously discussed, we observed a large diversity of iron acquisition genes in the present study. These findings indicate a broad, redundant, and adaptive siderophore capacity, consistent with systemically competent pathogenic profiles. Finally, the hlyE toxin, detected in the majority of the isolates, representing an additional virulence factor with important cytotoxic function. This finding goes against the findings of most studies with APEC strains, which generally do not report the presence of this toxin gene and instead describe the occurrence of vat (Awawdeh et al. 2024 , Ha et al. 2025 ). In recent literature, the only work to report the occurrence of hlyE in APEC was conducted by Saidenberg et al. ( 2024 ), and similar to this study, it evaluated APEC strains from poultry in Brazil. Such findings could suggest that hlyE is more prevalent in South America. However, further studies are necessary to confirm this hypothesis. Phenotypical and genotypical resistance Our findings reveal a widespread distribution of multidrug-resistant profiles combined with the presence of a genetic arsenal that reinforces this potential in avian E. coli , constituting a concerning scenario. The results observed amongst the phenotypic and genotypic findings indicates that a significant proportion of the strains not only actively express resistance but also carry determinants that may perpetuate this profile through horizontal gene transmission mechanisms. The high rates of resistance to traditional antimicrobials, including rifampicin, penicillins, and tetracyclines, not only confirm the weakening of traditional therapeutic efficacy in poultry production, but also suggests it is related to the presence of TEM, CTX-M , and tet resistance genes. The repeated patterns observed may suggest the existence of a selection pressure due to the prolonged use of antimicrobials in poultry farming. In this study, rifampicin exhibited the highest resistance rate, an expected finding, since this drug is reported to have low efficacy against E. coli when used as monotherapy (CLSI 2023 ; Wang et al. 2023 ). Phenotypically, high levels of total and intermediate resistance were found for the tested isolates, especially for β-lactam-based antimicrobials. The resistance could be connected with the presence of β-lactamase resistance genes, such as TEM-1, TEM-135, CTX-M-2, CTX-M-8 , and ampC , which are usually present in strains resistant to β-lactam-based antibiotic agents (Jalil et al. 2023 ), as well as variants such as TEM and CTX-M , found in penicillin-resistant strains (Ha et al. 2025 ). Tetracycline resistance related genes, tetA, tetB , and tetC , was observed in the tested strains that were also phenotypically resistant to the aforementioned antimicrobials. Therefore, our results further emphasize the strong connection between the presence of tet -type genes and the occurrence of phenotypic resistance. However, the correlation between the presence of sul -type genes and the prevalence of phenotypic resistance to sulfonamides is not as strong (Liu et al. 2024 ; Meng et al. 2024 ). This study did not identify carbapenemase-encoding genes, yet, some level of phenotypic resistance was observed which might connected to other resistance mechanisms such as the efflux pump mechanism that enables the regurgitation of substances (Rosa et al. 2024 ). The MCR-1.1 gene that is responsible for colistin resistance was observed in two strains, even though colistin was not part of the antimicrobials tested, the identification of this gene is a public health concern due to the potential for the spread of the resistance provided by it through plasmids from chicken pathogens to human pathogens (Liu et al. 2024 ). Conclusion The virulence and resistance profiles of the tested isolates were highly diverse and revealed a concerning infectious potential. The phenotypical information analyzed showed that most isolates presented multidrug resistance, which was confirmed by the presence of genes such as CTX-M, TEM, tet , and MCR-1.1 . Although the isolates did not present the standard APEC virulence markers, especially iss , they presented various other virulence genes associated with different methods of host colonization, adhesion, iron acquisition, toxicity and immune system evasion. Thus, these findings reinforce the atypical nature of these APEC strains and their potential to colonize, persist, and spread in different hosts. Our results highlight the importance of combined diagnostic and surveillance strategies and emphasize the health risk posed by these atypical strains. Declarations Ethical approval In accordance with Brazilian Federal Law No. 11.794/2008 no submission to an Ethics Committee on Animal Use was required. Funding This work was supported by the Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) (grant numbers IBPG-1469-2.00/22, IBPG-1363-500/21, BFP-0368-5.05/24, IBPG-1650-2.00/21, APQ-1199-5.05/22, APQ-1895-5.05/24, APQ-2138-5.05/24). The article processing fee for the publication of this research was covered by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (identifier ROR: 00x0ma614). For open access purposes, the authors have assigned the Creative Commons CC BY license to any accepted version of the article. Funding was also provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil – Finance Code 001 and a grant to author FSL (number 88887.615972/2021-00). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and experimentation were performed by ALS, FSL and MMC. Data analysis was conducted by ALS, BB, JJSG, and JFN. The first draft of the manuscript was written by ALS, edited by BASC reviewed by DSR and PIF. All authors read and approved the final manuscript. Acknowledgement We thank the Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) for financial support. We would also like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brasil - Finance code 001, for partially funding this study. The authors would like to thank the Rede de Ciências Ômicas (RECOM). The article processing fee for the publication of this research was covered by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (identifier ROR: 00x0ma614). For open access purposes, the authors have assigned the Creative Commons CC BY license to any accepted version of the article. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Ausubel MR, Brent RE, Kingston DD et al (1989) Current protocols in molecular biology. In: John Wiley & Sons (ed) Molecular Reproduction and Development, Wiley, Pennsylvania, pp 146-146 Awawdeh L, Forrest R, Turni C et al (2024) Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia. Aust Vet J. https://doi.org/10.1111/avj.13339 Barros MR, Silveira WD, Costa MM et al (2025) Prevalence of virulence genes in Escherichia coli isolates from poultry farms in Northeastern Brazil. Cienc Rural. https://doi.org/10.1590/0103-8478cr20240452 Bhambure SY, Costa EL, Gatty LIC et al (2024) Antibiotic resistance and virulence traits of Escherichia coli obtained from poultry waste. Braz J Microbiol. https://doi.org/10.1007/s42770-024-01367-1 ChiPlot (2025) ChiPlot software, version 7.0. https://www.chiplot.online/. Accessed 14 July 2025 Dawadi P, Bista B, Bista S (2021) Prevalence of colistin-resistant Escherichia coli from poultry in South Asian developing countries. Vet Med Int. https://doi.org/10.1155/2021/6398838 Denamur E, Clermont O, Bonacorsi S et al (2021) The population genetics of pathogenic Escherichia coli . Nat Rev Microbiol. https://doi.org/10.1038/s41579-020-0416-x Foroogh N, Rezvan M, Ahmad K et al (2021) Structural and functional characterization of the fimH adhesin of uropathogenic Escherichia coli and its novel applications. Microb Pathog. https://doi.org/10.1016/j.micpath.2021.105288 Ha EJ, Hong SM, Choi KS et al (2025) Evolution and zoonotic risk of O1:K1 and O2:K1 avian pathogenic Escherichia coli . Microbes Infect. https://doi.org/10.1016/j.micinf.2024.105462 Hasan RN, Jasim SA, Ali YH (2022) Detection of fimH, kpsMTII, hlyA and traT genes in Escherichia coli isolated from Iraqi patients with cystitis. Gene Rep. https://doi.org/10.1016/j.genrep.2021.101468 Ikeda M, Kobayashi T, Fujimoto F et al (2021) Prevalence of the iutA and ibeA genes in Escherichia coli isolates from severe and non-severe patients with bacteremic acute biliary tract infection. Gut Pathog. https://doi.org/10.1186/s13099-021-00429-1 Jalil A, Masood S, Ain Q et al (2023) High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from humid subtropical regions of Pakistan. J Glob Antimicrob Resist. https://doi.org/10.1016/j.jgar.2023.01.009 CLSI (2023) CLSI M100: performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute, Pennsylvania. Jhandai P, Mittal D, Gupta R et al (2025) Emerging avian pathogenic Escherichia coli serogroups, biofilm formation, ESBLs, integrons and in vivo pathogenicity in chicken. Microb Pathog. https://doi.org/10.1016/j.micpath.2025.107309 Johnson TJ, Wannemuehler Y, Doetkott C et al (2008) Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J Clin Microbiol. https://doi.org/10.1128/JCM.00816-08 Kazimierczak J, Pospiech K, Sowinska P et al (2025) Rapid detection of avian pathogenic Escherichia coli strains based on minimal virulence markers identified by whole-genome sequencing. BMC Microbiol. https://doi.org/10.1186/s12866-025-03861-4 Krumperman, PH (1983) Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. https://doi.org/10.1128/aem.46.1.165-170.1983 Liu C, Sun S, Sun Y et al (2024) Antibiotic resistance of Escherichia coli isolated from food and clinical environments in China from 2001 to 2020. Sci Total Environ. https://doi.org/10.1016/j.scitotenv.2024.173498 Lu Q, Zhang W, Luo L et al (2022) Genetic diversity and multidrug resistance of phylogenetic groups B2 and D in InPEC and ExPEC isolated from chickens in central China. BMC Microbiol. https://doi.org/10.1186/s12866-022-02469-2 Markey B, Leonard F, Archambault M et al (2013) Clinical veterinary microbiology. Elsevier, Edinburgh. Meng J, Wang W, Din, J et al (2024) Synergistic effect of matrine and berberine hydrochloride in treating colibacillosis caused by multidrug-resistant avian pathogenic Escherichia coli . Poult Sci. https://doi.org/10.1016/j.psj.2024.104151 Rosa DS, Oliveira SAS, Souza R. et al (2024) Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant Staphylococcus aureus . J Appl Microbiol. https://doi.org/10.1093/jambio/lxae072 Saidenberg ABS, Edslev SM, Hallstrom S et al (2024) Escherichia coli ST117: exploring the zoonotic hypothesis. Microbiol Spectr. https://doi.org/10.1128/spectrum.00466-24 Spiga L, Fansler RT, Perera YR et al (2023) Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection. Cell Host Microbe. https://doi.org/10.1016/j.chom.2023.08.018 Tivendale KA, Allen JL, Ginns CA et al (2004) Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic Escherichia coli . Infect Immun. https://doi.org/10.1128/IAI.72.11.6554-6560.2004 Wade JT, Reppas NB, Church GM et al (2005) Genomic analysis of LexA binding reveals permissive nature of the Escherichia coli genome and identifies unconventional target sites. Genes Dev. https://doi.org/10.1101/gad.1355605 Wang Y, Fu H, Shi X et al (2023) Genome-wide screen reveals cellular functions that counteract rifampicin lethality in Escherichia coli . Microbiol Spectr. https://doi.org/10.1128/spectrum.02895-23 Zhang R, Li D, Fang H et al (2025) Iron-dependent mechanisms in Acinetobacter baumannii: pathogenicity and resistance. JAC Antimicrob Resist. https://doi.org/10.1093/jacamr/dlaf039 Additional Declarations No competing interests reported. Supplementary Files TableS1suplementary.xlsx TableS2suplementary.xlsx Cite Share Download PDF Status: Published Journal Publication published 02 Apr, 2026 Read the published version in International Microbiology → Version 1 posted Editorial decision: Revision requested 04 Mar, 2026 Reviews received at journal 26 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviews received at journal 13 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor assigned by journal 11 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 03 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8781221","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591464279,"identity":"35b1a2b3-65cb-4d2a-bb16-30c6e8599c13","order_by":0,"name":"Angélica Liberalino da Silva","email":"","orcid":"","institution":"Federal Rural University of Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Angélica","middleName":"Liberalino da","lastName":"Silva","suffix":""},{"id":591464281,"identity":"a79a229d-55ab-4578-a83c-06710599c18b","order_by":1,"name":"Fabrina Sousa Luna","email":"","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":false,"prefix":"","firstName":"Fabrina","middleName":"Sousa","lastName":"Luna","suffix":""},{"id":591464285,"identity":"496bc646-3698-41a6-928e-ad12553745d5","order_by":2,"name":"Joel Fonseca Nogueira","email":"","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"Fonseca","lastName":"Nogueira","suffix":""},{"id":591464287,"identity":"4bfcbb4a-9adf-452d-ad02-dec7d060a029","order_by":3,"name":"Danillo Sales Rosa","email":"","orcid":"","institution":"Federal Rural University of Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Danillo","middleName":"Sales","lastName":"Rosa","suffix":""},{"id":591464289,"identity":"c4372341-5411-4d7d-80f9-be2ada3f7a64","order_by":4,"name":"Priscila Izidro Figueirêdo","email":"","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":false,"prefix":"","firstName":"Priscila","middleName":"Izidro","lastName":"Figueirêdo","suffix":""},{"id":591464290,"identity":"a1297d26-7969-40a2-827f-4cf40ed47000","order_by":5,"name":"Brendo Atila dos Santos da Cruz","email":"","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":false,"prefix":"","firstName":"Brendo","middleName":"Atila dos Santos da","lastName":"Cruz","suffix":""},{"id":591464292,"identity":"fc9246ef-2acd-4042-a5e1-de2db1542a27","order_by":6,"name":"João José Simoni Gouveia","email":"","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"José Simoni","lastName":"Gouveia","suffix":""},{"id":591464293,"identity":"5868953f-d2eb-4d77-b544-7e3960e0542e","order_by":7,"name":"Bertram Brenig","email":"","orcid":"","institution":"University of Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Bertram","middleName":"","lastName":"Brenig","suffix":""},{"id":591464299,"identity":"04acecaf-c8cd-4fe9-b707-f80be3b45f57","order_by":8,"name":"Mateus Matiuzzi Costa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYLCCBCBmgzBtgJix8QABDYwNcC0HGNLAAoS1wJkHGA5DaTxAd9rh5w8ettXZ87EfPvz5Q815u7Xth4G21NhE49JidjvNsCGxjY2ZjSctTeLAsdvJ284kArUcS8ttwKklAaSFh41NgseM4QDb7WSzA0AtjA2H8WhJ/wjUIsED1GL84cC/c8lm5x8S0pIDssVAAqjFQOJg2wE7sxsEbckpnJFwLsEA7JezfckJZjeAtiTg9Uv6ho8/yurs5dsPH/5Q8c3O3ux8+sMHH2pscGrBAIlglQnEKgcBe1IUj4JRMApGwcgAAC0JZBbtImFwAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Federal do Vale do São Francisco (UNIVASF)","correspondingAuthor":true,"prefix":"","firstName":"Mateus","middleName":"Matiuzzi","lastName":"Costa","suffix":""}],"badges":[],"createdAt":"2026-02-04 03:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8781221/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8781221/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10123-026-00811-6","type":"published","date":"2026-04-02T16:00:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102828012,"identity":"57bce186-e630-49ea-9639-435ca620b75f","added_by":"auto","created_at":"2026-02-17 09:20:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76253,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic antimicrobial susceptibility profile of \u003cem\u003eEscherichia coli\u003c/em\u003e isolates obtained from chickens on poultry farms. Bars represent the percentage of isolates classified as susceptible (green), intermediate (blue), or resistant (orange) to each antimicrobial agent tested, according to standardized interpretive criteria\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/11b4e63c22a2a02e35d98264.png"},{"id":102828010,"identity":"71a6db21-f68f-4e9d-83cc-836d9a09ec0a","added_by":"auto","created_at":"2026-02-17 09:20:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77755,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of Antimicrobial Resistance Profiles and IRMA Values of the \u003cem\u003eEscherichia coli\u003c/em\u003e Isolates. Rows represent antimicrobial agents and columns represent bacterial isolates. Red blocks indicate resistance (R), blue blocks indicate susceptibility (S), and yellow blocks indicate intermediate activity (I). Antimicrobials evaluated: GEN (gentamicin), AMK (amikacin), CEF (cephalexin), CFX (cefotaxime), CHL (chloramphenicol), FFC (florfenicol), CIP (ciprofloxacin), ENR (enrofloxacin), SXT (sulfamethoxazole–trimethoprim), NIT (nitrofurantoin), AMP (ampicillin), OXA (oxacillin), NAL (nalidixic acid), RIF (rifampicin), TET (tetracycline), DOX (doxycycline), ETP (ertapenem), IMP (imipenem) and MER (meropenem)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/2dd670f4ddca1c9dbc0ca6e9.png"},{"id":102962819,"identity":"46c9a795-4680-466a-8948-4c7986d810db","added_by":"auto","created_at":"2026-02-19 04:11:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":339687,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution and expression of resistance genes across all 28 isolates, according to Resistance Gene Identifier (RGI) (ChiPlot v7.0 2025). Each row corresponds to a gene, while columns represent individual isolates\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/41ee9afa12b45f836d1b1d88.png"},{"id":106344983,"identity":"566e3853-e22c-4d20-a873-6d590ae33a97","added_by":"auto","created_at":"2026-04-07 16:17:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":982460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/72b46d9c-d5e0-4f89-b3ae-9ef866ac74bb.pdf"},{"id":102962818,"identity":"b82b22d8-6092-452a-9653-449b49882fdf","added_by":"auto","created_at":"2026-02-19 04:11:29","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":56810,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1suplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/f8e384fd04ec0b42497ba874.xlsx"},{"id":102828014,"identity":"2652adf3-8b74-48f1-8e79-6ce69679fed8","added_by":"auto","created_at":"2026-02-17 09:20:54","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12109,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2suplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8781221/v1/cb2ffc0a95007a071a90d5d4.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenotypical and genetic virulence and resistance in atypical avian pathogenic Escherichia coli","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the most important sanitary problems that aviculture currently faces is the occurrence of colibacillosis, a disease caused by avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC) that can, in many cases, lead to significant economic and zootechnical losses (Denamur et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These \u003cem\u003eE. coli\u003c/em\u003e strains are a real threat to intensive production systems, as they are capable of causing local and systemic infections, especially in young chicks, which often leads to mortality and low production ratings (Ha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Jhandai et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, in recent decades, the issue of the spread of multidrug-resistant \u003cem\u003eE. coli\u003c/em\u003e has become a matter of particular concern and is considered a major challenge due to its potential to undermine the effectiveness of conventional antimicrobial therapy, making the control of such infections difficult, and increasing the risk of spreading resistance genes not only to other animals but also among humans (Hasan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ha et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother concern is the ability of \u003cem\u003eE. coli\u003c/em\u003e strains to develop antimicrobial resistance independently, as well as to acquire and exchange virulence and resistance genes through plasmids, transposons, and other mobile genetic elements with other bacteria, thereby promoting the sharing and persistence of such genes (Dawadi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consequently, poultry products may act as reservoirs of MDR APEC and other pathogenic strains, enabling their transmission to humans (Dawadi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present work aims to characterize the genotypical and phenotypical profile of \u003cem\u003eE. coli\u003c/em\u003e isolates from poultry farms in Santa Catarina, Brazil, analyzing both \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e data, to try to gain a deeper understanding of the mechanisms connected to the occurrence of colibacillosis outbreaks.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eBacterial isolates\u003c/p\u003e\n\u003cp\u003e28 lyophilized \u003cem\u003eE. coli\u003c/em\u003e bacterial isolates were donated by a veterinary diagnostic laboratory, Mercolab, having been previously obtained from colibacillosis outbreaks on poultry farms in Santa Catarina, Brazil. The isolates were registered in the National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen, no. A4E44C8). A standard strain, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, was used as a control for the phenotypic analysis.\u003c/p\u003e\n\u003cp\u003ePhenotypic Characterization\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIsolation and Identification of E. coli.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e isolates were rehydrated and cultivated in MacConkey agar, and further identification was performed through additional biochemical tests (Markey et al. 2013). Confirmed \u003cem\u003eE. coli isolates\u0026nbsp;\u003c/em\u003ewere stored in Brain Heart Infusion (BHI) broth with 40% glycerol, at -20 \u0026deg;C for subsequent molecular and antimicrobial resistance analyses.\u003c/p\u003e\n\u003cp id=\"_Toc201467547\"\u003e\u003cem\u003eAntimicrobial Susceptibility Test\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSusceptibility to antimicrobials was determined by disk diffusion method, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2023). 19 antibiotics, representing the following classes of antimicrobials, were used: \u0026beta;-lactams (penicillins (ampicillin 10\u0026micro;g, oxacillin 1\u0026micro;g), cephalosporins (cephalexin 30\u0026micro;g, cefotaxime 30\u0026micro;g), and carbapenems (ertapenem 10\u0026micro;g, imipenem 10\u0026micro;g, meropenem 10\u0026micro;g)), aminoglycosides (gentamicin 10\u0026micro;g, amikacin 30\u0026micro;g), fluoroquinolones (ciprofloxacin 5\u0026micro;g, enrofloxacin 5\u0026micro;g), folate pathway inhibitors ((sulfamethoxazole\u0026ndash;trimethoprim 30\u0026micro;g), phenicols (chloramphenicol 30\u0026micro;g, florfenicol 30\u0026micro;g), nitrofurans (nitrofurantoin 100\u0026micro;g), quinolones (nalidixic acid 30\u0026micro;g), tetracyclines (tetracycline 30\u0026micro;g, doxycycline 30\u0026micro;g), and rifamycins (rifampicin 5\u0026micro;g). The results were determined by the diameter (in mm) of the inhibition zone diameters and isolates were classified as \u0026ldquo;sensitive\u0026rdquo; (S), \u0026ldquo;intermediate\u0026rdquo; (I), or \u0026ldquo;resistant\u0026rdquo; (R) to the evaluated antimicrobials (CLSI 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe multiple antimicrobial resistance index (IRMA) was calculated based on the number of antimicrobial classes to which each isolate was resistant, divided by the total number of antimicrobials tested (n = 19), as adapted from the method described by Krumperman (1983). The class in which at least one antimicrobial exhibited a resistance profile was considered resistant. Results classified as intermediate were not considered for the calculation of the index.\u003c/p\u003e\n\u003cp\u003eGenotypic Characterization\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenomic DNA extraction\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMolecular characterization was performed by genomic DNA extractions through the method adapted from Ausubel et al\u003cem\u003e.\u003c/em\u003e (1989) and Wade et al\u003cem\u003e.\u003c/em\u003e (2005). For this purpose, the bacteria were cultured on BHI agar for 24 hours at 37 \u0026ordm;C. Using a calibrated 10 \u0026mu;L loop, colonies were added to a sterile microtube containing 300 \u0026mu;L of TE (Tris/HCl-EDTA, pH 8.0). The suspensions were homogenized using a vortex for 2 minutes, and then they were incubated for 10 minutes using a dry bath at 80 \u0026ordm;C to inactivate the samples. After that, 70 \u0026micro;L of 10% sodium dodecyl sulfate was added, and the homogenization was performed using a vortex again. Subsequently, 100 \u0026micro;L of 5M NaCl and 80 \u0026micro;L CTAB/NaCl (Cromoline) were added with stirring for two minutes. The samples remained in a dry bath at 65 \u0026ordm;C for 10 minutes. At this stage, 700 \u0026micro;L of chloroform/isoamyl alcohol in a ratio of 24:1 (Synth) was added, and the samples were homogenized by inversion for subsequent centrifugation at 11,750 x g for 5 minutes. Then, the first phase was transferred to another sterile microtube, adding 450 \u0026micro;L of isopropanol and homogenizing the solution. The samples were kept in contact with ice for 20 minutes, and then they were centrifuged at 11,750 x g for 15 minutes, after it the supernatant was discarded. 500 \u0026micro;L of 70% ethanol solution was added, and then the homogenization and centrifugation at 11,750 x g for 10 minutes were performed. After that, the supernatant was discarded, and the tubes were kept in a dry bath at 37 \u0026ordm;C. At last, 80 \u0026micro;L of water solution was added to elute the DNA, and then the samples were kept in a freezer at -20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eTotal DNA extraction was performed using the commercial HiYield\u0026trade; Genomic DNA Mini Kit (Real Genomics\u0026trade;). DNA concentration and integrity were assessed by spectrophotometry (NanoDrop\u0026reg;) and 1% agarose gel electrophoresis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAPEC genome sequencing\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWhole genome sequencing (WGS) of \u003cem\u003eE. coli\u003c/em\u003e isolates was performed through the Illumina MiSeq next-generation sequencing (NGS) platform. Libraries for whole-genome sequencing were prepared using Nextera XT DNA Library Prep Kit (Illumina\u0026reg;), in accordance with the standard protocol. DNA fragmentation and adapter addition were performed simultaneously through enzymatic tagging. A dual index was incorporated into each sample for multiplexing during sequencing. Sequencing was performed on the Illumina MiSeq platform using the v3 reagent kit, generating 300 bp (2\u0026times;300 bp) paired-end reads. A minimum coverage of 30\u0026times; was defined to ensure sturdiness of the genomes for subsequent bioinformatic analysis. Quality control on the raw FASTQ files was completed using FastQC. And residual adapters were removed with the Trimmomatic tool before \u003cem\u003ede novo\u003c/em\u003e genomic assembly and subsequent analytical steps.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn silico analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe genomic sequences of the 28 isolates were obtained by paired-end sequencing and subjected to \u003cem\u003ede novo\u003c/em\u003e assembly using Shovill software. Each assembly was executed with four threads, and the generated contigs were organized individually by sample. QUAST (version 5.3.0) was used to evaluated the quality of the assemblies, and the results were merged into a single report by the MultiQC software version 1.32. For the automated functional annotations of the genomes, Prokka software version 1.15.6 was used. Information on the sequencing locus and center was added, and the functions for detecting conserved RNAs and families were enabled. The screening of antimicrobial resistance genes was performed using two distinct and well-established approaches: the complete VFDB database and VirulenceFinder, both executed via ARIBA v.2.14.7, applying strict identity and coverage criteria. Using RGI v.6.0.5 (Resistance Gene Identifier), the annotated protein files (.faa) were processed against the CARD database, in protein mode, with eight threads and automatic cleaning of intermediate files. In parallel, DeepARG v.1.0.2 software was used with the LS model to identify genes in nucleotide sequence (.ffn), applying a minimum probability of 0.8 and alignment identity of at least 30%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, the ARIBA package was used for simultaneous screening of resistance genes (argannot bank), plasmids (PlasmidFinder), and virulence factors (VFDB and VirulenceFinder), with the banks previously prepared and updated. ARIBA analyses were performed from the raw fastq files, with ten threads per sample. MLST typing was performed using two independent methods: (i) through ARIBA, using \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e#1 and #2 schemes extracted from PubMLST, and compiling the results with the compileMLST.py script; and (ii) with the mlst command, using the contig files as input for cross-validation of the allelic profiles.\u003c/p\u003e\n\u003cp\u003eLastly, the results obtained from the gene annotation were converted into a binary matrix, which was then analyzed using the R environment, and analysis was conducted with the pheatmap library (v.1.0). Further information, such as the Multilocus Sequence Typing (MLST) data and the origin of the isolates, was included for the graphical analysis, which allowed for the generation of heatmaps for the visualization of the genetic data obtained from the isolates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePhenotypical susceptibility to antibiotics\u003c/p\u003e\n\u003cp\u003eDifferent levels of resistance were observed for all 19 antimicrobials tested, with the exception of imipenem. Based on the susceptibility profile (Fig. 1), the highest resistance rate was observed for rifampicin (96.42%) and oxacillin (67.85%). In contrast, the lowest resistance levels were recorded for florfenicol (7.14%) and nitrofurantoin (3.57%). The presence of intermediate resistance was more evident among carbapenems (ertapenem 10.71% and meropenem 10.71%) and tetracyclines (doxycycline 10.7% and tetracycline 3.57%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, it was possible to identify that 100% of the \u003cem\u003eE. coli\u003c/em\u003e isolates in this study exhibited resistance to at least one of the antimicrobials tested. In addition, based solely on IRMA values, 75.86% (22/28) of the isolates presented indices above 0.2 and were therefore classified as multidrug-resistant (MDR), as shown in Fig. 2. These isolates demonstrated concomitant resistance to a high number of the antimicrobials tested. High IRMA values indicate extensive diversity of antimicrobial classes to which these isolates are resistant, suggesting strong selective pressure in the environment of origin and strengthening the potential clinical and epidemiological risks presented by these bacteria, as it shows significantly reduced therapeutic options.\u003c/p\u003e\n\u003cp\u003eGenotypic virulence and resistance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral antimicrobial virulence and resistance genes were observed in the isolates analyzed. Due to the large volume of data, the complete results are available in the supplementary material (Table S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVirulence factors\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOut of the 28 isolates, nine isolates simultaneously presented the four classic virulence markers of the APEC panel \u0026ndash; \u003cem\u003eiutA, hlyF, ompT,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eiroN,\u0026nbsp;\u003c/em\u003ereinforcing their pathogenic potential as shown in Table 1. In addition, all of them carried genes with functions related to complement system evasion and survival in serum, such as \u003cem\u003etraT, ompA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;ibeB\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Most relevant virulence genes identified among the 28\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e isolated from poultry\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\" width=\"605\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFunctional Category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFrequency among isolates (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAPEC panel (core genes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ehlyF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e71.42%\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eiroN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eompT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e57.14%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eiutA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAdhesion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003efimH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.42%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIron acquisition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003efebB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003esitA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e71.42%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eiro\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003echuA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eiucA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eImmune evasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eompA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eibeB/ibeC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003e100%\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003etraT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eompT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e57.14%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eToxins\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ehlyE\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn spite of its importance in pathogenic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003estrains, such as APEC, the presence of the \u003cem\u003eiss\u003c/em\u003e gene was not detected in any of the isolates examined. The absence of this gene in all the analyzes carried out indicates that this gene may not be part of the genomes evaluated.\u003c/p\u003e\n\u003cp\u003eA few isolates (n=3) carried only three out of the five genes traditionally used as molecular indicators of APEC, rendering them beyond the most restrictive diagnostic limits for this group. However, each carried a number of other relevant virulence genes, such as adhesins (\u003cem\u003efimH, papC\u003c/em\u003e), siderophores (\u003cem\u003eiucA-D, fepA-D, chuA\u003c/em\u003e), and immune evasion factors (\u003cem\u003etraT, ompT\u003c/em\u003e), contributing to the pathogenic potential of these isolates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResistance factors\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSimilar to what was observed regarding virulence, the presence of numerous resistance genes was also remarkable. All genes detected are showcased in Fig. 3, as well as the intensity of their expression.\u003c/p\u003e\n\u003cp\u003eESBL genes were present in some isolates, the most frequent being \u003cem\u003eCTX-M-55\u003c/em\u003e (10.7%), \u003cem\u003eCTX-M-2\u003c/em\u003e (7.1%), and \u003cem\u003eCTX-M-8\u003c/em\u003e (3.6%), all of which were a part of the \u003cem\u003eCTX-M\u003c/em\u003e operon. \u003cem\u003eTEM\u003c/em\u003e gene variants were also observed in 12 isolates out of the 28 isolates (42.9%), and the most frequent one was \u003cem\u003eTEM-1\u003c/em\u003e (28.6%), followed by \u003cem\u003eTEM-244\u003c/em\u003e (10.7%) and less frequently \u003cem\u003eTEM-135\u003c/em\u003e (3.6%). \u003cem\u003eAmpC\u003c/em\u003e was present in 4 isolates (14.3%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor aminoglycoside resistance genes, the majority were detected. \u003cem\u003eANT(3\u0026apos;\u0026apos;)-IIa\u003c/em\u003e gene was the most prevalent, detected in ten isolates (35.7%). Other genes of significant frequency included \u003cem\u003eAPH(3\u0026apos;)-Ia\u003c/em\u003e (17.9%) and \u003cem\u003eaadA2\u003c/em\u003e (14.3%). Some forms of the acetyltransferase members of the \u003cem\u003eAAC(3)\u003c/em\u003e family were also found, although not as commonly. The quinolone resistance genes were also found, namely \u003cem\u003egyrA\u003c/em\u003e (39.2%) and \u003cem\u003eparC\u003c/em\u003e (14.3%), \u003cem\u003eqnrS1\u003c/em\u003e (10.34%) and \u003cem\u003eqnrB5\u003c/em\u003e (10.3%).\u003c/p\u003e\n\u003cp\u003e13 out of 28 isolates (46.4%) contained sulfonamide resistance genes, the most prevalent being \u003cem\u003esul2\u003c/em\u003e (28.6%) and \u003cem\u003esul1\u003c/em\u003e (14.3%), while \u003cem\u003esul3\u003c/em\u003e was present in a single isolate (3.6%). As far as tetracycline resistance genes are concerned, they occurred in 12 out of 28 isolates (42.9%), with the \u003cem\u003etet(A)\u0026nbsp;\u003c/em\u003egene prevailing in 11 isolates (39.3%), followed by four isolates (14.3%) in which the \u003cem\u003etet(B)\u0026nbsp;\u003c/em\u003evariant occurred, and the \u003cem\u003etetR\u003c/em\u003e regulatory gene in four samples. The \u003cem\u003edfrA\u003c/em\u003e variant responsible for resistance against trimethoprim was encountered in 11 out of 28 isolates (39.3%). The \u003cem\u003edfrA1\u003c/em\u003e was the most prevalent gene (17.9%). Other variants that were found included \u003cem\u003edfrA8, dfrA12, dfrA15b\u003c/em\u003e, and \u003cem\u003edfrA17\u003c/em\u003e. The presence of these genes, especially together with the sul genes, enhances the probability of resistance to the TMP-SMX therapeutic regimen.\u003c/p\u003e\n\u003cp\u003eGenes that encode resistance to phenicol were found in five isolates, with \u003cem\u003ecmlA6\u003c/em\u003e and \u003cem\u003efloR\u003c/em\u003e each found in two (7.1%) and \u003cem\u003ecatI\u003c/em\u003e found in one (3.6%). For the \u003cem\u003eMCR-1.1\u003c/em\u003e gene that induces colistin resistance, this was found in two isolates (7.1%).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we chose to highlight the genes considered most relevant from an epidemiological, clinical, and public health perspective, based on their frequency, association with multidrug resistance, or known role in pathogenicity.\u003c/p\u003e \u003cp\u003eVirulence profile\u003c/p\u003e \u003cp\u003eOur genotypic analysis of the isolates exposed a broad range of virulence genes, with profiles ranging from classical lineages compatible with APEC to atypical strains carrying alternative determinants with comparable pathogenic potential. As previously noted, nine of the 28 isolates analyzed presented the four classical markers of the APEC panel (\u003cem\u003eiutA, hlyF, ompT\u003c/em\u003e, and \u003cem\u003eiroN\u003c/em\u003e) defined by Johnson et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and therefore can be considered APEC. Although the five genes that compose the panel act as genetic markers for avian pathogenic \u003cem\u003eE. coli\u003c/em\u003e, it\u0026rsquo;s important to note that the profile of such strains is not solely defined by the presence of these genes. As stated by Kazimierczak et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), APEC strains are highly diverse and may present several virulence markers, frequently including iron acquisition, adhesins, invasins, protectins, and toxin genes.\u003c/p\u003e \u003cp\u003eIn this study, the APEC strains lacked the \u003cem\u003eiss\u003c/em\u003e gene, traditionally associated with increased serum resistance (Tivendale et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Although this may indicate a specific limitation in complement resistance, it appears to be compensated for by the combined action of other evasion genes such as \u003cem\u003etraT, ompA\u003c/em\u003e, and \u003cem\u003eibeB\u003c/em\u003e, all associated with complement evasion and serum survival (Ikeda et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The recurring nature of \u003cem\u003ecvaC\u003c/em\u003e, which is involved in the production of microcin (Bhambure et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), may also provide a competitive edge in challenging environments such as the intestinal tract of birds.\u003c/p\u003e \u003cp\u003eAdhesion genes, especially those related to type 1 fimbriae (\u003cem\u003efimA-fimH\u003c/em\u003e), were also present at a high frequency, indicating the functional or almost complete operon in most strains. The presence of fimbriae-related genes is a common feature in APEC strains, as described in recent studies on colibacillosis by Jalil et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Saidenberg et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), Bhambure et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), among others. These genes are responsible for the adhesion of the pathogen to the intestinal tract of the host, and their presence may indicate the horizontal acquisition of intestinal adhesion determinants (Foroogh et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), as described by the genomic plasticity of the strains in this study.\u003c/p\u003e \u003cp\u003eImmune evasion and tissue invasion ability were further enhanced by the presence of high numbers of \u003cem\u003etraT, ompA\u003c/em\u003e, and \u003cem\u003eibeB/ibeC\u003c/em\u003e genes. Yet, the number of these genes is vastly different among \u003cem\u003eE. coli\u003c/em\u003e populations and geographical areas, as observed in studies performed in China (Lu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and South Korea (Ha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The concurrency of these genes indicates an elevated potential for extracellular survival, systemic infection (Ikeda et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and colonization of protected sites (Hasan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIron acquisition, a critical process for bacterial survival and a cofactor for various cellular processes (Zhang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), was indicated by the presence of a wide and strong array of siderophore-related genes, including \u003cem\u003eiucA-D, chuA, sitABCD, iroBDE, febB-G\u003c/em\u003e, and \u003cem\u003eiroN\u003c/em\u003e. This array may contribute to host colonization and rapid bacterial growth (Spiga et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In recent studies on avian \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eiroN\u003c/em\u003e and \u003cem\u003eiutA\u003c/em\u003e are the most common iron acquisition-related genes, as part of the APEC panel, as reported by Meng et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Jhandai et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, as previously discussed, we observed a large diversity of iron acquisition genes in the present study. These findings indicate a broad, redundant, and adaptive siderophore capacity, consistent with systemically competent pathogenic profiles.\u003c/p\u003e \u003cp\u003eFinally, the \u003cem\u003ehlyE\u003c/em\u003e toxin, detected in the majority of the isolates, representing an additional virulence factor with important cytotoxic function. This finding goes against the findings of most studies with APEC strains, which generally do not report the presence of this toxin gene and instead describe the occurrence of \u003cem\u003evat\u003c/em\u003e (Awawdeh et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Ha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In recent literature, the only work to report the occurrence of \u003cem\u003ehlyE\u003c/em\u003e in APEC was conducted by Saidenberg et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and similar to this study, it evaluated APEC strains from poultry in Brazil. Such findings could suggest that \u003cem\u003ehlyE\u003c/em\u003e is more prevalent in South America. However, further studies are necessary to confirm this hypothesis.\u003c/p\u003e \u003cp\u003ePhenotypical and genotypical resistance\u003c/p\u003e \u003cp\u003eOur findings reveal a widespread distribution of multidrug-resistant profiles combined with the presence of a genetic arsenal that reinforces this potential in avian \u003cem\u003eE. coli\u003c/em\u003e, constituting a concerning scenario. The results observed amongst the phenotypic and genotypic findings indicates that a significant proportion of the strains not only actively express resistance but also carry determinants that may perpetuate this profile through horizontal gene transmission mechanisms. The high rates of resistance to traditional antimicrobials, including rifampicin, penicillins, and tetracyclines, not only confirm the weakening of traditional therapeutic efficacy in poultry production, but also suggests it is related to the presence of \u003cem\u003eTEM, CTX-M\u003c/em\u003e, and \u003cem\u003etet\u003c/em\u003e resistance genes. The repeated patterns observed may suggest the existence of a selection pressure due to the prolonged use of antimicrobials in poultry farming.\u003c/p\u003e \u003cp\u003eIn this study, rifampicin exhibited the highest resistance rate, an expected finding, since this drug is reported to have low efficacy against \u003cem\u003eE. coli\u003c/em\u003e when used as monotherapy (CLSI \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phenotypically, high levels of total and intermediate resistance were found for the tested isolates, especially for β-lactam-based antimicrobials. The resistance could be connected with the presence of β-lactamase resistance genes, such as \u003cem\u003eTEM-1, TEM-135, CTX-M-2, CTX-M-8\u003c/em\u003e, and \u003cem\u003eampC\u003c/em\u003e, which are usually present in strains resistant to β-lactam-based antibiotic agents (Jalil et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), as well as variants such as \u003cem\u003eTEM\u003c/em\u003e and \u003cem\u003eCTX-M\u003c/em\u003e, found in penicillin-resistant strains (Ha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTetracycline resistance related genes, \u003cem\u003etetA, tetB\u003c/em\u003e, and \u003cem\u003etetC\u003c/em\u003e, was observed in the tested strains that were also phenotypically resistant to the aforementioned antimicrobials. Therefore, our results further emphasize the strong connection between the presence of \u003cem\u003etet\u003c/em\u003e-type genes and the occurrence of phenotypic resistance. However, the correlation between the presence of \u003cem\u003esul\u003c/em\u003e-type genes and the prevalence of phenotypic resistance to sulfonamides is not as strong (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study did not identify carbapenemase-encoding genes, yet, some level of phenotypic resistance was observed which might connected to other resistance mechanisms such as the efflux pump mechanism that enables the regurgitation of substances (Rosa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eThe MCR-1.1\u003c/em\u003e gene that is responsible for colistin resistance was observed in two strains, even though colistin was not part of the antimicrobials tested, the identification of this gene is a public health concern due to the potential for the spread of the resistance provided by it through plasmids from chicken pathogens to human pathogens (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe virulence and resistance profiles of the tested isolates were highly diverse and revealed a concerning infectious potential. The phenotypical information analyzed showed that most isolates presented multidrug resistance, which was confirmed by the presence of genes such as \u003cem\u003eCTX-M, TEM, tet\u003c/em\u003e, and \u003cem\u003eMCR-1.1\u003c/em\u003e. Although the isolates did not present the standard APEC virulence markers, especially \u003cem\u003eiss\u003c/em\u003e, they presented various other virulence genes associated with different methods of host colonization, adhesion, iron acquisition, toxicity and immune system evasion. Thus, these findings reinforce the atypical nature of these APEC strains and their potential to colonize, persist, and spread in different hosts. Our results highlight the importance of combined diagnostic and surveillance strategies and emphasize the health risk posed by these atypical strains.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eIn accordance with Brazilian Federal Law No. 11.794/2008 no submission to an Ethics Committee on Animal Use was required.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Ci\u0026ecirc;ncia e Tecnologia de Pernambuco (FACEPE) (grant numbers IBPG-1469-2.00/22, IBPG-1363-500/21, BFP-0368-5.05/24, IBPG-1650-2.00/21, APQ-1199-5.05/22, APQ-1895-5.05/24, APQ-2138-5.05/24). The article processing fee for the publication of this research was covered by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - CAPES (identifier ROR: 00x0ma614). For open access purposes, the authors have assigned the Creative Commons CC BY license to any accepted version of the article. Funding was also provided by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior, Brazil \u0026ndash; Finance Code 001 and a grant to author FSL (number 88887.615972/2021-00).\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and experimentation were performed by ALS, FSL and MMC. Data analysis was conducted by ALS, BB, JJSG, and JFN. The first draft of the manuscript was written by ALS, edited by BASC reviewed by DSR and PIF. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Ci\u0026ecirc;ncia e Tecnologia de Pernambuco (FACEPE) for financial support. We would also like to thank the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) Brasil - Finance code 001, for partially funding this study. The authors would like to thank the Rede de Ci\u0026ecirc;ncias \u0026Ocirc;micas (RECOM). The article processing fee for the publication of this research was covered by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - CAPES (identifier ROR: 00x0ma614). For open access purposes, the authors have assigned the Creative Commons CC BY license to any accepted version of the article.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAusubel MR, Brent RE, Kingston DD et al (1989) Current protocols in molecular biology. In: John Wiley \u0026amp; Sons (ed) Molecular Reproduction and Development, Wiley, Pennsylvania, pp 146-146\u003c/li\u003e\n\u003cli\u003eAwawdeh L, Forrest R, Turni C et al (2024) Virulence-associated genes in faecal and clinical \u003cem\u003eEscherichia coli\u003c/em\u003e isolates cultured from broiler chickens in Australia. Aust Vet J. https://doi.org/10.1111/avj.13339\u003c/li\u003e\n\u003cli\u003eBarros MR, Silveira WD, Costa MM et al (2025) Prevalence of virulence genes in \u003cem\u003eEscherichia coli\u003c/em\u003e isolates from poultry farms in Northeastern Brazil. Cienc Rural. https://doi.org/10.1590/0103-8478cr20240452\u003c/li\u003e\n\u003cli\u003eBhambure SY, Costa EL, Gatty LIC et al (2024) Antibiotic resistance and virulence traits of Escherichia coli obtained from poultry waste. Braz J Microbiol. https://doi.org/10.1007/s42770-024-01367-1\u003c/li\u003e\n\u003cli\u003eChiPlot (2025) ChiPlot software, version 7.0. https://www.chiplot.online/. Accessed 14 July 2025\u003c/li\u003e\n\u003cli\u003eDawadi P, Bista B, Bista S (2021) Prevalence of colistin-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e from poultry in South Asian developing countries. Vet Med Int. https://doi.org/10.1155/2021/6398838\u003c/li\u003e\n\u003cli\u003eDenamur E, Clermont O, Bonacorsi S et al (2021) The population genetics of pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Nat Rev Microbiol. https://doi.org/10.1038/s41579-020-0416-x\u003c/li\u003e\n\u003cli\u003eForoogh N, Rezvan M, Ahmad K et al (2021) Structural and functional characterization of the fimH adhesin of uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e and its novel applications. Microb Pathog. https://doi.org/10.1016/j.micpath.2021.105288\u003c/li\u003e\n\u003cli\u003eHa EJ, Hong SM, Choi KS et al (2025) Evolution and zoonotic risk of O1:K1 and O2:K1 avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Microbes Infect. https://doi.org/10.1016/j.micinf.2024.105462\u003c/li\u003e\n\u003cli\u003eHasan RN, Jasim SA, Ali YH (2022) Detection of fimH, kpsMTII, hlyA and traT genes in \u003cem\u003eEscherichia coli\u003c/em\u003e isolated from Iraqi patients with cystitis. Gene Rep. https://doi.org/10.1016/j.genrep.2021.101468\u003c/li\u003e\n\u003cli\u003eIkeda M, Kobayashi T, Fujimoto F et al (2021) Prevalence of the iutA and ibeA genes in \u003cem\u003eEscherichia coli\u003c/em\u003e isolates from severe and non-severe patients with bacteremic acute biliary tract infection. Gut Pathog. https://doi.org/10.1186/s13099-021-00429-1\u003c/li\u003e\n\u003cli\u003eJalil A, Masood S, Ain Q et al (2023) High resistance of fluoroquinolone and macrolide reported in avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e isolates from humid subtropical regions of Pakistan. J Glob Antimicrob Resist. https://doi.org/10.1016/j.jgar.2023.01.009\u003c/li\u003e\n\u003cli\u003eCLSI (2023) CLSI M100: performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute, Pennsylvania.\u003c/li\u003e\n\u003cli\u003eJhandai P, Mittal D, Gupta R et al (2025) Emerging avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e serogroups, biofilm formation, ESBLs, integrons and in vivo pathogenicity in chicken. Microb Pathog. https://doi.org/10.1016/j.micpath.2025.107309\u003c/li\u003e\n\u003cli\u003eJohnson TJ, Wannemuehler Y, Doetkott C et al (2008) Identification of minimal predictors of avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e virulence for use as a rapid diagnostic tool. J Clin Microbiol. https://doi.org/10.1128/JCM.00816-08\u003c/li\u003e\n\u003cli\u003eKazimierczak J, Pospiech K, Sowinska P et al (2025) Rapid detection of avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e strains based on minimal virulence markers identified by whole-genome sequencing. BMC Microbiol. https://doi.org/10.1186/s12866-025-03861-4\u003c/li\u003e\n\u003cli\u003eKrumperman, PH (1983) Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. https://doi.org/10.1128/aem.46.1.165-170.1983\u003c/li\u003e\n\u003cli\u003eLiu C, Sun S, Sun Y et al (2024) Antibiotic resistance of \u003cem\u003eEscherichia coli\u003c/em\u003e isolated from food and clinical environments in China from 2001 to 2020. Sci Total Environ. https://doi.org/10.1016/j.scitotenv.2024.173498\u003c/li\u003e\n\u003cli\u003eLu Q, Zhang W, Luo L et al (2022) Genetic diversity and multidrug resistance of phylogenetic groups B2 and D in InPEC and ExPEC isolated from chickens in central China. BMC Microbiol. https://doi.org/10.1186/s12866-022-02469-2\u003c/li\u003e\n\u003cli\u003eMarkey B, Leonard F, Archambault M et al (2013) Clinical veterinary microbiology. Elsevier, Edinburgh.\u003c/li\u003e\n\u003cli\u003eMeng J, Wang W, Din, J et al (2024) Synergistic effect of matrine and berberine hydrochloride in treating colibacillosis caused by multidrug-resistant avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Poult Sci. https://doi.org/10.1016/j.psj.2024.104151\u003c/li\u003e\n\u003cli\u003eRosa DS, Oliveira SAS, Souza R. et al (2024) Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. J Appl Microbiol. https://doi.org/10.1093/jambio/lxae072\u003c/li\u003e\n\u003cli\u003eSaidenberg ABS, Edslev SM, Hallstrom S et al (2024) \u003cem\u003eEscherichia coli\u003c/em\u003e ST117: exploring the zoonotic hypothesis. Microbiol Spectr. https://doi.org/10.1128/spectrum.00466-24\u003c/li\u003e\n\u003cli\u003eSpiga L, Fansler RT, Perera YR et al (2023) Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection. Cell Host Microbe. https://doi.org/10.1016/j.chom.2023.08.018\u003c/li\u003e\n\u003cli\u003eTivendale KA, Allen JL, Ginns CA et al (2004) Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Infect Immun. https://doi.org/10.1128/IAI.72.11.6554-6560.2004\u003c/li\u003e\n\u003cli\u003eWade JT, Reppas NB, Church GM et al (2005) Genomic analysis of LexA binding reveals permissive nature of the \u003cem\u003eEscherichia coli\u003c/em\u003e genome and identifies unconventional target sites. Genes Dev. https://doi.org/10.1101/gad.1355605\u003c/li\u003e\n\u003cli\u003eWang Y, Fu H, Shi X et al (2023) Genome-wide screen reveals cellular functions that counteract rifampicin lethality in \u003cem\u003eEscherichia coli\u003c/em\u003e. Microbiol Spectr. https://doi.org/10.1128/spectrum.02895-23\u003c/li\u003e\n\u003cli\u003eZhang R, Li D, Fang H et al (2025) Iron-dependent mechanisms in Acinetobacter baumannii: pathogenicity and resistance. JAC Antimicrob Resist. https://doi.org/10.1093/jacamr/dlaf039\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"APEC, Colistin resistance, E. coli, MDR, Poultry","lastPublishedDoi":"10.21203/rs.3.rs-8781221/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8781221/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAvian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e is a threat to poultry production systems, as it is associated with infections, poor production ratings, and economic losses. The spread of multidrug-resistant bacterial strains has aggravated the situation. Therefore, this study aimed to characterize 28 \u003cem\u003eE. coli\u003c/em\u003e isolates from poultry farms in Brazil regarding virulence, as well as genetic and phenotypical resistance. Antimicrobial susceptibility test was carried out by the disk diffusion method, while the genomic information of the isolates was determined by whole-genome sequencing. Resistance was identified for all 19 antimicrobials tested, except imipenem. Elevated resistance was observed for rifampicin (96.4%) and oxacillin (67.9%); intermediate resistance for the carbapenems (ertapenem, meropenem, 10.7%), and tetracyclines (doxycycline, 10.7%) was also detected. Genotypic analyses showed that the most common resistance genes were \u003cem\u003etetA\u003c/em\u003e (39.3%) and \u003cem\u003egyrA\u003c/em\u003e (39.3%). Remarkably, the plasmid-mediated gene, \u003cem\u003eMCR-1.1\u003c/em\u003e, associated with colistin resistance, was detected in two isolates. The isolates tested did not carry the typical avian pathogenic \u003cem\u003eE. coli\u003c/em\u003e genetic virulence panel; however, they presented great genetic diversity with prominent pathogenic potential connected to mechanisms of adhesion (\u003cem\u003efimH\u003c/em\u003e, 96.4%), immune evasion (\u003cem\u003eompA\u003c/em\u003e and \u003cem\u003eibeB/C\u003c/em\u003e, 100%; \u003cem\u003etraT\u003c/em\u003e, 92.9%), and iron acquisition (\u003cem\u003efebB\u003c/em\u003e, 100%; \u003cem\u003eiroN\u003c/em\u003e, 60.7%). The overlap between genotypic and phenotypic information emphasizes the health risk imposed by such atypical strains and their dispersion capability amongst both animals and humans. Our findings contribute to a better understanding of the diversity of avian pathogenic \u003cem\u003eE. coli\u003c/em\u003e and reinforce the importance of combined diagnostic and surveillance strategies in the control of such bacteria.\u003c/p\u003e","manuscriptTitle":"Phenotypical and genetic virulence and resistance in atypical avian pathogenic Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 09:20:49","doi":"10.21203/rs.3.rs-8781221/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-04T14:53:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T14:26:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T09:02:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52536396771754325402826177713034261965","date":"2026-02-14T20:28:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T06:04:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216807987000982953798041072326819961498","date":"2026-02-13T05:46:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324268159818597904104595149357320334139","date":"2026-02-11T15:47:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T15:24:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T15:22:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T06:23:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2026-02-04T03:06:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"084b37c4-c784-42af-ba53-b5ad1017183a","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:15:50+00:00","versionOfRecord":{"articleIdentity":"rs-8781221","link":"https://doi.org/10.1007/s10123-026-00811-6","journal":{"identity":"international-microbiology","isVorOnly":false,"title":"International Microbiology"},"publishedOn":"2026-04-02 16:00:01","publishedOnDateReadable":"April 2nd, 2026"},"versionCreatedAt":"2026-02-17 09:20:49","video":"","vorDoi":"10.1007/s10123-026-00811-6","vorDoiUrl":"https://doi.org/10.1007/s10123-026-00811-6","workflowStages":[]},"version":"v1","identity":"rs-8781221","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8781221","identity":"rs-8781221","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}