Multidrug-resistant Escherichia coli isolated from free-range chickens in the Caatinga biome

Multidrug-resistant Escherichia coli isolated from free-range chickens in the Caatinga biome | 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 Short Report Multidrug-resistant Escherichia coli isolated from free-range chickens in the Caatinga biome Luise Canuto Sousa, José Diniz Souto Sobrinho, Bianca Lara Venâncio Godoy, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4360115/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Veterinary Research Communications → Version 1 posted 9 You are reading this latest preprint version Abstract Antimicrobial resistant Escherichia coli is a global health challenge in a One Health perspective. However, data on its emergence in the Caatinga biome are limited. This biome is exclusive to the Brazilian Northeast and offers unique epidemiological conditions that can influence the occurrence of infectious diseases and antimicrobial resistance. In this study, we assessed the carriage proportion, the antimicrobial susceptibility, and the population structure of cephalosporin-resistant E. coli in 300 cloacal swab samples of free-range chickens from three Brazilian states covered by Caatinga biome. The results showed that 44 (14.7%) samples were positive for cephalosporin-resistant E. coli , and Paraíba (PB) state had the highest frequency of isolates (68.2%). Genes encoding CTX-M or AmpC enzymes were identified in 30 (68.2%) and eight isolates (18.2%), respectively, comprising 31 E. coli . Overall, molecular typing by Xba I-PFGE revealed four clusters from two properties of the PB state composed by ESBL- and AmpC-producing E. coli carrying bla CTX−M−1−like and bla MIR−1/ACT−1 genes and belonging to different phylogenetic groups. There is a need for controlling antimicrobial resistance taking into account the genetic diversity of the strains and their implications for animal and public health, especially in free-range chickens reared in the Brazilian Caatinga biome. Antimicrobial resistance One Health Semiarid CTX-M AmpC Surveillance Figures Figure 1 Figure 2 Introduction Critical priority pathogens for which containing efforts must be done were listed by the World Health Organization (WHO) and include extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (Tacconelli et al. 2018 ). These organisms are spread through hospitals, the community, animals, and the environment, becoming a global health challenge in a One Health perspective due to their ability to colonize and infect different hosts through direct contact or food/water ingestion (Amos et al. 2018 ). The close relation between humans and poultry can lead to the transmission of zoonosis, and the excessive use of antibiotics in poultry farming contributes to the selection of resistant bacterial strains, which represents a growing threat to public health due to the reduced effectiveness of conventional treatments (Ramos et al. 2020 ). Several bacteria, such as Escherichia coli , colonize the gastrointestinal tract of humans and animals shortly after birth. Although they are not normally pathogenic to their hosts, the emergence of virulent strains has been a growing public health concern (Wang et al. 2024 ). Virulent and antimicrobial-resistant E. coli isolated from poultry and poultry meat are potential sources of contamination for humans (Casella et al. 2018 ). β-lactams are antimicrobials of a broad family that include cephalosporins, and resistance to third- and fourth-generation cephalosporins in E. coli is mainly due to production of ESBL and AmpC β-lactamases. ESBLs have been reported as one of the main resistance mechanisms in Enterobacterales , and ESBL/AmpC-producing E. coli have been increasingly detected, mainly from humans and healthy animals, especially poultry (Oliveira et al. 2024 ). Low rainfall and high temperatures characterize the semiarid region of Brazil and, when associated with the peculiarities of the existing vegetation, the Caatinga, a biome exclusive to the Brazilian Northeast with abundant wildlife, offers unique epidemiological conditions that can influence the occurrence of infectious diseases and antimicrobial resistance. We conducted a survey aimed to assess the carriage proportion, the antimicrobial susceptibility, and the population structure of cephalosporin-resistant E. coli in free-range chickens ( Gallus gallus domesticus ) in the Caatinga biome. Materials and Methods Study area and biological sample collection Between August 2021 and April 2022, 300 cloacal swab samples from free-range chickens were collected in urban and rural areas of three states (100 samples per state) in the Brazilian Northeast - Rio Grande do Norte (RN), Paraíba (PB) and Pernambuco (PE), covered by the Caatinga biome (Fig. 1 ), being six properties in RN, eight in PB and six in PE, with 15 animals sampled per property. The cloacal swabs were transported in Stuart media under refrigeration to the laboratory for microbiological analyses. Bacteriological culture and species identification Swab samples were placed onto MacConkey agar (KASVI, São José dos Pinhais, Paraná, Brazil) plates supplemented with 2 µg/mL cefotaxime and incubated at 37°C for up to 48 hours to check for bacterial growth (Mo et al. 2014 ). All E. coli colony morphology were selected and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Microflex LT MALDI-TOF MS, Bruker Daltonics, Bremen, Germany). E. coli phenotypic and genotypic antimicrobial resistance profiles The E. coli isolates were submitted to the antimicrobial susceptibility test using the Kirby-Bauer disk diffusion method as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2022), and categorized as multidrug-resistant (Magiorakos et al. 2012 ). The Escherichia coli ATCC 25922 standard strain was used as the quality control. Total DNA was extracted by boiling and screened for the presence of the most prevalent ESBL and AmpC genes families according to previous published protocols (Pérez-Pérez and Hanson 2002 ; Dallenne et al. 2010 ). Moreover, the ESBL/AmpC positive E. coli isolates were typed according to the phylogenetic group (Clermont et al. 2013 ) and by genome restriction using Xba I endonuclease followed by pulsed-field gel electrophoresis ( Xba I-PFGE) using the PulseNet protocol ( https://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c.pdf ). Genetic profiles were analyzed in BioNumerics v7.6 (Applied Maths-bioMérieux) using the Dice’s similarity coefficient and the UPGMA method for dendrogram construction and clustering. Isolates presenting ≥ 90% of genetic similarity were considered belonging to the same cluster. Results E. coli phenotypic antimicrobial resistance Of the 300 samples analyzed, 44 (14.7%) were positive for cephalosporin-resistant E. coli . The PB state had the highest frequency of isolates (30, 68.2%), followed by RN (eight, 18.2%) and PE (six, 13.6%). The antimicrobial resistance was most frequent for ceftriaxone (100%), ceftiofur (97%), cefepime (97%), aztreonam (91.4%), ampicillin (88.5%), ertapenem (85.7%) and tetracycline (62.8%). Thirty-five out of the 44 isolates (79.5%) were categorized as multidrug resistant (MDR). E. coli genotypic antimicrobial resistance Genes encoding CTX-M or AmpC enzymes were identified in 30 (68.2%) and eight isolates (18.2%), respectively, totaling 31 E. coli isolates. In 13 was not possible to identify the gene responsible for the cephalosporin-resistance. bla CTX−M−1−like genes were the most frequent, detected in 27 (84.4%) out of the 31 isolates, followed by bla MIR−1/ACT−1 (eight isolates; 25%) and bla CTX−M−2−like (three isolates; 9.4%); seven E. coli presented both bla CTX−M−1−like and bla MIR−1/ACT−1 . Among the 30 isolates from PB, 19 (63.3%) presented bla CTX−M−1−like genes, four (66.7%) out of six E. coli from PE carried such genes, and four (50%) out of eight isolates recovered in RN harbored bla CTX−M−1−like genes. bla CTX−M−2−like genes were detected in 6.7% (2/30 isolates) of the PB strains and 16.7% (1/6 isolates) of the PE strains, however, it was not identified in RN strains. bla MIR−1/ACT−1 genes were detected in the RN (12.5%; 1/8 isolates) and PB (23.3%; 7/30 isolates) states. E. coli phylogenetic groups and clusters Regarding the E. coli phylogenetic groups distribution among the 31 ESBL/AmpC positive isolates, it was found heterogeneity among the three states, with predominance of phylogroup A (12/31, 38.7%) followed by phylogroups D (9/31, 29%), F (5/31, 16.1%), E (2/31, 6.5%) and C (2/31, 6.5%); one (3.2%) isolate belonged to unknown phylogroup (Fig. 2 ). The molecular typing by Xba I-PFGE also revealed a general dissimilarity among the 31 isolates, with only eight grouping in four clusters (I, II, III and IV), all recovered from two properties of the PB state (Fig. 2 ). Cluster I was composed by two ESBL-producing E. coli isolates (XX08 and XX15) belonging to phylogroup D and carrying a bla CTX−M−1−like gene. Cluster II grouped two E. coli (XX05 and XX09) with distinct content of resistance genes: XX05 harbored both bla CTX−M−1−like and bla MIR−1/ACT−1 genes, while XX09 carried only the ESBL gene. The same occurred in cluster IV, where the isolate XX01 presented an ESBL and an AmpC gene, and the isolate XX14, only a bla CTX−M−1−like . Finally, cluster III comprised two isolates (SM02 and SM04) carrying bla CTX−M−1−like genes but belonging to different phylogenetic groups (A and D, respectively). Discussion In this survey, a relatively low frequency (14.7%) of chickens analyzed was positive for cephalosporin-resistant E. coli . This rate is variable in reports from similar sized chicken farms, from 11% in Thailand (Sudatip et al. 2023 ) to 30% in Brazil (Casella et al. 2018 ), which must represent regional variation. However, 70.5% of isolates were attested as ESBL/AmpC positive, which raises concerns since this bacterium is one of the main problems in Brazilian poultry farming, and a great meaning for public health since it is the main cause of community-acquired infections, especially urinary tract infections (García-Meniño et al. 2024 ). The antimicrobial resistance found in E. coli worldwide has had a major impact on human and animal populations regarding food safety and economy (Babines-Orozco et al. 2024 ). In poultry, the detection of MDR E. coli was reported in several countries, not only in poultry but also in its meat (Casella et al. 2018 ). In the context of the Caatinga biome, where free-range chickens are reared in poor hygiene conditions and without sanitary control, the occurrence of MDR E. coli warns of its spread in the animal-environment-human interface. The predominance of bla CTX−M−1−like genes was observed. The high prevalence of these genes in free-range chickens corroborates the findings of studies with poultry (Nahar et al. 2018 ; Baez et al. 2021 ). It is worth highlighting the report of a high prevalence of bla CTX−M in ESBL-producing E. coli in 91% of domestic poultry samples from Japan and in 100% of samples imported from Brazil (Nahar et al. 2018 ), which suggests the need for ongoing surveillance to monitor the prevalence and distribution of these genes in different geographic regions. The characterization of pathogenic strains is fundamental for epidemiological purposes. In the present study, similar isolates were identified in animals from the same farms in the Caatinga biome. These results show the potential for bacteria to spread in the environment and transmit AMR, which was already reported (Casella et al. 2018 ) and suggests possible localized dissemination or common sources of contamination. Dissimilarity between strains from different properties may indicate different sources of contamination or genetic variability of E. coli strains in the region, showing that disease outbreaks are often linked to more than one strain (Barbieri et al. 2021 ). Moreover, the presence of distinct E. coli strains carrying ESBL/AmpC genes in free-range chickens suggests the successful spread of plasmids through this hostile environment too. However, further studies must be conducted with these isolates and others collected from the entire environment in order to respond this question. The identification of E. coli strains belonging to phylogroup F is a relevant finding, considering the pathogenic and zoonotic implications of strains of this phylogroup (Mohammed et al. 2024 ). It has been shown that strains belonging to this phylogroup of avian origin share virulence genotypes and are closely related to extraintestinal pathogenic strains that cause infections in humans (Zhuge et al. 2020 ). In the Caatinga biome, free-range chickens are an important source of food and income for small farmers and local sales. However, the animals are usually reared in poor sanitary conditions and in contact with the environment and other animals, as well as made use of empirically medication. Hence, in view of the reported here, for controlling antimicrobial resistance in free-range chickens, an integrated approach is essential, such as the One Health guidelines (Ramos et al. 2020 ). This approach must take into account the genetic diversity of the strains and their implications for animal and public health, especially in free-range chickens reared in the Brazilian Caatinga biome, where the responsible authorities must act in favor of a population living in precarious situation. Declarations Author contributions Débora Luise Canuto de Sousa performed the sample collection, did the laboratory examination and wrote the manuscript; José Diniz de Souto Sobrinho, Bianca Lara Venâncio de Godoy, Domingos Andrade Neto and Giliel Rodrigues Leandro performed the sample collection and did the laboratory examination Investigation; Tiago Casella conceived the survey and corrected the manuscript; Sérgio Santos de Azevedo conceived the survey, did the project management and corrected the manuscript; Carolina de Sousa Américo Batista Santos conceived the survey, did the project management, corrected the manuscript and provided the project funding acquisition. All authors reviewed the manuscript. Funding This study was funded by the Research Support Foundation of the State of Paraíba (FAPESQ), grant number 47340.673.29278.09082021. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests All authors have read and approved the final manuscript. Its contents are solely the responsibility of the authors. All authors declare that they have no competing interests. Ethical Approval This investigation received the approval (number 02/2021) of the ethical committee of the Federal University of Campina Grande (UFCG), Brazil. The data were analyzed anonymously. Consent to participate Not applicable. Consent to publish Not applicable. References Amos GCA, Ploumakis C, Zhang L, Hawkey PM, Gaze WH, Wellington EMH (2018) The widespread dissemination of integrons throughout bacterial communities in a riverine system. ISME J 12:681-691 Babines-Orozco L, Balbuena-Alonso MG, Barrios-Villa E, Lozano-Zarain P, Martínez-Laguna Y, Rocha-Gracia RDC, Cortés-Cortés G (2024) Antimicrobial resistance in food-associated Escherichia coli in Mexico and Latin America. Biosci Microbiota Food Health 43:4-12 Baez M, Espinosa I, Collaud A, Miranda I, Montano DLN, Feria AL, Hernández-Fillor RE, Obregón D, Alfonso P, Perreten V (2021) Genetic features of extended-spectrum β-lactamase-producing Escherichia coli from poultry in Mayabeque province, Cuba. Antibiotics 10:107 Barbieri NL, Pimenta RL, Melo DA, Nolan LK, Souza MMS, Logue CM (2021) mcr -1 identified in fecal Escherichia coli and avian pathogenic E. coli (APEC) from Brazil. Front Microbiol 12:659613. Casella T, Haenni M, Madela NK, Andrade LK, Pradela LK, Andrade LN, Darini ALC, Madec J, Nogueira MCL (2018) Extended-spectrum cephalosporin-resistant Escherichia coli isolated from chickens and chicken meat in Brazil is associated with rare and complex resistance plasmids and pandemic ST lineages. J Antimicrob Chemother 73:3293-3297 Clermont O, Christenson JK, Denamur E, Gordon DM (2013) The Clermont Escherichia coli phylo‐typing method revisited: improvement of specificity and detection of new phylo‐groups. Environ Microbiol Rep 5:58-65 CLSI - Clinical and Laboratory Standards Institute (2022) Performance Standards for Antimicrobial Susceptibility Testing, M100, 32nd ed. Wayne, PA. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G (2010) Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae . J Antimicrob Chemother 65:490-495 García-Meniño I, García V, Lumbreras-Iglesias P, Fernández J, Mora A (2024) Fluoroquinolone resistance in complicated urinary tract infections: association with the increased occurrence and diversity of Escherichia coli of clonal complex 131, together with ST1193. Front Cell Infect Microbiol 14:1351618 Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268-281 Mohammed HN, Al Marjani MF, Authman SH (2024) Molecular identification and characterization and phylogenetic study in Escherichia coli in Baghdad province. Rev Res Med Microbiol. Advance online publication. https://doi.org/10.1097/MRM.0000000000000396 Mo SS, Norström M, Slettemeas JS, Løvland A, Urdahl AM, Sunde M (2014) Emergence of AmpC-producing Escherichia coli in the broiler production chain in a country with a low antimicrobial usage profile. Vet Microbiol 171:315-320 Nahar A, Awasthi SP, Hatanaka N, Okuno K, Hoang PH, Hassan J, Hinenoya A, Yamasaki S (2018) Prevalence and characteristics of extended-spectrum β-lactamase-producing Escherichia coli in domestic and imported chicken meats in Japan. J Vet Med Sci 80:510-517 Oliveira A, Dias C, Oliveira R, Almeida C, Fuciños P, Sillankorva S, Oliveira H (2024) Paving the way forward: Escherichia coli bacteriophages in a One Health approach. Crit Rev Microbiol 50 :87-104 Pérez-Pérez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153-2162 Ramos S, Silva V, Dapkevicius MDLE, Caniça M, Tejedor-Junco MT, Igrejas G, Poeta P (2020) Escherichia coli as commensal and pathogenic bacteria among food-producing animals: health implications of extended spectrum β-lactamase (ESBL) production. Animals 10:2239 Sudatip D, Mostacci N, Tiengrim S, Thamlikitkul V, Chasiri K, Kritiyakan A, Phanprasit W, Thinphovong C, Abdallah R, Baron SA, Rolain JM, Morand S, Oppliger A, Hilty M (2023) The risk of pig and chicken farming for carriage and transmission of Escherichia coli containing extended-spectrum beta-lactamase (ESBL) and mobile colistin resistance ( mcr ) genes in Thailand. Microb Genom 9:mgen000951 Tacconelli E, Carraca E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N, WHO Pathogens Priority List Working Group (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18:318-327 Wang Z, Jiang Z, Zhang Y, Wang C, Liu Z, Jia Z, Bhushan S, Yang J, Zhang Z (2024) Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function. PLoS Pathog 20:e1011926 Zhuge X, Zhou Z, Jiang M, Wang Z, Sun Y, Tang F, Xue F, Ren J, Dai J (2020) Chicken‐source Escherichia coli within phylogroup F shares virulence genotypes and is closely related to extraintestinal pathogenic E. coli causing human infections. Transbound Emerg Dis 68:880-895 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Veterinary Research Communications → Version 1 posted Editorial decision: Revision requested 19 May, 2024 Reviews received at journal 19 May, 2024 Reviewers agreed at journal 09 May, 2024 Reviews received at journal 06 May, 2024 Reviewers agreed at journal 03 May, 2024 Reviewers invited by journal 03 May, 2024 Editor assigned by journal 03 May, 2024 Submission checks completed at journal 03 May, 2024 First submitted to journal 02 May, 2024 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. 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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-4360115","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":300378120,"identity":"113867ce-2e9c-4a13-9878-7616639473a4","order_by":0,"name":"Luise Canuto Sousa","email":"","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":false,"prefix":"","firstName":"Luise","middleName":"Canuto","lastName":"Sousa","suffix":""},{"id":300378122,"identity":"31021fe5-0e09-4db7-a567-4abb468f995c","order_by":1,"name":"José Diniz Souto Sobrinho","email":"","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Diniz Souto","lastName":"Sobrinho","suffix":""},{"id":300378124,"identity":"67f78abb-5b15-406f-b776-ed7f55c1b5de","order_by":2,"name":"Bianca Lara Venâncio Godoy","email":"","orcid":"","institution":"Faculdade de Medicina de São José do Rio Preto","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"Lara Venâncio","lastName":"Godoy","suffix":""},{"id":300378126,"identity":"7c7fc77f-2ead-494f-8d9f-c928bc624ceb","order_by":3,"name":"Domingos Andrade Neto","email":"","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":false,"prefix":"","firstName":"Domingos","middleName":"Andrade","lastName":"Neto","suffix":""},{"id":300378128,"identity":"bbe22fa5-8fc6-4e73-8ccd-ef87dec9ce92","order_by":4,"name":"Giliel Rodrigues Leandro","email":"","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":false,"prefix":"","firstName":"Giliel","middleName":"Rodrigues","lastName":"Leandro","suffix":""},{"id":300378130,"identity":"cf318e99-125a-43e6-aac4-09bcbc6ff999","order_by":5,"name":"Tiago Casella","email":"","orcid":"","institution":"Faculdade de Medicina de São José do Rio Preto","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"","lastName":"Casella","suffix":""},{"id":300378134,"identity":"afb62eec-e170-4224-8f44-b57dcb824af0","order_by":6,"name":"Sérgio Santos Azevedo","email":"","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":false,"prefix":"","firstName":"Sérgio","middleName":"Santos","lastName":"Azevedo","suffix":""},{"id":300378140,"identity":"db16866f-ca2d-4ea7-a7db-60004138fc89","order_by":7,"name":"Carolina Sousa","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Campina Grande","correspondingAuthor":true,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Sousa","suffix":""}],"badges":[],"createdAt":"2024-05-02 15:52:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4360115/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4360115/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11259-024-10504-y","type":"published","date":"2024-08-19T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56154965,"identity":"f45aa77d-ce7b-4b95-9258-4c1ba4e6d971","added_by":"auto","created_at":"2024-05-09 07:59:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":451996,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Brazil highlighting the states and counties in which the biological samples were collected from free-range chickens.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4360115/v1/6bcaa7df6076a81018fabbf7.png"},{"id":56155493,"identity":"04cc6b77-5974-47f2-a1ec-f15f459a12a5","added_by":"auto","created_at":"2024-05-09 08:07:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153501,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram constructed based on the \u003cem\u003eXba\u003c/em\u003eI-PFGE typing method. AST, antimicrobial susceptibility profile. AMP, ampicillin; AMC, amoxicillin/clavulanic acid; CFO, cefoxitin; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; CTF, ceftiofur; CPM, cefepime; ATM, aztreonam; ETP, ertapenem; MER, meropenem; AMI, amikacin; GEN, gentamycin; NOR, norfloxacin; CLO, chloramphenicol; TET, tetracycline. The Brazilian states are represented as follows: PB, Paraíba; PE, Pernambuco; RN, Rio Grande do Norte.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4360115/v1/f3b993ccbcb4b3365f08df08.png"},{"id":63300048,"identity":"306a8501-a65a-4b7c-ab25-ae3aa9313c08","added_by":"auto","created_at":"2024-08-26 16:10:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":994623,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4360115/v1/6afc1321-da1d-4e04-b944-7b59393976a8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multidrug-resistant Escherichia coli isolated from free-range chickens in the Caatinga biome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCritical priority pathogens for which containing efforts must be done were listed by the World Health Organization (WHO) and include extended-spectrum β-lactamase (ESBL)-producing \u003cem\u003eEnterobacterales\u003c/em\u003e (Tacconelli et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These organisms are spread through hospitals, the community, animals, and the environment, becoming a global health challenge in a One Health perspective due to their ability to colonize and infect different hosts through direct contact or food/water ingestion (Amos et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The close relation between humans and poultry can lead to the transmission of zoonosis, and the excessive use of antibiotics in poultry farming contributes to the selection of resistant bacterial strains, which represents a growing threat to public health due to the reduced effectiveness of conventional treatments (Ramos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral bacteria, such as \u003cem\u003eEscherichia coli\u003c/em\u003e, colonize the gastrointestinal tract of humans and animals shortly after birth. Although they are not normally pathogenic to their hosts, the emergence of virulent strains has been a growing public health concern (Wang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Virulent and antimicrobial-resistant \u003cem\u003eE. coli\u003c/em\u003e isolated from poultry and poultry meat are potential sources of contamination for humans (Casella et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eβ-lactams are antimicrobials of a broad family that include cephalosporins, and resistance to third- and fourth-generation cephalosporins in \u003cem\u003eE. coli\u003c/em\u003e is mainly due to production of ESBL and AmpC β-lactamases. ESBLs have been reported as one of the main resistance mechanisms in \u003cem\u003eEnterobacterales\u003c/em\u003e, and ESBL/AmpC-producing \u003cem\u003eE. coli\u003c/em\u003e have been increasingly detected, mainly from humans and healthy animals, especially poultry (Oliveira et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLow rainfall and high temperatures characterize the semiarid region of Brazil and, when associated with the peculiarities of the existing vegetation, the Caatinga, a biome exclusive to the Brazilian Northeast with abundant wildlife, offers unique epidemiological conditions that can influence the occurrence of infectious diseases and antimicrobial resistance. We conducted a survey aimed to assess the carriage proportion, the antimicrobial susceptibility, and the population structure of cephalosporin-resistant \u003cem\u003eE. coli\u003c/em\u003e in free-range chickens (\u003cem\u003eGallus gallus domesticus\u003c/em\u003e) in the Caatinga biome.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and biological sample collection\u003c/h2\u003e \u003cp\u003eBetween August 2021 and April 2022, 300 cloacal swab samples from free-range chickens were collected in urban and rural areas of three states (100 samples per state) in the Brazilian Northeast - Rio Grande do Norte (RN), Para\u0026iacute;ba (PB) and Pernambuco (PE), covered by the Caatinga biome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), being six properties in RN, eight in PB and six in PE, with 15 animals sampled per property. The cloacal swabs were transported in Stuart media under refrigeration to the laboratory for microbiological analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBacteriological culture and species identification\u003c/h2\u003e \u003cp\u003eSwab samples were placed onto MacConkey agar (KASVI, S\u0026atilde;o Jos\u0026eacute; dos Pinhais, Paran\u0026aacute;, Brazil) plates supplemented with 2 \u0026micro;g/mL cefotaxime and incubated at 37\u0026deg;C for up to 48 hours to check for bacterial growth (Mo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All \u003cem\u003eE. coli\u003c/em\u003e colony morphology were selected and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Microflex LT MALDI-TOF MS, Bruker Daltonics, Bremen, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ephenotypic and genotypic antimicrobial resistance profiles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e isolates were submitted to the antimicrobial susceptibility test using the Kirby-Bauer disk diffusion method as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2022), and categorized as multidrug-resistant (Magiorakos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 standard strain was used as the quality control.\u003c/p\u003e \u003cp\u003eTotal DNA was extracted by boiling and screened for the presence of the most prevalent ESBL and AmpC genes families according to previous published protocols (P\u0026eacute;rez-P\u0026eacute;rez and Hanson \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Dallenne et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, the ESBL/AmpC positive \u003cem\u003eE. coli\u003c/em\u003e isolates were typed according to the phylogenetic group (Clermont et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and by genome restriction using \u003cem\u003eXba\u003c/em\u003eI endonuclease followed by pulsed-field gel electrophoresis (\u003cem\u003eXba\u003c/em\u003eI-PFGE) using the PulseNet protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c.pdf\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Genetic profiles were analyzed in BioNumerics v7.6 (Applied Maths-bioM\u0026eacute;rieux) using the Dice\u0026rsquo;s similarity coefficient and the UPGMA method for dendrogram construction and clustering. Isolates presenting\u0026thinsp;\u0026ge;\u0026thinsp;90% of genetic similarity were considered belonging to the same cluster.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ephenotypic antimicrobial resistance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOf the 300 samples analyzed, 44 (14.7%) were positive for cephalosporin-resistant \u003cem\u003eE. coli\u003c/em\u003e. The PB state had the highest frequency of isolates (30, 68.2%), followed by RN (eight, 18.2%) and PE (six, 13.6%). The antimicrobial resistance was most frequent for ceftriaxone (100%), ceftiofur (97%), cefepime (97%), aztreonam (91.4%), ampicillin (88.5%), ertapenem (85.7%) and tetracycline (62.8%). Thirty-five out of the 44 isolates (79.5%) were categorized as multidrug resistant (MDR).\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003egenotypic antimicrobial resistance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenes encoding CTX-M or AmpC enzymes were identified in 30 (68.2%) and eight isolates (18.2%), respectively, totaling 31 \u003cem\u003eE. coli\u003c/em\u003e isolates. In 13 was not possible to identify the gene responsible for the cephalosporin-resistance. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e genes were the most frequent, detected in 27 (84.4%) out of the 31 isolates, followed by \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;1/ACT\u0026minus;1\u003c/sub\u003e (eight isolates; 25%) and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;2\u0026minus;like\u003c/sub\u003e (three isolates; 9.4%); seven \u003cem\u003eE. coli\u003c/em\u003e presented both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;1/ACT\u0026minus;1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eAmong the 30 isolates from PB, 19 (63.3%) presented \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e genes, four (66.7%) out of six \u003cem\u003eE. coli\u003c/em\u003e from PE carried such genes, and four (50%) out of eight isolates recovered in RN harbored \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e genes. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;2\u0026minus;like\u003c/sub\u003e genes were detected in 6.7% (2/30 isolates) of the PB strains and 16.7% (1/6 isolates) of the PE strains, however, it was not identified in RN strains. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;1/ACT\u0026minus;1\u003c/sub\u003e genes were detected in the RN (12.5%; 1/8 isolates) and PB (23.3%; 7/30 isolates) states.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ephylogenetic groups and clusters\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRegarding the \u003cem\u003eE. coli\u003c/em\u003e phylogenetic groups distribution among the 31 ESBL/AmpC positive isolates, it was found heterogeneity among the three states, with predominance of phylogroup A (12/31, 38.7%) followed by phylogroups D (9/31, 29%), F (5/31, 16.1%), E (2/31, 6.5%) and C (2/31, 6.5%); one (3.2%) isolate belonged to unknown phylogroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe molecular typing by \u003cem\u003eXba\u003c/em\u003eI-PFGE also revealed a general dissimilarity among the 31 isolates, with only eight grouping in four clusters (I, II, III and IV), all recovered from two properties of the PB state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cluster I was composed by two ESBL-producing \u003cem\u003eE. coli\u003c/em\u003e isolates (XX08 and XX15) belonging to phylogroup D and carrying a \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e gene. Cluster II grouped two \u003cem\u003eE. coli\u003c/em\u003e (XX05 and XX09) with distinct content of resistance genes: XX05 harbored both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;1/ACT\u0026minus;1\u003c/sub\u003e genes, while XX09 carried only the ESBL gene. The same occurred in cluster IV, where the isolate XX01 presented an ESBL and an AmpC gene, and the isolate XX14, only a \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e. Finally, cluster III comprised two isolates (SM02 and SM04) carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e genes but belonging to different phylogenetic groups (A and D, respectively).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this survey, a relatively low frequency (14.7%) of chickens analyzed was positive for cephalosporin-resistant \u003cem\u003eE. coli\u003c/em\u003e. This rate is variable in reports from similar sized chicken farms, from 11% in Thailand (Sudatip et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) to 30% in Brazil (Casella et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which must represent regional variation. However, 70.5% of isolates were attested as ESBL/AmpC positive, which raises concerns since this bacterium is one of the main problems in Brazilian poultry farming, and a great meaning for public health since it is the main cause of community-acquired infections, especially urinary tract infections (Garc\u0026iacute;a-Meni\u0026ntilde;o et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The antimicrobial resistance found in \u003cem\u003eE. coli\u003c/em\u003e worldwide has had a major impact on human and animal populations regarding food safety and economy (Babines-Orozco et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In poultry, the detection of MDR \u003cem\u003eE. coli\u003c/em\u003e was reported in several countries, not only in poultry but also in its meat (Casella et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the context of the Caatinga biome, where free-range chickens are reared in poor hygiene conditions and without sanitary control, the occurrence of MDR \u003cem\u003eE. coli\u003c/em\u003e warns of its spread in the animal-environment-human interface.\u003c/p\u003e \u003cp\u003eThe predominance of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e genes was observed. The high prevalence of these genes in free-range chickens corroborates the findings of studies with poultry (Nahar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Baez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is worth highlighting the report of a high prevalence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e in ESBL-producing \u003cem\u003eE. coli\u003c/em\u003e in 91% of domestic poultry samples from Japan and in 100% of samples imported from Brazil (Nahar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which suggests the need for ongoing surveillance to monitor the prevalence and distribution of these genes in different geographic regions.\u003c/p\u003e \u003cp\u003eThe characterization of pathogenic strains is fundamental for epidemiological purposes. In the present study, similar isolates were identified in animals from the same farms in the Caatinga biome. These results show the potential for bacteria to spread in the environment and transmit AMR, which was already reported (Casella et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and suggests possible localized dissemination or common sources of contamination. Dissimilarity between strains from different properties may indicate different sources of contamination or genetic variability of \u003cem\u003eE. coli\u003c/em\u003e strains in the region, showing that disease outbreaks are often linked to more than one strain (Barbieri et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, the presence of distinct \u003cem\u003eE. coli\u003c/em\u003e strains carrying ESBL/AmpC genes in free-range chickens suggests the successful spread of plasmids through this hostile environment too. However, further studies must be conducted with these isolates and others collected from the entire environment in order to respond this question.\u003c/p\u003e \u003cp\u003eThe identification of \u003cem\u003eE. coli\u003c/em\u003e strains belonging to phylogroup F is a relevant finding, considering the pathogenic and zoonotic implications of strains of this phylogroup (Mohammed et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It has been shown that strains belonging to this phylogroup of avian origin share virulence genotypes and are closely related to extraintestinal pathogenic strains that cause infections in humans (Zhuge et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the Caatinga biome, free-range chickens are an important source of food and income for small farmers and local sales. However, the animals are usually reared in poor sanitary conditions and in contact with the environment and other animals, as well as made use of empirically medication. Hence, in view of the reported here, for controlling antimicrobial resistance in free-range chickens, an integrated approach is essential, such as the One Health guidelines (Ramos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This approach must take into account the genetic diversity of the strains and their implications for animal and public health, especially in free-range chickens reared in the Brazilian Caatinga biome, where the responsible authorities must act in favor of a population living in precarious situation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eD\u0026eacute;bora Luise Canuto de Sousa performed the sample collection, did the laboratory examination and wrote the manuscript; Jos\u0026eacute; Diniz de Souto Sobrinho, Bianca Lara Ven\u0026acirc;ncio de Godoy, Domingos Andrade Neto and Giliel Rodrigues Leandro performed the sample collection and did the laboratory examination Investigation; Tiago Casella conceived the survey and corrected the manuscript; S\u0026eacute;rgio Santos de Azevedo conceived the survey, did the project management and corrected the manuscript; Carolina de Sousa Am\u0026eacute;rico Batista Santos conceived the survey, did the project management, corrected the manuscript and provided the project funding acquisition. All authors reviewed the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was funded by the Research Support Foundation of the State of Para\u0026iacute;ba (FAPESQ), grant number 47340.673.29278.09082021.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e All authors have read and approved the final manuscript. Its contents are solely the responsibility of the authors. All authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eThis investigation received the approval (number 02/2021) of the ethical committee of the Federal University of Campina Grande (UFCG), Brazil. The data were analyzed anonymously.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to publish\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmos GCA, Ploumakis C, Zhang L, Hawkey PM, Gaze WH, Wellington EMH (2018) The widespread dissemination of integrons throughout bacterial communities in a riverine system. ISME J 12:681-691 \u003c/li\u003e\n\u003cli\u003eBabines-Orozco L, Balbuena-Alonso MG, Barrios-Villa E, Lozano-Zarain P, Mart\u0026iacute;nez-Laguna Y, Rocha-Gracia RDC, Cort\u0026eacute;s-Cort\u0026eacute;s G (2024) Antimicrobial resistance in food-associated \u003cem\u003eEscherichia coli\u003c/em\u003e in Mexico and Latin America. Biosci Microbiota Food Health 43:4-12 \u003c/li\u003e\n\u003cli\u003eBaez M, Espinosa I, Collaud A, Miranda I, Montano DLN, Feria AL, Hern\u0026aacute;ndez-Fillor RE, Obreg\u0026oacute;n D, Alfonso P, Perreten V (2021) Genetic features of extended-spectrum \u0026beta;-lactamase-producing \u003cem\u003eEscherichia coli\u003c/em\u003e from poultry in Mayabeque province, Cuba. Antibiotics 10:107 \u003c/li\u003e\n\u003cli\u003eBarbieri NL, Pimenta RL, Melo DA, Nolan LK, Souza MMS, Logue CM (2021) \u003cem\u003emcr\u003c/em\u003e-1 identified in fecal \u003cem\u003eEscherichia coli\u003c/em\u003e and avian pathogenic \u003cem\u003eE. coli\u003c/em\u003e (APEC) from Brazil. Front Microbiol 12:659613.\u003c/li\u003e\n\u003cli\u003eCasella T, Haenni M, Madela NK, Andrade LK, Pradela LK, Andrade LN, Darini ALC, Madec J, Nogueira MCL (2018) Extended-spectrum cephalosporin-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e isolated from chickens and chicken meat in Brazil is associated with rare and complex resistance plasmids and pandemic ST lineages. J Antimicrob Chemother 73:3293-3297\u003c/li\u003e\n\u003cli\u003eClermont O, Christenson JK, Denamur E, Gordon DM (2013) The Clermont \u003cem\u003eEscherichia coli\u003c/em\u003e phylo‐typing method revisited: improvement of specificity and detection of new phylo‐groups. Environ Microbiol Rep 5:58-65\u003c/li\u003e\n\u003cli\u003eCLSI - Clinical and Laboratory Standards Institute (2022) Performance Standards for Antimicrobial Susceptibility Testing, M100, 32nd ed. Wayne, PA.\u003c/li\u003e\n\u003cli\u003eDallenne C, Da Costa A, Decr\u0026eacute; D, Favier C, Arlet G (2010) Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. J Antimicrob Chemother 65:490-495\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Meni\u0026ntilde;o I, Garc\u0026iacute;a V, Lumbreras-Iglesias P, Fern\u0026aacute;ndez J, Mora A (2024) Fluoroquinolone resistance in complicated urinary tract infections: association with the increased occurrence and diversity of \u003cem\u003eEscherichia coli\u003c/em\u003e of clonal complex 131, together with ST1193. Front Cell Infect Microbiol 14:1351618\u003c/li\u003e\n\u003cli\u003eMagiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268-281\u003c/li\u003e\n\u003cli\u003eMohammed HN, Al Marjani MF, Authman SH (2024) Molecular identification and characterization and phylogenetic study in \u003cem\u003eEscherichia coli\u003c/em\u003e in Baghdad province. Rev Res Med Microbiol. Advance online publication. https://doi.org/10.1097/MRM.0000000000000396 \u003c/li\u003e\n\u003cli\u003eMo SS, Norstr\u0026ouml;m M, Slettemeas JS, L\u0026oslash;vland A, Urdahl AM, Sunde M (2014) Emergence of AmpC-producing \u003cem\u003eEscherichia coli\u003c/em\u003e in the broiler production chain in a country with a low antimicrobial usage profile. Vet Microbiol 171:315-320 \u003c/li\u003e\n\u003cli\u003eNahar A, Awasthi SP, Hatanaka N, Okuno K, Hoang PH, Hassan J, Hinenoya A, Yamasaki S (2018) Prevalence and characteristics of extended-spectrum \u0026beta;-lactamase-producing \u003cem\u003eEscherichia coli\u003c/em\u003e in domestic and imported chicken meats in Japan. J Vet Med Sci 80:510-517\u003c/li\u003e\n\u003cli\u003eOliveira A, Dias C, Oliveira R, Almeida C, Fuci\u0026ntilde;os P, Sillankorva S, Oliveira H (2024) Paving the way forward: \u003cem\u003eEscherichia coli\u003c/em\u003e bacteriophages in a One Health approach. Crit Rev Microbiol 50 :87-104\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-P\u0026eacute;rez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153-2162\u003c/li\u003e\n\u003cli\u003eRamos S, Silva V, Dapkevicius MDLE, Cani\u0026ccedil;a M, Tejedor-Junco MT, Igrejas G, Poeta P (2020) \u003cem\u003eEscherichia coli\u003c/em\u003e as commensal and pathogenic bacteria among food-producing animals: health implications of extended spectrum \u0026beta;-lactamase (ESBL) production. Animals 10:2239 \u003c/li\u003e\n\u003cli\u003eSudatip D, Mostacci N, Tiengrim S, Thamlikitkul V, Chasiri K, Kritiyakan A, Phanprasit W, Thinphovong C, Abdallah R, Baron SA, Rolain JM, Morand S, Oppliger A, Hilty M (2023) The risk of pig and chicken farming for carriage and transmission of \u003cem\u003eEscherichia coli\u003c/em\u003e containing extended-spectrum beta-lactamase (ESBL) and mobile colistin resistance (\u003cem\u003emcr\u003c/em\u003e) genes in Thailand. Microb Genom 9:mgen000951\u003c/li\u003e\n\u003cli\u003eTacconelli E, Carraca E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N, WHO Pathogens Priority List Working Group (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18:318-327\u003c/li\u003e\n\u003cli\u003eWang Z, Jiang Z, Zhang Y, Wang C, Liu Z, Jia Z, Bhushan S, Yang J, Zhang Z (2024) Exosomes derived from bladder epithelial cells infected with uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e increase the severity of urinary tract infections (UTIs) by impairing macrophage function. PLoS Pathog 20:e1011926\u003c/li\u003e\n\u003cli\u003eZhuge X, Zhou Z, Jiang M, Wang Z, Sun Y, Tang F, Xue F, Ren J, Dai J (2020) Chicken‐source \u003cem\u003eEscherichia coli\u003c/em\u003e within phylogroup F shares virulence genotypes and is closely related to extraintestinal pathogenic \u003cem\u003eE. coli\u003c/em\u003e causing human infections. Transbound Emerg Dis 68:880-895\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":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antimicrobial resistance, One Health, Semiarid, CTX-M, AmpC, Surveillance","lastPublishedDoi":"10.21203/rs.3.rs-4360115/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4360115/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistant \u003cem\u003eEscherichia coli\u003c/em\u003e is a global health challenge in a One Health perspective. However, data on its emergence in the Caatinga biome are limited. This biome is exclusive to the Brazilian Northeast and offers unique epidemiological conditions that can influence the occurrence of infectious diseases and antimicrobial resistance. In this study, we assessed the carriage proportion, the antimicrobial susceptibility, and the population structure of cephalosporin-resistant \u003cem\u003eE. coli\u003c/em\u003e in 300 cloacal swab samples of free-range chickens from three Brazilian states covered by Caatinga biome. The results showed that 44 (14.7%) samples were positive for cephalosporin-resistant \u003cem\u003eE. coli\u003c/em\u003e, and Para\u0026iacute;ba (PB) state had the highest frequency of isolates (68.2%). Genes encoding CTX-M or AmpC enzymes were identified in 30 (68.2%) and eight isolates (18.2%), respectively, comprising 31 \u003cem\u003eE. coli\u003c/em\u003e. Overall, molecular typing by \u003cem\u003eXba\u003c/em\u003eI-PFGE revealed four clusters from two properties of the PB state composed by ESBL- and AmpC-producing \u003cem\u003eE. coli\u003c/em\u003e carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u0026minus;1\u0026minus;like\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;1/ACT\u0026minus;1\u003c/sub\u003e genes and belonging to different phylogenetic groups. There is a need for controlling antimicrobial resistance taking into account the genetic diversity of the strains and their implications for animal and public health, especially in free-range chickens reared in the Brazilian Caatinga biome.\u003c/p\u003e","manuscriptTitle":"Multidrug-resistant Escherichia coli isolated from free-range chickens in the Caatinga biome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-09 07:59:28","doi":"10.21203/rs.3.rs-4360115/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-19T21:42:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-19T08:27:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332171886888016215952194658553261582667","date":"2024-05-09T05:35:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-06T18:27:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136847713370448941547188438930765472669","date":"2024-05-03T23:36:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-03T23:34:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-03T19:12:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-03T19:12:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2024-05-02T15:51:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"025060c2-5a04-4eb3-aef9-89b182dcbbb5","owner":[],"postedDate":"May 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T15:59:33+00:00","versionOfRecord":{"articleIdentity":"rs-4360115","link":"https://doi.org/10.1007/s11259-024-10504-y","journal":{"identity":"veterinary-research-communications","isVorOnly":false,"title":"Veterinary Research Communications"},"publishedOn":"2024-08-19 15:57:00","publishedOnDateReadable":"August 19th, 2024"},"versionCreatedAt":"2024-05-09 07:59:28","video":"","vorDoi":"10.1007/s11259-024-10504-y","vorDoiUrl":"https://doi.org/10.1007/s11259-024-10504-y","workflowStages":[]},"version":"v1","identity":"rs-4360115","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4360115","identity":"rs-4360115","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}