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

doi:10.21203/rs.3.rs-4360115/v1

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

https://doi.org/10.21203/rs.3.rs-4360115/v1

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published 19 Aug, 2024

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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 XbaI-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

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.

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 XbaI endonuclease followed by pulsed-field gel electrophoresis (XbaI-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.

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 XbaI-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).

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.

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.

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No competing interests reported.

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

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Abstract

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Introduction

Materials and Methods

Study area and biological sample collection

Bacteriological culture and species identification

Results

Discussion

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