This study aimed to understand the origin and to explain the maintenance of extended-spectrum β-lactamase (ESBL) Enterobacteriaceae isolated from food-producing animals in a third-generation cephalosporin (3GC)-free farm. Volume 3 - 2024 | https://doi.org/10.3389/frabi.2024.1367936 This article is part of the Research TopicAntimicrobial Resistance in food-producing environment: a One Health approachView all 8 articles Introduction: This study aimed to understand the origin and to explain the maintenance of extended-spectrum β-lactamase (ESBL) Enterobacteriaceae isolated from food-producing animals in a third-generation cephalosporin (3GC)-free farm Methods: Culture and molecular approaches were used to test molecules other than 3GC such as antibiotics (tetracycline and oxytetracycline) and antioxidant (butylated hydroxytoluene) as sources of selective pressure Whole-genome sequencing using short read (Illumina™) and long read (Nanopore™) technologies was performed on 34 genomes In silico gene screening and comparative analyses were used to characterize the genetic determinants of resistance and the genomic relatedness among isolates Results: Our analysis unveiled a low diversity among the animal ESBL-producing strains coli ST3268 was recurrently isolated from both flies (n = 9) and cattle (n = 5) coli ST3268/blaCTX-M-15/blaTEM-1B have accumulated multiple plasmids and genes thereby representing a reservoir of resistance and virulence factors Our findings suggest that flies could act as effective mechanical vectors for antimicrobial gene transfer and are capable of transporting resistant bacteria across different environments and to multiple hosts facilitating the spread of pathogenic traits A significantly higher mean minimum inhibitory concentration of oxytetracycline (841.4 ± 323.5 mg/L vs coli and blaCTX-M-15 gene overexpression in oxytetracycline-treated vs p = 0.024) confirmed oxytetracycline as a source of selective pressure in ESBL E coli in a farm without 3GC use is probably due to an as yet undefined human origin of Enterobacteriaceae blaCTX-M-15 gene transmission to animals in close contact with cattle farm workers and the maintenance of the local ESBL E coli reservoir by a high fly diversity and oxytetracycline selective pressure These findings highlight the critical need for stringent vector control to mitigate antimicrobial resistance spread for preserving public health Addressing this issue necessitates a multifaceted approach combining microbial genetics Antimicrobial resistance (AMR) is currently one of the most important public health problems in the world (O’Neill, 2014). It has dramatically increased morbidity and mortality in both humans and animals (Eurosurveillance editorial team, 2015). The emergence of AMR is mainly due to the selective pressure of antibiotics used in both human and veterinary medicine (Nóbrega and Brocchi, 2014) Our study focuses on a cattle farm with a hotspot of ESBL E. coli blaCTX-M-15 carriers despite rational antimicrobial use and the absence of 3GC treatments (Gruel et al., 2021) coli was significantly higher in this farm than in other farms (47.1% vs we demonstrated the role of animal food production systems as a reservoir of mobile genetic elements carrying multiple resistance determinants and maintenance of resistance were not established and further studies are warranted to better define the genetic background of ESBL E coli isolates and the context of antibiotic resistance in Guadeloupe especially in food-producing animals not exposed to third-generation cephalosporins Mechanisms other than the selective pressure of these antimicrobials in the emergence of antibiotic resistance remain to be elucidated We investigate the hypothesis that other selective pressures such as oxytetracycline and environmental factors may play a role in the persistence of ESBL Enterobacteriaceae we explore the potential for human–animal transfer as a source of AMR This work aims to elucidate the origins and maintenance mechanisms of AMR in cattle potentially offering insights into mitigation strategies that address these resistance pathways at the ecosystem level A total of 16 farms were visited and sampled between February 2018 and November 2019 (Supplementary Data Set S1). We focused our investigations on one farm, number 13, which had the highest rate of ESBL E. coli (Gruel et al., 2021) 74 samples were collected only once at that farm Fresh fecal samples were randomly collected from cattle living in the stall (n = 32) or in the field (n = 13) and from stalled goats (n = 10) immediately after defecation We did not actually sample manure or goat feed Flies that landed around cattle feces (n = 1) or goat breastfeeding food (pool n = 4) and adult mosquitoes in unused goat feeders (pool n = 1) were trapped using a 6-V mechanical aspirator The mechanical aspiration technique used allowed the collection of pools of several flies: around cattle feces (n = 1) yielded 42 flies and goat breastfeeding food (n = 4) yielded 34 flies A total of 157 flies were collected from six samples Drinking water (n = 3) and untreated agricultural water (n = 2) were sampled Wastewater samples (n = 3) were collected downstream of the administration building and pellets (n = 1) were collected aseptically All samples were stored and transported in sterile cups or bags on ice to the laboratory of the Institut Pasteur within 4 h Minimum inhibitory concentration (MIC) values were used to compare the relative resistance levels of ESBL isolates with those of non-ESBL isolates. The MIC was determined using the EUCAST reference broth microdilution method (https://www.eucast.org/publications_and_documents/consultations/) and antioxidant (butylated hydroxytoluene) molecules were tested Serial dilutions were inoculated with a pure bacterial suspension at 0.5 McFarland turbidity within 2 h of preparation the optical density at 620 nm (OD620) was measured using a microplate reader (Multiscan™ FC The MICs were read as the lowest concentrations that produced no visible growth coli ATCC 25922 was used as the control strain The listed MIC values presented are the mean of three independent experiments To assess the selective advantage of ESBL E. coli under oxytetracycline, ivermectin, and copper selective pressure, blaCTX-M-15 gene expression was quantified and compared between treated and untreated isolates. The blaCTX-M-15 gene expression was determined in 14 ESBL E. coli isolates using a two-step RT-qPCR strategy described in detail in Supplementary Material M1 bacterial samples were obtained from overnight-cultured ESBL and non-ESBL E coli in Luria–Bertani broth media supplemented or not with oxytetracycline at a subinhibitory concentration The bacterial density was measured by using a photometer and pelleted to adjust the concentration to 108 cells/mL Total RNA was extracted immediately using the NucleoSpin® RNA isolation kit following the manufacturer’s recommendations (Macherey-Nagel) A maximum of 2 µg of RNA was then reverse-transcribed to the corresponding cDNA using the SuperScript™ VILO™ Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions cDNA was then used in qPCR using the TaqMan™ Gene Expression Master Mix and thanks to a 7500 Real-Time PCR system (Thermo Fisher Scientific) a standard curve was generated in duplicate using a 10-fold serial dilution of a quantification calibrator of untreated E The 2‐ΔΔCT algorithm was used to estimate the relative expression level of blaCTX-M-15 transcripts for the two populations studied using the RQ application module on the Thermo Fisher Cloud Each real-time PCR run included the gene expression measurements of the endogenous 16S rRNA gene and the target blaCTX-M-15 gene in the corresponding samples libraries were constructed from 1 μg of unfragmented bacterial gDNA following the protocol instructions for native barcoded genomic DNA (using EXP-NBD104 The final library was loaded onto a R9.4.1 flow cell (FLO-MIN106D) according to the manufacturer’s instructions and run on a laptop (MinKNOW Core v3.6.5) Single-flow cell sequencing data from multiplexed barcoded isolates were run on the MinION for 48 h Base calling of MinION raw signals was performed Fastq files were extracted and split by barcode De novo genome assembly was performed using a hybrid strategy on combined nanopore long reads and previously available Illumina short reads The fully resolved assemblies were generated and visualized Quality control of nanopore data was performed The plasmids were aligned graphically and annotated Mobilization module characterization was performed Table 1 Description of samples and ESBL enterobacteria collected from the farm environment Corresponding β-lactamase-associated resistance-coding genes are indicated by black squares and plasmids by black circles The IncFIB [F-:A-:B42] and IncFIB [F-:A-:B70] replicon sequence types were found in ESBL E The IncN-pST3 replicon type was found only in cattle and fly ESBL E Table 2 Distribution of blaCTX-M gene and replicon type in extended-spectrum β-lactamase isolates Figure 2 Schematic representation showing two combinations of the ultrastructural genetic background of the ST3268.1 ESBL E and EC338) and ST3268.2 (n = 2 isolates: BCA26.1 and EC347) Plasmids are shown as circles annotated for replicon Supercoiled chromosomal DNA is schematically shown in red truncated IncN_1_AY046276 replicon (247 bp) complete IncN_1_AY046276-pST3 replicon (512 bp) some of which are mobilizable with multiple associated resistance and virulence genes The collection of ST3268 isolates from other geographical origins found on Enterobase presents only ESBL producers (n = 22) (Supplementary Figure S4) This sequence type was identified in many countries and was also found in wild and domestic animals with a blaCTX-M-15 gene and the IncF replicon [F-:A-:B42] was only identified in farm number 13 No clonal relationship was found between the Guadeloupean isolates and those identified internationally No difference in blaCTX-M-15 gene expression was observed with the ivermectin and copper treatments Figure 3 Modulation of blaCTX-M-15 gene expression under in vitro selective pressure Relative quantification of blaCTX-M-15 gene expression under oxytetracycline treatment in ESBL E Error bars represent the standard deviation of at least two independent experiments Error bars indicate the range between RQ min and RQ max ** Statistically significant p-value < 0.05 The two largest clusters (ST3268 and ST2015) contained eight to 10 ESBL E coli harboring a blaCTX-M-1 (ST2015) or a blaCTX-M-15 (ST3268) gene coli tends to show a higher proportion of unclustered isolates These results reflect sporadically acquired isolates from different lineages rather than the active spread of major clones Cluster ST3268 showed a close genomic relationship between 10 CTX-M-15 producing E coli ST3268/blaCTX-M-15/blaTEM-1B have accumulated and maintained multiple plasmids and genes thereby representing an extensive reservoir of resistance and virulence factors Our results suggest that flies could act as vectors and highlight a clear link between cattle and flies in the spread of CTX-M-15 producing E This underscores the role of flies in increasing the risk of transmission of such resistance factors from livestock to the wider environment This refined statement underscores the importance of understanding these dynamics in addressing the spread of antibiotic resistance Figure 4 Core genome comparative analysis of 68 E. coli isolates from food-producing animals and flies. Maximum likelihood phylogenetic tree of 68 E. coli isolated from farms in Guadeloupe between 2018 and 2019. The farm number refers to our previous reference number (Gruel et al., 2021) Associated hosts and antimicrobial susceptibility phenotypes are indicated by vertical colored stripes The resistant phenotype is assigned to isolates that are resistant to at least one of the 17 antimicrobials tested and ST155) represent groups of similar core genomes (≤25 SNP) Only β-lactamase-encoding genes are indicated by black squares Plasmid replicons are indicated by a black circle harboring resistance genes common to both cattle and flies is significant It suggests that vectors such as flies may play a role in the maintenance and spread of novel and important resistance genes with potential implications for both animal and human health This reinforces the need for integrated veterinary and public health surveillance and control strategies we advocate improved farm hygiene and waste management practices the use of biosecurity measures such as insect screens and zappers and further research into environmentally friendly insect control methods Due to the hygienic measures taken after our visit the persistence of antibiotic resistance genes and their vectors can be expected in the absence of antibiotic selection pressure Other means of reducing plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes that they carry Differences in plasmid characteristics between samples highlight the complexity of transmission dynamics Our study contributes to the understanding of how resistance genes spread with implications for approaches to monitoring and controlling AMR on farms and the importance of considering a variety of genetic vehicles in these processes The coexistence of multiple resistant and mobilizable plasmids has serious implications for both the agricultural ecosystem and public health It demonstrates the ability of pathogens to evolve rapidly in response to environmental pressures and the need for comprehensive genomic surveillance strategies to monitor and understand this genetic exchange coli in a 3GC-free environment suggests that alternative selective pressures may be at play It highlights the possibility of other contributing factors such as the use of different antimicrobials such as oxytetracycline Our work calls for a re-evaluation of the current understanding of AMR transmission and highlights the need to consider a wider range of selective pressures The identification of oxytetracycline as a potential selective agent for ESBL-producing bacteria highlights the need for comprehensive stewardship that includes all antibiotics not just those thought to directly select for resistance It contributes to a more nuanced approach to antibiotic use in agriculture While the design of this study primarily focused on investigating the role of the human compartment through the analysis of wastewater we acknowledge the opportunity to extend our research by exploring ESBL Enterobacteriaceae presence among farm workers Such an extension would not only complement our current findings but also offer a more comprehensive understanding of contamination origins thereby enhancing the robustness of proposed risk mitigation strategies the integrity and relevance of the results presented remain intact including longitudinal monitoring of strains among farm workers further strengthening the study’s impact on preventing the emergence and spread of ESBL clones in such environments We demonstrated that the high level of ESBL E coli in a farm without 3GC use is likely due to the maintenance of the local ESBL E coli animal reservoir by a high fly diversity and oxytetracycline pressure This is the first observation of multiple E coli IncFIB/IncN::blaCTX-M-15/blaTEM-1B replicon plasmids clustering in animals While the likely human origin of this plasmid observed in flies and cattle remains to be clarified our study highlights the importance of considering environmental factors and antibiotic stewardship in managing antimicrobial resistance contribute to the selection of resistance genes requiring a comprehensive strategy that includes monitoring drug use regulating potential environmental contributors to AMR and implementing biosecurity to reduce vector spread These findings call for a One Health approach that integrates human and environmental health to inform policy and improve agricultural practices The datasets presented in this study can be found in online repositories The names of the repository/repositories and accession number(s) can be found below: BioProject As fecal samples were taken from animals in the field after excretion “use of live animals for scientific purposes” (within the meaning of the Rural Code Art R214-87 and following) was not relevant; no invasive procedure was conducted on live animals the project was considered to be outside the scope of the regulations on animal experimentation by the chairman of the Ethics Committee on animal experimentation of the Antilles and Guyane (registered with the French Ministry of Higher Education according to French national law for the protection of animals (No which reproduces European directive 2010/63/UE on the protection of animals used for experimental and other scientific purposes no ethics committee approval was deemed necessary according to Article 7.1 on recommendations for animal welfare and Article 7.8 on use of animals in research and education of the World Organization for Animal Health Terrestrial Animal Health Code The entity responsible for the animals was the owners Sampling of animals feces was authorized verbally by the owners The studies were conducted in accordance with the local legislation and institutional requirements Written informed consent was obtained from the owners for the participation of their animals in this study J-CB: Writing – review & editing The author(s) declare that financial support was received for the research This work was supported by the European Union on the Guadeloupe Region under the European Research and Development Fund (ERDF) 2014–2020 program (2018-FED-1084) GG is funded by a PhD fellowship from the Guadeloupe Region The funders had no role in the study design We would like to thank all the technicians and students who participated in this project at the Institut Pasteur de la Guadeloupe The authors would like to thank Ludovic Arthein and Alain Farant (PTEA Guadeloupe) for their help with sampling during the field work on the farm Guadeloupe) for his useful contribution to the development of the scripts supported by France Génomique (ANR-10-INBS-09) and IBISA The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/frabi.2024.1367936/full#supplementary-material Antibiotic resistance in Escherichia coli in husbandry animals: the African perspective doi: 10.1128/microbiolspec.MTBP-0019-2016 CrossRef Full Text | Google Scholar PlasmidFinder and in silico pMLST: identification and typing of plasmid replicons in whole-genome sequencing (WGS) VFDB: a reference database for bacterial virulence factors Prevalence and distribution of extended-spectrum β-lactamase and AmpC-producing Escherichia coli in two New Zealand dairy farm environments Persistence of transferable extended-spectrum-β-lactamase resistance in the absence of antibiotic pressure Occurrence of ESBL-producing escherichia coli in livestock and farm workers in mecklenburg-western pomerania Phylogeny.fr: robust phylogenetic analysis for the non-specialist Presence of ESBL/ampC -producing escherichia coli in the broiler production pyramid: A descriptive study Eurosurveillance editorial team (2015) WHO member states adopt global action plan on antimicrobial resistance PubMed Abstract | CrossRef Full Text | Google Scholar Extended-spectrum β-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals and their putative impact on public health: a global perspective doi: 10.1111/j.1469-0691.2012.03850.x Reexamining the association of ampC variants with enterobacter species in the context of updated taxonomy DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Role of flies in the maintenance of antimicrobial resistance in farm environments Prevalence of antimicrobial resistant and extended-spectrum beta-lactamase-producing escherichia coli in dairy cattle farms in east tennessee High Prevalence of bla (CTXM-1)/IncI1-Iγ/ST3 Plasmids in Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates Collected From Domestic Animals in Guadeloupe (French West Indies) Antimicrobial use and resistance in Escherichia coli from healthy food-producing animals in Guadeloupe Guyomard-Rabenirina S. 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Metrics details Angiostrongylus cantonensis (rat lungworm) is the main pathogen responsible for eosinophilic meningitis in humans was already present in many countries around the world before it appeared in the West Indies in the late 1980s In the French territories in the Caribbean and northern South America the first cases of human neuroangiostrongyliasis were reported in Martinique In order to better characterize angiostrongyliasis in Guadeloupe particularly its geographical origin and route of introduction we undertook molecular characterization of adult worms of Angiostrongylus cantonensis and its intermediate host Achatina fulica Genomic DNA of adult Angiostrongylus cantonensis and Achatina fulica was extracted and amplified by polymerase chain reaction (PCR) targeting the mitochondrial genes cytochrome B and C for A The PCR products were sequenced and studied by phylogenetic analysis Cytochrome B and cytochrome C molecular markers indicate a monophyletic lineage of A These results confirm the recent introduction of both Angiostrongylus cantonensis and Achatina fulica into Guadeloupe Achatina fulica in Guadeloupe shares a common origin with those in Barbados and New Caledonia while Angiostrongylus cantonensis in Guadeloupe shares a common origin with those in Brazil but only Angiostrongylus cantonensis and Angiostrongylus costaricensis are known to infect humans Humans are accidental hosts and also do not allow completion of the parasite’s life cycle Infection occurs by consumption of raw or undercooked intermediate or paratenic hosts little information is available about the intermediate hosts of A After the emergence of cases of neuroangiostrongyliasis in Guadeloupe Achatina fulica were collected in 7 municipalities on Guadeloupe (Vieux-Habitant Saint-Anne and Capesterre de Marie-Galante) to evaluate the prevalence of Angiostrongylus cantonensis Approximately 38% of these snails were infected with A Although their presence has been reported for several years in the Caribbean few molecular data are available for either Angiostrongylus cantonensis or Achatina fulica in the region To determine the geographic origin and route of introduction of Angiostrongylus cantonensis and to better define the role of Achatina fulica in the emergence of A we undertook molecular characterization of both A Geographic distribution of Angiostrongylus cantonensis in rats on Guadeloupe and outlying islets. Red sectors on the pie charts indicate positive cases and green sectors negative cases. The yellow snails indicate the sites at which Achatina fulica were captured. The original map is available under Creative Commons license https://commons.wikimedia.org/wiki/File:Guadeloupe_map.JPG and the newly designed primers 5′-TTAGTTTRCATTGTGCTGG-3' and 5′-CATCAAAGACTAATACCAG-3' for cytC The expected amplicon sizes were 853 base pairs (bp) for cytB and 748 bp for cytC PCR was performed in a final volume of 50 μL under the following conditions: 5 μL of 10X buffer; 2 μL of 25 mM MgCl2; 2 μL of 5 mM deoxynucleotide triphosphate; 2 μL of bovine serum albumin; 3 μL of dimethyl sulphoxide; 0.25 μL of Taq DNA polymerase at 5U/μL (Eurobiotaq France); 1 μL of each of the two primers at 20 μM; 28.75 μL of ultrapure water; and 5 μL of DNA The PCR reaction and the size of the resulting fragments were monitored by migration on 1.5% agarose gel in 1× Tris–acetate-ethylenediaminetetraacetic acid buffer A total of 10 μL of PCR products mixed with 2 μL of 5× bromophenol blue was placed in each well Migration was performed in 1× Tris–acetate-ethylenediaminetetraacetic acid buffer at 90 V for 45 min Amplicons were purified and sequenced by the Sanger method The experimental sequences obtained from the mitochondrial cytB and cytC genes of Angiostrongylus cantonensis and the 16S rRNA gene of Achatina fulica were first examined for quality control and compared with published sequences in the National Center for Biotechnology Information (NCBI) GenBank database The 71 new experimental sequences generated for this study are available in GenBank A chi-squared test was used to compare the prevalence of A cantonensis infection in the two rat species (R and Student’s t-test was used to compare the prevalence of A cantonensis among municipalities on Guadeloupe Odds ratios and 95% confidence intervals were calculated R software version 4.1.2 (2021-11-01) was used for statistical analysis and P-values < 0.05 were considered significant Statistically significant differences were observed between the numbers of rats that were positive and those that were negative for A cantonensis in the municipalities (t = − 3.9645 None of the rats caught on the three offshore islets (Kahouanne Labiche and Christophe) were infected with A Maximum likelihood phylogenetic unrooted tree based on cytochrome B (cytB) sequences of Angiostrongylus cantonensis adult worms Two sequences of Angiostrongylus malaysiensis were used as an outgroup Labels are coloured according to the country of isolation The scale indicates the length of the branches representing the number of nucleotide substitutions per site Maximum-likelihood phylogenetic unrooted tree based on cytochrome C (cytC) sequences of adult Angiostrongylus cantonensis worms Angiostrongylus malaysiensis was used as an outgroup Labels are coloured according to the area of isolation The scale indicates the length of the branches given as the number of nucleotide substitutions per site The phylogenetic tree (Fig. 4) based on the partial sequence of the 16S rRNA gene of A. fulica (243 pb) was constructed with the same technique as above. A total of seventy-one 16S rRNA gene sequences of A. fulica from Guadeloupe were compared with the 37 sequences from 10 countries and areas in the NCBI GenBank database. Maximum-likelihood phylogenetic unrooted tree based on the rRNA 16S gene of Achatina fulica snails Labels are coloured according to the country or area of isolation The scale indicates the length of the branches given by the amount of genetic change in nucleotide substitutions per site Countries and administrative areas including Tanzania the United Arab Emirates and India show diversity among samples fulica from Martinique to confirm that they all differ from those from Guadeloupe Because of its recent introduction into the Caribbean Achatina fulica may have either introduced Angiostrongylus cantonensis into the area at the time of its arrival or have amplified its presence if rats brought it in If different geographic origins are clearly proven for Achatina fulica and Angiostrongylus cantonensis fulica could not have been responsible for the introduction of the parasite It is more likely that Achatina fulica played a role in the introduction of Angiostrongylus cantonensis if they both originated from the same country or region as no specimens of Angiostrongylus cantonensis have been sequenced for either New Caledonia or Barbados it is impossible to determine whether individuals of this parasite and Achatina fulica in Guadeloupe originate from the same region Our results indicate a common origin of Achatina fulica found on Barbados whereas Angiostrongylus cantonensis on Guadeloupe shares a common origin with those found in Hawaii due to a lack of information on these two species from different parts of the world it is not possible to clearly determine the precise origin of Angiostrongylus cantonensis found in Guadeloupe and if Achatina fulica played a role in its introduction there sequences of Angiostrongylus cantonensis from Barbados and New Caledonia are needed as are sequences of this species and Achatina fulica from West Africa verify the absence of genetic variation among our samples and enable comparison of the whole genome sequence of Angiostrongylus cantonensis the present study needs to be complemented by further work in which sequences of A cantonensis and Achatina fulica from Martinique and various localities in Africa are included The 20 partial Angiostrongylus cantonensis cytB sequences cantonensis sequences cytC and 71 Achatina fulica rRNA 16S sequences have been deposited in GenBank and are available under BioSample accession numbers OQ238771–OQ238790 Annotated catalogue of species of Angiostrongylus and the related genera Gallegostrongylus Rodentocaulus and Stefanskostrongylus (Nematoda: Metastrongyloidea sp.: new species found parasitizing coatis (Nasua nasua) in an urban protected area in Brazil Angiostrongylus cantonensis (Chen) (Nematoda: Metastrongylidae) Paratenic hosts of Angiostrongylus cantonensis and their relation to human neuroangiostrongyliasis globally Presence of Angiostrongylus cantonensis in Madagascar the presence of Angiostrongylus cantonensis in islands of the Indian Ocean and the probable role of the giant African snail in dispersal of the parasite to the Pacific Islands Angiostrongylosis in the Pacific and Southeast Asia The first record of Angiostrongylus cantonensis from Egypt First record of Angiostrongylus cantonensis in Cuba The finding of Angiostrongylus cantonensis in rats in New Orleans An outbreak of eosinophilic meningitis caused by Angiostrongylus cantonensis in travelers returning from the Caribbean Eosinophilic meningitis beyond the Pacific Basin: the global dispersal of a peridomestic zoonosis caused by Angiostrongylus cantonensis and distribution of Angiostrongylus cantonensis Geographic range expansion for rat lungworm in North America Central nervous system manifestations of Angiostrongylus cantonensis infection Fatal autochthonous eosinophilic meningitis in a Jamaican child caused by Angiostrongylus cantonensis Human Angiostrongylus cantonensis: an update Angiostrongyliasis in Thailand: epidemiology and laboratory investigations Angiostrongylus cantonensis eosinophilic meningitis: a clinical study of 42 consecutive cases in French Polynesia Angiostrongylus cantonensis infection on Mayotte Island Alicata disease: neuroinfestation by Angiostrongylus cantonensis in Recife Angiostrongylus cantonensis of central nervous system First cases of Angiostrongylus cantonensis infection reported in Martinique Two giant African land snail species spread to Martinique Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia and its application to field epidemiological studies Restricted genetic variation in populations of Achatina (Lissachatina) fulica outside of East Africa and the Indian Ocean Islands points to the Indian Ocean Islands as the earliest known common source Improved molecular detection of Angiostrongylus cantonensis in mollusks and other environmental samples with a species-specific internal transcribed spacer 1-based TaqMan assay Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT KaruBioNet: a network and discussion group for a better collaboration and structuring of bioinformatics in Guadeloupe (French West Indies) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies MAFFT multiple sequence alignment software version 7: improvements in performance and usability Survey of Angiostrongylus cantonensis in rats and giant African land snails in Phitsanulok province Thailand High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: a closer look at life cycle traits and patterns of infection in wild rats (Rattus spp) Recherches écophysiologiques sur le régime alimentaire de l’escargot petit-gris Low diversity of Angiostrongylus cantonensis complete mitochondrial DNA sequences from Australia French Polynesia and the Canary Islands revealed using whole genome next-generation sequencing The occurrence of nematodes of the subfamily Angiostrongylinae in Viet-Nam and the question of geographical origin of Parastrongylus cantonensis (Chen Evaluation of haplotype diversity of Achatina fulica (Lissachatina) [Bowdich] from Indian sub-continent by means of 16S rDNA sequence and its phylogenetic relationships with other global populations Fine-scale geographical sampling and molecular characterization of the giant African land snail in its invasive range in Asia shows low genetic diversity new haplotypes and the emergence of another haplotype from the Indian Ocean Islands Download references The authors thank Emmanuel Albina at the French Agricultural Research Centre for International Development and Simone Mège at the National Park of Guadeloupe for collecting the rats and sending us the samples This work was supported by a European Regional Development Fund grant financed by the European Union and Guadeloupe Region (grant ID 2018-FED-1084) Team Host-Pathogen Interactions and Immunity to Infection GG and AT performed the sampling and drafted the manuscript the molecular analysis of the samples and the bioinformatics analysis DC contributed to the realization of the figures and the validation of the bioinformatics analysis SF and CD critically reviewed the manuscript All the authors have read and approved the final version of the manuscript The captures made within the framework of this project were authorised by the Department of Performance Funding and Contracting with Research Organizations Department of Regulated Research Practices Animals Used for Scientific Purposes Unit—AFiS This study does not contain any material requiring consent for publication The authors declare that they have no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations unless otherwise stated in a credit line to the data Download citation DOI: https://doi.org/10.1186/s13071-023-05872-4 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Volume 12 - 2021 | https://doi.org/10.3389/fmicb.2021.628058 This article is part of the Research TopicAntimicrobials in Wildlife and the EnvironmentView all 12 articles Species belonging to Enterobacter cloacae complex have been isolated in numerous environments and samples of various origins They are also involved in opportunistic infections in plants Previous prospection in Guadeloupe (French West Indies) indicated a high frequency of E cloacae complex strains resistant to third-generation cephalosporins (3GCs) in a local lizard population (Anolis marmoratus) but knowledge of the distribution and resistance of these strains in humans and the environment is limited The aim of this study was to compare the distribution and antibiotic susceptibility pattern of E cloacae complex members from different sources in a “one health” approach and to find possible explanations for the high level of resistance in non-human samples cloacae complex strains were collected between January 2017 and the end of 2018 from anoles Isolates were characterized by the heat-shock protein 60 gene-fragment typing method and whole-genome sequencing was conducted on the most frequent clusters (i.e. The prevalence of resistance to 3GCs was relatively high (56/346 The associated resistance mechanism was related to an AmpC overproduction; however most of the resistant strains (40/62) produced an extended-spectrum beta-lactamase No relation was found between resistance in isolates from wild anoles (35/168) and human activities Specific core-genome phylogenetic analysis highlighted an important diversity in this bacterial population and no wide circulation among the different compartments the mutations responsible for resistance to 3GCs cloacae complex isolates are probably due to environmental factors that favor the selection of these resistant strains We conducted this study to investigate the distribution of ECC hsp60 clusters isolated from various sources in Guadeloupe and to characterize their antibiotic susceptibility patterns in a “one health” approach Genomic analysis was conducted on the main clusters in clinical samples (C-VI and C-VIII) and in different biotopes to compare the strains and to investigate the high level of 3GC-R in non-human samples clinical ECC isolates obtained during routine bacteriological diagnostics were collected prospectively from patients admitted to the University Hospital of Guadeloupe a 900-bed teaching hospital in Pointe-à-Pitre/Les Abymes and the results of antibiotic susceptibility analysis were recorded anonymously Isolates were considered to be hospital-acquired if there were collected from patients hospitalized for more than 48 h after admission The others were notified as to be community-acquired Human samples were taken in accordance with the requirements of the local ethics committee and did not interfere with laboratory organization (reference A5_19_12_05_TRAMID) ECC strains were isolated from water catchment areas marmoratus and farm animals sampled at different sites in Guadeloupe which corresponded to drinking water before treatment in collaboration with the regional health agency and the hygiene laboratory of the Pasteur Institute of Guadeloupe Most of these sampling points were located in Basse-Terre All samples were transported rapidly to the laboratory stored at 5 ± 3°C and analyzed within 4 h A maximum of five presumptive ECC colonies from each plate were isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Shimadzu Biotech Production of ESBL was detected with the double-disk synergy test according to CA-SFM/EUCAST recommendations Total bacterial DNA was initially extracted from pure cultures with the Qiagen QIAamp DNA minikit (Qiagen Extended-spectrum beta-lactamase-encoding genes were screened by polymerase chain reaction (PCR). On the basis of previous epidemiological evidence for ECC in the community, PCR was performed only for blaCTX–M group 1 (Dallenne et al., 2010; Guyomard-Rabenirina, 2016). Amplicons were sequenced at Eurofins (Eurofins Genomic SAS, Les Ullis, France). Resistance genes were identified from the ResFinder database (Zankari et al., 2012) When multiple strains were found in the same sample only one in each cluster or resistance phenotype profile against beta-lactam antibiotics was conserved for the analysis An in-house Perl script was used to extract results from files generated by Snippy and to create a table of the numbers of non-synonymous and synonymous gene mutations in the ECC sequences retrieved from the same host but with different antibiotic resistance profiles The analyses and data collection were performed with Microsoft Access 2003 Pearson’s χ2 or Fisher’s exact test was used P values < 0.05 were considered significant Prevalence of resistance to each tested antibiotic of E cloacae complex and third-generation cephalosporin-resistant (3GC-R) strains in the Anolis population Most of human isolates were hospital-acquired (95/107) and 3GC-R were mainly associated to ESBL production (40/62; Table 1 and Supplementary Table 2) Genes encoding for ESBL were identified in all clinical strains positive in the double-disk synergy test (40/107 Amplicon sequencing revealed a blaCTX–M–15 gene in 38 isolates (95.0%) WGS allowed us to identify a blaGES–7 gene on the two last ESBL producers (GENC084 One strain of human origin carried a blaOXA–48 gene (GENC133) Co-resistance against beta-lactams (including 3GCs) and other antibiotic families was observed mainly in human associated strains and was usually to fluoroquinolones (41/62, 66.2%; Table 1) Co-resistance to gentamicin and trimethoprim–sulfamethoxazole was also found frequently in 29 3GC-R clinical strains (46.8%) but not in samples from other sources Only one strain from a raw water sample was resistant to nalidixic acid (ECC403) and one strain from livestock was notified resistant to trimethoprim–sulfamethoxazole and tigecycline (ECC408) cloacae complex (ECC) members in samples of different origin Antibiotic resistance profiles are indicated by gray triangles; AKN As observed in the C-VI phylogenetic tree, some strains of different origins were genetically related. ST90 was recovered from three different A. marmoratus, one water sample, and one human (two strains from the same patient, l in Figure 2 and Supplementary Table 4; mean SNP among isolates n = 1283 Most of these strains presented a CoP (5/6) Two other STs found in wild fauna and human samples clustered together: ST50 225 SNPs) and ST304 (ECC273–GENC100 The human strain GENC117 shared the same target sequence of seven housekeeping genes with ECC386 isolated from water and a difference of 123 SNPs (i.e. One strain from Anolis clustered with an isolate from water with a difference of only 101 SNPs (ECC443–ECC426) Two strains isolated from two Anolis 28 km apart clustered with a difference of only 81 SNPs (ECC140–ECC300) human and non-human isolates expressed a similar number of virulence genes (mean twenty-one) All sequenced C-VIII strains (n = 86) harbored genes involved in the salmochelin siderophore system (iroB while this system was not found in the C-VI population and ybtX) was identified in two non-human strains belonging to ST90 (ECC169 and ECC403) This study of ECC diversity in samples from different compartments in Guadeloupe showed by hsp60 typing analysis that six clusters (C-IV, -VI, -VIII, -IX, -XI, and -XII) are identified in more than two thirds of the strains (228/313, 72.8%), and most are present in all sample types. Although this typing method is limited and focus on only one gene (Hoffmann and Roggenkamp, 2003; Wu et al., 2020) it indicates a high degree of diversity of this bacterial complex which is present in a wide variety of compartments and a possible new successful ESBL-producing lineage ST344 clones were found in three fresh products but from two different market stands This study also showed a high frequency of 3GC-R ECC members in non-human samples such as livestock and fresh local produce, and confirmed previous observations on the local anole population (Guyomard-Rabenirina, 2016) Three hypotheses have been proposed to explain this high prevalence of CoP ECC strains in these compartments The first one was a human origin of these resistant strains with the spread of successful lineages among compartments; however hsp60 clusters distribution within each sample type and the WGS analysis of C-VI and -VIII Although some whole-genomes sequenced strains isolated from humans although we did not estimate the mutation rate in our non-human samples due to the presence of various cultivable genus and species in agar plates Our findings highlight the widely diverse distribution of ECC members in non-human and human samples We found a high prevalence of 3GC-R ECC in non-human samples None of our hypotheses could explain this prevalence These results suggest that this characteristic confers a selective advantage for these strains The 313 partial hsp60 sequences are available in Supplementary Table 2 with strain details, while the whole-genome sequences of C-VI, clade A (n = 42) and C-VIII (n = 86) are deposited in GenBank under BioSample accession numbers SAMN15680734 to SAMN15680861 (Supplementary Table 3) The parts of this project involving human participants were reviewed and approved by Commission de recherche éthique Direction de la recherche et de l’innovation Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements The animal study was reviewed and approved by the Committee for Ethics in animal experiments of the French West Indies and Guyana (reference 971-2016-12-20-001) Written informed consent for participation was not obtained from the owners because feces were collected from livestock animals at the slaughterhouse Only the municipality origin of screened animals batchs was recorded All the authors critically revised and approved the final version of the manuscript financed by the European Union and Guadeloupe Region (Grant ID: 2018-FED-1084) We are grateful to all the technicians and students for their participation in this project at the ARS (Agence Régionale de Santé) the Pasteur Institute of Guadeloupe and the “Plateforme de microbiologie mutualisée.” We thank the curator team of the E cloacae typing PubMLST database for their co-operation We also express our gratitude to the employees of the slaughterhouse in Le Moule and all the farmers and fruit and vegetable merchants for their collaboration The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.628058/full#supplementary-material List of ECC partial hps60 sequences included in this figure (associated cluster: C or UD LEDN00000000; UD5: AP019007.1; and UD6: MTKD00000000 Supplementary Table 1 | Details of non-clinical samples: sampling date Supplementary Table 2 | Details of strains: hsp60 partial sequence and results for antibiotic resistance Resistance profiles were obtained against 16 antibiotics Intermediate or resistant results are labeled “R”; “s” corresponds to a susceptible phenotype Supplementary Table 3 | Details of genetic content of sequenced E xiangfangensis (C-VI – clade A; n = 42) and E steigerwaltii (C-VIII – clade B; n = 86) Supplementary Table 4 | Cephalosporinase gene complex mutation. ∗Clone numbers refer to letters used in Figures 1, 2 and correspond to two strains from the same sample with different resistance profile against beta-lactam antibiotics: wild-type or cephalosporinase overproduction Each strain is considered to be a clone that differs by < 45 single nucleotide polymorphisms (SNPs) The abbreviation “N/S” after the gene names indicates the number of non-synonymous mutations (N) versus the number of synonymous mutations (S) in comparison to the reference data Please note that only the different non-synonymous mutations between strains from the same sample have been conserved in the observation column third-generation cephalosporin resistant; ECC Pseudomonas aeruginosa and Enterobacter spp.; CoP a novel oligotroph isolated from leaf soil SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing Novel Enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing Comprehensive genome analysis of carbapenemase-producing Enterobacter spp.: New insights into phylogeny VFDB 2016: hierarchical and refined dataset for big data analysis – 10 years on AlienTrimmer removes adapter oligonucleotides with high sensitivity in short-insert paired-end reads Commentary on Turner (2014) Assessment of insert sizes and adapter content in FASTQ data from NexteraXT libraries Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae ClonalFrameML: Efficient inference of recombination in whole bacterial genomes Association of novel nonsynonymous single nucleotide polymorphisms in AmpD with cephalosporin resistance and phylogenetic variations in AmpC and OmpC in Enterobacter cloacae isolates that are highly resistant to carbapenems Elective distribution of resistance to beta-lactams among Enterobacter cloacae genetic clusters Genetic background of CTX-M-15-producing Enterobacter hormaechei ST114 and Citrobacter freundii ST265 co-infecting a free-living green turtle (Chelonia mydas) Antimicrobial-resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital Complex regulation pathways of AmpC-mediated β-lactam resistance in Enterobacter cloacae complex QUAST: Quality assessment tool for genome assemblies Guyomard-Rabenirina Résistance aux antibiotiques des Entérobactéries en Guadeloupe: Importance en milieu communautaire et diffusion environnementale Saint-Claude: Université des Antilles Google Scholar Guyomard-Rabenirina Antimicrobial resistance in wildlife in Guadeloupe (French West Indies): Distribution of a single blaCTX–M–1 Incl1/ST3 among humans and wild animals High prevalence of international ESBL CTX-M-15-producing Enterobacter cloacae ST114 clone in animals Phenotypic and molecular characterization of antimicrobial resistance in Enterobacter spp Population genetics of the nomenspecies Enterobacter cloacae three new subspecies of clinical importance Unraveling the role of vegetables in spreading antimicrobial-resistant bacteria: A need for quantitative risk assessment Complete genome of the onion pathogen Enterobacter cloacae EcWSU1 MLST reveals potentially high-risk international clones of Enterobacter cloacae Open-access bacterial population genomics: BIGSdb software the PubMLST.org website and their applications [version 1; referees: 2 approved] Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system Complete genome sequence of Enterobacter sp IIT-BT 08?: A potential microbial strain for high rate hydrogen production Species-specific mutation rates for ampC derepression in Enterobacterales with chromosomally encoded inducible AmpC β-lactamase Effect of ampC derepression on cefepime MIC in Enterobacterales with chromosomally encoded inducible AmpC β-lactamase Prevalences of the Enterobacter cloacae complex and its phylogenetic derivatives in the nosocomial environment Interactive tree of life (ITOL) v4: Recent updates and new developments Complete genome sequence of the endophytic Enterobacter cloacae subsp Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest Genomics population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX–M–55 along with sophisticated iron acquisition strategies Infections caused by naturally AmpC-producing Enterobacteriaceae: Can we use third-generation cephalosporins Genomic diversity within the Enterobacter cloacae complex Yersiniabactin reduces the respiratory oxidative stress response of innate immune cells Enterobacter bugandensis: A novel enterobacterial species associated with severe clinical infection Genomic epidemiology of global carbapenemase-producing Enterobacter spp. Dissemination of extended-spectrum-β-lactamase-producing Enterobacter cloacae complex from a hospital to the nearby environment in Guadeloupe (French West Indies): ST114 lineage coding for a successful IncHI2/ST1 plasmid Federal funding for the study of antimicrobial resistance in nosocomial pathogens a variant of Acinetobacter genomic island 1 (AGI1) in a French clinical isolate belonging to the Enterobacter cloacae complex BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs Antimicrobial and herbal drug resistance in enteric bacteria isolated from faecal droppings of common house lizard/gecko (Hemidactylus frenatus) Multi-drug resistant Enterobacter bugandensis species isolated from the international space station and comparative genomic analyses with human pathogenic strains RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies Natural antibiotic susceptibility of strains of the Enterobacter cloacae complex CrossRef Full Text | Google Scholar and Enterobacter muelleri is a later heterotypic synonym of Enterobacter asburiae based on computational analysis of sequenced Enterobacter genomes Complete genome of Enterobacter sichuanensis strain SGAir0282 isolated from air in Singapore Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables World Health Organization WHO publishes list of bacteria for which new antibiotics are urgently needed Google Scholar Precise species identification for Enterobacter: A genome sequence-based study with reporting of two novel species Characterization of the population structure drug resistance mechanisms and plasmids of the community-associated Enterobacter cloacae complex in China Characterization of a blaNDM–1-carrying IncHI5 plasmid from Enterobacter cloacae complex of food-producing animal origin Talarmin A and Guyomard-Rabenirina S (2021) Wide Distribution and Specific Resistance Pattern to Third-Generation Cephalosporins of Enterobacter cloacae Complex Members in Humans and in the Environment in Guadeloupe (French West Indies) Copyright © 2021 Pot, Reynaud, Couvin, Ducat, Ferdinand, Gravey, Gruel, Guérin, Malpote, Breurec, Talarmin and Guyomard-Rabenirina. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Matthieu Pot, bXBvdEBwYXN0ZXVyLWd1YWRlbG91cGUuZnI= VP sales Florian Couvin and CEO Thierry Bouchet a French startup specializing in power electronics solutions based on GaN technologies the country's research institute for specialized technology in micro and nano technologies in Grenoble Some subscribers prefer to save their log-in information so they do not have to enter their User ID and Password each time they visit the site check the 'Save my User ID and Password' box in the log-in section This will save the password on the computer you're using to access the site your new go-to podcast to spice up your weekday mornings with relevant news and behind-the-scenes from Brussels and beyond From the economy to the climate and the EU's role in world affairs this talk show sheds light on European affairs and the issues that impact on our daily lives as Europeans Tune in to understand the ins and outs of European politics Dare to imagine the future with business and tech visionaries Deep dive conversations with business leaders Euronews Tech Talks goes beyond discussions to explore the impact of new technologies on our lives the podcast provides valuable insights into the intersection of technology and society Europe's water is under increasing pressure floods are taking their toll on our drinking water Join us on a journey around Europe to see why protecting ecosystems matters and to discover some of the best water solutions an animated explainer series and live debate - find out why Water Matters We give you the latest climate facts from the world’s leading source analyse the trends and explain how our planet is changing We meet the experts on the front line of climate change who explore new strategies to mitigate and adapt One person has been killed as low pressure system The Isle de France region saw severe flooding and record high water levels The departement Seine-et-Marne in Isle de France was most severely impacted by the heavy rainfall flooding houses and blocking several roads A red flood alert announced on Thursday remains in place there as further rainfall and potential flooding is expected in the coming days The ex-hurricane also drenched most of Belgium although the country saw only limited damage The Ardennes in the south received most of the rain A flood in the town of Couvin led to the activation of the communal emergency plan as a precautionary measure local officials there remain vigilant as the water levels in the Eau Noire You don't have permission to access the page you requested What is this page?The website you are visiting is protected.For security reasons this page cannot be displayed A moss-covered, square-cement building blends into the woodlands landscape outside the village of Brûly-de-Pesche in the South of the Province of Namur. The unsuspecting building was once the bunker accompanying Hitler's temporary headquarters for three weeks in June, 1940, prior to his invasion of France. Only a few kilometers from France, the site is discreet but ideally located. From there, Hitler orchestrated the battle of France from June 6 and on. Known by the code name Wolfsschlucht, meaning Wolf's Gorge, the site still bears the scars of Hitler's stay and has the original concrete bunker, as well as two reconstructed Bavarian-style chalets in which the Fuhrer stayed. The two chalets are now exhibition centers. One of the buildings has a 20-minute film about Hitler's arrival and photographs charting the German occupation of the area. The other chalet is dedicated to the local resistance effort. Touch screens, videos, films, and educational signs in French, Dutch and English, tell the story of Hitler’s short time here. Numerous objects and accounts from the locals, resistance fighters, and descendants provide locals’ perspectives on the traumatic event. The site is open to the public every day from 10:30 a.m. to 3:30 p.m. Under the streets of La Rochelle, a former wartime bunker holds some surprising secrets. A mammoth WWII anti-air defense tower has been repurposed as a hotel with a rooftop garden. This museum in the small German city offers a unique glimpse into World War II history. Witness to Nazi occupation, this bunker has managed to soldier on to this day. This cone-shaped bomb shelter, modeled on German design, is a rare WWII relic that never saw use but still looms over the harbor. Both the World War II Allied Forces and NATO have used this modern bunker underneath Malta’s capital. At this concrete bunker on the Trondheim fjord, secrets lie above and below the water's surface. These secretive tunnels were once the site of a Royal Air Force fuel reserve depot. Volume 11 - 2020 | https://doi.org/10.3389/fmicb.2020.01524 This article is part of the Research TopicPlasmid Transfer: Mechanisms, Ecology, Evolution, and ApplicationsView all 34 articles Limited data are available on the contribution of wildlife to the spread of antibacterial resistance We determined the prevalence of resistance to antibiotics in Escherichia coli isolates collected from wild animals in 2013 and 2014 and the genetic basis for resistance to third-generation cephalosporin in Guadeloupe We recovered 52 antibiotic-resistant (AR) E coli strains from 48 of the 884 (5.4%) wild animals tested (46 iguanas Rodents had higher rates of carriage (n = 38 10.3%) than reptiles and birds (2.4% and 1.1% A significant association (p < 0.001) was found between the degree of anthropization and the frequency of AR E The carriage rate of ciprofloxacin- and cefotaxime-resistant isolates was 0.7% (6/884) and 1.5% (13/884) and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing E two genetically unrelated isolates being found in one bird These isolates and 20 human invasive ESBL E coli isolates collected in Guadeloupe during the same period were investigated by whole genome sequencing blaCTX–M–1 was the only ESBL gene shared by three animal classes (humans The blaCTX–M–1 gene and most of the antimicrobial resistance genes were present in a large conjugative IncI1 plasmid that was highly similar (>99% nucleotide identity) to ESBL-carrying plasmids found in several countries in Europe and in Australia Although the prevalence of ESBL-producing E coli isolates was very low in wild animals it is of concern that the well-conserved IncI1 plasmid-carrying blaCTX–M–1 is widespread and occurs in various E Most ESBL-producing bacterial pathogens in wild animals are E because of its ubiquity and its ability to acquire the AR gene through mobile genetic elements Rats and mice proliferate on the island because of poor waste management and crops (fruit trees Birds such as bananaquit are also found around dwellings The primary objective of our study was to determine the prevalence of AR E The secondary objectives were to determine the influence of human activities on the spread of AR E to use whole genome sequencing to identify the genetic background of ESBL-producing E coli collected from wild animals and the genetic basis for resistance to 3GC and to compare the genomic features of isolates with those from humans collected during the same period We sampled 884 animals between February 2013 and February 2014 at sites throughout Guadeloupe and nearby islands (Les Saintes, Marie-Galante, La Désirade, and Petite-Terre) identified with the geographical information system Q-GIS (Figure 1) A single cloacal swab was taken from 181 birds at 46 sampling sites and from 289 anoles at 76 sampling sites Feces from 46 iguanas living in colonies were collected at six sampling sites fragments of colon were collected during dissection of 368 rodents trapped live at 35 sampling sites and placed in sterile water Species were identified according to morphological criteria Map of Guadeloupe and location of sampling sites All the procedures were approved by the regional environment and housing agencies and by the Guadeloupe National Park The project was also approved by the Committee for Ethics in animal experiments of the French West Indies and Guyana (references 69-2012-4; 69-2012-6; 69-2012-7) Animals were cared for and used according to the French decree No which meets European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes In order to investigate the association between carriage of AR bacteria and proximity to human activities sampling sites were classified into two groups according to their degree of anthropization with Q-GIS: (i) wilderness with no human presence or countryside with limited human activities and (ii) human-perturbed landscapes with a matrix of agriculture and livestock activities and urban and suburban areas with high levels of human activity Buffered peptone water was added to the samples, which were then shaken manually. After incubation overnight at 37°C, 100 μL of the suspension was inoculated onto three lactose-TTC-agars supplemented with Tergitol-7, each containing a different antibiotic: 2 mg/L of ampicillin, 2 mg/L of cefotaxime, or 1 mg/L of ciprofloxacin. They were incubated at 37°C for 24 h (SFM and EUCAST, 2014) Presumptive Enterobacteriaceae colonies on lactose-TTC-agar (orange colonies Gram-negative bacilli) were isolated randomly and identified by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry on an Axima Performance (Shimadzu Corp. Colony morphology was investigated as a means of screening isolates with different antibiotic susceptibilities Three colonies were identified randomly for each identical morphology Whole genome sequencing was performed at the “Plateforme de microbiologie mutualisée” of the Pasteur International Bioresources Network (Institut Pasteur coli isolates collected from wild animals for this study and on all human ESBL-producing E coli isolates (n = 20) recovered during the same period from patients admitted to the University Hospital of Guadeloupe for community-acquired (n = 4) and nosocomial (n = 16) bloodstream infections Isolates were considered to be community-acquired if recovered by culture from a sample obtained within 48 h after admission The study protocol was approved by the Ethics Committee of the University Hospital of Guadeloupe (reference A5_19_12_05_TRAMID) Previously described polymerase chain reaction (PCR) methods were used to screen for genes encoding plasmid-encoded blaCTX–M, blaTEM, and blaSHV beta-lactamase genes (Guyomard-Rabenirina et al., 2016) Microsoft Access 2003 was used for data entry and Stata Version 10 for statistical analysis the χ2 test (or Fisher’s exact test when appropriate) was used to compare categorical data We considered p-values < 0.05 to be significant Wild animal species sampled and the prevalence of antibiotic resistant- and extended spectrum beta-lactamase producing-Escherichia coli from respective host species from Guadeloupe (French West Indies) coli isolates in wild animals was 0.7% (6/884) 65.4%) were resistant to at least three classes of antibiotics and were thus classified as multi-drug-resistant coli isolates were characterized by high rates of resistance to amoxicillin (n = 46 26.9%) and by moderate rates of resistance to nalidixic acid (n = 11 We recovered 34 resistance phenotypes; the two most frequent were to amoxicillin and ticarcillin (n = 5) and to amoxicillin 8 (0.9% of all 884 animals) were positive in the double-disk synergy test indicating the presence of an ESBL gene; 5 were from 5 rats No ESBL gene was recovered among the six isolates with a negative double-disk synergy test using PCR We observed no significant difference in the number of ESBL-producing E coli according to the degree of anthropization (p = 0.12) probably because few isolates were recovered and extended-spectrum beta-lactamase producing-Escherichia coli according to the level of anthropization Maximum likelihood phylogenetic analysis based on 47 690 single nucleotide polymorphisms (SNPs) in the core genomes (3082 genes, global alignment 1784574 bp) of the 28 ESBL-producing E. coli revealed clustering into three main branches (Figure 2 and Supplementary Figures S1, S2) The first branch consisted mainly of sequence types (STs) ST131 isolates (n = 11) and 1 ST95 and 1 ST1193 isolated from humans; no ST131 E This cluster was the least polymorphic (mean SNP between isolates n = 3747 A second branch grouped four clinical samples (ST349 and 2 ST69) and 1 ST117 isolated from a rat (mean SNP between isolates n = 17363 The last branch consisted of six isolates found in wild animals (ST6914 ST10) and three clinical samples (2 ST410 and ST124) (mean SNP between isolates n = 10422 The isolates from humans and wild animals within each cluster were genetically unrelated and no ST was shared between wild animals and humans Maximum likelihood (ML) phylogenetic tree of extended-spectrum beta-lactamase-producing Escherichia coli isolates based on multiple sequence alignments of the 3082 core genome loci Sequence type (ST) is indicated for each isolate Bootstrap values > 60 are indicated on nodes Hosts and ESBL genes are indicated by vertical colored strips Other antibiotic resistance genes characterized by ResFinder are indicated by black dots; antimicrobial resistance profiles are represented by triangles: gray for resistance and no triangle for susceptibility to corresponding antimicrobial agents Of the isolates carrying blaCTX–M–1 Two ST196 isolates (EC1 and EC7) from birds and rats trapped at different sites were almost identical with four core genome multilocus sequence typing allelic mismatches Venn diagram of the antibiotic resistance genes (A) and of the plasmid incompatibility groups recovered from extended-spectrum beta-lactamase-producing Escherichia coli isolates from humans and wildlife (B) Syntenic analysis of 4 ST3/IncI1/CTX-M1 plasmids versus reference plasmid pESBL20150178 The innermost black ring 1 represents the reference sequence of pEC1 The subsequent rings correspond to GC skew and pairwise comparisons with pESBL20150178 The last 2 rings represent a genetic map of pEC1: antibiotic resistance genes are indicated by red boxes During the search for this type of IncI1/blaCTX–M–1/ST3 plasmid in the PLSDB database, 24 plasmids (from 83 410 to 122 616 bp) sampled only from E. coli were identified that shared >99% nucleotide identity with pESBL20150178. All plasmids were defined as IncI1/blaCTX–M–1/ST3 with the aadA5/dfrA17/sul2 region (Supplementary Table S3) All but one (from Australia) plasmid were recovered in isolates from Europe (Denmark All plasmids except four collected from humans were identified in isolates from livestock a synanthropic species living in urban and periurban areas a commensal species closely linked to people and their movements but which readily establishes itself in undeveloped areas Although most of the AR E. coli were multi-drug resistant, ESBL-producing E. coli were isolated from only 0.9% of the wild animals and especially rodents (5 of the 8 strains). This frequency is in the lower range of values reported previously for any species (Dolejska and Papagiannitsis, 2018) we found the first case of ESBL-producing E Its epidemiological relevance may be even greater coli isolates from animal and human sources Its higher frequency in animals than in humans suggests an animal contribution to the CTX-M-1 reservoir in humans through the spread of this specific IncI1/blaCTX–M–1/ST3 plasmid Further studies should be conducted in Guadeloupe on the role of livestock and domestic animals in its spread to humans the spread of such a gene–plasmid combination in humans and animals will continue despite a reduction in antibiotic use coli carriage in wild animals in Guadeloupe in 2013 and 2014 were recovered our study indicate that well-conserved IncI1/blaCTX–M–1/ST3 plasmids are spread across wide geographical distances and occur in different E The datasets generated for this study can be found in the NCBI with accession number PRJNA600948 The studies involving human participants were reviewed and approved by the Ethics Committee of the University Hospital of Guadeloupe The animal study was reviewed and approved by the Committee for Ethics in animal experiments of the French West Indies and Guyana (references 69-2012-4; 69-2012-6; 69-2012-7) and SB analyzed the data and wrote the manuscript All authors critically revised the manuscript This work has been supported by a FEDER grant, financed by the European Union and Guadeloupe Region [Programme Opérationnel FEDER-Guadeloupe-Conseil Régional 2014–2020, Grant number 2018-FED-1084 (MALIN 2, https://www.projet-malin.fr)] We thank all the technicians and students involved in this work at the Institut Pasteur of Guadeloupe at the University Hospital of Pointe-à-Pitre The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.01524/full#supplementary-material FIGURE S1 | Maximum likelihood (ML) unrooted phylogenetic tree of extended-spectrum beta-lactamase-producing Escherichia coli isolates based on multiple sequence alignments of the 3082 core genome loci FIGURE S2 | Maximum likelihood (ML) phylogenetic tree of extended-spectrum beta-lactamase-producing Escherichia coli isolates based on multiple sequence alignments of the 3082 core genome loci versus plasmidic incompatibility groups Bootstrap values >60 are indicated on nodes Plasmidic incompatibility groups characterized by plasmid finder are indicated by black dots FIGURE S3 | Alignment of IncI1/blaCTX–M–1/ST3 plasmids from the PLSDB database listed in Supplementary Table S3 with the four plasmids sequenced in this study: pEC1 TABLE S1 | Molecular characteristics of extended-spectrum beta-lactamase-producing Escherichia coli isolates from wild animals and humans in Guadeloupe (French West Indies) TABLE S2 | Characteristics of extended-spectrum beta-lactamase-producing Escherichia coli isolates from wild animals and humans in Guadeloupe (French West 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Breurec S (2020) Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single blaCTX–M–1/IncI1/ST3 Plasmid Among Humans and Wild Animals Copyright © 2020 Guyomard-Rabenirina, Reynaud, Pot, Albina, Couvin, Ducat, Gruel, Ferdinand, Legreneur, Le Hello, Malpote, Sadikalay, Talarmin and Breurec. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Stephanie Guyomard-Rabenirina, c2d1eW9tYXJkQHBhc3RldXItZ3VhZGVsb3VwZS5mcg== †These authors have contributed equally to this work The supermarket chain has unveiled plans to open 23 new stores in the country by the end of February 2022 The new stores are expected to create as many as 500 new jobs in the country and the plans are in line with Lidl’s expansion strategy to provide high quality local ingredients and produce to as many customers as possible the company is also renovating a number of existing stores including those located in Wallonia and Brussels Aubange and Tongeren will open 24 February and Lidl will open stores in Sint-Genesius-Rode Lidl believes that their special consideration to local produce has impacted their positive performance in the Belgian market commented: “We pay extra attention to local products Half of our fruit and vegetables and 90 per cent of our meat come from Belgium 100 per cent of our milk and eggs come from our country.” Contact us: info@rli.uk.com Départs : Alex Attardo (Oreye), Abil Kondi (Blegny), Manu Papalino (Vyle-Tharoul), Yannick Pauletti (Sprimont), Arnaud Fransquet ( ?), Kevin Syroit (Libramont), Hugues Praillet (Bas-Oha), David Lambrechts ( ?), Stefano Henrot (Huy), Evans Kondogbia (Sprimont), Alex Magnetico (Cité Sport), Thomas Masselin (Vyle-Tharoul), Taormina, J. 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