Next Article in Journal
Genomic Characterization and Phylogenetic Analysis of SARS-CoV-2 in Libya
Previous Article in Journal
Elucidating the Relations between Gut Bacterial Composition and the Plasma and Fecal Metabolomes of Antibiotic Treated Wistar Rats
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microbiology Research
Volume 12
Issue 1
10.3390/microbiolres12010009
microbiolres-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Brief Report

Multidrug Resistance Dissemination in Escherichia coli Isolated from Wild Animals: Bacterial Clones and Plasmid Complicity

by
Carolina Sabença
1,2,3,
Gilberto Igrejas
2,3,4,
Patrícia Poeta
1,4,
Frédéric Robin
5,6,
Richard Bonnet
5,6 and
Racha Beyrouthy
5,6,*
1
MicroART-Antibiotic Resistance Team, Department of Veterinary Sciences, University of Trá-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
2
Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
3
Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
4
Associated Laboratory for Green Chemistry, University NOVA of Lisbon, 1099-085 Caparica, Portugal
5
Institut National de la Santé et de la Recherche Médicale (UMR 1071), Institut National de la Recherche Agronomique (USC-2018), Université Clermont Auvergne, 63000 Clermont-Ferrand, France
6
Centre National de Référence de la Résistance aux Antibiotiques, Centre de Biologie, Centre Hospitalier Universitaire, 63003 Clermont-Ferrand, France
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2021, 12(1), 123-137; https://doi.org/10.3390/microbiolres12010009
Submission received: 17 December 2020 / Revised: 14 February 2021 / Accepted: 23 February 2021 / Published: 2 March 2021
Browse Figures
Versions Notes

Abstract

:
Objectives. Epidemiological data concerning third-generation cephalosporin (3GC) resistance in wild fauna are scarce. The aim of this study was to characterize the resistance genes, their genetic context, and clonal relatedness in 17 Escherichia coli resistant to 3GC isolated from wild animals. Methods. The isolates were characterized by short-read whole genome sequencing, and long-read sequencing was used for the hybrid assembly of plasmid sequences. Results. The 3GC resistance gene most identified in the isolates was the extended-spectrum β-lactamases (ESBL)-encoding gene blaCTX-M-1 (82.3%), followed by blaCTX-M-32 (5.9%), blaCTX-M-14 (5.9%), and blaSHV-12 (5.9%). E. coli isolates mainly belonged to the sequence types (STs) rarely reported from humans. The single nucleotide polymorphism (SNP)-based typing showed that most E. coli genomes from wild animals (wild boars, birds of prey, and buzzards) formed clonal clusters (<5 SNPs), showing a clonal dissemination crossing species boundaries. blaCTX-M-1-harboring IncI1-ST3 plasmid was the predominant ESBL-encoding plasmid (76.4%) in wild animal isolates. Plasmid comparison revealed a 110-kb self-transferable plasmid consisting of a conserved backbone and two variable regions involved in antimicrobial resistance and in interaction with recipient cells during conjugation. Conclusion. Our results highlighted the unexpected clonal dissemination of blaCTX-M-1-encoding clones and the complicity of IncI1-ST3 plasmid in the spread of blaCTX-M-1 within wild fauna.

1. Introduction

Antibiotics overuse is a key factor in the emergence of antimicrobial resistance (AMR) mechanisms, especially extended-spectrum β-lactamases (ESBL) [1]. ESBLs are enzymes that confer resistance to penicillins and cephalosporins, including third-generation cephalosporins (3GC), classified by the World Health Organization (WHO) as critically important antimicrobial agents in human medicine [2].
Antimicrobial resistance is also a complex and multifaceted threat to humans, animals, and the environment. Advanced knowledge on AMR has demonstrated that it not only affects the human sector but is also widely distributed across animals and the environment [3]. A major cause of the AMR burden is the capability of AMR to transmit within and between individuals, including across sectors. There is a great concern that contacts with animals may enhance the risk of ESC-E/CP-E acquisition by humans [3]. An increase in ESBL-producing bacteria carriage was demonstrated for farmers and employees in contact with broilers [4].
The occurrence of antibiotic-resistant bacteria in wild fauna has also been reported, with the first description of chloramphenicol-resistant Escherichia coli isolates in Japanese wild birds in 1977 [5,6]. The ESBL-producing E. coli isolates from wild animals were first reported in Portugal in 2006 [7]. Since then, the occurrence of ESBL-producing bacteria within diverse animal species, including urban and wild birds with no evident exposure to antimicrobial drugs, has been recently reported across different geographical areas [6,8,9]. On the other hand, wild animals, especially migratory birds, have been identified as potential reservoirs for ESBL, AmpC β-lactamase, carbapenemase, and colistin resistance genes in Enterobacteriaceae [6,10]. Because of their frequent presence in close proximity to human-influenced environments, such as urban areas, landfills, wastewater, and livestock facilities, they can be colonized by resistant bacteria and then disseminate them over long distances, thus constituting a possible transmission factor of resistant bacteria that can contaminate animal food and natural water reservoirs [9,11]. However, few data were collected on the spread of resistant bacteria in wildlife.
CTX-M-type are the most common plasmid-mediated ESBL among Enterobacteriaceae isolates of human and veterinary origin worldwide [12,13,14,15,16]. The CTX-M-1-encoding gene blaCTX-M-1 has been frequently associated with IncI1 and IncN plasmids in Enterobacteriaceae of human and veterinary origin [17,18,19], and it has been mainly associated with IncI1 plasmids in wildlife in Europe [20,21]. In addition, IncI1 plasmids can also carry other ESBL-encoding genes such as blaTEM-52 and blaSHV-12 in wild animals [6,10]. However, genomic data for ESBL-encoding plasmids in wild animals are scarce [22,23].
This study aimed to characterize a collection of 17 ESBL-producing E. coli isolates collected in distinct regions of Portugal from wild animals with different eating habits to analyze the clonal diversity of isolates and the ESBL genetic context. The results showed both an unexpected clonal dissemination of ESBL-producing E. coli and the epidemic spread of ESBL-encoding plasmids in wild fauna.

2. Materials and Methods

2.1. Bacterial Isolates

A total of 17 ESBL-producing E. coli isolates from different species of wild animals with different eating habits living in natural parks and reserves in distinct regions of Portugal were included in this study, ranging from carnivores such as birds of prey (n = 4), buzzards (n = 4), lynx (n = 2), and wolves (n = 2) to omnivores such as wild boar (n = 3) and seagulls (n = 1) and herbivores such as a bat (n = 1) (Table 1) [24,25,26,27,28,29,30].

2.2. Antibiotic Susceptibility Testing

Antimicrobial susceptibility testing was assessed with the disc (Bio-Rad, Marnes-la-Coquette, France) diffusion method according to the European Committee on Antimicrobial Susceptibility Testing guidelines (http://www.eucast.org/ (accessed on 1 December 2020)). The molecules tested were ampicillin (10 µg), amoxicillin-clavulanate combination (20–10 µg), ticarcillin (75 µg), piperacillin (30 µg), piperacillin-tazobactam combination (30–6 µg), cephalexin (30 µg), cefuroxime (30 µg), cefixime (5 µg), cefoxitin (30 µg), cefotaxime (5 µg), ceftazidime (10 µg), cefepime (30 µg), aztreonam (30 µg), ertapenem (10 µg), imipenem (10 µg), mecillinam (10 µg), tobramycin (10 µg), amikacin (30 µg), gentamicin (10 µg), netilmicin (10 µg), nalidixic acid (30 µg), norfloxacin (10 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), trimethoprim (5 µg), trimethoprim-sulfamethoxazole combination (1.25–23.75 µg), temocillin (30 µg), and fosfomycin (200 µg). ESBL production was detected by the double-disc synergy test using cefotaxime, an amoxicillin-clavulanate combination, and ceftazidime (http://www.eucast.org/ (accessed on 1 December 2020)). E. coli ATCC 25,922 was used as a reference strain.

2.3. Whole-Genome Sequencing

Whole-genome sequencing (WGS) was performed using the next-generation sequencing platform of the teaching hospital of Clermont-Ferrand, France. DNA was extracted with a DNeasy UltraClean Microbial kit (Qiagen). The libraries were prepared with a Nextera XT Kit (Illumina, San Diego, CA, USA), and sequenced by the Illumina MiSeq system generating 2 × 301-base pair (bp) paired-end reads. The Fastp v0.19.10 [31] was used for quality filtering of Illumina reads and SPAdes [32] for short reads assembly. The average sequencing depth was ≥75×; the number of assembled contigs ranged between 69 and 154, and genome sizes between 4,567,913 and 5,210,322 nucleotides. The raw reads have been deposited in the European Nucleotide Archive (ENA) under the project accession number PRJEB36175.

Genomic Data Analysis

E. coli phylogroups and multi-locus sequence typing (MLST) were determined in silico according to ClermonTyping method [33] and Achtman’s MLST scheme [34]. The molecular typing of isolates was performed by core genome SNP-based typing (cgSNP) and core genome MLST (cgMLST). BactSNP was used to perform cgSNP using the E. coli EC958 genome [35] as reference (genome size: 5,162,892 bases) as previously described [36,37]. After the filtration of recombination zones detected by Gubbins [38], a phylogenetic tree was inferred from the resulting alignment by maximum likelihood using RAxML [39]. The cgMLST was performed according to the Enterobase cgMLST scheme (https://enterobase.warwick.ac.uk (accessed on 4 February 2021)) and allele calling with package chewBBACA [40]. The antibiotic resistant genes were identified by alignment against a database including the online databases CARD [41], Resfinder [42], and the NCBI National Database of Antibiotic Resistant Organisms (https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/ (accessed on 1 December 2020)) using a 95% minimum threshold for the breadth of coverage and identity percentage, as previously described [43].

2.4. Plasmid Analysis

The plasmid content of bacteria and the size of plasmids were determined using plasmid DNA extracted using the method of Kado and Liu [44]. The PCR-based replicon typing (PBRT) scheme (Diatheva, Fano, Italy) and Southern blots using probes specific for the IncI1, IncN, and IncK replicons and CTX-M-1, CTX-M-14, and SHV-12-encoding genes were used to localize the ESBL-encoding genes in plasmids, as previously described [45]. The PCR-generated probes derived from blaCTX-M-harboring sequenced strains and PBRT kit controls (Diatheva, Fano, Italy). The experimental results were confirmed in silico from WGS using plasmidFinder and the plasmid MLST typing, as previously described [46]. Long reads were obtained from E. coli isolates 2-101 (birds of prey) and J31 (wild boar) using the ligation sequencing kit 1D SQK-LSK109 with the barcoding extension kit EXP-NBD 104 according to the Oxford Nanopore Technologies protocols (Oxford, United Kingdom). The same DNA extract was used for sequencing across Illumina and Nanopore platforms to minimize biological variability introduced in bacterial culture. A flowcell FLO-MIN106 connected to the MinION sequencing device was used to sequence the library during 48 h. Real-time base calling of MinION reads was performed with the MinIT and integrated Guppy software to produce fastQ files. The reads size from Nanopore varied between 120 and 69,617 bases for the J31 dataset (39,647 reads with a median of average quality score per read of Q15—base call accuracy: 97%—and an average size of 11,691 bases corresponding to a total of 463,529,496 bases) and between 107 and 87,164 bases for 2-101 dataset (85,034 reads with a median of average quality score per read of Q15—base call accuracy: 97%—and an average size of 10,090 bases corresponding to a total of 857,958,899 bases). These long reads were de novo assembled with the Illumina short reads using Unicycler v. 0.4.7 [47]. The average sequencing depth of the hybrid assembly was 573× (minimum 139×), and 169× (minimum 30×) for J31 isolate and 370× (minimuFm 89×) and 110× (minimum 20×) for 2-101 isolate from Nanopore and Illumina reads, respectively. Plasmids annotation was performed by the National Center for Biotechnology Information prokaryotic genome annotation pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ (accessed on 4 May 2020), project accession number PRJEB36175).

3. Results

3.1. Resistance Phenotype and Resistome

The E. coli isolates exhibited a positive synergy test and resistance to penicillin and cephalosporins, including the oxyimino-cephalosporins cefotaxime or ceftazidime, in agreement with ESBL production. A total of 41% of the isolates were resistant to all quinolones, 76.4% to trimethoprim, and 82.3% to sulphonamides. All the tested isolates were susceptible to amikacin, and only one isolate was resistant to gentamicin. The antimicrobial susceptibility profiles are listed in Supplementary Table S1.
WGS analysis detected ESBL-encoding genes in all isolates (Table 1). The predominant ESBL gene was blaCTX-M-1 (82.3%), followed by the point variants blaCTX-M-32 (5.9%), blaCTX-M-14 (5.9%), and blaSHV-12 (5.9%). Analysis of gyrA and parC mutations responsible for quinolone resistance showed the predominance of the double mutations gyrA (S83L and D87N) associated with parC mutation (S80I; 35.3%). The gene qnrS1 that decreases quinolone susceptibility was identified in 23.5% of the isolates. The most prevalent aminoglycoside resistance genes were the genes aadA (aadA1, aadA2, and aadA5), strA, and strB that affect susceptibility to streptomycin. Only one gene known to affect the susceptibility to gentamicin was detected (aac(3)-VIa; 5.9%), and none affecting amikacin were detected. Overall, the content in resistance genes agreed with the resistance phenotype.

3.2. Whole-Genome Typing of E. coli Isolates

Most E. coli isolates belonged to phylogroup A (41.2%); the others belonged to phylogroups C (29.4%), B1 (23.5%), and F (5.9%). Nine STs were identified among the isolates, and 70% of the wild animal isolates were clustered in four STs: ST48 (n = 4), ST1998 (n = 4), ST1800 (n = 2) and ST155 (n = 2) (Table 1). None of these isolates belonged to the predominant STs that carry ESBLs in humans [48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64], suggesting that the dissemination of ESBLs in wild animals is not due to human strains.
The core genome alignment consisted of 3,499,887 bp, with the isolates differing from each other by 0 to 77,550 SNPs. Three clonal clusters comprising 2 to 4 isolates differed from each other by less than 5 SNPs, while the others diverged by 1831 to 77,550 SNPs (Figure 1a). To provide an unbiased phylogeny, recombination zones were removed. The resulting alignment consisted of 568,547 nucleotides with 0 or 1 SNP for the clonal isolates (≤5 SNPs) and of 211 to 32,758 SNPs for the other isolates. The clonality relationships were also inferred by a cgMLST scheme including 2513 conserved loci (Figure 1b). In agreement with the cgSNP approach, the isolates differed from each other by 0 or 3 alleles for the clonal isolates (≤5 alleles) and by 258 to 2375 alleles for the other isolates (Figure 1b). The clonal clusters belonged to ST48, ST1800, and ST1198, and corresponding isolates were isolated from carnivore birds and a wild boar. These results reveal the clonal dissemination of ESBL-producing E. coli strains for 10 out of 17 isolates in wild fauna.

3.3. CTX-M-1-Encoding Plasmids

Hybridization experiments revealed that ~110-kb IncI1 plasmids were mainly associated with blaCTX-M-1 (n = 13) and, to a lesser extent, with blaSHV-12 (n = 1). Furthermore, blaCTX-M-1 and blaCTX-M-32 were also localized on ~50-kb IncN plasmid in isolates GV23 and W151, respectively. The probe specific for the blaCTX-M-14 gene hybridized with a ~90-kb IncK plasmid in L16 isolate. The in silico analysis of WGS performed with plasmidFinder identified seven replicons among the isolates, with IncF and IncI1-ST3 replicons among the predominant ones (88.2%). The experimental and in silico plasmid analysis results are listed in the Supplementary Table S2.
The genetic context of the predominant ESBL-encoding gene blaCTX-M-1 was further investigated. Based on short-read mapping and WGS assembly, blaCTX-M-1 was localized in large contigs (size ranged between 38,271–88,238 bp), including the replicon origin IncI1 (60%) or at least a fragment of the region previously called shufflon that is specific to IncI1 plasmids [65,66]. These data agreed with the hybridization experiments and the detection of plasmid IncI1-ST3 in the 13 blaCTX-M-1 isolates. The mapping of corresponding reads against IncI1 reference plasmid R64 confirmed the results and showed a highly similar structure of IncI1-ST3 plasmids except in two variable regions, including the shufflon.
While the ESBL-producing plasmid IncI1-ST3 has already been sequenced in strains isolated from human and human-influenced habitats [67,68], data that have been collected from wild fauna are scarce [23]. The isolates J31 (wild boar) and 2-101 (bird of prey) were, therefore, sequenced to closure by Nanopore sequencing. The hybrid assembly of the J31 dataset generated five circular contigs (sizes: 4871 kb, 50 kb, 108 kb, 126 kb, and 160 kb), including the complete and circular IncI1-ST3 plasmid pJ31 harboring blaCTX-M-1 (108,661 bases, accession number: CP053788). The hybrid assembly of the 2-101 dataset generated a linear contig (size: 150 kb) and two circular contigs (sizes: 4759 kb and 108 kb), including complete and circular IncI1-ST3 plasmid p2-101 harboring blaCTX-M-1 (108,661 bases, accession number: CP053786).
These data confirmed the presence of two variable regions. The first variable region was located downstream of the gene repZ and flanked by an ISSbo1 and ISVsa3, two IS91-like elements in plasmids p2-101 and pJ31. This region (12.725 kb) harbored a class 1 integrase gene int1, two resistance gene cassettes (dfrA17 and aadA5), IS26, and truncated IS5075 insertion sequences and sul2 (Figure 2a,b), as previously reported IncI1-ST3 plasmids (e.g., KJ484638, MH846978, and LT985235), but was absent in plasmid of wolf’s strain W4 (Table 1).
The second variable region, the shufflon, was located between the conserved genes rci and pilVA and usually consisted of four segments A to D randomly rearranged by the activity of the rci-encoded recombinase [69]. In contrast to IncI1 reference plasmid R64, shufflon segment D was absent in the InI1-ST3 plasmids, and the remaining segments exhibited different organizations in the circularized plasmids p2-101 and pJ31 (Figure 2b). An additional level of rearrangement was provided by the presence of insertion sequence ISEcp1 associated with blaCTX-M-1. The element ISEcp1-blaCTX-M-1, which is well-known to be mobile, was flanked by direct repeats (TTTTTA) and was inserted in the same site within the shufflon segment B. However, this block ISEcp1-blaCTX-M-1-B exhibited two different orientations in plasmids p2-101 and pJ31 (Figure 2b). Since the shufflon segments are involved in the synthesis of PilV adhesin of the type IV conjugative pilus, the insertion of ISEcp1-blaCTX-M-1 associated with the shuffling of segments could provide diversity in PilV adhesin and, therefore, modify the recognition and the binding of recipient cell during IncI1-ST3 conjugation [65]. The resistance element ISEcp1-blaCTX-M-1 could thereby modulate the transfer efficiency of IncI1-ST3 blaCTX-M-1 plasmids and may be, therefore, involved their successful spread in a broad diversity of E. coli lineages.

4. Discussion

This work aimed to analyze at the genomic level ESBL-producing E. coli collected from wild animals with different eating habits and living in natural parks and reserves or other rural conservation areas in distinct regions of Portugal [24,25,26,27,28,29,30]. All ESBL-producing E. coli strains were resistant to penicillin, cephalosporin 1st generation (cephalexin), and cefuroxime and 94% were resistant to cefotaxime. The isolates also exhibited associated resistances to quinolones, fluoroquinolones, sulfonamides, and trimethoprim, showing that multidrug-resistant bacteria have emerged in wild birds, as previously reported [6], and in a broad range of other wildlife species.
The blaCTX-M-1 gene was the predominant ESBL-encoding gene (82.3%), followed by blaCTX-M-14 (5.9%), and blaCTX-M-32 (5.9%). In addition to blaCTX-M genes, we detected the blaSHV-12 gene, previously reported in isolates from humans, livestock, companion animals, and wild birds [6,70,71,72,73]. We also detected genes blaOXA-1 and blaTEM-1, which have been reported in waterbirds on the Baltic sea coast of Poland [20] and from wild boars and Barbary macaques [74], respectively. Except for the absence of blaCTX-M-15, these results were also consistent with those previously reported from wild birds [6], in which the blaCTX-M-1 and blaCTX-M-15 genes were the most widespread ESBL genes, followed by blaCTX-M-14 and blaCTX-M-32. The blaCTX-M-15 gene is the predominant ESBL in humans [6]. The absence of blaCTX-M-15 in our study may reflect a weak anthropization of the wild animals included in this study. Previous reports identified the impact of the human-influenced habitat in the diffusion of the antimicrobial resistance determinant in the wildlife, especially in urban areas where wild birds can live and feed [6,10,75]. Our isolates were collected from natural parks and reserves or other rural conservation areas [24,25,26,27,28,29,30], which are less influenced by human activity than urban areas.
E. coli ST131 and ST405 are the major STs associated with the rapid worldwide rise in blaCTX-M genes, including blaCTX-M-15 [76] and other CTX-M genes, such as blaCTX-M-14 and blaCTX-M-1 [77]. None of our blaCTX-M-positive isolates belonged to ST131 or ST405, showing that the dissemination of blaCTX-M genes in our isolates is not related to well-known clonal strains, in contrast to previous reports about wildlife [6,10]. Furthermore, E. coli isolates from this study essentially belonged to novel STs (ST1998, ST1800, ST10430), STs rarely reported in human and human-influenced habitats (ST361, ST155, ST117, ST710) and/or STs reported in wild animals (ST117 and ST155) [10]. A striking finding from this study is the clonal dissemination of ESBL-producing E. coli in wild fauna. The isolates belonged to ST48, ST1800, and ST1198 formed clusters that diverged by less than 5 SNPs, suggesting direct and/or indirect transmissions between wild animals. This contrasts with data obtained from human isolates, in which ESBLs are often observed in distantly related E. coli isolates [48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64]. Overall, the genetic background of isolates suggests that ESBL spread in the wild animals of this study is not directly linked to contacts with humans or human-influenced habitats and is marked by direct or indirect transmission within wild fauna despite a probable low antibiotic selection pressure.
Consequently, anthropization is probably not the main spreading factor of blaCTX-M-1, which was observed in five different species (bird of prey, bat, wolf, buzzard, wild boar) out of the seven wild animal species included in this study. 92.8% of the gene blaCTX-M-1 was encoded by the Incl1-ST3 plasmid. Incl1 is one of the most common plasmid families in Enterobacteriaceae, in both animals and humans [6,10,78], and one of the major plasmids contributing to the dissemination of the blaCTX-M-1 gene in animals, the environment, and humans [79]. On the Baltic coast of Poland, ESBL-producing bacteria isolated from waterbirds predominantly had the blaCTX-M-1 gene located on plasmid Incl1 [20]. Similarly, in the Netherlands, the blaCTX-M-1 gene was carried by Incl1 plasmids among E. coli isolates from several wild birds [21]. This association was also more recently detected in ESBL-producing E. coli from various wild animals isolated in Guadeloupe [23]. The identification of the epidemic plasmids IncI1-ST3 encoding blaCTX-M-1 in samples of different sources, including humans, animals, and the environment, further underlined the role of horizontal transfer in the dissemination of resistance genes [80]. Since the IncI1 shufflon segments are involved in the synthesis of PilV adhesin of the type IV conjugative pilus, the insertion of ISEcp1-blaCTX-M-1 associated with the shuffling of segments could modify the recognition and the binding of recipient cells during IncI1-ST3 conjugation [65], modulate the transfer efficiency of IncI1-ST3 blaCTX-M-1 plasmids, and may be, therefore, involved in their successful spread in a broad diversity of E. coli lineages even in an antibiotic-free ecosystem.

5. Conclusions

It emerges from this study that the clonal dissemination of ESBL-producing E. coli strains in wild fauna crosses the species barriers. It is worth noting the moderate diversity of ESBLs detected in our E. coli isolates. The blaCTX-M-1 gene, associated with tetracyclines, fluoroquinolones, aminoglycosides, sulphonamides, and trimethoprim-encoding resistance genes, was the predominant resistance mechanism observed in our isolates, which belonged to unusual E. coli lineages collected from wild animals. Our results also underline the key role of the epidemic plasmid IncI1-ST3 in the diffusion of CTX-M-1 in areas with low or no antibiotic exposure and weak anthropization. The present study is a step forward to understanding the dissemination of antibiotic resistance in the wild ecosystem. More surveillance programs focusing on the spread of antibiotic resistance mechanisms at the interface of ecosystems, including the wildlife, are needed to better assess the emergence of antibiotic resistance in the context of the “one health” approach.

Supplementary Materials

The following are available online at https://www.mdpi.com/2036-7481/12/1/9/s1, Table S1: Antimicrobial susceptibility profiles of Escherichia coli isolates collected from different wild animals., Table S2: Plasmid analysis of Escherichia coli isolates based on experimental and whole genome sequencing results.

Author Contributions

Conceptualization, R.B. (Racha Beyrouthy), G.I., and R.B. (Richard Bonnet); methodology, R.B. (Racha Beyrouthy), R.B. (Richard Bonnet); software, R.B. (Racha Beyrouthy).; validation, R.B. (Richard Bonnet), G.I., F.R., P.P., R.B. (Racha Beyrouthy); formal analysis, R.B. (Richard Bonnet), G.I., F.R., P.P., R.B. (Racha Beyrouthy); investigation, C.S., R.B. (Racha Beyrouthy); resources, R.B. (Richard Bonnet), G.I., data curation, R.B. (Racha Beyrouthy); writing—original draft preparation R.B. (Racha Beyrouthy), C.S., R.B. (Richard Bonnet), G.I., writing—review and editing, R.B. (Richard Bonnet), G.I., F.R., R.B. (Racha Beyrouthy); supervision, R.B. (Racha Beyrouthy), R.B. (Richard Bonnet), G.I.; project administration, R.B. (Racha Beyrouthy)., R.B. (Richard Bonnet), G.I.; funding acquisition, R.B. (Racha Beyrouthy), R.B. (Richard Bonnet), G.I., P.P. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Ministère de la Recherche et de la Technologie, Inserm (UMR 1071), INRAe (USC-2018), Santé Publique France; the Centre Hospitalier Regional Universitaire de Clermont-Ferrand; the French government’s IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25); and the JPIAMR program TransComp-ESC-R of the European Commission. We also thank the ‘Espectrometria de massa e sequenciação aplicadas à microbiologia clínica moderna: pesquisa e identificação de biomarcadores em espécies bacterianas multiresistentes’ (Programa de Acções Universitárias Integradas Luso-Francesas, Ação n°: TC-14/2017) and the Associate Laboratory for Green Chemistry—LAQV, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The Illumina raw sequence reads have been deposited in the European Nucleotide Archive under the project accession number PRJEB36175. The pJ31 and p2-101 complete plasmid sequence were deposited in GenBank under the accession number CP053788 and CP053786, respectively.

Acknowledgments

We thank Alexis Pontvianne and Lucie Pourpuech for their technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Roca, I.; Akova, M.; Baquero, F.; Carlet, J.; Cavaleri, M.; Coenen, S.; Cohen, J.; Findlay, D.; Gyssens, I.; Heure, O.E.; et al. The global threat of antimicrobial resistance: Science for intervention. New Microbes New Infect. 2015, 6, 22–29. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. WHO. WHO List of Critically Important Antimicrobials for Human Medicine (WHO CIA List); 5th revision; World Health Organization: Geneva, Switzerland, 2017. [Google Scholar]
  3. Hernando-Amado, S.; Coque, T.M.; Baquero, F.; Martínez, J.L. Defining and combating antibiotic resistance from One Health and Global Health perspectives. Nat. Microbiol. 2019, 4, 1432–1442. [Google Scholar] [CrossRef]
  4. Huijbers, P.M.C.; Graat, E.A.M.; Haenen, A.P.J.; van Santen, M.G.; van Essen-Zandbergen, A.; Mevius, D.J.; van Duijkeren, E.; van Hoek, A.H.A.M. Extended-spectrum and AmpC β-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: Prevalence, risk factors and molecular characteristics. J. Antimicrob. Chemother. 2014, 69, 2669–2675. [Google Scholar] [CrossRef] [PubMed]
  5. Sato, G.; Oka, C.; Asagi, M.; Ishiguro, N. Detection of conjugative R plasmids conferring chloramphenicol resistance in Escherichia coli isolated from domestic and feral pigeons and crows. Zent. Bakteriol. Parasitenkd. Infekt. Hyg. 1978, 241, 407–417. [Google Scholar] [PubMed]
  6. Wang, J.; Ma, Z.B.; Zeng, Z.L.; Yang, X.W.; Huang, Y.; Liu, J.H. The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes. Zool. Res. 2017, 38, 55–80. [Google Scholar] [CrossRef] [Green Version]
  7. Costa, D.; Poeta, P.; Sáenz, Y.; Vinué, L.; Rojo-Bezares, B.; Jouini, A.; Zarazaga, M.; Rodrigues, J.; Torres, C. Detection of Escherichia coli harbouring extended-spectrum β-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J. Antimicrob. Chemother. 2006, 58, 1311–1312. [Google Scholar] [CrossRef] [PubMed]
  8. Poirel, L.; Potron, A.; De La Cuesta, C.; Cleary, T.; Nordmann, P.; Munoz-Price, L.S. Wild coastline birds as reservoirs of broad-spectrum-β-lactamase- producing Enterobacteriaceae in Miami Beach, Florida. Antimicrob. Agents Chemother. 2012, 56, 2756–2758. [Google Scholar] [CrossRef] [Green Version]
  9. Stedt, J.; Bonnedahl, J.; Hernandez, J.; Waldenström, J.; McMahon, B.J.; Tolf, C.; Olsen, B.; Drobni, M. Carriage of CTX-M type extended spectrum β-lactamases (ESBLs) in gulls across Europe. Acta Vet. Scand. 2015, 57, 4–11. [Google Scholar] [CrossRef] [Green Version]
  10. Dolejska, M.; Papagiannitsis, C.C. Plasmid-mediated resistance is going wild. Plasmid 2018, 99, 99–111. [Google Scholar] [CrossRef]
  11. Greig, J.; Rajić, A.; Young, I.; Mascarenhas, M.; Waddell, L.; Lejeune, J. A scoping review of the role of wildlife in the transmission of bacterial pathogens and antimicrobial resistance to the food chain. Zoonoses Public Health 2015, 62, 269–284. [Google Scholar] [CrossRef]
  12. Haenni, M.; Beyrouthy, R.; Lupo, A.; Châtre, P.; Madec, J.Y.; Bonnet, R. Epidemic spread of Escherichia coli ST744 isolates carrying mcr-3 and blaCTX-M-55 in cattle in France. J. Antimicrob. Chemother. 2018, 73, 533–536. [Google Scholar] [CrossRef]
  13. Robin, F.; Beyrouthy, R.; Bonacorsi, S.; Aissa, N.; Bret, L.; Brieu, N.; Cattoir, V.; Chapuis, A.; Chardon, H.; Degand, N.; et al. Inventory of Extended-Spectrum-B- Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study. Antimicrob. Agents Chemother. 2017, 61, e01911-16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Wellington, E.M.H.; Boxall, A.B.A.; Cross, P.; Feil, E.J.; Gaze, W.H.; Hawkey, P.M.; Johnson-Rollings, A.S.; Jones, D.L.; Lee, N.M.; Otten, W.; et al. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. Lancet Infect. Dis. 2013, 13, 155–165. [Google Scholar] [CrossRef]
  15. Pietsch, M.; Eller, C.; Wendt, C.; Holfelder, M.; Falgenhauer, L.; Fruth, A.; Grössl, T.; Leistner, R.; Valenza, G.; Werner, G.; et al. Molecular characterisation of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from hospital and ambulatory patients in Germany. Vet. Microbiol. 2017, 200, 130–137. [Google Scholar] [CrossRef]
  16. Liu, X.; Thungrat, K.; Boothe, D.M. Occurrence of oxa-48 carbapenemase and other β-lactamase genes in esbl-producing multidrug resistant: Escherichia coli from dogs and cats in the united states, 2009–2013. Front. Microbiol. 2016, 7. [Google Scholar] [CrossRef] [Green Version]
  17. Madec, J.Y.; Haenni, M.; Métayer, V.; Saras, E.; Nicolas-Chanoine, M.H. High prevalence of the animal-associated blaCTX-M-1 IncI1/ST3 plasmid in human Escherichia coli isolates. Antimicrob. Agents Chemother. 2015, 59, 5860–5861. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Jakobsen, L.; Bortolaia, V.; Bielak, E.; Moodley, A.; Olsen, S.S.; Hansen, D.S.; Frimodt-Møller, N.; Guardabassi, L.; Hasman, H. Limited similarity between plasmids encoding CTX-M-1 β-lactamase in Escherichia coli from humans, pigs, cattle, organic poultry layers and horses in Denmark. J. Glob. Antimicrob. Resist. 2015, 3, 132–136. [Google Scholar] [CrossRef] [PubMed]
  19. Carattoli, A. Resistance plasmid families in Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 2227–2238. [Google Scholar] [CrossRef] [Green Version]
  20. Literak, I.; Dolejska, M.; Janoszowska, D.; Hrusakova, J.; Meissner, W.; Rzyska, H.; Bzoma, S.; Cizek, A. Antibiotic-resistant Escherichia coli bacteria, including strains with genes encoding the extended-spectrum beta-lactamase and QnrS, in waterbirds on the baltic sea coast of Poland. Appl. Environ. Microbiol. 2010, 76, 8126–8134. [Google Scholar] [CrossRef] [Green Version]
  21. Veldman, K.; van Tulden, P.; Kant, A.; Testerink, J.; Mevius, D. Characteristics of cefotaxime-resistant Escherichia coli from wild birds in the Netherlands. Appl. Environ. Microbiol. 2013, 79, 7556–7561. [Google Scholar] [CrossRef] [Green Version]
  22. Schaufler, K.; Nowak, K.; Düx, A.; Semmler, T.; Villa, L.; Kourouma, L.; Bangoura, K.; Wieler, L.H.; Leendertz, F.H.; Guenther, S. Clinically relevant ESBL-producing K. pneumoniae ST307 and E. coli ST38 in an urban West African rat population. Front. Microbiol. 2018, 9, 150. [Google Scholar] [CrossRef] [PubMed]
  23. Guyomard-Rabenirina, S.; Reynaud, Y.; Pot, M.; Albina, E.; Couvin, D.; Ducat, C.; Gruel, G.; Ferdinand, S.; Legreneur, P.; Le Hello, S.; et al. Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single blaCTX–M–1/IncI1/ST3 Plasmid Among Humans and Wild Animals. Front. Microbiol. 2020, 11, 1–11. [Google Scholar] [CrossRef] [PubMed]
  24. Poeta, P.; Radhouani, H.; Igrejas, G.; Gonçalves, A.; Carvalho, C.; Rodrigues, J.; Vinué, L.; Somalo, S.; Torres, C. Seagulls of the Berlengas natural reserve of Portugal as carriers of fecal Escherichia coli harboring CTX-M and TEM extended-spectrum beta-lactamases. Appl. Environ. Microbiol. 2008, 74, 7439–7441. [Google Scholar] [CrossRef] [Green Version]
  25. Radhouani, H.; Pinto, L.; Coelho, C.; Gonçalves, A.; Sargo, R.; Torres, C.; Igrejas, G.; Poeta, P. Detection of Escherichia coli harbouring extended-spectrum β-lactamases of the CTX-M classes in faecal samples of common buzzards (Buteo buteo). J. Antimicrob. Chemother. 2010, 65, 171–173. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Gonçalves, A.; Igrejas, G.; Radhouani, H.; Estepa, V.; Alcaide, E.; Zorrilla, I.; Serra, R.; Torres, C.; Poeta, P. Detection of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of Iberian lynx. Lett. Appl. Microbiol. 2012, 54, 73–77. [Google Scholar] [CrossRef]
  27. Pinto, L.; Radhouani, H.; Coelho, C.; Da Costa, P.M.; Simões, R.; Brandão, R.M.L.; Torres, C.; Igrejas, G.; Poeta, P. Genetic detection of extended-spectrum β-lactamase-containing Escherichia coli isolates from birds of prey from Serra da Estrela natural reserve in Portugal. Appl. Environ. Microbiol. 2010, 76, 4118–4120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Gonçalves, A.; Igrejas, G.; Radhouani, H.; Estepa, V.; Pacheco, R.; Monteiro, R.; Brito, F.; Guerra, A.; Petrucci-Fonseca, F.; Torres, C.; et al. Iberian Wolf as a Reservoir of Extended-Spectrum β-Lactamase-Producing Escherichia coli of the TEM, SHV, and CTX-M Groups. Microb. Drug Resist. 2012, 18, 215–219. [Google Scholar] [CrossRef]
  29. Poeta, P.; Radhouani, H.; Pinto, L.; Martinho, A.; Rego, V.; Rodrigues, R.; Gonçalves, A.; Rodrigues, J.; Estepa, V.; Torres, C.; et al. Wild boars as reservoirs of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli of different phylogenetic groups. J. Basic Microbiol. 2009, 49, 584–588. [Google Scholar] [CrossRef] [PubMed]
  30. Garcês, A.; Correia, S.; Amorim, F.; Pereira, J.E.; Igrejas, G.; Poeta, P. First report on extended-spectrum beta-lactamase (ESBL) producing Escherichia coli from European free-tailed bats (Tadarida teniotis) in Portugal: A one-health approach of a hidden contamination problem. J. Hazard. Mater. 2017, 370, 219–224. [Google Scholar] [CrossRef]
  31. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef]
  32. Nurk, S.; Bankevich, A.; Antipov, D.; Gurevich, A.A.; Korobeynikov, A.; Lapidus, A.; Prjibelski, A.D.; Pyshkin, A.; Sirotkin, A.; Sirotkin, Y.; et al. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. J. Comput. Mol. Cell Biol. 2013, 20, 714–737. [Google Scholar] [CrossRef] [Green Version]
  33. Beghain, J.; Bridier-Nahmias, A.; Le Nagard, H.; Denamur, E.; Clermont, O. ClermonTyping: An easy-to-use and accurate in silico method for Escherichia genus strain phylotyping. Microb. Genom. 2018, 4. [Google Scholar] [CrossRef] [PubMed]
  34. Wirth, T.; Falush, D.; Lan, R.; Colles, F.; Mensa, P.; Wieler, L.H.; Karch, H.; Reeves, P.R.; Maiden, M.C.J.; Ochman, H.; et al. Sex and virulence in Escherichia coli: An evolutionary perspective. Mol. Microbiol. 2006, 60, 1136–1151. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Totsika, M.; Beatson, S.A.; Sarkar, S.; Phan, M.D.; Petty, N.K.; Bachmann, N.; Szubert, M.; Sidjabat, H.E.; Paterson, D.L.; Upton, M.; et al. Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: Genome analysis and virulence mechanisms. PLoS ONE 2011, 6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Phan, M.D.; Peters, K.M.; Sarkar, S.; Lukowski, S.W.; Allsopp, L.P.; Moriel, D.G.; Achard, M.E.S.; Totsika, M.; Marshall, V.M.; Upton, M.; et al. The Serum Resistome of a Globally Disseminated Multidrug Resistant Uropathogenic Escherichia coli Clone. PLoS Genet. 2013, 9. [Google Scholar] [CrossRef] [Green Version]
  37. Ben Zakour, N.L.; Alsheikh-Hussain, A.S.; Ashcroft, M.M.; Khanh Nhu, N.T.; Roberts, L.W.; Stanton-Cook, M.; Schembri, M.A.; Beatsona, S.A. Sequential acquisition of virulence and fluoroquinolone resistance has shaped the evolution of Escherichia coli ST131. mBio 2016, 7, 1–11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Croucher, N.J.; Page, A.J.; Connor, T.R.; Delaney, A.J.; Keane, J.A.; Bentley, S.D.; Parkhill, J.; Harris, S.R. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015, 43, e15. [Google Scholar] [CrossRef] [Green Version]
  39. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bionformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef] [PubMed]
  40. Silva, M.; Machado, M.P.; Silva, D.N.; Rossi, M.; Moran-Gilad, J.; Santos, S.; Ramirez, M.; Carriço, J.A. chewBBACA: A complete suite for gene-by-gene schema creation and strain identification. Microb. Genom. 2018, 4, 1–7. [Google Scholar] [CrossRef] [PubMed]
  41. Jia, B.; Raphenya, A.R.; Alcock, B.; Waglechner, N.; Guo, P.; Tsang, K.K.; Lago, B.A.; Dave, B.M.; Pereira, S.; Sharma, N.; et al. CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017, 45, 566–573. [Google Scholar] [CrossRef] [PubMed]
  42. Zankari, E.; Hasman, H.; Cosentino, S.; Vestergaard, M.; Rasmussen, S.; Lund, O.; Aarestrup, F.M.; Larsen, M.V. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 2012, 67, 2640–2644. [Google Scholar] [CrossRef] [PubMed]
  43. Beyrouthy, R.; Barets, M.; Marion, E.; Dananché, C.; Dauwalder, O.; Robin, F.; Gauthier, L.; Jousset, A.; Dortet, L.; Guérin, F.; et al. Novel enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains. Emerg. Infect. Dis. 2018, 24, 1505–1515. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Kado, C.I.; Liu, S.T. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 1981, 145, 1365–1373. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Beyrouthy, R.; Robin, F.; Fouad, D.; Mallat, H.; Hamze, M.; Bonnet, R. Carbapenemase and virulence factors of Enterobacteriaceae in North Lebanon between 2008 and 2012: Evolution via endemic spread of OXA-48. J. Antimicrob. Chemother. 2014, 69, 2699–2705. [Google Scholar] [CrossRef] [PubMed]
  46. Carattoli, A.; Zankari, E.; García-fernández, A.; Larsen, V.; Lund, O.; Villa, L.; Aarestrup, M.; Hasman, H. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing. Antimicrob. Agents Chemother. 2014, 58, 3895–3903. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Wick, R.R.; Judd, L.M.; Gorrie, C.L.; Holt, K.E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 2017, 13, 1–22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Ahmed, S.F.; Ali, M.M.M.; Mohamed, Z.K.; Moussa, T.A.; Klena, J.D. Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community. Ann. Clin. Microbiol. Antimicrob. 2014, 13, 1–8. [Google Scholar] [CrossRef] [Green Version]
  49. Nicolas-chanoine, M.H.; Gruson, C.; Bialek-Davenet, S.; Bertrand, X.; Thomas-Jean, F.; Bert, F.; Moyat, M.; Meiller, E.; Marcon, E.; Danchin, N.; et al. 10-fold increase (2006-11) in the rate of healthy subjects with extended-spectrum β-lactamase-producing Escherichia coli faecal carriage in a parisian check-up centre. J. Antimicrob. Chemother. 2013, 68, 562–568. [Google Scholar] [CrossRef]
  50. Aibinu, I.; Odugbemi, T.; Koenig, W.; Ghebremedhin, B. Sequence Type ST131 and ST10 Complex (ST617) predominant among CTX-M-15-producing Escherichia coli isolates from Nigeria. Clin. Microbiol. Infect. 2012, 18, E49–E51. [Google Scholar] [CrossRef] [Green Version]
  51. Mshana, S.E.; Imirzalioglu, C.; Hain, T.; Domann, E.; Lyamuya, E.F.; Chakraborty, T. Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary hospital in Tanzania. Clin. Microbiol. Infect. 2011, 17, 1279–1282. [Google Scholar] [CrossRef] [Green Version]
  52. Van Der Bij, A.K.; Peirano, G.; Goessens, W.H.F.; Van Der Vorm, E.R.; Van Westreenen, M.; Pitout, J.D.D. Clinical and molecular characteristics of extended-spectrum-β- lactamase-producing Escherichia coli causing bacteremia in the Rotterdam Area, Netherlands. Antimicrob. Agents Chemother. 2011, 55, 3576–3578. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Correia, S.; Pacheco, R.; Radhouani, H.; Diniz, J.C.; Ponce, P.; Jones-Dias, D.; Caniça, M.; Igrejas, G.; Poeta, P. High prevalence of ESBL-producing Escherichia coli isolates among hemodialysis patients in Portugal: Appearance of ST410 with the blaCTX-M-14 gene. Diagn. Microbiol. Infect. Dis. 2012, 74, 423–425. [Google Scholar] [CrossRef]
  54. Horner, C.; Fawley, W.; Morris, K.; Parnell, P.; Denton, M.; Wilcox, M. Escherichia coli bacteraemia: 2 years of prospective regional surveillance (2010-12). J. Antimicrob. Chemother. 2014, 69, 59–66. [Google Scholar] [CrossRef] [Green Version]
  55. Wu, G.; Day, M.J.; Mafura, M.T.; Nunez-Garcia, J.; Fenner, J.J.; Sharma, M.; van Essen-Zandbergen, A.; Rodríguez, I.; Dierikx, C.; Kadlec, K.; et al. Comparative Analysis of ESBL-Positive Escherichia coli Isolates from Animals and Humans from the UK, The Netherlands and Germany. PLoS ONE 2013, 8, 1–10. [Google Scholar] [CrossRef] [Green Version]
  56. Coque, T.M.; Novais, A.; Carattoli, A.; Poirel, L.; Pitout, J.; Peixe, L.; Baquero, F.; Canton, R.; Nordmann, P. Dissemination of clonally related E.coli strains expressing ESBL CTX-M-15. Emerg. Infect. Dis. 2008, 14, 195–200. [Google Scholar] [CrossRef]
  57. Doi, Y.; Park, Y.S.; Rivera, J.I.; Adams-Haduch, J.M.; Hingwe, A.; Sordillo, E.M.; Lewis, J.S.; Howard, W.J.; Johnson, L.E.; Polsky, B.; et al. Community-associated extended-spectrum -lactamase-producing Escherichia coli infection in the United States. Clin. Infect. Dis. 2013, 56, 641–648. [Google Scholar] [CrossRef]
  58. Cao, X.; Cavaco, L.M.; Lv, Y.; Li, Y.; Zheng, B.; Wang, P.; Hasman, H.; Liu, Y.; Aarestrup, F.M. Molecular characterization and antimicrobial susceptibility testing of Escherichia coli isolates from patients with urinary tract infections in 20 Chinese Hospitals. J. Clin. Microbiol. 2011, 49, 2496–2501. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Matsumura, Y.; Yamamoto, M.; Nagao, M.; Hotta, G.; Matsushima, A.; Ito, Y.; Takakura, S.; Ichiyama, S. Emergence and spread of B2-st131-o25b, B2-st131-o16 and d-st405 clonal groups among extended-spectrum-β-lactamase-producing escherichia coli in Japan. J. Antimicrob. Chemother. 2012, 67, 2612–2620. [Google Scholar] [CrossRef] [Green Version]
  60. Rodrigues, C.; Machado, E.; Fernandes, S.; Peixe, L.; Novais, Â. An update on faecal carriage of ESBL-producing Enterobacteriaceae by Portuguese healthy humans: Detection of the H30 subclone of B2-ST131 Escherichia coli producing CTX-M-27. J. Antimicrob. Chemother. 2016, 71, 1120–1122. [Google Scholar] [CrossRef] [Green Version]
  61. Blanco, J.; Mora, A.; Mamani, R.; López, C.; Blanco, M.; Dahbi, G.; Herrera, A.; Blanco, J.E.; Alonso, M.P.; García-Garrote, F.; et al. National survey of Escherichia coli causing extraintestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain. J. Antimicrob. Chemother. 2011, 66, 2011–2021. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Ojer-Usoz, E.; González, D.; Vitas, A.I. Clonal diversity of ESBL-producing Escherichia coli isolated from environmental, human and food samples. Int. J. Environ. Res. Public Health 2017, 14, 10–12. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Banerjee, R.; Johnston, B.; Lohse, C.; Chattopadhyay, S.; Tchesnokova, V.; Sokurenko, E.V.; Johnson, J.R. The clonal distribution and diversity of extraintestinal Escherichia coli isolates vary according to patient characteristics. Antimicrob. Agents Chemother. 2013, 57, 5912–5917. [Google Scholar] [CrossRef] [Green Version]
  64. Giufrè, M.; Graziani, C.; Accogli, M.; Luzzi, I.; Busani, L.; Cerquetti, M. Escherichia coli of human and avian origin: Detection of clonal groups associated with fluoroquinolone and multidrug resistance in Italy. J. Antimicrob. Chemother. 2012, 67, 860–867. [Google Scholar] [CrossRef] [Green Version]
  65. Ishiwa, A.; Komano, T. Thin pilus PilV adhesins of plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells. J. Bacteriol. 2003, 185, 5192–5199. [Google Scholar] [CrossRef] [Green Version]
  66. Ishiwa, A.; Komano, T. PilV adhesins of plasmid R64 thin pili specifically bind to the lipopolysaccharides of recipient cells. J. Mol. Biol. 2004, 343, 615–625. [Google Scholar] [CrossRef]
  67. Abraham, S.; Kirkwood, R.N.; Laird, T.; Saputra, S.; Mitchell, T.; Singh, M.; Linn, B.; Abraham, R.J.; Pang, S.; Gordon, D.M.; et al. Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-bla CTXM-1 plasmid among Escherichia coli in pigs. ISME J. 2018, 12, 2352–2362. [Google Scholar] [CrossRef] [Green Version]
  68. Valcek, A.; Roer, L.; Overballe-Petersen, S.; Hansen, F.; Bortolaia, V.; Leekitcharoenphon, P.; Korsgaard, H.B.; Seyfarth, A.M.; Hendriksen, R.S.; Hasman, H.; et al. IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin. J. Antimicrob. Chemother. 2019, 74, 2171–2175. [Google Scholar] [CrossRef]
  69. Brouwer, M.S.M.; Tagg, K.A.; Mevius, D.J.; Iredell, J.R.; Bossers, A.; Smith, H.E.; Partridge, S.R. IncI shufflons: Assembly issues in the next-generation sequencing era. Plasmid 2015, 80, 111–117. [Google Scholar] [CrossRef] [PubMed]
  70. Carattoli, A.; Lovari, S.; Franco, A.; Cordaro, G.; Di Matteo, P.; Battisti, A. Extended-spectrum β-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003. Antimicrob. Agents Chemother. 2005, 49, 833–835. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Smet, A.; Martel, A.; Persoons, D.; Dewulf, J.; Heyndrickx, M.; Herman, L.; Haesebrouck, F.; Butaye, P. Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: Molecular aspects, mobility and impact on public health. FEMS Microbiol. Rev. 2010, 34, 295–316. [Google Scholar] [CrossRef] [Green Version]
  72. Hordijk, J.; Schoormans, A.; Kwakernaak, M.; Duim, B.; Broens, E.; Dierikx, C.; Mevius, D.; Wagenaar, J.A. High prevalence of fecal carriage of extended-spectrum β-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs. Front. Microbiol. 2013, 4, 1–5. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Blanco, J.; Mora, A.; Mamani, R.; López, C.; Blanco, M.; Dahbi, G.; Herrera, A.; Marzoa, J.; Fernández, V.; De La Cruz, F.; et al. Four main virotypes among extended-spectrum-β-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, epidemiological, and clinical characteristics. J. Clin. Microbiol. 2013, 51, 3358–3367. [Google Scholar] [CrossRef] [Green Version]
  74. Bachiri, T.; Bakour, S.; Ladjouzi, R.; Thongpan, L.; Rolain, J.M.; Touati, A. High rates of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in wild boars and Barbary macaques in Algeria. J. Glob. Antimicrob. Resist. 2017. [Google Scholar] [CrossRef] [PubMed]
  75. Bonnedahl, J.; Järhult, J.D. Antibiotic resistance in wild birds. Upsala J. Med. Sci. 2014, 119, 113–116. [Google Scholar] [CrossRef] [Green Version]
  76. Peirano, G.; Pitout, J.D.D. Molecular epidemiology of Escherichia coli producing CTX-M β-lactamases: The worldwide emergence of clone ST131 O25:H4. Int. J. Antimicrob. Agents 2010, 35, 316–321. [Google Scholar] [CrossRef] [PubMed]
  77. Naseer, U.; Sundsfjord, A. The CTX-M Conundrum: Dissemination of Plasmids and Escherichia coli Clones. Microb. Drug Resist. 2011, 17, 83–97. [Google Scholar] [CrossRef]
  78. Carattoli, A.; Bertini, A.; Villa, L.; Falbo, V.; Hopkins, K.L.; Threlfall, E.J. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 2005, 63, 219–228. [Google Scholar] [CrossRef] [PubMed]
  79. Zurfluh, K.; Jakobi, G.; Stephan, R.; Hächler, H.; Nüesch-Inderbinen, M. Replicon typing of plasmids carrying bla CTX-M-1 in Enterobacteriaceae of animal, environmental and human origin. Front. Microbiol. 2014, 5, 555. [Google Scholar] [CrossRef]
  80. Carattoli, A.; Villa, L.; Fortini, D.; García-Fernández, A. Contemporary IncI1 plasmids involved in the transmission and spread of antimicrobial resistance in Enterobacteriaceae. Plasmid 2018. [Google Scholar] [CrossRef] [Green Version]
Microbiolres 12 00009 g001 550
Figure 1. The core genome of extended-spectrum β-lactamases (ESBL)-encoding E. coli isolates analyzed by the cgSNP (a) and cgMLST (b) approaches. The dendrogram from cgSNP was inferred from recombination-free core genome SNPs by the maximum likelihood method (scale: Number of mutations per site). The dendrogram from cgMLST was inferred by the neighbor-joining method (scale: Number of differences in allele). The bootstrap values indicated for the main nodes were calculated from 100 replicates. The clusters corresponding to E. coli phylogroups A, B1, B2, and D are indicated. The number of differences in allele or SNP are indicated for the clonal isolates according to the cgMLST and cgSNP approaches (1 before recombination filtration, and 2 after recombination filtration). The clonal relatedness of the strains was determined using less than five variant sites as clonality criteria.
Figure 1. The core genome of extended-spectrum β-lactamases (ESBL)-encoding E. coli isolates analyzed by the cgSNP (a) and cgMLST (b) approaches. The dendrogram from cgSNP was inferred from recombination-free core genome SNPs by the maximum likelihood method (scale: Number of mutations per site). The dendrogram from cgMLST was inferred by the neighbor-joining method (scale: Number of differences in allele). The bootstrap values indicated for the main nodes were calculated from 100 replicates. The clusters corresponding to E. coli phylogroups A, B1, B2, and D are indicated. The number of differences in allele or SNP are indicated for the clonal isolates according to the cgMLST and cgSNP approaches (1 before recombination filtration, and 2 after recombination filtration). The clonal relatedness of the strains was determined using less than five variant sites as clonality criteria.
Microbiolres 12 00009 g001
Microbiolres 12 00009 g002a 550Microbiolres 12 00009 g002b 550
Figure 2. A CTX-M-1-encoding IncI1-ST3 plasmid recovered from E. coli isolated in wild animals. (a) Schematic representation of the complete plasmid pJ31 (CP053788) from a wild boar. The two outer rings represent the forward and reverse open reading frames, respectively. (b) Comparative sequence analysis of p2-101 (CP053786), and pJ31 (CP053788) with the typical IncI1-ST3 blaCTX-M-1 plasmid p04-021 (MH847038) and the reference IncI1 plasmid R64 (AP005147). Vertical blocks between sequences indicate regions of shared similarity shaded according to BLASTn sequence identity (>95% nucleotide similarity).
Figure 2. A CTX-M-1-encoding IncI1-ST3 plasmid recovered from E. coli isolated in wild animals. (a) Schematic representation of the complete plasmid pJ31 (CP053788) from a wild boar. The two outer rings represent the forward and reverse open reading frames, respectively. (b) Comparative sequence analysis of p2-101 (CP053786), and pJ31 (CP053788) with the typical IncI1-ST3 blaCTX-M-1 plasmid p04-021 (MH847038) and the reference IncI1 plasmid R64 (AP005147). Vertical blocks between sequences indicate regions of shared similarity shaded according to BLASTn sequence identity (>95% nucleotide similarity).
Microbiolres 12 00009 g002aMicrobiolres 12 00009 g002b
Table
Table 1. Metadata and key features of Escherichia coli isolates collected from the stool of different wild animals.
Table 1. Metadata and key features of Escherichia coli isolates collected from the stool of different wild animals.
Strain IDSourceCountry/YearPhylogroup/STESBL Gene/Replicon aPlasmid-Mediated Resistance Genes bChromosome-Mediated Resistance Genes b
TB63BatPortugal/2014B1/ST155blaCTX-M-1/IncI1-ST3aadA5, dfrA17, sul2gyrA-S83L
1-102Bird of preyPortugal/2008C/ST1998blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), gyrA-S83L-D87N, parC-S80I
13-103Bird of preyPortugal/2008B1/ST1800blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R)gyrA-S83L-D87N, parC-S80I
2-101Bird of preyPortugal/2008C/ST1998blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), gyrA-S83L-D87N, parC-S80I
21-101Bird of preyPortugal/2008C/ST1998blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R)gyrA-S83L-D87N, parC-S80I
BU10ABuzzardPortugal/2007A/ST48blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), qnrS1, strAB, aadA1, dfrA1, sul1
BU10BBuzzardPortugal/2007A/ST48blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), qnrS1, strAB, aadA1, dfrA1, sul1
BU22ABuzzardPortugal/2007B1/ST1800blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R)gyrA-S83L-D87N, parC-S80I
BU41ABuzzardPortugal/2007A/ST48blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), qnrS1, strAB, aadA1, dfrA1, sul1
L16LynxSpain/2010C/ST23blaCTX-M-14/IncKaadA1, aac(3)-VIa, tet(A), tet(R), sul1
L98LynxSpain/2010F/ST117blaSHV-12/IncI1-ST3aadA1, aadA2, cmlA1, sul3, strA, dfrA5gyrA-S83L
GV23SeagullPortugal/2007A/ST710blaCTX-M-1/IncN-ST1 blaOXA-1, aadA1, catA1, sul1, tet(B), tet(R)
J31Wild boarPortugal/2006A/ST48blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R), qnrS1, strAB, aadA1, dfrA1, sul1
J64Wild boarPortugal/2006C/ST1998blaCTX-M-1/IncI1-ST3blaTEM-1, aadA5, dfrA17, sul2, tet(A), tet(R)gyrA-S83L-D87N, parC-S80I
J69Wild boarPortugal/2006A/ST10430blaCTX-M-1/IncI1-ST3aadA5, dfrA17, sul2gyrA-S83L, parC-S80R, tet(B), tet(R)
W151WolfPortugal/2008A/ST361blaCTX-M-32/IncN-ST1
W4WolfPortugal/2008B1/ST155blaCTX-M-1/IncI1-ST3tet(A), tet(R)gyrA-S83L
a The association of ESBL genes with replicons was determined from Southern blot experiments and in silico analysis of WGS; b deduced from in silico NGS data analysis.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Sabença, C.; Igrejas, G.; Poeta, P.; Robin, F.; Bonnet, R.; Beyrouthy, R. Multidrug Resistance Dissemination in Escherichia coli Isolated from Wild Animals: Bacterial Clones and Plasmid Complicity. Microbiol. Res. 2021, 12, 123-137. https://doi.org/10.3390/microbiolres12010009

AMA Style

Sabença C, Igrejas G, Poeta P, Robin F, Bonnet R, Beyrouthy R. Multidrug Resistance Dissemination in Escherichia coli Isolated from Wild Animals: Bacterial Clones and Plasmid Complicity. Microbiology Research. 2021; 12(1):123-137. https://doi.org/10.3390/microbiolres12010009

Chicago/Turabian Style

Sabença, Carolina, Gilberto Igrejas, Patrícia Poeta, Frédéric Robin, Richard Bonnet, and Racha Beyrouthy. 2021. "Multidrug Resistance Dissemination in Escherichia coli Isolated from Wild Animals: Bacterial Clones and Plasmid Complicity" Microbiology Research 12, no. 1: 123-137. https://doi.org/10.3390/microbiolres12010009

Article Metrics

Back to TopTop