https://orcid.org/0000-0003-1401-2961
Figures
Abstract
In the present study, the presence of the Enterobacterales, Staphylococcus spp., Mammaliicoccus spp., and Enterococcus spp. in cloacal samples of nestling ospreys (Pandion haliaetus), a fish-eating specialist, from Mono Lake, California, USA was examined by a multiphasic approach, including antimicrobial and biocide susceptibility testing, genotyping, and whole genome sequencing of selected isolates. The most commonly detected species was Escherichia coli, followed by Mammaliicoccus sciuri, Staphylococcus delphini, Enterococcus faecalis, Enterococcus faecium, Hafnia alvei, Klebsiella pneumoniae, Citrobacter braakii and single isolates of Edwardsiella tarda, Edwardsiella albertii, Klebsiella aerogenes, Plesiomonas shigelloides and Staphylococcus pseudintermedius. Multi-drug resistance (MDR) was observed in two E. coli isolates and in an Enterococcus faecium isolate. The MDR blaCTX-M-55-positive E. coli belonged to the pandemic clone ST58. The results of the present study suggest that nestling ospreys are exposed to MDR bacteria, possibly through the ingestion of contaminated fish. Ospreys may be good biosentinels for the presence of these microorganisms and antibiotic resistance in the local environment and the risk for other wildlife, livestock and humans.
Citation: Loncaric I, Szostak MP, Cabal-Rosel A, Grünzweil OM, Riegelnegg A, Misic D, et al. (2024) Molecular characterization, virulence and antimicrobial and biocidal susceptibility of selected bacteria isolated from the cloaca of nestling ospreys (Pandion haliaetus) from Mono Lake, California, USA. PLoS ONE 19(9): e0311306. https://doi.org/10.1371/journal.pone.0311306
Editor: Gabriel Trueba, Universidad San Francisco de Quito, ECUADOR
Received: May 28, 2024; Accepted: September 6, 2024; Published: September 27, 2024
Copyright: © 2024 Loncaric et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The osprey (Pandion haliaetus) is a large, specialized fish-eating bird of prey with a worldwide distribution [1]. The species is associated mainly with lakes, rivers, seacoasts, and other water bodies present in a wide range of habitats, where they can capture prey and breed [2]. With its unique features in its osteology, pelvic musculature, the distribution of feather tracts, and results of modern molecular genetics studies, the osprey is broadly accepted to be a unique, monotypic, and distinct taxon that differs from other diurnal birds of prey [3]. For more detailed information on the ecology and natural history of the species, see [2, 4].
The North American continental subspecies (P. haliaetus carolinensis) was severely affected by DDT and other organochlorine pesticides in the past century, as these pollutants interfere with calcium metabolism, causing eggshell thinning, embryo mortality, and as a result negatively impacted hatching and breeding success of birds [5, 6]. Between 1950 and 1970 several osprey populations in North America collapsed [2]. In California, ospreys were also declining since the early 1900s, due to habitat loss, shooting and contamination. In California, ospreys originally nested along the Pacific coastline [7–10] and along the major inland lakes [11]. By the late eighties, and once the osprey’s population started to recover as the result of the banning of DDT and other organochlorine pesticides, new areas started being colonized by the species, facilitated sometimes by the presence of new nesting sites such as anthropogenic structures, and/or food availability in the form of seeded fish in inland lakes [4, 12]. One of these recently colonized nesting areas is Mono Lake in California.
By 1985, ospreys were observed for the first-time nesting in the Mono Lake, an inland, alkaline, endorheic water body located in Mono County, eastern central California [13]. In this lake, the presence of calcium carbonate towers, locally called “tufas” that emerged as a result of the reduction in water levels provided new available nesting sites for the species. Ospreys build these nests on the top of the tufa, which are frequently located offshore and surrounded by water, making access to the nest to mammalian predators almost impossible. As a result, breeding success in this area is usually high [12], Bloom unpublished data). In 2021 and 2022, approximately 15–17 pairs were nesting in this lake (A. Lewis pers. com, Bloom unpublished data). This unique and relatively small osprey population relies on fish captured in rivers and lakes located nearby, usually between <1 km to 11 km, as Mono Lake does not support any fish population due to its hypersaline alkaline waters [12]. This outside fish supply for ospreys relies on the presence of rainbow trout, (Oncorhynchus mykiss) and cutthroat trout (Oncorhynchus clarkia) stocked in the streams and nearby lakes by the California Department of Fish & Wildlife with the goal of supplementing local fish populations to support fishing and human consumption [12, 14]. Fish stocked come from hatchery farms located all along north and central California.
Due to their predatory feeding habits, raptors have been a subject of interest for the study of comparative gastrointestinal anatomy and physiology among birds [15]. Important differences in gastrointestinal length, size, presence of crop, ceca length and size, among others have been identified for different taxa among this polyphyletic group of predatory birds [15]. Being a specialized fish eater, numerous studies have reported hunting behavior and success, type, and frequency of different fish species in osprey’s diet [4]. Ospreys are unique birds of prey due to their specialized feeding and foraging ecology that is accompanied by unique physiological and anatomical features [2]. However, studies on the gastrointestinal physiology, including gastrointestinal microbiota composition and the presence of antimicrobial resistant bacteria in this specialist raptor are scarce [16]. Several published reports have highlighted the importance of gastrointestinal microbiota in animal species, including birds [17, 18]. These studies on raptor’s microbiome have been focused on scavengers [19–21], on mammal eaters [16] and even on highly specialized species such as honey buzzards (Pernis apivorus) [22] and lesser kestrels (Falco naumanni) [23], in most of these cases by investigating birds admitted to rehabilitation centers [16]. Studies on wild raptors are relatively uncommon [16]. Furthermore, the presence of enteropathogens that may show antimicrobial resistance have been demonstrated by isolation and/or molecular characterization of antibiotic-resistant bacteria in the respiratory and gastrointestinal system of several species of raptors worldwide [24–27]. However, there is a paucity of studies conducted on Ospreys. In the only study where antimicrobial resistance was investigated for ospreys, multi-drug-resistant Escherichia (E.) coli and tetracycline-resistant Enterococcus (E.) faecalis were not isolated [25]. The aim of the present study was to investigate the presence of Enterobacterales, including isolation of β-lactamase-producing determinants and Salmonella spp., Enterococcus spp., Staphylococcus spp., and Mammaliicoccus spp. in the cloaca of nestling osprey population of Mono Lake, California, U.S.A. Furthermore, molecular characterization, antimicrobial and biocidal susceptibility testing was performed on the isolated bacteria.
Material and methods
Ethics statement
Capture, handling, and sampling of nestling ospreys were done by Peter H. Bloom, Ashli Lewis, and Miguel D. Saggese under permits provided by the California Department of Fish and Wildlife (Permit #S-190320004-19036-001) and the United States Fish and Wildlife Service (Permit #20431) and approved by the Western University of Health Sciences Institutional Animal Care and Use Committee.
Study area and animal samples
Mono Lake is located in Mono County, central western California (38.0128° N, 118.9762° W). Its origin dates up to around one million years ago, in a high desert basin located in the Eastern Sierra Nevada Mountain range. Its endorheic waters are filled by water from rainfall and tributary streams. The highly alkaline waters do not support many life forms, except shrimp and flies, but no fish are found there [28]. The high levels of calcium carbonate can precipitate in these waters creating underwater formations locally known as tufas which can reach several meters of height above the water in and on the periphery of the lake as result of a decrease in water levels. For a detailed description of Mono Lake see [29]. For additional information on tufas formation see [30].
As result of the monitoring efforts started by the California State Parks on this population of ospreys, since 2000, nestlings produced on this lake were banded. On July 13, 2021, nests were accessed by boat between 7:00 AM-2:00 PM. Nests were climbed using a ladder to confirm content, and when present, to take nestlings down for sampling and banding. On each nest site, nests were accessed and inspected for content. Nestling ospreys were gently removed from their nests and temporarily handled for physical examination and sample collection using classical raptor handling techniques [31]. Nestlings were identified with color, alphanumeric, bands on their legs. Upon physical examination, cloacal swabs were taken by carefully introducing a cotton swab in the cloaca of these birds, gently rotated for a few seconds, and saved in Amies Transport Medium (Copan Diagnostics Inc, Murrieta, CA). Swabs were kept refrigerated at 4°C until arrival to the laboratory for bacterial isolation, with the exception during the flight between USA and Austria.
Bacterial isolates
The isolation of bacteria, antimicrobial susceptibility testing, and molecular characterization with the exception of whole genome sequencing were performed at the University of Veterinary Medicine Vienna. For isolation of Enterobacterales, each sample was preincubated at 37°C overnight in buffered peptone water (BPW) (Merck, Germany). An aliquot of 200 μL of the incubated sample was cultured at 37°C overnight in BD™ MacConkey Broth (BD, Heidelberg, Germany) and then cultivated at 37°C overnight on BD™ MacConkey II Agar (Becton Dickinson (BD), Heidelberg, Germany). Selective isolation of β-lactamase-producing Enterobacterales and selective isolation of Salmonella spp. was performed as described previously (Grünzweil et al., 2021). The isolation of Staphylococcus spp. and Mammaliicoccus spp. was conducted by incubating each sample at 37°C overnight in tryptic soy broth (BD, Heidelberg, Germany) with 6.5% (w/v) NaCl and subsequently streaked onto BD™ Columbia CNA Agar with 5% Sheep Blood, Improved II (BD, Heidelberg, Germany). In addition, the isolation of methicillin-resistant staphylococci and mammaliicocci was achieved as recently described (Schauer et al., 2021). For the isolation of Enterococcus spp., each sample was incubated at 37°C in BBL™ Enterococcosel™ Broth (BD, Heidelberg, Germany) and then streaked onto BBL™ Enterococcosel™ Agar (BD, Heidelberg, Germany) and incubated at 37°C for 24 h. The respective colonies of isolates of interest showed the typical colony appearance for the abovementioned bacteria and were identified to the species level by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany) were saved at -80°C for further analyses.
Antimicrobial and biocide susceptibility testing
Biocide susceptibility testing was performed at was performed at Department of Functional Food Products Development, Faculty of Biotechnology and Food Science, Wroclaw University of Environmental and Life Sciences. All susceptibility tests were conducted by agar disk-diffusion method according to CLSI, 2021 standards [32]. For Enterobacterales, the following antimicrobial agents (all from Becton Dickinson, Heidelberg, Germany) were used: ampicillin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), tobramycin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), tetracycline (30 μg), doxycycline (30 μg), chloramphenicol (30 μg), and fosfomycin (200 μg). Susceptibility testing of staphylococci and mammaliicocci was performed with the following antimicrobial agents: penicillin (10 units), cefoxitin (30 μg), ciprofloxacin (5 μg), amikacin (30 μg), gentamicin (10 μg), tetracycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), chloramphenicol (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), rifampicin (5 μg), and linezolid (30 μg). Penicillin (10 units), vancomycin (30 μg), teicoplanin (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), erythromycin (15 μg), chloramphenicol (30 μg), nitrofurantoin (300 μg), rifampicin (5 μg), linezolid (30 μg) were used for susceptibility testing of enterococci. Staphylococcal interpretative criteria have also been applied for mammaliicocci. E. coli ATCC® 25922 and Staphylococcus (S.) aureus ATCC® 25923 served as the quality control strains. AmpC-hyperproducing isolates (stably de-repressed) of Citrobacter spp. were examined as previously described [33].
Biocide susceptibility testing was performed according to the previously established protocol [34]. Benzalkonium chloride (Acros Organics, Geel, Belgium, 21541), representing the quaternary ammonium compounds, was tested at concentration ranges 0.000015%–0.016%, chlorhexidine (Sigma-Aldrich, Schnelldorf, Germany, 55-56-1), representing cationic compounds, was tested at concentration ranges 0.000015%–0.002%, glutardialdehyde (Chempur, Piekary Slaskie, Poland, 424610240), representing aldehydes, was tested at concentration ranges 0.0075%–1%, and isopropanol (99.9%, PHPU Eurochem BGD, Tarnow, Poland), representing alcohols, was tested at concentration ranges 1–14%. The method was performed in 96-wells U-bottom polystyrene microtiter plates (Sarstedt, Numbrecht, Germany, 82.1582.001). The bacterial inoculum was prepared as previously described [34] using Trypticasein soy broth (Biomaxima, Lublin, Poland, PS 23–500). The final concentration of bacteria inoculated into the wells was 2.5–5 × 105 CFU/mL.
Molecular identification and typing methods
Species identification of staphylococci and mammaliicocci was performed by rpoB sequencing [35]. The revisited Clermont method determined the phylogroup of the E. coli isolates [36]. Clonal relatedness of E. coli isolates was assessed by two-locus sequence typing of combined data of fumC and fimH sequences, as described previously [37], using CHTyper [38] hosted at the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/chtyper/, accessed on 1. April 2024), as well as Escherichia/Shigella database hosted on EnteroBase (https://enterobase.warwick.ac.uk/species/index/ecoli) [39]. Clonal relatedness of Mammaliicoccus (M.) sciuri isolates was characterized by MLST as previously described [40]. Briefly, internal segments of the seven house-keeping genes ack, aroE, ftsZ, glpK, gmk, pta1 and tpiA were amplified. The PCR reactions were performed starting with 94°C for 300 s, followed by 30 cycles of 30 s at 94°C, 30 s at the respective annealing temperatures (55°C except for ftsZ (53°C)), 1 min at 72°C and a final extension at 72°C for 5 min. The amplicons were further purified and amplicons were Sanger sequenced with the same primer pair used for amplification at LGC Genomics Berlin, Germany. Allelic numbers and profiles were assigned using the MLST database hosted on PubMLST (https://pubmlst.org/organisms/mammaliicoccus-sciuri, accessed on 1. April 2024) [41]. The spa typing of S. pseudintermedius was performed as described previously [42].
Virulence-associated genes
Detection and analysis of virulence-associated genes of E. coli isolates was performed using custom-made microarrays from INTER-ARRAY (INTER-ARRAY by fzmb GmbH, Bad Langensalza, Germany) according to manufacturer’s instructions [33, 43]. For the complete list of virulence-associated genes analyzed see S1 Table.
Whole-Genome Sequencing (WGS)
Whole genome sequencing was performed at Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene. Fifteen isolates (E. coli (n = 5), Citrobacter (C.) braakii (n = 2), M. sciuri (n = 2), Edwardsiella (E.) alberti, E. faecalis, E. faecium, Klebsiella (K.) aerogenes, S. pseudintermedius and S. delphini (each n = 1) were selected based on their resistance and/or virulence load to be analyzed by WGS. Bacterial DNA was isolated using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany). Ready-to-sequence libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, United States). Isolates were paired-end-sequenced using the Illumina MiSeq platform with a read length of 2 × 300 bp [44]. De novo-assembly of raw reads was performed using SPAdes v.3.9.0 [45], and WGS data analysis was performed with SeqSphere+ software (Ridom, Münster, Germany). The assembled contigs were analyzed in the JSpecies workspace using the ANIb (average nucleotide identity via BLAST) analysis tool [46]. To assess the genetic relatedness of the examined isolates, classical MLST data were extracted from the corresponding database. Sequence types (ST) of E. coli and E. albertii were assigned using the Escherichia/Shigella database hosted on EnteroBase. STs of C. braakii, K. aerogenes, M. sciuri, E. faecalis, E. faecium, and S. pseudintermedius were determined using databases hosted at PubMLST (C. braakii: https://pubmlst.org/organisms/citrobacter-spp, K. aerogenes: https://pubmlst.org/organisms/klebsiella-aerogenes, M. sciuri: https://pubmlst.org/organisms/mammaliicoccus-sciuri, Ent. faecalis: https://pubmlst.org/organisms/enterococcus-faecalis, Ent. faecium: https://pubmlst.org/organisms/enterococcus-faecium, and S. pseudintermedius: https://pubmlst.org/organisms/staphylococcus-pseudintermedius) [41]. In addition, E. coli phylotypes were extracted from WGS by Clermont Typing (http://clermontyping.iame-research.center/, accessed on 1. April 2024) [47, 48]. CH types were characterized as mentioned above, and serogenotypes were analyzed by SerotypeFinder (https://cge.cbs.dtu.dk/services/SerotypeFinder/, accessed on 1. April 2024) [49]. To identify acquired resistance genes and/or chromosomal mutations, the Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca/home, accessed on 1. April 2024) [50], as well as ResFinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/, accessed on 1. April 2024) [51], were used. Genes associated with biocide resistance were compared with the BacMet database (Antibacterial Biocide and Metal Resistance Genes Database, http://bacmet.biomedicine.gu.se/, accessed on 1. April 2024) [52]. Virulence genes were identified using VirulenceFinder 2.0 (https://cge.cbs.dtu.dk/services/VirulenceFinder/, accessed on 1. April 2024) [53] as well as the Virulence Factor Database (VFDB; http://www.mgc.ac.cn/VFs/, v) [54]. The presence of plasmids was assessed using PlasmidFinder 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/, accessed on 1. April 2024) [55]. Probability prediction of the location of a given bla resistance gene in Enterobacterales was achieved by applying mlplasmids trained on E. coli or Klebsiella [56]. Posterior probability scores (ppp) >0.7 and a minimum contig length of 1000 bp indicate that a given contig is plasmid-derived. For genes with ppp scores below 0.7, BLAST searches were performed for the respective contig sequence. If the BLAST search listed only plasmids first with 100% coverage and identities, a plasmid location was assumed. Gene prediction and annotation were performed using Genome Annotation Service PATRIC [57]. The genomes of WGS isolates were deposited under PRJNA1047016 in the NCBI BioProject database.
Results
Birds
A total of 18 nestling Ospreys, from ten different nests were banded and sampled. Age ranged from 3 to 6.5 weeks. All birds were found in overall good physical condition, alert, and responsive to handling.
Bacterial isolates
In total, 44 isolates from 18 cloacal samples were obtained. The most commonly identified species was E. coli (n = 13), followed by M. sciuri (n = 6), S. delphini (n = 5), E. faecalis (n = 5), E. faecium (n = 4), Hafnia alvei (n = 2), Klebsiella (K.) pneumoniae (n = 2), C. braakii (n = 2), and singletons E. tarda, E. albertii, K. aerogenes, Plesiomonas (P.) shigelloides and S. pseudintermedius.
Antimicrobial and biocide susceptibility testing and detection of resistance genes
Ten out of 13 E. coli isolates were susceptible to all tested antimicrobial agents. The remaining E. coli isolates (20-1a, 21-ctx and 22c-ctx) displayed an ESBL phenotype and two of them (21-ctx and 22c-ctx) were multi-drug resistant (MDR) [58]. Isolate 20 1a/P0 was resistant to β-lactam antibiotics ampicillin, amoxicillin/clavulanic acid and cefotaxime and carried blaEC. The gene blaCTX-M-55 was observed in both multi-drug resistant isolates. In addition, the detection of non-β-lactamase genes reflected well the phenotypic resistance profiles of the respective isolates. Thus, the isolate 21ctx that was resistant to tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole and ciprofloxacin carried tet(A), floR, sul3, dfrA14, and the analysis of the quinolone resistance-determining regions (QRDR) revealed mutations in gyrA, parC and parE. Besides ampicillin, amoxycillin/clavulanic acid, cefotaxime and ceftazidime, the second blaCTX-M-55 positive isolate (22c-ctx/P8) was also resistant to gentamicin, tobramycin and chloramphenicol and harbored corresponding aac(3)-IIa and floR resistance genes (Table 1). Among other Enterobacterales isolates, resistance to 3rd generation of cephalosporins (cefotaxime and ceftazidime) was observed in K. aerogenes isolate 22b ctx, which displayed both, an AmpC and ESBL phenotype. In addition, the K. aerogenes isolate was resistant to fosfomycin. WGS revealed that this isolate harbored ampC (EC 3.5.2.6) β-lactamase, ampH (encodes β-lactam binding protein AmpH) and fosA5 genes. C. braakii isolate 22a-ctx was identified as a stably de-repressed AmpC-producer (Table 1). The mlplasmids analyses could not clearly predict whether the blaCTX-M-55 gene, which is identical in both isolates, is plasmid-encoded (ppp< 0.7). However, BLAST analyses of the respective contigs identified exclusively E. coli plasmids with 100% identity over the full contig length (4.27 and 5.83 kb, respectively). Another approach for an in-silico plasmid assembly from WGS data (see Virulence-assoc. Genes) also indicates the blaCTX-M-55 gene to be encoded on a large plasmid. Gene blaEC-1149 BLDB (http://bldb.eu; last accessed 1. April 2024 [59] detected in E. coli isolate 20 1a is predicted as chromosome-encoded (ppp 0.055 0.945) as well as ampC (EC 3.5.2.6) detected in K. aerogenes isolate 22b ctx (ppp 0.008). Two out of six M. sciuri isolates were resistant to clindamycin solely (sal(A)). Among staphylococci, the S. pseudintermedius isolate 19a was resistant to penicillin (blaZ). Resistance to rifampicin was observed in four E. faecalis isolates. E. faecalis isolate 17b as additionally resistant to tetracycline. WGS analysis revealed that this isolate carried the gene tet(M), and no known mutation in gene rpoB. Rifampicin resistance was also observed in three E. faecium.
E. faecium isolate 22b/P8 was MDR to penicillin, ampicillin, ciprofloxacin, erythromycin, tetracycline (tet(S)) and rifampicin resistance (Table 2) and carried a hybrid-like pbp5 gene of S8/R13-type 8 [60]. The same S/R-type is found in E. faecium strains EnGen35, EnGen21 and EnGen52 which show variable ampicillin MICs of 1, 8 and 128 ug/ml, respectively, so that also pbp5 mRNA levels and PBP5 abundance could play a role.
The E. faecium isolate 22b, which had an elevated BAC MIC (0.0005), a gene coding for a small multi-drug resistance (SMR) transporter, which mediates resistance to quaternary ammonium compounds (QACs) was detected by analyzing WGS data (Table 2).
Molecular typing methods
Most E. coli isolates were assigned to the group B1 (n = 8), followed by group A (n = 3), and singletons B2, whereas E. albertii belonged to chuAalbertii. Among E. coli isolates, the fumC and fimH (CH) clonotyping divided E. coli and E. albertii isolates into 12 distinct CH clonotypes. Two isolates, 11/M1 and 12/ML shared the same CH clonotype (CH1961-38). The same applies for isolates 20/P0 and 20-1a/P0 (CH11-41). All other E. coli isolates were singletons. WGS based serogenotyping disclosed all analyzed E. coli isolates into distinct serogenotypes: O15:H2, O145:H11, O45:H30, O8:H25 and O8:H28. WGS based MLST (Achtman 7 loci MLST) detected five different STs among the five sequenced E. coli isolates: ST20, ST58, ST162, ST361 and ST1968, whereas the E. albertii isolate was assigned to the new ST12692. Both C. braakii isolates (22a ctx; 22e) belonged to ST109 and the K. aerogenes isolate 22b ctx to the new ST242. Among six examined M. sciuri, four STs were detected: ST30 (n = 2) and three new ST112, ST113 (n = 2) and ST119. Sequence types of single E. faecalis (ST116), E. faecium (ST355), and S. pseudintermedius (new ST2199) were extracted from WGS data (Table 2). The S. pseudintermedius isolate belonged to spa type t23. PlasmidFinder identified seven different replicon types IncFIB(AP001918), IncFIC(FII), IncI1-I(Alpha), p0111, IncFII, ColpVC and IncFIA among E. coli isolates. IncFIB(AP001918) was detected in E. albertii. Finally, repUS43 replicon was identified in E. faecalis.
Characterization of virulence-associated genes
In all eight E. coli isolates screened by the DNA microarray-based method, the adhesion gene fimH was detected. In addition, E. coli isolate 9 carried cnf1 gene. WGS analysis of virulence-associated genes associated with E. coli isolates predicted the combination of fimH, papC and iucD characterizing the uropathogenic strains (UPEC) pathotype in the blaCTX-M-55-positive isolate (22c-ctx). The presence of eae and the absence of the bfp operon (encoding the bundle forming pilus—BFP) was detected in E. coli isolate 14 as well in E. albertii isolate 8 characterizing an atypical enteropathogenic E. coli (aEPEC). WGS-based analysis of virulence-associated genes of other selected bacteria revealed various virulence traits in all examined genomes (Tables 3 and 4).
Analysis of WGS of E. coli isolate 22c ctx by VirulenceFinder detected several virulence traits like iutA/iucA, sitA, iss, ompT and hlyF, which are generally associated with virulence of avian pathogenic E. coli (APEC). When comparing the WGS data with plasmids from APEC strains using GeneiousPrime, more than 55% of the 174,240-bp plasmid pAPEC-O1-ColBM was encoded by contigs of strain 22c-ctx. Moreover, the aerobactin siderophore system (iutA/iucABCD), the iron/manganese transport system sitABCD, the increased serum survival gene iss, the same RepFIIA and RepFIC-like replicons reported from pAPEC-O1-ColBM, as well as the Colicin B and Colicin M region, all genes were located on contigs which were categorized as highly plasmid-associated rated by mlplasmids (ppp>0.7).
A BLAST search of the concatenated plasmid-predicted contigs of E. coli 22c-ctx/P8 identified multiple >100-kb Enterobacteriaceae plasmids. When using the best-listed pTREC1 of E. coli as a backbone for alignment, over 90% of the 128,358-bp plasmid could be reconstructed by WGS contigs of both E. coli isolates, 22c-ctx/P8 and 21ctx/P7. Interestingly, this also placed an AMR island on the predicted plasmid, consisting of sul3, blaCTX-M-55, aadA, qacL, aac(3)-IIe and aph(3´)-Ia.
Discussion
In the present study, a population of ospreys at Mono Lake, California, was investigated for the presence of Enterobacterales, Staphylococcus spp., Mammaliicoccus spp., and Enterococcus spp. with special consideration of their antimicrobial resistance profile and their virulence-associated genes, which was analyzed by microarray analysis and WGS.
To the best of the authors’ knowledge, this is the first detailed investigation on the molecular characterization, virulence, and antimicrobial and biocide susceptibility of selected bacteria isolated from the cloacae of nestling ospreys, and from ospreys nesting in Mono Lake, California, USA. Ospreys, at very early age in life, appear to harbor a wide range of bacteria in their gastrointestinal tract, as reflected by the cloacal specimens examined here. Little is known about the establishment of raptors’ gastrointestinal microbiome since hatching to fledging. This study indicates that even at 3 to 6.5 weeks of age, ospreys, specialized fish-eating birds of prey, are exposed to at least several species of Enterobacteria and Gram-positive cocci. Some species, like C. braakii, a member of Citrobacter freundii complex and P. shigelloides are ubiquitous bacteria found in water, fish, birds, and other vertebrates, including humans [61, 62]. Prey or water seems to be a likely source of these bacteria for ospreys [63]
Enterobacteria are commonly found in fish [64], being the water used in aquaculture potentially contaminated with pathogens [63]. However, the presence of multi-drug resistant isolates in Enterobacterales, such as E. coli, K. aerogenes, and Gram-positive cocci such as E. faecium, suggest an anthropogenic source (spill over), either from human waste or more likely by the use of antimicrobial agents in fish farming that has led to the emergence of resistant strains.
Mono Lake is an endorreic lake that receives the tributary waters of many creeks originated at >3,000 m in the Sierra Nevada, such as Lee Vining creek, Rush creek and Mill creek. Domestic animals, wild animals and human activities occur around them. These creeks can be a potential source of microorganisms and being a potential source of these and other bacteria for ospreys. The physical and chemical properties of Mono Lake support the presence of several Proteobacteria, although Enterobacterales have not been reported in previous studies on the microbial composition of this endorreic lake [65, 66].
Prey, in this case stocking fish, appear to be a more likely candidate as the source of these microorganisms for ospreys. Fish are stocked on many rivers, creeks, and lakes of California with the purpose of supplying water bodies for fishing activities. Ospreys nesting in Mono Lake prey upon fish found in rivers, creeks and hatcheries located far from their nesting sites in Mono Lake. Presence of wildlife, livestock, and human waste are potential sources of bacteria in lakes [65], fish [67] and may be the source of these microorganisms for Ospreys.
Among the 44 isolates characterized, four isolates displayed multi-drug resistance pheno- and genotypes (E. coli (n = 2), K. aerogenes and E. faecium). Both multi-drug resistant E. coli isolates exhibited an ESBL phenotype, carried blaCTX-M-55 and belonged to ST58 and ST361. The blaCTX-M-55 gene was first discovered in ESBL-producing E. coli and K. pneumoniae in Thailand in 2006 [68]. The blaCTX-M-55 gene belongs to the blaCTX-M-1 group and carries an A77V substitution relative to blaCTX-M-15. The blaCTX-M-55 gene is common in Enterobacterales in the Asian continent, especially in E. coli of human and animal origins [69]. Since the first detection, blaCTX-M-55 has been identified in Enterobacterales of human and animal (including wildlife) origin as well from environmental samples from around the globe [70]. E. coli ST58 is a pandemic clone isolated from humans and animal hosts [71]. E. coli ST58 belongs to the clonal complex (CC) 151, and it has been recognized mainly as uropathogenic E. coli (UPEC), and as such, it belongs to extraintestinal pathogenic E. coli (ExPEC). [71] recently analyzed a genome collection of 752 whole-genome sequences of E. coli ST58 isolates that originated from different hosts, including 93 originating from wild animals. In that study, a large ST58 sub-lineage was identified that is characterized by the near-ubiquitous carriage of ColV plasmids, which carry virulence-associated genes and genes typical of the Yersiniabactin High Pathogenicity Island [72]. Several genes were highly associated with this sub-lineage: ugd and galF, both encode enzymes involved in outer membrane lipopolysaccharide biosynthesis, two prophage integrase intA genes, the mlrA gene, a regulator of curli biosynthesis and biofilm formation and fyuA, a marker gene for Yersiniabactin High Pathogenicity Island (HPI) [71]. All genes except intA were detected in our isolate 22c ctx.
When performing a genome similarity search using PATRIC, the two closest homologs to the Osprey isolate 22c ctx were two human isolates, Ec 908541 (917/1000 K-mer counts), an ExPEC isolate from a human UTI patient, and Ec UPEC-203 (914/1000 K-mer counts), both with an ST58 background. Interestingly, among UPEC strains, the ST58 sequence type is rather rare, the PubMLST listed only five out of 592 UPEC strains as of ST58.
The APEC pathotype is associated with large plasmids, such as the 124-kb plasmid pAPEC-O103-ColBM, which carries virulence gene clusters of the ColV pathogenicity island and occasionally a multi-drug-resistant (MDR) island [73, 74]. ColBM and ColV plasmids seem to contribute to avian colibacillosis but also to septicemias, neonatal meningitis and UTI infections in humans [75]. Historically, the first ColBM plasmids were isolated from human UPEC isolates. Nowadays, it is speculated that ColBM plasmids may have evolved from ColV plasmids [73]. Even though E. coli isolates belonging to ST361 were isolated from different hosts including humans and carried different resistance genes (https://enterobase.warwick.ac.uk/species/index/ecoli, last accessed 1. April 2024), the combination of ST361 and blaCTX-M-55 was rarely observed. Three ST361 strains carrying blaCTX-M-55 were recently isolated from sheep in the USA [76]. ESBL E. coli belonging to phylogroup A and ST361 have already been detected in wild animals (wolf (Canis lupus)) in Portugal but harbored blaCTX-M-32 [77]. The combination of ST361 and blaCTX-M-55 has not been observed in wildlife yet (https://enterobase.warwick.ac.uk/species/index/ecoli, last accessed 1. April 2024). Besides two multi-drug resistant E. coli isolates carrying blaCTX-M-55, isolate 20 1a displayed an ESBL phenotype and harbored blaEC-1149, belonging to phylogroup A and ST1968. The blaEC family class belongs to C β-lactamases (cephalosporinases) and were termed as extended-spectrum AmpC β-lactamases (ESAC) [78], which are chromosome-borne [79]. E. coli isolates carrying ESAC are rarely reported from animals [80], and were not associated with ospreys yet. ST1968 has nineteen entries in the Enterobase Escherichia/Shigella Database (https://enterobase.warwick.ac.uk/species/index/ecoli, last accessed 1. April 2024), of which none was associated with wildlife or carry blaEC gene. Even though two Escherichia isolates were non-resistant, they are of particular interest due to their composition of virulence gened. E. albertii isolate ID 8 E5 with the new sequence type ST12692 and chuAlbertii phylogroup and E. coli isolate ID 14 M4 belonging to ST20-B1 harbored among other an eae gene, one of the most highly polymorphic genes of the locus of enterocyte effacement island (LEE), which encodes the adhesin intimin [81]. LEE has the ability to induce intestinal histopathology termed as attaching and effacing’ (A/E), which is characteristic for Enteropathogenic E. coli (EPEC) infections. Based on the absence of the bfp operon (encoding the bundle forming pilus—BFP), both strains are characterized as atypical EPEC (aEPEC) [82]. aEPEC isolates of E. coli and E. albertii are associated with human diarrhea worldwide and were isolated from domesticated and wild animals, but were not associated with colonization or infection in our sampled ospreys [83–86].
Among other Enterobacterales, K. aerogenes isolate ID 22b ctx belonged to new ST242 and displayed a multi-drug resistant phenotype. Interestingly, this isolate displayed both an AmpC (due to intrinsic cephalosporinase) as well as an ESBL phenotype. None of β-lactamases were detected. The alterations in major porins of OmpK36 were detected. The altered OmpK36 could be responsible either for a decrease of porin expression or a loss of porin that could lead in the MIC-increase of β-lactams [87, 88]. Antimicrobial-resistant K. aerogenes is rarely reported to be associated with wildlife. Very recently, K. aerogenes was isolated from Syncerus caffer and Gorilla gorilla gorilla from Gabon, but these isolates did not display acquired resistance [89]. There are no entries in the K. aerogenes PubMLST database associated with wildlife (https://pubmlst.org/organisms/klebsiella-aerogenes, last accessed 1. April 2024).
Besides the isolation of multi-drug and antimicrobial-resistant Enterobacterales, the isolation of multi-drug resistant isolates among Gram-positive bacteria, is an important finding. E. faecium isolate 22b/P8 that belonged to ST355 displayed a multi-drug-resistance phenotype. There are four entries of ST355 in the E. faecium PubMLST database (https://pubmlst.org/organisms/enterococcus-faecium, last accessed 1. April 2024) with no known host. ST355 is a double locus variant of CC17, which is a major human clonal lineage [90]. Reports on antimicrobial resistance in E. faecium isolated from wild animals and especially associated with the birds of prey are scarce. Multi-drug resistant E. faecium were isolated in various wild animals, including wild boars (Sus scrofa) [91] in Portugal, wolves (Canis lupus) in Italy [92], from different wild birds in Poland [93], wild rabbits from Portugal and Tunisia [94–96], buzzards (Buteo buteo) from Portugal [97], Iberian wolf (Canis lupus signatus) [98] and wild birds of Azores Archipelago [99]. E. faecium isolates that were not susceptible to at least one antimicrobial agent in one or two classes of antimicrobial agents could be also detected among wild animals. The majority of E. faecium isolates observed in those studies were resistant to tetracyclines and macrolides (reviewed in [90]). Ampicillin resistance detected in 22b ST355 as well as tetracycline resistance mediated by tet(S) gene was rarely observed in E. faecium associated with wildlife (all above-mentioned references). The tet(S) gene was identified among various Gram-positive genera (i.e. Enterococcus, Lactococcus, Lactobacillus, Staphylococcus, Streptococcus) (https://faculty.washington.edu/marilynr/tetweb3.pdf). Contrary to tet(S), the tet(M) gene that was identified in 17 E. faecalis is the most frequent tet gene in Enterococcus [90]. 17 E. faecalis belonged to ST116. ST116 is an infrequent clone and has been associated with human clinical specimens, poultry, the environment, and very recently with wild Magellanic penguins (Spheniscus magellanicus) from Cidreira Beach, Brasil (https://pubmlst.org/organisms/enterococcus-faecalis, [100].
S. pseudintermedius and S. delphini are animal-associated staphylococci. Both species belong to S. intermedius-group and are commonly isolated from domesticated and wild animals [42, 101, 102]. Mammaliiccoccus genus, another animal-associated genus, was recently separated from Staphylococcus into a new genus [103]. Among staphylococci and mammaliicocci, the resistance was observed only to penicillin mediated by blaZ-blaI-blaR1 operon in S. pseudintermedius, and to clindamycin mediated by sal(A) in M. sciuri. The blaZ gene was observed in many staphylococci and mammaliicocci from different animal species including wildlife [101]. The sal(A) gene confer resistance to lincosamides, pleuromutilines, streptogramin A was first described in M. sciuri but later on also in S. haemolyticus, S. epidermidis and S. xylosus [101]. After performing MLST either by sequencing of seven loci or after extracting the data from genome assembly, all of the analyzed M. sciuri and S. pseudintermedius resulted in a new sequence type. This is interesting and deserves to be monitored in the future to elucidate whether this particular osprey population at Mono Lake harbored distinct clonal lineages of S. pseudintermedius and M. sciuri.
The principal limitations of our study reside in the low sample size (n = 18) and a single isolated population of ospreys investigated. A greater sample size would have provided a more diverse picture of the examined bacteria in nestling ospreys. In addition, a greater number of populations would have provided information on the geospatial variability of this globally distributed raptor. Another issue is that the samples originated from nestlings. Since a gastrointestinal microbiome is known to vary among nestling and adult raptors [104, 105], our results may not necessarily mirror the situation at different life stages nor for ospreys in other geographic locations. Even though trapping and sampling adults is a very difficult task due to the nature of ospreys, this deserves to be discussed in the future. However, our study provides basic data on the development of nestlings’ microbiota for this unique raptor species at an early life stage. The presence of virulent and AMR bacteria in nestlings of a raptor species could be a potential threat to them. Antibiotic exposure is a primary driver of AMR in bacteria. However, other factors released into the environment, such as heavy metals or biocides, can also contribute to the selection and emergence of AMR from wildlife [106]. Given the potential role of biocides in selecting for resistance, biocide susceptibility testing should be conducted in conjunction with antimicrobial susceptibility testing. Furthermore, analyzing the diversity of virulence genes offer valuable insights into the evolutionary factors that have influenced bacterial pathogenicity. Ultimately, studying bacteria from wildlife contributes to a deeper understanding of the interconnectedness of human, animal, and environmental health, as emphasized by the One Health approach.
Several isolates in this study may be considered virulent and potentially pathogenic for animals and humans. Exposure to these bacteria early in life when the immune system is not fully developed might become a source of primary or secondary infection for birds, other animals, and even humans. Strict following of guidelines for handling wild birds should be followed when handling this and other raptor species. The present study contributes to the growing evidence that antimicrobial-resistant bacteria are currently part of the microbiome of wild animals, even at very early stages in life. Ospreys may be good biosentinels for the presence of these microorganisms and antibiotic resistance in the environment. Moreover, antimicrobial resistance is evolving and spreading easily via genes frequently encoded on mobile genetic elements among bacteria. Therefore, the present study’s results emphasize the need for a One Health approach to tackle the global antimicrobial resistance crisis. A longitudinal study is warranted to elucidate whether the presence of antibiotic-resistant bacteria in this population represents transient colonization or a persistent phenomenon.
Supporting information
S1 Table. The complete list of virulence-associated genes analyzed using DNA microarray-based technology.
https://doi.org/10.1371/journal.pone.0311306.s001
(XLSX)
Acknowledgments
We would also like to express our thank to Anna Stöger and Michael Steinbrecher for their technical assistance.
References
- 1.
Ferguson-Lees J, Christie DA. Raptors of the world. New York: Houghton Mifflin Company; 2001.
- 2.
Poole AF. Ospreys: The Revival of a Global Raptor. Johns Hopkins University Press; 2019.
- 3.
Mindell DP, Fuchs J, Johnson JA. Phylogeny, Taxonomy, and Geographic Diversity of Diurnal Raptors: Falconiformes, Accipitriformes, and Cathartiformes. Birds of Prey. Cham: Springer International Publishing; 2018. pp. 3–32. https://doi.org/10.1007/978-3-319-73745-4_1
- 4.
Bierregaard RO, Poole AF, Martell MS, Pyle P, Patten MA. Osprey (Pandion haliaetus). In: Rodewald PG, editor. Birds of the World. Cornell Lab of Ornithology; 2020. https://doi.org/10.2173/bow.osprey.01
- 5.
Newton I. Population Limitations in Birds. USA: Academic Press, Elsevier; 1998.
- 6.
Newton I. Population Ecology of Raptors. Vermillion, South Dakota, USA: Buteo Books; 1979.
- 7. Linton C. Notes from San Clemente Island. Condor. 1908;10: 82–86.
- 8. Grinnell J Jr, Miller A. The distribution of the birds of California. Condor. 1986;88: 482–482.
- 9.
Zeiner D, Laundenslayer W, Mayer K, White M. California’s wildlife. Sacramento: California Department of Fish and Game, Sacramento, CA USA.; 1988.
- 10. Houghton L, Rymon L. Nesting Distribution and Population Status of Us Ospreys 1994. Journal of Raptor Research. 1997;31: 44–53.
- 11. Anderson DW, Suchanek TH, Eagles-Smith CA, Cahill TM. Mercury residues and productivity in osprey and grebes from a mine-dominated ecosystem. Ecol Appl. 2008;18: A227–38. pmid:19475927
- 12. Fields LE, Pagel JE. Osprey Occupancy of Mono Lake—Unique Habitat in Eastern California. Journal of Raptor Research. 2016;50: 97–102.
- 13.
Carle D. Mono Lake Viewpoint. Artemisia Press; 1992.
- 14. CDWC. California Department of Fish and Wildlife, CDWC. In: https://wildlife.ca.gov/Fishing/Hatcheries. 2023.
- 15.
Houston DC, Duke GE. Physiology. In: Bird D, Bildstein K, editors. Raptor Research and Management Techniques. 2007.
- 16. Oliveira BCM, Murray M, Tseng F, Widmer G. The fecal microbiota of wild and captive raptors. Anim Microbiome. 2020;2: 15. pmid:33499952
- 17. Kohl KD. Diversity and function of the avian gut microbiota. J Comp Physiol B. 2012;182: 591–602. pmid:22246239
- 18. Waite DW, Taylor MW. Exploring the avian gut microbiota: current trends and future directions. Front Microbiol. 2015;6: 673. pmid:26191057
- 19. Roggenbuck M, Bærholm Schnell I, Blom N, Bælum J, Bertelsen MF, Sicheritz-Pontén T, et al. The microbiome of New World vultures. Nat Commun. 2014;5: 5498. pmid:25423494
- 20. Zepeda Mendoza ML, Roggenbuck M, Manzano Vargas K, Hansen LH, Brunak S, Gilbert MTP, et al. Protective role of the vulture facial skin and gut microbiomes aid adaptation to scavenging. Acta Vet Scand. 2018;60: 61. pmid:30309375
- 21. Oakley BB, Melgarejo T, Bloom PH, Abedi N, Blumhagen E, Saggese MD. Emerging Pathogenic Gammaproteobacteria Wohlfahrtiimonas chitiniclastica and Ignatzschineria Species in a Turkey Vulture (Cathartes aura). J Avian Med Surg. 2021;35. pmid:34677026
- 22. Nagai K, Tokita K-I, Ono H, Uchida K, Sakamoto F, Higuchi H. Hindgut Bacterial Flora Analysis in Oriental Honey Buzzard (Pernis ptilorhynchus). Zoolog Sci. 2019;36: 77–81. pmid:31116541
- 23. Costanzo A, Ambrosini R, Franzetti A, Romano A, Cecere JG, Morganti M, et al. The cloacal microbiome of a cavity-nesting raptor, the lesser kestrel (Falco naumanni). PeerJ. 2022;10: e13927. pmid:36221261
- 24. Blanco G. Supplementary feeding as a source of multiresistant Salmonella in endangered Egyptian vultures. Transbound Emerg Dis. 2018;65: 806–816. pmid:29333678
- 25. Hathcock T, Poudel A, Kang Y, Butaye P, Raiford D, Mobley T, et al. Multidrug-Resistant Escherichia coli and Tetracycline-Resistant Enterococcus faecalis in Wild Raptors of Alabama and Georgia, USA. J Wildl Dis. 2019;55: 482–487. pmid:30265589
- 26. Handrova L, Kmet V. Antibiotic resistance and virulence factors of Escherichia coli from eagles and goshawks. J Environ Sci Health B. 2019;54: 605–614. pmid:31046564
- 27. Prandi I, Bellato A, Nebbia P, Stella MC, Ala U, von Degerfeld MM, et al. Antibiotic resistant Escherichia coli in wild birds hospitalised in a wildlife rescue centre. Comp Immunol Microbiol Infect Dis. 2023;93: 101945. pmid:36621272
- 28. Wiens JA, Patten DT, Botkin DB. Assessing Ecological Impact Assessment: Lessons from Mono Lake, California. Ecol Appl. 1993;3: 595–609. pmid:27759296
- 29.
Tierney T. Geology of the Mono Basin (revised ed.). Lee Vining, California: Kutsavi Press, Mono Lake Committee; 2000.
- 30. Dunn J. The origin of the deposits of tufa in Mono Lake [California]. J Sediment Petrol. 1953;23: 18–23.
- 31.
Arent L, Martell M. Care and management of captive raptors. University of Minnesota, Saint Paul. USA; 1996.
- 32.
CLSI. M100 Performance Standards for Antimicrobial Susceptibility Testing, 34nd Edition. Clinical Laboratory Standard Institute. 2024.
- 33. Grünzweil OM, Palmer L, Cabal A, Szostak MP, Ruppitsch W, Kornschober C, et al. Presence of β-lactamase-producing enterobacterales and salmonella isolates in marine mammals. Int J Mol Sci. 2021;22. pmid:34072783
- 34. Schug AR, Bartel A, Scholtzek AD, Meurer M, Brombach J, Hensel V, et al. Biocide susceptibility testing of bacteria: Development of a broth microdilution method. Vet Microbiol. 2020;248: 108791. pmid:32827921
- 35. Mellmann A, Becker K, von Eiff C, Keckevoet U, Schumann P, Harmsen D. Sequencing and Staphylococci Identification. Emerg Infect Dis. 2006;12: 333–336. pmid:16494767
- 36. Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5. pmid:23757131
- 37. Weissman SJ, Johnson JR, Tchesnokova V, Billig M, Dykhuizen D, Riddell K, et al. High-resolution two-locus clonal typing of extraintestinal pathogenic Escherichia coli. Appl Environ Microbiol. 2012;78: 1353–60. pmid:22226951
- 38. Roer L, Johannesen TB, Hansen F, Stegger M, Tchesnokova V, Sokurenko E, et al. CHTyper, a Web Tool for Subtyping of Extraintestinal Pathogenic Escherichia coli Based on the fumC and fimH Alleles. J Clin Microbiol. 2018;56. pmid:29436420
- 39. Zhou Z, Alikhan N-F, Mohamed K, Fan Y, Achtman M. The EnteroBase user’s guide, with case studies on Salmonella transmissions, Yersinia pestis phylogeny, and Escherichia core genomic diversity. Genome Res. 2020;30: 138–152. pmid:31809257
- 40. Schauer B, Szostak MP, Ehricht R, Monecke S, Feßler AT, Schwarz S, et al. Diversity of methicillin-resistant coagulase-negative Staphylococcus spp. and methicillin-resistant Mammaliicoccus spp. isolated from ruminants and New World camelids. Vet Microbiol. 2021;254: 109005. pmid:33582485
- 41. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3: 124. pmid:30345391
- 42. Perreten V, Kadlec K, Schwarz S, Gronlund Andersson U, Finn M, Greko C, et al. Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. Journal of Antimicrobial Chemotherapy. 2010;65: 1145–1154. pmid:20348087
- 43. Bernreiter-Hofer T, Schwarz L, Müller E, Cabal-Rosel A, Korus M, Misic D, et al. The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates. Microorganisms. 2021;9: 1676. pmid:34442755
- 44. Lepuschitz S, Huhulescu S, Hyden P, Springer B, Rattei T, Allerberger F, et al. Characterization of a community-acquired-MRSA USA300 isolate from a river sample in Austria and whole genome sequence based comparison to a diverse collection of USA300 isolates. Sci Rep. 2018;8. pmid:29930324
- 45. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology. 2012;19. pmid:22506599
- 46. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpeciesWS: A web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32. pmid:26576653
- 47. 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. pmid:29916797
- 48. Clermont O, Dixit OVA, Vangchhia B, Condamine B, Dion S, Bridier-Nahmias A, et al. Characterization and rapid identification of phylogroup G in Escherichia coli, a lineage with high virulence and antibiotic resistance potential. Environ Microbiol. 2019;21: 3107–3117. pmid:31188527
- 49. Joensen KG, Tetzschner AMM, Iguchi A, Aarestrup FM, Scheutz F. Rapid and Easy In Silico Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data. J Clin Microbiol. 2015;53: 2410–2426. pmid:25972421
- 50. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 2019. pmid:31665441
- 51. Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, et al. ResFinder 4.0 for predictions of phenotypes from genotypes. Journal of Antimicrobial Chemotherapy. 2020;75. pmid:32780112
- 52. Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DGJ. BacMet: antibacterial biocide and metal resistance genes database. Nucleic Acids Res. 2014;42: D737–D743. pmid:24304895
- 53. Malberg Tetzschner AM, Johnson JR, Johnston BD, Lund O, Scheutz F. In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data. J Clin Microbiol. 2020;58. pmid:32669379
- 54. Liu B, Zheng D, Zhou S, Chen L, Yang J. VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res. 2022;50: D912–D917. pmid:34850947
- 55. Carattoli A, Zankari E, Garciá-Fernández A, Larsen MV, Lund O, Villa L, et al. In Silico detection and typing of plasmids using plasmidfinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother. 2014;58. pmid:24777092
- 56. Arredondo-Alonso S, Rogers MRC, Braat JC, Verschuuren TD, Top J, Corander J, et al. Mlplasmids: A user-friendly tool to predict plasmid- and chromosome-derived sequences for single species. Microb Genom. 2018;4. pmid:30383524
- 57. Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, et al. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res. 2014;42. pmid:24225323
- 58. Sweeney MT, Lubbers B V., Schwarz S, Watts JL. Applying definitions for multidrug resistance, extensive drug resistance and pandrug resistance to clinically significant livestock and companion animal bacterial pathogens. Journal of Antimicrobial Chemotherapy. 2018;73. pmid:29481657
- 59. Naas T, Oueslati S, Bonnin RA, Dabos ML, Zavala A, Dortet L, et al. Beta-lactamase database (BLDB)—structure and function. J Enzyme Inhib Med Chem. 2017;32: 917–919. pmid:28719998
- 60. Montealegre MC, Roh JH, Rae M, Davlieva MG, Singh K V., Shamoo Y, et al. Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance. Antimicrob Agents Chemother. 2017;61. pmid:27821450
- 61. BRENNER DJ, GRIMONT PAD, STEIGERWALT AG, FANNING GR, AGERON E, RIDDLE CF. Classification of Citrobacteria by DNA Hybridization: Designation of Citrobacter farmeri sp. nov., Citrobacter youngae sp. nov., Citrobacter braakii sp. nov., Citrobacter werkmanii sp. nov., Citrobacter sedlakii sp. nov., and Three Unnamed Citrobacter Genomospecies. Int J Syst Bacteriol. 1993;43: 645–658. pmid:8240948
- 62. Janda JM, Abbott SL, McIver CJ. Plesiomonas shigelloides Revisited. Clin Microbiol Rev. 2016;29: 349–74. pmid:26960939
- 63. Cabello FC, Godfrey HP, Buschmann AH, Dölz HJ. Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. Lancet Infect Dis. 2016;16: e127–e133. pmid:27083976
- 64. Diwan AD, Harke SN, Gopalkrishna, Panche AN. Aquaculture industry prospective from gut microbiome of fish and shellfish: An overview. J Anim Physiol Anim Nutr (Berl). 2022;106: 441–469. pmid:34355428
- 65. Zhu H, Yuan F, Yuan Z, Liu R, Xie F, Huang L, et al. Monitoring of Poyang lake water for sewage contamination using human enteric viruses as an indicator. Virol J. 2018;15: 3. pmid:29301542
- 66. Phillips AA, Speth DR, Miller LG, Wang XT, Wu F, Medeiros PM, et al. Microbial succession and dynamics in meromictic Mono Lake, California. Geobiology. 2021;19: 376–393. pmid:33629529
- 67. Marathe NP, Gaikwad SS, Vaishampayan AA, Rasane MH, Shouche YS, Gade WN. Mossambicus tilapia (Oreochromis mossambicus) collected from water bodies impacted by urban waste carries extended-spectrum beta-lactamases and integron-bearing gut bacteria. J Biosci. 2016;41: 341–346. pmid:27581926
- 68. Kiratisin P, Apisarnthanarak A, Saifon P, Laesripa C, Kitphati R, Mundy LM. The emergence of a novel ceftazidime-resistant CTX-M extended-spectrum β-lactamase, CTX-M-55, in both community-onset and hospital-acquired infections in Thailand. Diagn Microbiol Infect Dis. 2007;58: 349–355. pmid:17449211
- 69. Lupo A, Saras E, Madec J-Y, Haenni M. Emergence of blaCTX-M-55 associated with fosA, rmtB and mcr gene variants in Escherichia coli from various animal species in France. Journal of Antimicrobial Chemotherapy. 2018;73: 867–872. pmid:29340602
- 70. Yang J-T, Zhang L-J, Lu Y, Zhang R-M, Jiang H-X. Genomic Insights into Global bla CTX-M-55 -Positive Escherichia coli Epidemiology and Transmission Characteristics. Microbiol Spectr. 2023;11. pmid:37358409
- 71. Reid CJ, Cummins ML, Börjesson S, Brouwer MSM, Hasman H, Hammerum AM, et al. A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58. Nat Commun. 2022;13: 683. pmid:35115531
- 72. Galardini M, Clermont O, Baron A, Busby B, Dion S, Schubert S, et al. Major role of iron uptake systems in the intrinsic extra-intestinal virulence of the genus Escherichia revealed by a genome-wide association study. PLoS Genet. 2020;16: e1009065. pmid:33112851
- 73. Johnson TJ, Johnson SJ, Nolan LK. Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids. J Bacteriol. 2006;188: 5975–5983. pmid:16885466
- 74. Skyberg JA, Johnson TJ, Johnson JR, Clabots C, Logue CM, Nolan LK. Acquisition of Avian Pathogenic Escherichia coli Plasmids by a Commensal E. coli Isolate Enhances Its Abilities To Kill Chicken Embryos, Grow in Human Urine, and Colonize the Murine Kidney. Infect Immun. 2006;74: 6287–6292. pmid:16954398
- 75. Johnson TJ, Jordan D, Kariyawasam S, Stell AL, Bell NP, Wannemuehler YM, et al. Sequence Analysis and Characterization of a Transferable Hybrid Plasmid Encoding Multidrug Resistance and Enabling Zoonotic Potential for Extraintestinal Escherichia coli. Infect Immun. 2010;78: 1931–1942. pmid:20160015
- 76. Atlaw NA, Keelara S, Correa M, Foster D, Gebreyes W, Aidara-Kane A, et al. Identification of CTX-M Type ESBL E. coli from Sheep and Their Abattoir Environment Using Whole-Genome Sequencing. Pathogens. 2021;10: 1480. pmid:34832635
- 77. 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 (Pavia). 2021;12: 123–137.
- 78. Hanson ND. AmpC -lactamases: what do we need to know for the future? Journal of Antimicrobial Chemotherapy. 2003;52: 2–4. pmid:12775673
- 79. Mammeri H, Eb F, Berkani A, Nordmann P. Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered in a French hospital. Journal of Antimicrobial Chemotherapy. 2008;61: 498–503. pmid:18250231
- 80. Haenni M, Châtre P, Madec J-Y. Emergence of Escherichia coli producing extended-spectrum AmpC β-lactamases (ESAC) in animals. Front Microbiol. 2014;5. pmid:24592257
- 81. Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2: 123–140. pmid:15040260
- 82. Trabulsi LR, Keller R, Gomes TAT. Typical and Atypical Enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8: 508–513. pmid:11996687
- 83. Hernandes RT, Elias WP, Vieira MAM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297: 137–149. pmid:19527295
- 84. Chandran A, Mazumder A. Prevalence of Diarrhea-Associated Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Animal Hosts. Appl Environ Microbiol. 2013;79: 7371–7380. pmid:24056456
- 85. Yamamoto D, Hernandes RT, Liberatore AMA, Abe CM, Souza RB de, Romão FT, et al. Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites. PLoS One. 2017;12: e0171385. pmid:28178312
- 86. Adorján A, Thuma Á, Könyves L, Tóth I. First isolation of atypical enteropathogenic Escherichia coli from geese (Anser anser domestica) and first description of atypical EPEC from turkeys and pigeons in Hungary. BMC Vet Res. 2021;17: 263. pmid:34353312
- 87. Martínez-Martínez L, Pascual A, Hernández-Allés S, Alvarez-Díaz D, Suárez AI, Tran J, et al. Roles of β-Lactamases and Porins in Activities of Carbapenems and Cephalosporins against Klebsiella pneumoniae. Antimicrob Agents Chemother. 1999;43: 1669–1673. pmid:10390220
- 88. Davin-Regli A, Lavigne J-P, Pagès J-M. Enterobacter spp.: Update on Taxonomy, Clinical Aspects, and Emerging Antimicrobial Resistance. Clin Microbiol Rev. 2019;32. pmid:31315895
- 89. Mbehang Nguema PP, Onanga R, Ndong Atome GR, Tewa JJ, Mabika Mabika A, Muandze Nzambe JU, et al. High level of intrinsic phenotypic antimicrobial resistance in enterobacteria from terrestrial wildlife in Gabonese national parks. PLoS One. 2021;16: e0257994. pmid:34637441
- 90. Torres C, Alonso CA, Ruiz-Ripa L, León-Sampedro R, Del Campo R, Coque TM. Antimicrobial Resistance in Enterococcus spp. of animal origin. Microbiol Spectr. 2018;6. pmid:30051804
- 91. Poeta P, Costa D, Igrejas G, Rodrigues J, Torres C. Phenotypic and genotypic characterization of antimicrobial resistance in faecal enterococci from wild boars (Sus scrofa). Vet Microbiol. 2007;125: 368–374. pmid:17658226
- 92. Dec M, Stępień-Pyśniak D, Gnat S, Fratini F, Urban-Chmiel R, Cerri D, et al. Antibiotic Susceptibility and Virulence Genes in Enterococcus Isolates from Wild Mammals Living in Tuscany, Italy. Microbial Drug Resistance. 2020;26: 505–519. pmid:31663834
- 93. Stępień-Pyśniak D, Hauschild T, Dec M, Marek A, Urban-Chmiel R. Clonal Structure and Antibiotic Resistance of Enterococcus spp. from Wild Birds in Poland. Microbial Drug Resistance. 2019;25: 1227–1237. pmid:31107150
- 94. Figueiredo N, Radhouani H, Gonçalves A, Rodrigues J, Carvalho C, Igrejas G, et al. Genetic characterization of vancomycin-resistant enterococci isolates from wild rabbits. J Basic Microbiol. 2009;49: 491–494. pmid:19322837
- 95. Silva N, Igrejas G, Figueiredo N, Gonçalves A, Radhouani H, Rodrigues J, et al. Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus). Science of The Total Environment. 2010;408: 4871–4876. pmid:20624632
- 96. Lengliz S, Abbassi MS, Rehaiem A, Ben Chehida N, Najar T. Characterization of bacteriocinogenic Enterococcus isolates from wild and laboratory rabbits for the selection of autochthonous probiotic strains in Tunisia. J Appl Microbiol. 2021;131: 1474–1486. pmid:33629433
- 97. Radhouani H, Poeta P, Gonçalves A, Pacheco R, Sargo R, Igrejas G. Wild birds as biological indicators of environmental pollution: antimicrobial resistance patterns of Escherichia coli and enterococci isolated from common buzzards (Buteo buteo). J Med Microbiol. 2012;61: 837–843. pmid:22403140
- 98. Gonçalves A, Igrejas G, Radhouani H, López M, Guerra A, Petrucci-Fonseca F, et al. Detection of vancomycin-resistant enterococci from faecal samples of Iberian wolf and Iberian lynx, including Enterococcus faecium strains of CC17 and the new singleton ST573. Science of The Total Environment. 2011;410–411: 266–268. pmid:22018960
- 99. Santos T, Silva N, Igrejas G, Rodrigues P, Micael J, Rodrigues T, et al. Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago. Anaerobe. 2013;24: 25–31. pmid:24047647
- 100. Prichula J, Van Tyne D, Schwartzman J, Sant’Anna FH, Pereira RI, da Cunha GR, et al. Enterococci from Wild Magellanic Penguins (Spheniscus magellanicus) as an Indicator of Marine Ecosystem Health and Human Impact. Appl Environ Microbiol. 2020;86. pmid:32737129
- 101. Schwarz S, Feßler AT, Loncaric I, Wu C, Kadlec K, Wang Y, et al. Antimicrobial Resistance among Staphylococci of Animal Origin. Microbiol Spectr. 2018;6. pmid:29992898
- 102. Vrbovská V, Sedláček I, Zeman M, Švec P, Kovařovic V, Šedo O, et al. Characterization of Staphylococcus intermedius Group Isolates Associated with Animals from Antarctica and Emended Description of Staphylococcus delphini. Microorganisms. 2020;8: 204. pmid:32024111
- 103. Madhaiyan M, Wirth JS, Saravanan VS. Phylogenomic analyses of the Staphylococcaceae family suggest the reclassification of five species within the genus Staphylococcus as heterotypic synonyms, the promotion of five subspecies to novel species, the taxonomic reassignment of five Staphylococcus species to Mammaliicoccus gen. nov., and the formal assignment of Nosocomiicoccus to the family Staphylococcaceae. Int J Syst Evol Microbiol. 2020;70: 5926–5936. pmid:33052802
- 104. Taylor MJ, Mannan RW, U’Ren JM, Garber NP, Gallery RE, Arnold AE. Age-related variation in the oral microbiome of urban Cooper’s hawks (Accipiter cooperii). BMC Microbiol. 2019;19: 47. pmid:30791867
- 105. Zhou L, Huo X, Liu B, Wu H, Feng J. Comparative Analysis of the Gut Microbial Communities of the Eurasian Kestrel (Falco tinnunculus) at Different Developmental Stages. Front Microbiol. 2020;11. pmid:33391209
- 106. Larsson DGJ, Andremont A, Bengtsson-Palme J, Brandt KK, de Roda Husman AM, Fagerstedt P, et al. Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance. Environ Int. 2018;117: 132–138. pmid:29747082