Genomic Features of an MDR Escherichia coli ST5506 Harboring an IncHI2/In229/blaCTX-M-2 Array Isolated from a Migratory Black Skimmer

Migratory birds have contributed to the dissemination of multidrug-resistant (MDR) bacteria across the continents. A CTX-M-2-producing Escherichia coli was isolated from a black skimmer (Rynchops niger) in Southeast Brazil. The whole genome was sequenced using the Illumina NextSeq platform and de novo assembled by CLC. Bioinformatic analyses were carried out using tools from the Center for Genomic Epidemiology. The genome size was estimated at 4.9 Mb, with 4790 coding sequences. A wide resistome was detected, with genes encoding resistance to several clinically significant antimicrobials, heavy metals, and biocides. The blaCTX-M-2 gene was inserted in an In229 class 1 integron inside a ∆TnAs3 transposon located in an IncHI2/ST2 plasmid. The strain was assigned to ST5506, CH type fumC19/fimH32, serotype O8:K87, and phylogroup B1. Virulence genes associated with survival in acid conditions, increased serum survival, and adherence were also identified. These data highlight the role of migratory seabirds as reservoirs and carriers of antimicrobial resistance determinants and can help to elucidate the antimicrobial resistance dynamics under a One Health perspective.


Introduction
Extended-spectrum β-lactamase (ESBL)-producing Enterobacterales have been considered one of the greatest threats to human health, being listed as critical priority pathogens by the World Health Organization (WHO) [1].Despite greater emphasis being placed on the human clinical sphere, the One Health approach draws attention to the need for studies involving other ecological spheres, given the interdependence among humans, animals, and the environment [2].Dissemination of antimicrobial resistance between these different sectors can occur through direct transmission of resistant bacteria or through genetic exchanges, mainly by plasmids and other mobile genetic elements, as integrons and transposons [3].In this regard, whole-genome sequencing can be a valuable tool for surveillance of antimicrobial resistance, as it allows a deep understanding of the genetic basis of resistance mechanisms, evolution, and dissemination [4].
Since wild animals can also be exposed to human-associated multidrug-resistant (MDR) pathogens, there is a global trend for monitoring the dissemination of medically relevant bacteria in wildlife populations [5].Recently, migratory birds have been recognized as important reservoirs and vectors for spreading ESBL-producers across the globe [5,6].When contacting contaminated environments, these animals can incorporate antimicrobial resistant bacteria, as ESBL-producing Escherichia coli, in the gut microbiota; then, it can be spread over distant geographic locations, according to the migratory route [7].
Black skimmers (Rynchops niger) are widespread piscivorous seabirds that inhabit sandy shorelines, being commonly found in the Nearctic and Neotropical regions [8].Due to their migration behavior, they appear seasonally in large numbers in both the Pacific and Atlantic coasts of South America and are frequently observed in bays, estuaries, beaches, and shallow lagoons, where they prey on populations of small fish [8].In this study, we aimed to analyze the genomic features of an MDR ESBL-producing E. coli strain recovered from the gut microbiota of a migratory black skimmer.

Materials and Methods
In March 2016, as part of a surveillance study on antimicrobial-resistant Gram-negative bacteria among wild animals, a cloacal swab sample was collected from a black skimmer (R. niger), at the admission time to a wildlife rehabilitation center.The bird was rescued by firefighters at a beach on the southeast coast of Brazil (−23.985143,−46.309956) and presented a fracture of the right humerus.Until the sample collection time, no medication had been administered to the bird.The sample was inoculated on MacConkey agar plates supplemented with ceftriaxone (2 µg/mL), colistin (2 µg/mL) and ciprofloxacin (1 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA), for screening of bacteria resistant to cephalosporins, polymyxins, and fluoroquinolones, respectively.Agar plates were incubated for 18-24 h, at 35 • C. The bacterial isolate was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).Antimicrobial susceptibility was determined by disk diffusion and E-test methods [9], and the colistin susceptibility test was performed by the broth microdilution method, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [9].E. coli 25922 was used as the quality control strain.
No ethical approval was required for this study.Biological sample collection was authorized by the Authorization System and Information on Biodiversity (SISBIO license number 55804-2).
In summary, we report an international E. coli clone, isolated from a migratory seabird, carrying a wide resistome against clinically significant antimicrobials, heavy metals, and biocides, which should be considered a critical epidemiological issue.The broad resistome identified in this WHO critical priority pathogen is likely related to an environment impacted by anthropogenic activities [34].In addition, these data highlight the importance of monitoring migratory seabirds as reservoirs and carriers of such bacteria and genetic determinants and their role as sentinels of ecosystem health [35].Last but not least, this study provides valuable genomic information that might be useful in understanding the antimicrobial resistance dynamics under a One Health perspective.

Table 1 .
Phenotypic and genomic data from Escherichia coli strain ICBTMS1.