International clones of extended‐spectrum β‐lactamase (CTX‐M)‐producing Escherichia coli in peri‐urban wild animals, Brazil

Abstract CTX‐M‐type extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli clones have been increasingly reported worldwide. In this regard, although discussions of transmission routes of these bacteria are in evidence, molecular data are lacking to elucidate the epidemiological impacts of ESBL producers in wild animals. In this study, we have screened 90 wild animals living in a surrounding area of São Paulo, the largest metropolitan city in South America, to monitor the presence of multidrug‐resistant (MDR) Gram‐negative bacteria. Using a genomic approach, we have analysed eight ceftriaxone‐resistant E. coli. Resistome analyses revealed that all E. coli strains carried bla CTX‐M‐type genes, prevalent in human infections, besides other clinically relevant resistance genes to aminoglycosides, β‐lactams, phenicols, tetracyclines, sulphonamides, trimethoprim, fosfomycin and quinolones. Additionally, E. coli strains belonged to international sequence types (STs) ST38, ST58, ST212, ST744, ST1158 and ST1251, and carried several virulence‐associated genes. Our findings suggest spread and adaptation of international clones of CTX‐M‐producing E. coli beyond urban settings, including wildlife from shared environments.


| INTRODUC TI ON
The spread of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales has been broadly reported worldwide (Brolund, 2014;Fernandes et al., 2018;Pardon et al., 2015). In this respect, a number of interlinked factors, such as food animals, environmental sources, human migration and access to basic sanitation in highly populated cities, are contributing for the accelerated dissemination of these bacteria in urban and wild environments (Radhouani et al., 2014;Sacramento et al., 2018;Sellera, Fernandes, Moura, Carvalho, & Lincopan, 2018).
While the exposure to polluted environments constitutes a risk factor for humans to acquire multidrug-resistant (MDR) bacteria, recent studies have pointed out that it could also have implications for wildlife (Cerdà-Cuéllar et al., 2019;Sellera, 2019;Wang et al., 2017). In fact, although this matter remains poorly addressed under ecological perspectives, the scientific community and nature conservation authorities have begun to see wild animals as reservoirs and potential disseminators of ESBL-producing bacteria (Ardiles-Villegas, González-Acuña, Waldenström, Olsen, & Hernández, 2011;Cerdà-Cuéllar et al., 2019;Sellera, 2019;Wang et al., 2017).

| RE SULTS AND D ISCUSS I ON
In this study, eight ceftriaxone-resistant E. coli isolates (8/90; 8.88%) were recovered from five birds (one owl and four vultures) and three mammals (coatis). MDR profiles, defined as resistant to three or more classes of antibiotics (Magiorakos et al., 2012), were evidenced in six isolates (ECPET11, ECPET31, ECPET36, ICBUR6, ICBUR15 and ICBUR20). ECPET3 displayed resistance only to cephalosporins and aztreonam, whereas ECPET13 was resistant to cephalosporins, aztreonam and nalidixic acid. Additionally, ESBL production was confirmed by double-disc synergy test (DDST), and PCR analysis revealed the presence of bla CTX-M -type genes in all eight bacterial isolates (Table 1). No MCR-1-positive or carbapenemase-producing bacteria were identified.
Schematic representations of the genetic contexts surrounding bla CTX-M -type genes in E. coli strains are presented in Figure 1.

Regarding other plasmids identified in this study, IncN and IncHI2
have been related to the spread of bla CTX-M-1 and bla CTX-M-9 , respectively, and IncQ1 or IncX plasmid has been responsible by dissemination of carbapenemase encoding genes Mollenkopf et al., 2017;Paul et al., 2017;Zhao & Hu, 2013).
In this study, genomic analysis identified E. coli strains belonging to international ST38, ST58, ST212, ST744, ST1158 and ST1251 (Table 1). The global distribution of these E. coli clones is presented in Figures 2 and 3. The broadly distributed E. coli ST38 and ST744 have been reported in wildlife, farm animals and human samples from Europe, Africa, Asia, Australia and America, in general associated with the production of clinically significant beta-lactamases  was recovered from food animals (Vogt et al., 2014). Regarding E.
coli ST1251, fluoroquinolone-resistant strains have been reported in animal faeces and wastewater (Jamborova et al., 2015;Varela, Macedo, Nunes, & Manaia, 2015), as well as mcr-1-harbouring strains from food animals (Zurfluh et al., 2017). Escherichia coli belonging to ST58 has been globally reported from a variety of sources in- ESBL-producing E. coli in wild animals begun to be documented in 2006, in Portugal (Costa et al., 2006), and then were rapidly observed in other countries from Europe, Africa, Asia, South America, North America and Australia (Allen et al., 2010;Wang et al., 2017).
Predominantly, E. coli-and K. pneumoniae-producing CTX-M seem to be the most adapted to these hosts; however, the identification of ESBL genes in other different species of Enterobacterales has already been reported (Wang et al., 2017). In most of cases, animals became colonized in gastrointestinal tract without any evidences of infection, contributing for the silent dissemination of these critically important pathogens in natural environments.
A widely debated example is the occurrence of ESBL-producing bacteria in migratory birds, which are probably involved in the spread of these pathogens through long distances, including natural reserves and pelagic areas with low anthropogenic impact (Ardiles-Villegas et al., 2011;Cerdà-Cuéllar et al., 2019).
Otherwise, the role of peri-urban wild animals as disseminators of bacterial pathogens has been so far neglected. In this study, all animals sampled lived in the transboundary area of São Paulo city, the most populated metropolitan region of Brazil, with about 21.5 million inhabitants, and one of the ten most populous metropolitan regions in the world. Even though the source of these bacterial isolates remains uncertain, wildlife is not directly exposed to antibiotics in most cases and other anthropogenic pathways of transmission, such as contact to contaminated water and predation of infected animals, should be considered (Wang et al., 2017). Yet, it is important to take in account that some highly polluted rivers cross this area, where KPC-2-and ESBL-producing K. pneumoniae isolates from water samples were previously reported Oliveira et al., 2014).  Figure S1). Remarkably, these strains also shared identical serotype, resistome and plasmidome. These findings suggest an adaptation of CTX-M-producing E. coli into the wildlife food chain and the versatility of these bacteria to colonize different hosts. Indeed, interspecific interactions among wild animals colonized by ESBL producers represent an incommensurable threat to ecosystem maintenance, since Enterobacteriaceae constitutes the gut microbiota of most endothermic animals (Madoshi et al., 2016). Thus, antimicrobial resistance must also be viewed as an ecological problem (Fuentes-Castillo et al., 2019).
In conclusion, anthropogenic activities have been contributing for the dissemination of ESBL-producing bacteria in wildlife. The occurrence of ESBL-producing bacteria in peri-urban wild animals from highly populated cities is a critical issue and deserves special attention. Therefore, continuous epidemiological and genomic surveillance studies are urgently required to determine routes of transmission of these bacteria in wildlife. Finally, while humans can negatively affect nature environments for contributing to the spread of MDR bacteria, animals could also disseminate these pathogens to humans in a continuous cycle. which kindly supplied antibiotic discs for antimicrobial susceptibility testing, and the CEFAP-Genial by whole-genome sequencing support.

E TH I C A L S TATEM ENT
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CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.