3.4. Virulome of ESBL-positive E. coli ST58 colonizing Brazilian merganser is associated with a virulent behavior
Virulome analysis of ESBL-producing E. coli PMPU strain highlighted virulence factors, including adherence factors (fim, eaeH , lpfAO113 , csgBCDEFG ), invasins (iss ,ibeBC ), cytolytic pore-forming toxin (hlyE ), iron acquisition systems (entBCEFS , fepABCD ) and chemotaxis (cheABRMWYZ , motAB ), among other virulence factors commonly found in commensal and pathogenic E. coli strains (Table 1). The virulent potential of PMPU strain was confirmed in the G. mellonella infection model, where strains inoculated at 1 × 105 CFU killed 100% of wax moth larvae within 50h, showing a more virulent behavior than E. coli ATCC 25922, but no more than hypervirulent meningitis-causing E. coli MNEC RS218 (Figure S1). G. mellonella has been successfully utilized as anin vivo model to assess the pathogenic potential of clinically important bacterial pathogen. Therefore, responses to bacterial infections observed in this model could closely mimics responses displayed by mammalian models (Jander, Rahme, & Ausubel, 2000; Kavanagh & Reeves, 2004; Lange et al., 2019). In this study, virulent performance of E. coli PMPU strain was correlated with virulence factors commonly identified in pathogenic E. coli lineages from humans and poultry, highlighting adherence factors (fimBCEFGHI, eaeH , lpfAO113 , csgBCDEFG ) (Osek, Weiner & Hartland, 2003; Dale & Woodford, 2015; Torres, 2016; Sarowska et al., 2019), invasins (iss , ibeBC ) (Sarowska et al., 2019), toxin (hlyE ) (Wyborn et al., 2004), iron acquisition systems (entBCEFS , fepABCD ) (Torres, 2016; Robinson, Heffernan, & Henderson, 2018) and chemotaxis factors (cheABRMWYZ ,motAB ) (Pettersen, Mosevoll, Lindemann, & Wiker, 2016). In this regard, adherence factors and invasins found in the E. coli PMPU strain may have contributed to the colonization in different tissues of the bird; and, the cytolytic pore-forming toxin hlyE could be related to hemodynamic disturbances and tissue damage found in the histopathology (Oscarssonet al, 1999; Lai et al, 2000; Lithgow, Haider, Roberts, & Green, 2007). On the other hand, the immature immune system in a 8-day-old Brazilian merganser, the artificial incubation conditions (Ruiz-Castellano et al., 2016), as well as use of disinfectants may contributed to the selection of a virulent E. coli resistant to a wide range of antibiotics and disinfectants, establishing a disseminated infection with a fatal end. In order to avoid new infections due toE. coli widely resistant to antimicrobials and disinfectants, a cleaning of the environments was carried out using peracetic acid concentrated at 0.2%. After this, no new cases of deaths occurred due to this bacterium.
Virulent pathogens resistant to an increasing number of antimicrobials cause thousands of deaths in the human population each year (Gu et al. , 2018; Cassini et al. , 2019; Centers for Disease Control, 2019). In this concern, wildlife plays an important role in the epidemiology of antibiotic-resistant pathogens in the environment (Alcalá et al. , 2016; Vittecoq et al., 2016; Sevilla et al. , 2020). However, little is known about the impact of these MDR-pathogens on wildlife, especially on threatened wildlife species (Gonçalves et al. , 2012; Larsson et al. , 2018; Ramey & Ahlstrom, 2020). In this study, we isolated a MDR ESBL-producingE. coli with virulent behavior, belonging to international clone ST58 and serotype O102:H30, causing fatal infection in a critically endangered Brazilian merganser. Of note, a MDR colistin-resistantE. coli ST58 was recently isolated from a polluted mangrove ecosystem in Brazil (Sacramento et al. , 2018), being able to become a serious threat to the associated wildlife and human population.
A better integration of environmental and wildlife issues is necessary to a successful One Health approach for global AMR crisis (White & Hughes, 2019). In this context, to understand epidemiologically the evolution and adaptation of AMR, wildlife veterinarians must increasingly report the challenges that arise when treating antimicrobial-resistant pathogenic bacteria in wildlife species. Herein, we report a fatal colibacillosis by MDR-ESBL-producing E. coli in critically endangered Brazilian merganser, highlighting that besides colonization, critical priority pathogens are threatening wildlife.