RESULTS AND DISCUSSION
The Gram-negative bacilli were identified as belonging to theEnterobacter cloacae complex (E11R strain) by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The E11R strain displayed a multidrug-resistant (MDR) profile (Magiorakos et al., 2012) to amoxicillin/clavulanic acid, aztreonam, cefotaxime, ceftriaxone, cefepime, cefoxitin, ceftiofur, ciprofloxacin, enrofloxacin, chloramphenicol, fosfomycin, gentamicin, sulfamethoxazole/trimethoprim and tetracycline; remaining susceptible to ertapenem, imipenem, meropenem and amikacin. Furthermore, E11R strain exhibited resistance to colistin (MIC, 16 µg/mL), whereas ESBL production was detected by the DDST.
Genomic analysis identified the E11R strain as E. kobei , confirming a wide resistome, with genes conferring resistance to colistin [mcr-9.1 ], β-lactams [bla CTX-M-15, bla ACT-9,bla OXA-1 and bla TEM-1B], aminoglycosides [aac(3)-IIa , aadA1 , aph(3”)-Iband aph(6)-Id ], trimethoprim [dfrA14 ], tetracycline [tetA ], quinolones [aac(6’)-Ib-cr andqnrB1 ], fosfomycin [fosA ], sulphonamide [sul2 ], and phenicols [catA1 and catB3 ]. IncHI2 and IncHI2A replicons were detected, and analysis of the genetic environment confirmed that mcr-9.1 was flanked by IS903Band IS26 upstream and downstream, respectively (Figure 1) (Kieffer et al., 2019; Lin et al., 2020; Tyson et al., 2020; Yuan et al., 2019).
In the last years, colistin has been used as a last-resort for the treatment of infections caused by multidrug-resistant and/or carbapenem-resistant Gram-negative bacteria (El-Sayed Ahmed et al., 2020). However, the previous and extensive use of colistin in production animals, as a growth promoter or for prophylaxis, has been recognized as a responsible factor for the emergence and rapid dissemination of mobile colistin resistance (mcr ) genes (Rhouma, Beaudry, & Letellier, 2016). In this respect, since the description of mcr-1 , nine additional mcr homologues have been described, with several gene variants occurring worldwide (El-Sayed Ahmed et al., 2020; Wang et al., 2020).
The mcr-9.1 allele was identified for the first time inSalmonella Typhimurium isolated from a human patient (Carroll et al., 2019), and currently has been reported worldwide with a rapid dissemination among Enterobacterales from human, food, poultry, swine and horse samples (Figure 2) (Börjesson et al., 2020; Carroll et al., 2019; El-Sayed Ahmed et al., 2020; Faccone et al., 2020; Khalifa et al., 2020; Li et al., 2020; Ling et al., 2020; Osei Sekyere, Maningi, Modipane, & Mbelle, 2020; Saidenberg et al., 2020; Wang et al., 2020). Recently, two novel variants, mcr-9.2 and mcr-9.3 , have been identified in Enterobacter hormaechei subsp. xiangfangensis(GenBank accession number: MN164032.1) andKlebsiella pneumoniae (GenBank accession number: MT505326.1) isolates, respectively. The environmental dissemination of critical priority pathogens has been considered a serious threat to ecosystem maintenance (de Carvalho et al., 2020; Sevilla et al., 2020). The exposure to polluted environments could also substantially increase the risk for marine populations acquire such bacteria (Power et al., 2016). Specifically in Brazilian coast, the occurrence of MCR-type, ESBL and/or carbapenemases has been documented in recreational waters (Campana, Montezzi, Paschoal, & Picão, 2017; Fernandes et al., 2017; Paschoal et al., 2017; Sellera et al., 2017a), beach sand samples (Furlan, dos Santos, Ramos, Gallo, & Stehling, 2020), and mangrove waters (Sacramento et al., 2018). More critically, their occurrence colonizing or infecting marine host have begun to be documented (Goldberg et al., 2019; Sellera et al., 2017b; Sellera et al., 2018).
In this study, we report the emergence of mcr-9.1 in an ESBL-producing E. kobei isolated from an infected free-living dolphin. In this regard, the Franciscana dolphin is considered the most threatened small cetacean in the southwestern Atlantic Ocean, which includes the coasts of Brazil, Uruguay, and Argentina (Sucunza, Danilewicz, Cremer, Andriolo, & Zerbini, 2018). Due to their coastal habits, these animals have been frequently exposed to different degrees of anthropogenic impacts, including fisheries by catch and habitat degradation (Sucunza, Danilewicz, Cremer, Andriolo, & Zerbini, 2018). Consequently, this species is currently listed as vulnerable to extinction at both global and regional levels (Zerbini, Secchi, Crespo, Danilewicz, & Reeves, 2017). Therefore, the environmental dissemination of antibiotic-resistant critical-priority pathogens may have serious implications for endangered wild animals. In this regard, while occurrence of CTX-M-15-producing E. coli has been reported in captive dolphins (Manageiro et al., 2015), we demonstrated that this type of pathogen can also threaten free-living dolphins. Particularly,E. kobei has never been isolated from animal infections. In fact,E. kobei is member of the E. cloacae complex that includes other five species (i.e., E. asburiae , E. cloacae ,E. dissolvens , E. hormaechei and E. nimipressuralis ) recognized as important nosocomial pathogens (Mezzatesta, Gona, & Stefani, 2012).
In summary, we report the emergence of MCR-9-producing bacteria in marine wildlife. Considering that oceanic environments and human and animal health are strictly connected, the dissemination of clinically important MDR pathogens in marine ecosystems must be viewed as serious One Health problem. Finally, since multidrug-resistant pathogens have begun to be associated with fatal cases of infections in endangered animals (Fuentes-Castillo et al, 2020), continued surveillance of MCR- and ESBL-producing bacteria in marine ecosystems should be globally performed for a better comprehension of the transmission pathways and clinical impacts on marine wildlife.