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.