Colistin‐resistant Entero bacter kobei carrying mcr‐9.1 and bla CTX‐M‐15 infecting a critically endangered franciscana dolphin (Pontoporia blainvillei), Brazil

Abstract The emergence of mobile mcr genes mediating resistance to colistin is a critical public health issue that has hindered the treatment of serious infections caused by multidrug‐resistant pathogens in humans and other animals. We report the emergence of the mcr‐9.1 gene in a polymyxin‐resistant extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacter kobei infecting a free‐living franciscana dolphin (Pontoporia blainvillei), threatened with extinction in South America. Genomic analysis confirmed the presence of genes conferring resistance to clinically relevant β‐lactam [bla CTX‐M‐15, bla ACT‐9, bla OXA‐1 and bla TEM‐1B], aminoglycoside [aac(3)‐IIa, aadA1, aph(3'')‐Ib and aph(6)‐Id], trimethoprim [dfrA14], tetracycline [tetA], quinolone [aac(6')‐Ib‐cr and qnrB1], fosfomycin [fosA], sulphonamide [sul2] and phenicol [catA1 and catB3] antibiotics. The identification of mcr‐9.1 in a CTX‐M‐15‐producing pathogen infecting a critically endangered animal is of serious concern, which should be interpreted as a sign of further spread of critical priority pathogens and their resistance genes in threatened ecosystems.

The franciscana dolphin (Pontoporia blainvillei) is considered the most threatened small cetacean in the south-western Atlantic Ocean, which includes the coasts of Brazil, Uruguay and Argentina (Sucunza et al., 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 et al., 2018). Consequently, this species is currently listed as vulnerable to extinction by the International Union for Conservation of Nature (Cunha et al., 2014;Zerbini et al., 2017), and as critically endangered by the Red Book of Threatened Species of Fauna, Brazil (ICMBio, 2018).
In this study, we report the emergence of mcr-9.1 in an ESBLproducing E. kobei infecting a free-living franciscana dolphin in Brazil. Additionally, an epidemiological landscape of global distribution of MCR-9-producing Enterobacterales circulating at humananimal interface is presented.

| MATERIAL S AND ME THODS
In December 2019, a female neonate franciscana was found stranded alive in Mambucaba Beach, in Angra dos Reis (−23.027184, −44.518130), located in the Southern coast of Rio de Janeiro state, Brazil ( Figure S1). The animal was rescued by the staff of the Santos Basin Beach Monitoring Project (PMP-BS), presenting excoriations on the head and with part of the umbilical cord still present. The dolphin was closely monitored, receiving intensive care and bottlefeeding with a special dolphin formula every 3 hr. However, after 11 hr in captivity, the animal began to exhibit clinical signs of shock leading to death. In order to determine the main causes of death, necropsy was performed, where histopathological analysis of fixed lung tissue revealed severe pneumonia. Additionally, bacteriological culture of respiratory exudate collected through the spiracle was positive for Gram-negative bacilli.
Escherichia coli ATCC 25,922 was used as control strain. Bacterial conjugation for the mcr-9.1-positive E. kobei isolate was done in a liquid and solid mating-out assay (Lampkowska et al., 2008), using the azide-resistant E. coli C600 as recipient.
Genomic DNA was extracted and used to construct a pairedend library, which was sequenced using the NextSeq 550 platform (Illumina), using paired-end reads (150 bp). De novo genome assembly and contig annotation was carried out using CLC Genomics
Genomic analysis identified the E11R strain as E. kobei, confirming a wide resistome, with genes conferring resistance to colistin IS903B. On the other hand, the region downstream of mcr-9.1 included wbuC (encoding a cupin fold metalloprotein), but no genes encoding the two-component system qseC-qseB, which has been associated with the expression of mcr-9 in other Enterobacterales (Kananizadeh et al., 2020;Kieffer et al., 2019). Conjugation attempts to evaluate the transferability of the mcr-9.1 gene were unsuccessful. In this regard, absence of the qseC-qseB genes in the E. kobei  ), Sweden (22, Börjesson et al., 2020 and the United States of America (23, Carrol et al, 2019;24, Chavda et al., 2019;25, Tyson et al., 2020), from human and nonhuman sources 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 the rapid dissemination of mobile colistin resistance (mcr) genes (Rhouma et al., 2016). In this respect, since the detection of mcr-1, nine additional mcr homologues have been described, with several gene variants occurring worldwide .
The mcr-9.1 allele was identified for the first time in Salmonella 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, pets, swine and horse samples (Figure 2). Recently, two novel variants, mcr-9.2 and mcr-9.3, have been identified in Enterobacter hormaechei subsp.
In this study, we report the emergence of mcr-9.1 in an ESBLproducing E. kobei isolated from an infected free-living franciscana dolphin, a species critically endangered by anthropogenic activities in Brazil (Cunha et al., 2014;ICMBio, 2018). The occurrence of CTX-M-15-producing E. coli has been reported in captive dolphins (Manageiro et al., 2015), and now, we demonstrated that this type of pathogen can also threaten free-living dolphins, which may have serious implications for wild populations and associated ecosystems.
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 multidrugresistant 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. to Saquarema/RJ, is a requirement set by the Brazilian Institute of the Environment (IBAMA) for the environmental licensing of the oil and natural gas production and transport by Petrobras.

CO N FLI C T O F I NTE R E S T S
No potential conflict of interest was reported by the authors.

E TH I C A L A PPROVA L
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required for this specific study. The licences and research permit for monitoring programme and the biological sampling were issued by the Brazilian government (IBAMA-ABIO 624/2015); all animal handling procedures and protocols followed the required ethics and welfare practices.