MATERIALS AND METHODS
In December 2019, a female neonate Franciscana dolphin 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 with excoriations on
the head and with part of the umbilical cord. The dolphin was closely
monitored, receiving intensive care and bottle-feeding with a special
dolphin formula every 3 hours. However, after 11 hours in captivity, the
animal began to exhibit clinical signs of shock with subsequent 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.
Antimicrobial susceptibility testing was performed by the disc diffusion
method (CLSI, 2020), including amoxicillin/clavulanic acid, aztreonam,
cefotaxime, ceftriaxone, cefepime, cefoxitin, ceftiofur, ciprofloxacin,
enrofloxacin, chloramphenicol, fosfomycin, amikacin, gentamicin,
ertapenem, imipenem, meropenem, sulfamethoxazole/trimethoprim and
tetracycline. In addition, colistin susceptibility testing was performed
by broth microdilution method
(http://www.eucast.org). ESBL production was
screened by the double‐disc synergy test (DDST; CLSI, 2020).
Genomic DNA was extracted and used to construct a paired-end library,
which was sequenced using the NextSeq 550 platform (Illumina, San Diego,
CA, USA), using paired-end reads (150 bp). De novo genome
assembly and contig annotation was carried out using CLC Genomics
Workbench 12.0.3. Prediction of bacterial species, resistome and
plasmidome was performed using fast K-mer algorithm KmerFinder 3.2
(Larsen et al, 2014), ResFinder 3.2 (Zankari et al. , 2012), and
PlasmidFinder 2.1 (Carattoli et al. , 2014) databases,
respectively (http://www.genomicepidemiology.org/).