Resistance profiles and genes of Enterobacteriaceae from cetaceans stranded in Philippine waters from 2018-2019 provide clues on the extent of antimicrobial resistance in the marine environment

With the premise that cetaceans are sentinels for understanding the extent of antimicrobial resistance in the marine environment, we determined the phenotypic and genotypic antibiotic resistance profiles of the Enterobacteriaceae isolated from cetaceans (representing twelve cetacean species) that stranded in Philippine waters from 2018-2019. The phenotypic identifications and antibiotic susceptibility profiles of the isolates were determined through VITEK 2 system while their genotypic identifications were confirmed through 16S rRNA gene sequencing. Targeted antibiotics for profiling phenotypic resistance include penicillins, cephalosporins, carbapenems, quinolones, polymyxins and folate pathway inhibitors while detected antibiotic resistance genes (ARGs) for evaluating genotypic resistance include: (1) ampicillins (blaAmpC); (2) cephalosporins (blaAmpC blaTEM, blaSHV, and blaCTX-M); (4) carbapenem (blaKPC); (4) polymyxins (mcr-1) and (5) sulphonamides (sul1, and sul2). Percent resistances (% R), percent susceptibilities (% S) and multiple antibiotic resistance (MAR) index values were computed. Eighty-six Enterobacteriaceae were isolated from the exhaled breath condensate and swab samples of 19 stranded cetaceans. These isolates were confirmed to belong to the following genera: Escherichia (39.53%), Enterobacter (26.74%), Klebsiella (24.41%), Citrobacter (5.81%), Morganella (1.16%), Pantoea (1.16%) and Providencia (1.16%). Overall, 35/86 (40.70%) of the isolates exhibited acquired resistances against cephalosporins (i.e., cefuroxime, 26/86 or 30.23%), polymyxins (i.e., colistin, 6/86 or 6.97%), folate-pathway inhibitors (i.e., trimethoprim-sulfamethoxazole,5/86 or 5.82%), ampicillin (3/86 or 3.49%), and cefoxitin (2/86 or 2.32%), while the lowest resistance (1.16% of isolates) were resistant against amoxicillin-clavulanic acid, piperacillin and imipenem. Moreover, 40.70% of the isolates were characterized as multidrug-resistant (2.33%) and extensively drug-resistant (38.37%) while 5/86 (5.81%) of the isolates had MAR indices greater than 0.2. Six out of seven (85.71%) of the targeted ARGs responsible for the resistance types for ampicillins, cephalosporins, polymyxins and sulphonamides (i.e., blaAmpC, blaSHV blaTEM, mcr-1, sul1 and sul2, respectively) were detected in 48.57% of isolates. Antibiotic susceptibility testing revealed that a considerable portion of the isolates exhibited acquired resistance to selected antibiotics and were categorized as multidrug-resistant (MDR) or extremely drug-resistant (XDR). As for genotypic resistance, six out of seven target antibiotic resistance genes (ARGs) responsible for resistance to ampicillins, cephalosporins, polymyxins, and sulfonamides were detected in nearly half of the isolates with acquired resistance. Considering the habitat ranges of the source animals, this indicates the extent of reach of antibiotics and/or ARGs in the marine environment, and pelagic migratory cetaceans may play an important role in their dissemination.