Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia

Abstract Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired β-lactamases, including extended-spectrum-β-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel ( Mytilus galloprovincialis ) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and β-lactam/β-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC ( bla FOX , bla CMY , bla MOX , bla LAT , bla BIL , bla DHA , bla ACC , bla MIR , bla ACT) , ESBLs ( bla TEM , bla SHV , bla CTX-M , bla PER , bla VEB , bla GES/IBC , bla OXA ) and integrases ( intI1 , intI2 , intI3 ). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila ) isolates, with bla CTX-M-15 gene identified in 19 and bla SHV-12 in 12 isolates. Among them, 10 isolates simultaneously harboured bla CTX-M-15 and bla SHV-12 , while 3 isolates additionally carried an AmpC β-lactamase bla FOX-2 gene. bla PER-1 gene was identified in a single isolate also harbouring the bla CTX-M-15 gene. While bla SHV-12 was chromosomally encoded, bla CTX-M-15 was located on conjugative IncFIB-type plasmids of ~ 40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates. To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.