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

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.