Aims: Manila clam (Ruditapes philippinarum) is one of the most popular seafood in Korea, owing to their unique taste and nutritional value. This study aimed to disclose the antibiotic and heavy metal resistance characteristics of Aeromonas spp. isolated from marketed Manila clam in Korea. Methods and results: A total of 36 Aeromonas spp. strains were isolated and subjected to two tests: an antibiotic disk diffusion test to determine their resistance to antibiotics, and a broth dilution test to determine their resistance to heavy metals. PCR-based amplification was performed to detect the resistance genes. A high level of resistance to ampicillin (100%) and cephalothin (89%) was observed, while 42, 39, 36 and 36% of the isolates were resistant to oxytetracycline, imipenem, nalidixic acid and tetracycline respectively. In addition, among the tested heavy metals, cadmium (Cd) recorded the highest resistance rate (61%), followed by chromium (Cr) (50%), lead (Pb) (47%) and copper (Cu) (37%). However, mercury (Hg) resistance was not observed. PCRs revealed the occurrence of bla TEM , bla SHV , bla CTX-M , qnrS, tetB, tetE, aac(6 0 )-Ib, strA-strB and intI1 genes among 100, 31, 31, 78, 78, 89, 25, 50 and 72% of the isolates respectively. Moreover, heavy metal resistance genes, copA, merA and czcA were detected in 25, 47 and 61% of the isolates respectively. Conclusions: The results suggest the importance of multi-drug and heavy metal-resistant aeromonads in Manila clam to assess the consumer safety and public health. Significance and Impact of the Study: This study is the first to elaborate on the importance of multi-drug and heavy metal-resistant aeromonads in Manila clam. Particularly, the presence of extended-spectrum-b-lactamase genes and other antibiotic resistance genes intensifies the possible health risks and may complicate therapeutic treatments upon infection, while heavy metal resistance suggests possible heavy metal exposure.

Aeromonas sp. are opportunistic pathogenic bacteria which are associated with various diseases in ornamental fish, aquaculture raised species and wild fisheries. In our study, antimicrobial resistance patterns, antimicrobial resistance genes and class 1 integron gene cassettes of 52 guppy‐borne Aeromonas sp. were examined. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. Every isolate was resistant to at least four antimicrobials in disc diffusion test. The resistance to amoxicillin, nalidixic acid and oxytetracycline was 100% among the tested isolates. 92·30, 76·92, 71·15, 51·92, 51·92 and 50·00% of the isolates were resistant to ampicillin, rifampicin, imipenem, cephalothin, tetracycline and trimethoprim respectively. The multiple antibiotic resistance index values ranged from 0·28 to 0·67. PCR amplification of antimicrobial resistance genes implied the occurrence of tetracycline resistance (tetA (65·39%), tetE (25·00%) and tetB (15·38%)), plasmid‐mediated quinolone resistance (qnrS (26·92%) and qnrB (17·31%)) and aminoglycoside resistance (aphaAI‐IAB (7·69%) and aac (6′)‐Ib (3·84%)) genes in the isolates. The IntI gene was positive for 36·54% of the isolates and four class 1 integron gene cassette profiles (aadA2, qacE2‐orfD, aadA2‐catB2 and dfrA12‐aadA2) were identified. These data suggest that ornamental guppy can be a reservoir of multidrug‐resistant Aeromonas sp. which comprise different antimicrobial resistance genes and class 1 integrons. Significance and Impact of the Study Antimicrobial resistance genes and integron gene cassettes of ornamental fish‐borne aeromonads are poorly studied. The antimicrobial resistance patterns, antimicrobial resistance genes and class 1 integron gene cassettes of Aeromonas sp. isolated from ornamental guppy were characterized for the first time in Korea. The incidence of different antimicrobial resistance genes and class 1 integron gene cassettes were observed in multidrug‐resistant Aeromonas isolates. This result suggests that better management practices are necessary to prevent and address the serious consequences of indiscriminate and inappropriate antimicrobial use, and the distribution of multidrug‐resistant bacteria.

Aeromonas spp. are Gram‐negative opportunistic bacteria which have been commonly associated with fish diseases. In this study, antibiogram, antimicrobial resistance genes and integrons of 43 zebrafish‐borne Aeromonas spp. were studied. The isolates were identified as six Aeromonas species (A. veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), A. hydrophila (n = 8), A. caviae (n = 3), A. enteropelogenes (n = 2) and A. dhakensis (n = 1)). Antibiogram of the isolates indicated that most of them were resistant to amoxicillin (100·00%), nalidixic acid (100·00%), oxytetracycline (100·00%), ampicillin (93·02%), tetracycline (74·42%), rifampicin (67·44%) and imipenem (65·15%). Multiple antimicrobial resistance (MAR) index values ranged from 0·19–0·44 to 90·70% isolates showed multidrug resistance. PCR of antimicrobial resistance genes revealed that the tetracycline resistance gene (tetA) was the most predominant (67·44%) among the isolates. The qnrS (53·49%), tetB (30·23%), tetE (30·23%), qnrB (23·26%) and aac(6’)‐Ib‐cr (4·65%) genes were also detected. Class 1 integrase (IntI1) gene was found in 46·51% of the isolates. Two types of class 1 integron gene cassette profiles (qacG‐aadA6‐qacG and drfA1) were identified. The results showed that zebrafish‐borne aeromonads can harbour different types of antimicrobial resistance genes and class 1 integrons. Significance and Impact of the Study Aeromonas spp. are important pathogens found in diverse environments. Antimicrobial resistance genes and integrons of ornamental fish‐borne Aeromonas spp. are not well studied. The antibiogram, antimicrobial resistance genes and class 1 integrons of Aeromonas spp. isolated from zebrafish were characterized for the first time in Korea. The prevalence of tetracycline resistance genes, plasmid‐mediated quinolone resistance genes and class 1 integron gene cassettes were observed among the isolates. The qacG‐aadA6‐qacG gene cassette was identified for the first time in Aeromonas spp. The results suggest that the wise use of antimicrobials is necessary for the better management of the ornamental fish.

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Significance and Impact of the Study: Aeromonas sp. are known as common pathogenic bacteria isolated from seafood. The virulence factors and antimicrobial resistance profiles of mussel-borne Aeromonas sp. are poorly understood. This study demonstrated for the first time the existence of virulence markers and antimicrobial resistance of Aeromonas sp. from mussels in Korea. Majority of the isolates were positive for phenotypic virulence characteristics and harboured several virulence genes which reveal the potential virulence of mussel-borne Aeromonas sp. Multiple antimicrobial resistance was also observed among the isolates. Our study highlights the importance of food safety standards in mussel consumption. AbstractHard-shelled mussel (Mytilus coruscus) is a popular seafood in Korea. This study aimed to determine the virulence markers and antimicrobial resistance patterns of 33 Aeromonas strains isolated from mussels. The isolates were identified as A. salmonicida (n = 14), A. veronii (n = 9), A. enteropelogenes (n = 4), A. caviae (n = 3), A. allosaccharophila (n = 2) and A. bivalvium (n = 1) by gyrB gene sequencing. The sequence divergence between and within the species ranged from 3Á70 to 10Á40% and 0-1Á50% respectively. Every species formed a distinct group in a neighbour-joining phylogenetic tree. The DNase, gelatinase, caseinase, b-haemolysis, biofilm and lipase activities were observed in 33 (100Á00%), 31 (93Á93%), 30 (90Á90%), 27 (81Á81%), 21 (63Á63%) and 17 (51Á51%) isolates respectively. The virulence genes were detected by PCR in the following frequencies: fla (90Á09%), aer (87Á88%), hlyA (87Á88%), ahyB (81Á19%), gcaT (75Á76%), ser (69Á70%), lip (66Á67%), alt (57Á58%), ast (51Á51%) and act (21Á21%). Every isolate was resistant to at least three of 18 antimicrobials in the disk diffusion test. The multiple antimicrobial resistance index values ranged from 0Á11 to 0Á44 among the isolates. Our study suggests that mussels can be a potential reservoir of virulent and multidrugresistant Aeromonas sp.

Significance and Impact of the Study: This study summarized the virulence and antimicrobial resistance in Aeromonas spp. associated with shellfish. This review may help to increase consumer awareness about the possible health risk of shellfish borne Aeromonas spp. infections and emphasizes the importance of alternative or regulated control methods in Aeromonas spp. infections as most of the antimicrobials are less effective against them.

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