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Vibrio harveyi is a significant cause of infection in both marine animals and humans. It has been reported frequently in seafood-borne infections worldwide. This study was conducted to determine th...

Hard-shelled mussel (Mytilus coruscus) is a popular seafood in South Korea because of its delicacy and high nutritional value. Our study aimed to identify antimicrobial and heavy-metal resistance determinants in Aeromonas isolates from marketed hard-shelled mussel in South Korea. A total of 33 Aeromonas species were isolated, and antimicrobial disk diffusion test was done to observe antimicrobial resistance patterns. In addition, broth microdilution test was performed to determine resistance to heavy-metals. PCR amplification was done to detect resistance genes. High resistance to amoxicillin (100.0%), ampicillin (93.9%), rifampicin (78.8%), and cephalothin (48.5%) was observed where least resistance to other antimicrobials was also detected. In addition, the isolates showed high resistance to cadmium (Cd) (57.6%), and 42.4% and 27.3% were resistant to chromium (Cr) and copper (Cu). The occurrence of antimicrobial resistance genes, such as blaTEM, blaSHV, blaCTX-M, tetB, tetE, and intI1 genes, was observed in 9 (27.3%), 8 (24.2%), 8 (24.2%), 6 (18.2%), 5 (15.2%), and 9 (27.3%) isolates, respectively. Also, heavy-metal resistance genes, czcA, copA, and merA were detected in 17 (51.5%), 11 (33.3%), and 7 (21.2%) of the isolates, respectively. The results suggest that mussels are a reservoir of multidrug and heavy-metal-resistant Aeromonas spp.

Aeromonas species are bacterial pathogens that cause significant economic losses in the aquaculture industry worldwide. They are widely distributed in aquatic environments and cause several diseases in both humans and aquatic animals. The presence of various virulent Aeromonas spp. in aquatic environments predisposes infections in aquatic animals and humans. Concern about the transfer of pathogens from fish to humans also increased with the substantial increase in seafood consumption. Aeromonas spp. are also primary human pathogens that cause local and systemic infections, both in immunologically compromised and immunologically competent hosts. The most common Aeromonas spp. causing infections in aquatic animals and humans are A. hydrophila, A. salmonicida, A. caviae, and A. veronii biotype sobria. The ability of Aeromonas spp. to produce a variety of virulence factors enhances their pathogenic ability. Literature has supplied evidence for the presence of various virulence factors, including proteases, enterotoxins, hemolysin, and toxin genes of Aeromonas spp. in aquatic environments. The high prevalence of Aeromonas spp. in the aquatic environment is also a threat to public health. Because the Aeromonas spp. infections in humans are generally the result of ingestion or exposure to contaminated food and water. This review summarizes the recently published information on various virulence factors and virulence genes of Aeromonas spp. isolated from various aquatic environments, including seawater, freshwater, wastewater, and drinking water. It is also intended to highlight the risks associated with Aeromonas species’ virulence properties for both aquaculture and public health.

Human infections caused by the bacterial pathogens transmitted from pet-turtles are becoming very common and getting more importance as the turtles are considered unsafe pet animals, mainly for children and immunocompromised people. Pet-turtles are known as the reservoir of different bacterial species as their intestinal microflora. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose pet-turtles to bacterial infections. The presence of different virulence genes leads to the virulent potential of bacteria. The virulent bacteria can cause infections in turtles and humans, if the turtle owners or shopkeepers don't practice proper sanitation while handling turtles. The aim of this review paper was to provide an overview of different bacterial species isolated from pet-turtles for awareness-raising about potential health risks related to raise pet-turtles.

Aeromonas spp. are associated with seafood-related outbreaks worldwide. In seafood industry, shellfish play a major role in global seafood production. With this emerging trend of shellfish consumption, shellfish-related bacterial infections are being reported frequently. Aeromonas spp. are natural contaminants found in shellfish. Although 36 species have been identified, some species including Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria have dragged major attention as foodborne pathogenic bacteria. The ability to elaborate a variety of virulence factors of Aeromonas spp. contributes to the pathogenic activities. Also, emerging antimicrobial resistance in Aeromonas spp. has become a huge challenge in seafood industry. Furthermore, multidrug resistance increases the risk of consumer health. Studies have supplied pieces of evidence about the emerging health risk of Aeromonas spp. isolated from seafood. Therefore, the present review was intended to highlight the prevalence, virulence and antimicrobial resistance of Aeromonas spp. isolated from various types of shellfish.

Vibrio spp. are associated with water and seafood-related outbreaks worldwide. They are naturally present in aquatic environments such as seawater, brackish water and freshwater environments. These aquatic environments serve as the main reservoirs of antimicrobial-resistant genes and promote the transfer of antimicrobial-resistant bacterial species to aquatic animals and humans through the aquatic food chain. Vibrio spp. are known as etiological agents of cholera and non-cholera Vibrio infections in humans and animals. Antimicrobial-resistant Vibrio species have become a huge threat in regard to treating Vibrio infections in aquaculture and public health. Most of the Vibrio spp. possess resistance towards the commonly used antimicrobials, including β-lactams, aminoglycosides, tetracyclines, sulphonamides, quinolones and macrolides. The aim of this review is to summarize the antimicrobial resistance properties of Vibrio spp. isolated from aquatic environments to provide awareness about potential health risks related to Vibrio infections in aquaculture and public health.

Antimicrobial resistance properties of 32 Aeromonas strains isolated from fresh scallops (Patinopecten yessoensis (Jay)) marketed in Korea were assessed. All the Aeromonas spp., including A. salmonicida, were mesophilic and grew very well at 37°C. The isolates were tested for susceptibility to 19 antimicrobials belonging to eight antimicrobial classes. All isolates were multi-drug resistant, which means they were resistant to five or more antimicrobials. Higher resistance rates (≥ 50%) were observed for ampicillin, piperacillin, cephalothin, imipenem, meropenem, trimethoprim-sulfamethoxazole, tetracycline, oxytetracycline, and nalidixic acid while intermediate resistance was also determined. PCR assays revealed the presence of many antimicrobial resistance genes among the isolates in varying combinations. Among them, some isolates harbored higher numbers of resistant genes, e.g., A. veronii-V1 (aac(6’)-Ib, tetE, qnrS, IntI1), A. salmonicida–SL10 (IntI1, blaCTX, aac(3’)-Ib, aac(6’)-Ib, qnrS), A. hydrophila–H13 (IntI1, blaTEM, qnrS, aac(6’)-Ib, strA-strB). However, neither the blaSHV, blaIMP, tetB, qnrA, qnrB, and aphAI-IAB genes nor class1 integrons were detected in any of the isolates. Discrepancies between phenotypic and genetic resistance traits were observed in some isolates. With respect to outcomes, scallops are proposed as a source of multi-drug resistant Aeromonas spp. that harbor antimicrobial resistant genes.

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. 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. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as β-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL(−1) at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4–11), temperature (4–50 °C), and salinity (0.1–3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 μg mL(−1)), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-β, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00178-z) contains supplementary material, which is available to authorized users.

Aims The object of this study was to identify potential health concerns of the Aeromons spp. isolated from marketed Pacific abalone (Haliotis discus hannai) with respect to their virulence and antimicrobial resistance patterns. Methods and results We identified 29 strains of aeromonads consisting of five species; Aeromonas hydrophila (n = 9), Aeromonas enteropelogenes (n = 14), Aeromonas veronii (n = 3), Aeromonas salmonicida (n = 2) and Aeromonas sobria (n = 1), by employing series of biochemical tests and gene sequencing. In the phenotypic virulence assays, all isolates showed gelatinase and caseinase activities, while lipase formation (69%), phospholipase production (90%), DNase formation (82%), slime production (49%) and haemolysis activity (α = 18% and β = 82%) were also detected among isolates. Prevalence of virulence genes; aerA (100%), fla (66%), ahyB (73%), act (52%), alt (42%), ast (35%), ser (52%), gcat (69%), ascV (43%), hlyA (83%), lip (52%) and exu (59%) were detected by PCR assays. In disc diffusion test, 100% resistance was detected against ampicillin while cephalothin, rifampicin, oxytetracycline, colistine sulphate, nalidixic acid and piperaciliin were resisted by 86, 73, 42, 35, 28, 20 and 20% of the isolates respectively. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) indices ≥ 0·2. Conclusions Our findings suggest that the potential health risk posed by the abalone-borne Aeromonas spp. should not be underestimated. Significance and impact of the study This is the first time to evaluate possible public health risks upon consumption of abalone harbored Aeromonas spp. and also to isolate potential pathogenic and multidrug-resistant Aeromonas spp. from Pacific abalone in Korea.

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.

Development of antibiotic resistance in bacteria has challenged significantly in both veterinary and human medicine. In this study, we analyzed the potential risk of pet turtles harboring tribe Proteeae as a source of quinolone-resistant determinants, including plasmid-mediated quinolone resistance (PMQR) genes and target gene alterations in the quinolone resistance-determining region (QRDR). Antimicrobial susceptibility of 54 Proteeae isolates against ciprofloxacin, ofloxacin, levofloxacin, and nalidixic acid was examined. The PMQR genes and QRDR alterations were identified using conventional PCR assays and sequencing. Four isolates were resistant to all quinolones tested in this study. Nine isolates showed resistance to nalidixic acid and showed either intermediate resistance or susceptibility to other tested quinolones. All isolates resistant to one or more tested quinolones harbored mutations in gyrB and some also had gyrA and parC mutations. Of 54, 12 Proteeae isolates displayed the novel E466D, N440T, Q411S, and F417L mutations in gyrB. Among the PMQR genes, 41 (76%) isolates harbored the qnrD gene with the highest prevalence, whereas aac(6')Ib-cr, qnrS, qnrA, and qnrB genes were detected in 28 (52%), 9 (17.0%), 7 (13.0%), and 1 (1.9%) study isolates, respectively. The QRDR analysis of selected mutants revealed that increasing quinolone selective pressure led to a predominance of gyrA mutants. All results indicate that a healthy pet turtle can play as a potential reservoir for quinolone-resistant Proteeae, which it might cause public health risk on pet owners.

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.

As opportunistic pathogens, Aeromonas spp. can cause a multitude of infectious diseases in ornamental fish. In the present study, virulence properties and multiple antimicrobial resistance in 65 goldfish-borne Aeromonas spp. were investigated. The isolates were identified as A. hydrophila (n = 30), A. veronii biovar veronii (n = 32), and A. caviae (n = 3) by gyrB gene sequencing, and each of the species formed distinct clusters in the neighbor-joining phylogenetic tree. The virulence-related genes were detected by Polymerase chain reaction (PCR) assay in the following proportions: aer (87.7%), ahyB (86.2%), fla (83.1%), ascV (67.7%), ser (67.7%), act (66.2%), ast (61.5%), alt (56.9%), gcaT (53.8%), and lip (49.2%). According to the in vivo challenge test with A. hydrophila and A. veronii biovar veronii, the strains (AH1 and AV1) harboring the highest number of genes displayed LD50 values 2.2 × 106 and 9.8 × 106 CFU fish−1 whereas the strains (AH30 and AV32) harboring the lowest number of genes showed LD50 values 1.2 × 107 and 4.6 × 107 CFU fish−1, respectively. A negative relationship (r = −0.85) was found between the frequency of virulence genes and LD50 values. In antimicrobial disk diffusion test, each of the isolates was resistant to at least 4 antimicrobials. The multiple antimicrobial resistance index values ranged from 0.22 to 0.56. Our results suggest that ornamental goldfish can be the reservoir of virulent and multidrug resistant motile aeromonads.

Bacterial biofilm formation is one of the dynamic processes, which facilitates bacteria cells to attach to a surface and accumulate as a colony. With the help of biofilm formation, pathogenic bacteria can survive by adapting to their external environment. These bacterial colonies have several resistance properties with a higher survival rate in the environment. Especially, pathogenic bacteria can grow as biofilms and can be protected from antimicrobial compounds and other substances. In aquaculture, biofilm formation by pathogenic bacteria has emerged with an increased infection rate in aquatic animals. Studies show that Vibrio anguillarum, V. parahaemolyticus, V. alginolyticus, V. harveyi, V. campbellii, V. fischeri, Aeromonas hydrophila, A. salmonicida, Yersinia ruckeri, Flavobacterium columnare, F. psychrophilum, Piscirickettsia salmonis, Edwardsiella tarda, E. ictaluri, E. piscicida, Streptococcus parauberis, and S. iniae can survive in the environment by transforming their planktonic form to biofilm form. Therefore, the present review was intended to highlight the principles behind biofilm formation, major biofilm-forming pathogenic bacteria found in aquaculture systems, gene expression of those bacterial biofilms and possible controlling methods. In addition, the possibility of these pathogenic bacteria can be a serious threat to aquaculture systems.

Hard-shelled mussel (

Aeromonas spp. are ubiquitous bacteria that cause diseases in fish and other aquatic animals. They are the natural inhabitants of different aquatic environments, such as freshwater, brackishwater and marinewater. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose fish to Aeromonas infection. In ornamental fish, motile Aeromonas spp. are known as aetiological agents of motile aeromonad infections, which cause significant mortality in fish and economic loss in the ornamental fish industry. The existence of different virulence factors leads to the virulence potential of motile Aeromonas spp. There are several antimicrobials used to treat bacterial infections in ornamental fish. However, the extensive use of antimicrobials in the ornamental fish industry causes multidrug resistance. This article reviewed a multitude of virulence factors that are related to the ornamental fish-borne Aeromonas pathogenicity and the antimicrobial resistance determinants related to the multidrug resistance phenotypes of motile Aeromonas spp. in ornamental fish.

Salmonella enterica subsp. enterica is a part of the natural bacterial biota of pet turtles which can opportunistically cause human salmonellosis. In this study, virulence, antimicrobial resistance gene properties, and class 1 integron gene cassettes of 35 S. enterica subsp. enterica strains were investigated. The isolates were identified as S. Pomona (n = 9), S. Paratyphi (n = 7), S. Typhimurium (n = 5), S. Thompson (n = 5), S. Stanley (n = 4), S. Braenderup (n = 2), S. Kentucky (n = 1), S. Singapore (n = 1), and S. Potsdam (n = 1), respectively. By polymerase chain reaction (PCR) of virulence genes, invA and stn genes were detected in every isolate. The fimH and pefA genes were detected in 15 (42.86%) and 14 (40.00%) isolates, respectively. However, none of the isolates harbored fimA and spvC genes. In antimicrobial susceptibility testing, the resistance rates were observed against ampicillin (40.00%), cephalothin (34.00%), streptomycin (20.00%), and chloramphenicol (10.00%). The presence of streptomycin resistance encoding strA-strB gene was found in 12 (40.00%) isolates. Class 1 integron gene cassettes, qacE2 and orfD were identified in 8 (22.86%) strains. The results revealed that pet turtle-borne S. enterica subsp. enterica can harbor a considerable number of virulence, antimicrobial resistance genes, and class 1 integrons represent a potential risk for pet turtle keepers which must not be underestimated.

As the most consumed shrimp variety, white-leg shrimp (Litopenaeus vannamei) owns a high market demand in Korea. This study sought to screen the frozen white-leg shrimp for Aeromonas spp. harboring antimicrobial and heavy metal resistance characteristics. A total of 44 Aeromonas spp. strains were isolated and tested for antibiotic susceptibility and heavy metal tolerance followed by PCR-based detection of resistance genes and integrons. It was observed that resistance to ampicillin and oxacillin was 100% among isolates. Besides, 95%, 89%, 86%, 80%, 66%, and 43% of the isolates were resistant to nalidixic acid, tetracycline, cephalothin, streptomycin, trimethoprim-sulfamethoxazole, and imipenem, respectively, and less resistance to other antibiotics was also observed. Cr resistance was the highest (91%) among five heavy metals tested, whereas 57%, 32%, 20%, and 9% of the isolates were tolerant to Cu, Pb, Cd, and Hg, respectively. The PCR assays implied the presence of qnrB, qnrS, tetA, tetE, aac(6')-Ib, and aphAI-IAB, and intI1 genes among 80%, 77%, 18%, 30%, 9%, 0.25%, and 82% of the isolates, respectively. There were 35 (80%) integron 1-positive isolates harboring qacE2, dfrA1, orfC, orfD, aadB, catB3, oxa-10, and aadA1 genes in varying combinations. In addition, heavy metal resistance genes, CopA, merA, and CzcA were positive in 9%, 7%, and 27% of the isolates, respectively. According to these outcomes, the frozen white-leg shrimp in Korean markets can be suggested as a source of multidrug and heavy metal-resistant Aeromonas spp. that carries genetic determinants.

Aeromonas spp. are opportunistic pathogenic bacteria associated with a multitude of diseases in ornamental fish. In this study, virulence properties and antibiotic resistance patterns of 43 Aeromonas strains isolated from 46 zebrafish were investigated. The isolates were identified as Aeromonas veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), Aeromonas hydrophila (n = 8), A. caviae (n = 3), Aeromonas enteropelogenes (n = 2) and Aeromonas dhakensis (n = 1) by gyrB gene sequencing. The sequence divergence within and between the species ranged from 0-5·80% and 4·90-8·00%. Each species formed a distinct group in a neighbour-joining phylogenetic tree. The lipase production, biofilm formation, DNase activity, gelatinase production, caseinase production and β-hemolysis were phenotypically observed in 34 (79·07%), 33 (74·74%), 30 (69·77%), 25 (58·14%), 22 (51·18%) and 21 (48·84%) isolates. The virulence genes were detected by polymerase chain reaction (PCR) in following frequencies- aer (86·05%), hlyA (83·72%), gcaT (83·72%), lip (72·09%), act (67·44%), fla (65·12%), ascV (58·14%), ast (55·81%), ser (41·86%), ahyB (39·53%) and alt (25·58%). Every isolate was resistant to at least four antibiotics in disk diffusion test. The multiple antibiotic resistance (MAR) index values ranged from 0·22-0·50 among the isolates. Our study suggests that zebrafish can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are Gram-negative and facultative anaerobic bacteria which are ubiquitous in aquatic environments. Virulence properties and antibiotic resistance of ornamental fish-borne Aeromonas spp. are poorly understood. The virulence factors as well as multiple antibiotic resistance profiles of zebrafish-borne Aeromonas spp. were characterized for the first time in Korea. Most of the isolates were positive for phenotypic virulence traits and harboured several virulence genes revealing the virulence potential of zebrafish-borne Aeromonas spp. Additionally, the high multiple antibiotic resistance (MAR) index values displayed by the isolates highlight the necessity of responsible use of antibiotics in the ornamental fish industry.

Proteeae is a tribe which consists of three genera: Proteus, Providencia and Morganella. The objective of this study was to determine antimicrobial resistance profile, virulence genotype and class 1 and 2 integrons in Proteeae isolated from pet turtles and to determine the impact of antibiotic resistance on virulence and antimicrobial resistance genes. Integron-positive isolates were used to detect their gene cassette array. Sixty four Proteeae were isolated and all were resistant to macrolides (100%). Among 64 isolates 56, 52, 36 and 25 were resistant to nitrofurans, β-lactams, tetracycline and aminoglycoside respectively. Sixteen (25%) isolates were positive for intI1 while 14 (21·87%) were positive for integrase 2 (intI2). Eleven (17·18%) isolates were positive for class 1 variable region while 7 (10·93%) were positive for class 2 variable region. IMP27, a novel metallo β-lactamase gene was found in Providencia isolates. Proteus sp. were positive for every tested virulence genes and UreC gene was detected in 48·44% followed by zapA (17·19%), mrpA (17·19%) and hlyA (14·06%) genes. In this study, integron associated-antibiotic resistance genes have been identified in Proteeae isolates in a considerable range representing clear threats to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, multidrug-resistant Proteeae isolates had several antibiotic resistance genes. Integrons are important contributors to the development and dissemination of antibiotic resistance. We could detect both class 1 and 2 integrons and several gene cassette arrays in class 1 integron. The gene cassette arrays of the Class 2 integrons contained IMP27-dfrA1-aadA1-catB2-ybeA-ybgA in two isolates. To the best of our knowledge this is the first study to report detection of IMP27 in Providencia rettgeri isolates. All results indicate that healthy pet turtles act as potential reservoirs for Proteeae species with zoonotic potential.

Aims Yesso scallop (Patinopecten yessoensis) is a popular seafood in Korea. Aeromonas spp., well-known pathogenic bacteria, has been reported in some molluscan shellfish, but it has not been studied in scallops so far. Therefore, we aimed to isolate, identify and characterize the Aeromonas spp. isolated from marketed Yesso scallops to estimate their potential risk to public health. Methods and results Thirty-two Aeromonas spp. including A. hydrophila (n = 13), A. salmonicida (n = 11), A. media (n = 3), A. caviae (n = 2), A. veronii (n = 2) and A. enteropelogenes (n = 1) were isolated from 105 marketed scallops and tested for phenotypic pathogenicity, virulence genes and antimicrobial susceptibility. Mean total bacterial count of scallop meat was 1·34 × 104 CFU per gram. Slime production and lipase tests were positive in 97% of the isolates while DNase, protease, gelatinase, phospholipase and haemolysis were shown by 88, 88, 81, 88 and 72% of the isolates respectively. Eleven virulence genes were detected among Aeromonas spp. (act (75%), alt (59%), ast (47%), aerA (78%), lip (59%), ahyB (94%), ser (75%), hlyA (75%), fla (64%), gcat (84%) and ascV (23%)), and exu was negative in all isolates. Aeromonas hydrophila and A. salmonicida harboured ≥7 virulence genes and positive for enterotoxin genes, act, alt and ast. All the isolates were multidrug resistant and 100% resistant to ampicillin, colistin, vancomycin and cephalothin. Also, 30, 31, 20, 21, 29, 24, 27 and 27 of the isolates were resistant to piperacillin, clindamycin, erythromycin, nalidixic acid, imipenem, meropenem, trimethoprim-sulfamethoxazole and rifampicin respectively. Conclusions It is obvious with our results that the Aeromonas spp. isolated from Yesso scallops are highly virulent and potentially pathogenic, whereas the multidrug resistance further expedite their importance. Significance and impact of the study To our knowledge, this is the first study reporting Aeromonas spp. in scallop. This implies that not only the common varieties like oysters, but other bivalves can also harbour potentially pathogenic aeromonads which may have impacts on consumer health.

Proteus spp. are widely recognized as opportunistic pathogens causing urinary tract and septic infections in humans and animals. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance genes and mutations in the quinolone resistance determining region in association with the detection of quinolone susceptibility of 24 strains of pet turtle-borne Proteus spp. Susceptibility of 4 antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined by disk diffusion and minimum inhibitory concentration test. Six isolates were resistant to nalidixic acid showing either intermediate resistance or resistance to other quinolones. All nalidixic acid, resistant isolates harbored mutations in gyrB (N440T/A401G/Q411S). Two of the isolates had both gyrA (S83I) and parC (S80I) mutations. Twenty-one isolates were positive for the presence of plasmid-mediated quinolone resistance genes; the qnrD gene had the highest prevalence with 19 (79.2%), while qnrS, qnrA, qnrB, and aac(6′), Ib-cr genes were present in 9 (37.5%), 2 (8.3%), 1 (4.2%), and 11 (45.8%) isolates, respectively. These results suggest that pet turtle-associated Proteus spp. should be considered a potential source of antimicrobial resistance determinants.

Fish can be potentially co-infected by two or more bacterial strains, which can make synergistic influence on the virulence of infection. In this study, two opportunistic and multidrug resistant Aeromonas strains were isolated from wounds of morbid zebrafish with typical deep skin lesions similar to Motile Aeromonas Septicemia. Isolates were genetically identified as A. hydrophila and A. veronii by 16 S rRNA sequencing and phylogenetic analysis. Both isolates were positive for virulent genes (aerA, lip, ser, exu gcaT) and selected phenotypic tests (DNase, protease, gelatinase, lipase, biofilm production and β-haemolysis). A. hydrophila and A. veronii had strong antibiotic resistance against ampicillin, tetracycline, nalidixic acid, kanamycin, erythromycin, clindamycin and trimethoprim-sulfamethoxazole. Histopathological studies revealed that co-infection causes severe necrosis and hypertrophy in the muscles, kidney and liver of zebrafish. Naturally co-infected zebrafish showed highly induced tnf-α, il-1β, il-6, il-12, ifn, ifn-γ, cxcl18 b and ccl34a.4 at transcription level compared to healthy fish, suggesting virulence factors may activate immune and inflammatory responses of zebrafish. Experimentally infected zebrafish showed significantly higher mortality under co-infection with A. hydrohila and A. veronii (87%), followed by individual challenge of A. hydrophila (72%) or A. veronii (67%) suggesting that virulence of A. hydrophila have greater pathogenicity than A. veronii during co-infection.

The antibacterial activity of lime (Citrus aurantifolia) essential oil (LEO) and limonene was tested against seven Gram-negative and nine Gram-positive fish pathogenic bacteria isolated from cultured olive flounder, Paralichthys olivaceus (Temminck & Schlegel) in Korea. Limonene was >99% concentrated and LEO consisted of eleven chemical compounds including 56.22% of limonene. Disk diffusion assay, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests were done. LEO and limonene inhibited the growth of both Gram-negative and Gram-positive bacteria. LEO and limonene (MBC/MIC= 2-8) were both bactericidal and bacteriostatic for the strains tested. In every fish pathogenic bacteria, the inhibition zone diameter (IZD) increased in proportion to the oil concentration and the maximum effect was found at 100% (V/V) concentrations of LEO and limonene. The antibiogram pattern indicated that all the bacterial strains, excluding three strains of S. iniae (S186, S530, and S131), showed resistance to one or more antibiotics. The percentage of the relative inhibition zone diameter (RIZD %) exhibited high values at higher concentrations of all the agents. Since antibacterial activities of LEO and limonene were considerably effective against fish pathogenic bacteria, they could be used as alternatives to treat bacterial infections in aquaculture. CORRESPONDING AUTHOR: H. N.K.S. Pathirana, S. H.M.P. Wimalasena, B. C.J. De Silva, S. Hossain, G.-J. Heo [+] Laboratory of Aquatic Animal Medicine, Veterinary Medical Center and College of Veterinary Medicine Chungbuk National University, Chungdae-ro 1, Seowon-gu, Cheongju, Chungbuk 28644, Korea e-mail: gjheo@cbu.ac.kr

In this study, we performed genotype analysis based on multi-locus sequence typing (MLST) and antibiotic resistance of 27 pirAB-harboring Vibrio parahaemolyticus (Vp AHPND) isolates recovered from white leg shrimp (Penaeus vannamei) from acute hepatopancreatic necrosis disease (AHPND) affected farms (in 2016, n = 22) and retail markets (in 2016, n = 5) of Korea. Most Vp AHPND isolates were resistant to colistin, ampicillin, and streptomycin but susceptible to other antibiotics. The comparison of their antibiotic resistance to that of non-Vp AHPND isolates revealed a significantly higher resistance of Vp AHPND isolates to streptomycin. No differences, however, were observed in the minimal inhibitory concentration median (MIC50), 90th percentile (MIC90), and average multiple antibiotic resistance (MAR) index between the two groups. In MLST analysis, sequence types could not be defined for 19 isolates owing to the failure of recA gene sequencing. Seven new STs were found in five and two isolates from retail markets and AHPND affected farms, respectively. Based on the MLST-based phylogenetic tree, all isolates were divided into eight clades; 22 isolates from AHPND affected farms were grouped into three clades, and five isolates from retail markets formed singleton clades. These data suggest that most AHPND outbreaks in Korea may be related to Vp AHPND isolates originating from a common clone through the acquisition of pirAB. Further, the detection of Vp AHPND from retail shrimp samples suggests that the pathogen may be widely distributed in shrimp aquaculture and/or the environment of Korea.

The usage of essential oils as antimicrobial agents is gaining attention. Besides, pet turtles were known to harbor a range of pathogenic bacteria while the turtle keeping is a growing trend worldwide.The current study examined the antimicrobial activity of lemon grass oil (LGO) against seven species of Gram negative bacteria namely; Aeromonas hydrophila, A. caviae, Citrobacter freundii, Salmonella enterica, Edwardsiella tarda, Pseudomonas aeruginosa, and Proteus mirabilis isolated from three popular species of pet turtles. Along with the results of disc diffusion, minimum inhibitory and minimum bactericidal concentration (MIC and MBC) tests, LGO was detected as effective against 6 species of bacteria excluding P. aeruginosa. MIC of LGO for the strains except P. aeruginosa ranged from 0.016 to 0.5% (V/V). The lowest MIC recorded in the E. tarda strain followed by A. hydrophilla, C. freundii, P. mirabilis, and S. enterica. Interestingly, all the bacterial species except E. tarda were showing high multiple antimicrobial resistance (MAR) index values ranging from 0.36 to 0.91 upon the 11 antibiotics tested although they were sensitive to LGO.

Objectives The aim of this study was to characterise Aeromonas spp. isolated from popular species of pet turtle to assess the potential risk of pet turtles as a source of target gene alterations in the quinolone resistance-determining region (QRDR) and transferable plasmid-mediated quinolone resistance (PMQR) genes. Methods Twenty-five isolates comprising four species, namely Aeromonas enteropelogenes, Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii, were obtained from healthy pet turtles. The antimicrobial susceptibility of the isolates to nalidixic acid, ciprofloxacin and ofloxacin was examined by minimum inhibitory concentration (MIC) determination. QRDR substitutions and PMQR genes were detected using conventional PCR assays and sequencing. Results Although more than one-half of the isolates were resistant to nalidixic acid (14/25; 56%), most were susceptible to ciprofloxacin and ofloxacin. In QRDR substitution analysis, gyrA Ser-83 → Ile substitution was predominant among A. enteropelogenes isolates, whilst two isolates of A. caviae displayed a novel Asp-95 → Pro substitution. With regard to parC, Ser-80 → Ile substitution was noted in all species except A. veronii. Furthermore, qnrS, qnrB and aac(6′)-Ib-cr genes were detected in 68% (17/25), 8% (2/25) and 8% (2/25) of the isolates, respectively; 86% (12/14) of A. enteropelogenes isolates harboured a qnrS gene. Unexpectedly, quinolone resistance determinants were also detected in some isolates that were phenotypically susceptible to the tested quinolones. Conclusions The current study reveals the mismatch phenomenon between quinolone resistance phenotype and genotype of turtle-borne aeromonads and suggests that susceptible isolates might be a potential risk source for storage and transmission of resistance genes.

The present study aimed to investigate the incidence, virulence and antibiotic properties in Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea. A total of 32 Vibrio spp. isolates including V. parahaemolyticus (n = 4), V. alginolyticus (n = 11), V. diabolicus (n = 14) and V. harveyi (n = 3) were detected using gyrB sequencing. The phenotypic pathogenicity revealed that the DNase, amylase and phospholipase activities were 100%, while lipase, slime production, gelatinase and caseinase were detected in 72, 88, 88 and 81% of the isolates respectively. The PCR amplification for the detection of V. parahaemolyticus species-specific tdh, tlh, trh and toxR genes were positive in 4 (13%), 16 (50%), 0 (0%) and 4 (13%) isolates respectively. The V. alginolytuicus species-specific tdh, tlh, trh, toxR and vac genes were carried by 15 (47%), 29 (91%), 0 (0%), 15 (47%) and 25 (78%) of the isolates respectively. In addition, multidrug resistance was observed by 27 (84%) isolates, whereas higher resistant rates were observed against ampicillin, piperacillin, streptomycin and cephalothin. The occurrence of blaCTX (78%), blaTEM (40%), blaSHV (22%) and aac(6')-Ib (94%) were prevalent, while strAB, tetB, aphAI-IAB, intl1 and aadA1 gene cassettes were also detected. The results signify the potential health risks resulting from the consumption of raw cockles in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios are well known to cause human infections following consumption of raw or undercooked seafood. This phenomenon has undoubtedly increased the number of health issues over the past few years in Korea. Among the identified Vibrio spp., we could detect V. diabolicus and V. harveyi for the first time in marketed cockles in Korea. The presence of species-specific genes (tdh-VA, tlh-VP, tlh-VA and toxR-VA) in V. diabolicus exhibits the close genetic affinity among V. parahaemolyticus and V. alginolyticus. Furthermore, the prevalence of extended-spectrum β-lactamases (ESBLs) and other antibiotic resistance genes along with multidrug resistance signifies the potential threat for consumers.

Hard-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 3·70 to 10·40% and 0-1·50% respectively. Every species formed a distinct group in a neighbour-joining phylogenetic tree. The DNase, gelatinase, caseinase, β-haemolysis, biofilm and lipase activities were observed in 33 (100·00%), 31 (93·93%), 30 (90·90%), 27 (81·81%), 21 (63·63%) and 17 (51·51%) isolates respectively. The virulence genes were detected by PCR in the following frequencies: fla (90·09%), aer (87·88%), hlyA (87·88%), ahyB (81·19%), gcaT (75·76%), ser (69·70%), lip (66·67%), alt (57·58%), ast (51·51%) and act (21·21%). Every isolate was resistant to at least three of 18 antimicrobials in the disk diffusion test. The multiple antimicrobial resistance index values ranged from 0·11 to 0·44 among the isolates. Our study suggests that mussels can be a potential reservoir of virulent and multidrug-resistant Aeromonas sp. 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.

Antibacterial activity of cinnamon ( Cinnamomum zeylanicum ) essential oil (CEO) and cinnamaldehyde was tested against seven Gram negative and nine Gram positive fish pathogenic bacteria isolated from cultured olive flounder ( Paralichthys olivaceus ) in Korea. Disk diffusion assay, MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests were done. Cinnamon and cinnamaldehyde inhibited the growth of both Gram negative and Gram positive bacteria. Cinnamaldehyde showed lower MIC values than CEO which ranged from 0.001 to 0.015% (V/V). CEO (MBC/MIC 2-4) was bactericidal for all tested strains while cinnamaldehyde was bactericidal for all except 6 strains. For all fish pathogenic bacteria, the inhibition zone diameter (IZD) increased in proportion to the oil concentration and the maximum effect was found at 100% (V/V) concentration of CEO and cinnamaldehyde. The antibiogram pattern indicated that all the bacterial strains excluding three strains of Streptococcus iniae (S186, S530 and S131) showed resistance to one or more antibiotics. Higher percentages of relative inhibition zone diameter (RIZD %) were exhibited at higher concentrations of CEO and cinnamaldehyde. Since antibacterial activities of CEO and cinnamaldehyde were considerably effective against fish pathogenic bacteria, these could be used as alternatives to treat bacterial infections in aquaculture. Keywords: Antibacterial activity, Cinnamon essential oil, Cinnamaldehyde, Fish pathogenic bacteria, Olive flounder

The present study assessed the occurrence, virulence determinants and antimicrobial susceptibility patterns of Vibrio spp. isolated from live Manila clam (Ruditapes philippinarum). A total of 31 Vibrio spp. including 27 V. diabolicus, two V. fluvialis, one V. alginolyticus and one V. antiquarius were isolated and identified. Phenotypic detection of DNase, lipase, phospholipase, amylase and caseinase activities was 100%; and 87% gelatinase, 45% slime production and 6% haemolysin activities were also observed. The prevalence of toxin-related virulence genes for collagenase (94%), toxR (100%), tlh (68%), and VPI (71%) was detected by PCR. Additionally, two V. fluvialis isolates carried F-toxR and hupo genes. Moreover, 61% of the isolates showed multiple antimicrobial resistance indices >0·2. The resistance rates of ampicillin, piperacillin, colistin sulfate, rifampicin, and cephalothin were 100, 81, 71, 77 and 68% respectively. The prevalence of blaCTX-M (87%), blaTEM (55%) and Int1 (90%) genes was observed, whereas blaSHV, strA-strB, tetA, tetB, and aadA2 gene cassette were reported in varying combinations. However, armA, aac(3)-IIa and quinolone resistance genes (qnrA, qnrB, qnrS) were not amplified. Thus, the virulence along with extended-spectrum β-lactamases (ESBLs) and other antimicrobial resistance genes in multidrug-resistant Manila clam-borne vibrios may pose a public health threat for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: In Korea, eating raw seafood is considered a great delicacy. This has negatively affected the public by increasing health issues over the years due to the ingestion of vibrios. For the first time, we could identify Vibrio diabolicus and Vibrio antiquarius in marketed Manila clams in Korea. The prevalent Vibrio diabolicus isolates demonstrated the Vibrio parahaemolyticus and Vibrio cholerae homologous virulence genes (toxR, tlh, and VPI). Additionally, the abundance of extended-spectrum β-lactamases (ESBLs) and integron (IntI1) harboured by Manila clam-borne vibrios elucidate the potential health risk for consumers and may complicate health treatments in the case of infection.

Klebsiella sp. are responsible for a multitude of infectious diseases in both humans and animals. In this study, phylogenetic relationships, virulence and antimicrobial resistance gene properties of 16 Klebsiella sp. isolated from 49 pet turtles were investigated. The isolates including Klebsiella oxytoca (n = 13) and Klebsiella pneumoniae (n = 3) were identified using 16S rRNA gene sequencing and each species formed distinct clusters in the neighbour-joining phylogenetic tree. The prevalence of virulence genes including ureC (100%) and kfu (68·75%) was observed among the isolates using Polymerase chain reaction (PCR) assay. The fimH, mrkD and rmpA genes were detected in all K. pneumoniae while these were absent in every K. oxytoca isolate. In antimicrobial susceptibility testing, high resistance rates were observed against ampicillin (100%) and cephalothin (62·50%). The resistance rates against imipenem, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and ciprofloxacin were 12·50, 12·50, 12·50, 6·25 and 6·25% respectively. The presence of antimicrobial resistance genes such as plasmid-mediated quinolone resistance (PMQR) [qnrB (37·50%), qnrA (31·25%), qnrS (12·50%) and aac(6')-Ib-cr (12·50%)], extended-spectrum β-lactamase (ESBL) [blaCTX-M (18·75%)], β-lactamase [blaSHV-1 (18·75%)] and tetracycline resistance [tetE (12·50%)] was observed. The results revealed that pet turtle-borne Klebsiella sp. may carry different types of virulence and antimicrobial resistance genes which represents a potential threat to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Klebsiella sp. are nonmotile Gram-negative bacteria that are found in different environments. The virulence and antimicrobial resistance properties of pet turtle-borne Klebsiella sp. have not been studied before. Phylogenetic relationships, virulence traits and antimicrobial resistance profiles of pet turtle-borne Klebsiella sp. were characterized for the first time in Korea. Multiple virulence and antimicrobial resistance genes were observed among the isolates. The occurrence of virulence and antimicrobial resistance determinants in Klebsiella sp. may represent a potential threat to public health.

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 blaTEM, blaSHV, blaCTX‐M, qnrS, tetB, tetE, aac(6′)‐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‐β‐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.

To investigate the potential enterotoxicity and antimicrobial resistance of aeromonads from pet turtles as a risk for human infection, one hundred and two Aeromonas spp. were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%). Their potential enterotoxicities were evaluated by PCR assays for detecting genes encoding cytotoxic enterotoxin (act) and two cytotonic enterotoxins (alt and ast). 75.8% of A. hydrophila isolates exhibited the act+/alt+/ast+ genotype, whereas 94.4% of A. enteropelogenes isolates were determined to be act-/alt-/ast-. In an antimicrobial susceptibility test, most isolates were susceptible to all tested antibiotics except amoxicillin, ampicillin, cephalothin, chloramphenicol and tetracycline. Non-susceptible isolates to penicillins (ampicillin and amoxicillin) and fluoroquinolones (ciprofloxacin and norfloxacin) were frequently observed among the A. enteropelogenes isolates. Few isolates were resistant to imipenem, amikacin, ceftriaxone and cefotaxime. Collectively, these results suggest that pet turtles may pose a public health risk of infection by enterotoxigenic and antimicrobial resistant Aeromonas strains.