Aeromonas spp. Isolated from Pacific Abalone (Haliotis discus hannai) Marketed in Korea: Antimicrobial and Heavy-Metal Resistance Properties.

Antimicrobial and heavy-metal resistance of 29 Aeromonas spp. (Aeromonas hydrophila n = 9, Aeromonas enteropelogenes n = 14, Aeromonas veronii n = 3, Aeromonas salmonicida n = 2, and Aeromonas sobria n = 1) isolated from Pacific abalone marketed in Korea were analyzed. All isolates were found to be resistant against ampicillin. High level of resistant to cephalothin (86%), rifampicin (73%), imipenem (42%), and oxytetracycline (35%) were also detected. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) index ≥ 0.2. The PCR assays implied the presence of qnrS, qnrB, qnrA, tetB, tetA, aac (3')- IIa, aac(6')-Ib, aphAI-IAB, blaCTX, blaTEM, and intI1 genes among 76%, 28%, 14%, 17%, 3%, 3%, 41%, 10%, 41%, 28%, and 66% of the isolates, respectively. Class 1 integron gene cassette profiles aadA1(3%) and aadA2 (3%) were also identified. Lead (Pb) resistance was the highest (69%) among 5 heavy metals tested, whereas 38%, 27%, and 20% of the isolates were resistant to Cadmium (Cd), Chromium (Cr), and Copper (Cu), respectively. Heavy-metal resistance genes, CopA, CzcA, and merA were positive in 83%, 75%, and 41% of the isolates, respectively. In conclusion, observed genotypic and phenotypic resistance profiles of Aeromonas spp. against antimicrobials and heavy metals reveal the ability of serving as a source of antimicrobials and heavy-metal-resistant traits.

Prevalence of virulence and antimicrobial resistance genes in Aeromonas species isolated from marketed cockles (Tegillarca granosa) in Korea.

This study aimed to examine incidence, virulence and antimicrobial properties in Aeromonas spp. isolated from cockles (Tegillarca granosa) in Korea. Firstly, genomic DNA was extracted from 32 Aeromonas spp. isolates, and PCR screening for virulence, antimicrobial resistance genes was carried out. The disk diffusion assay was used to examine antimicrobial susceptibility. Aeromonas spp. isolates comprised, A. hydrophila (n = 8), A. veronii (n = 15), A. media (n = 2), A. salmonicida (n = 2), A. allosaccharophila (n = 1), A. bestiarum (n = 1), A. culicicola (n = 1), A. enteropelogenes (n = 1) and A. rivipollensis (n = 1). High prevalence of virulence-related genes reported as; act (69%), alt (47%), ast (41%), aerA (56%), lip (50%), ahyB (47%), ser (28%), fla (66%), gcat (44%), ascV (50%) and hlyA (72%). All isolates were multidrug resistant, while highest resistance level observed for ampicillin (100%), followed by imipenem (81%), rifampicin (78%), cephalothin (72%), piperacillin (47%) and Colistin sulfate (31%). The presence of blaSHV , blaCTX , tetE, aac(6')-Ib, strA-strB, qnrS, qnrB and IntI1 genes were reported in varying combinations. Nevertheless, blaTEM , blaIMP , tetA, tetB, qnrA, qnrB and aphAI-IAB genes and the class1 integron were not detected. The high occurrence of virulence and antimicrobial resistance genes in cockles reveals that it can be a potential health risk source for consumers.

Manila clam (Ruditapes philippinarum) marketed in Korea as a source of vibrios harbouring virulence and ß-lactam resistance genes.

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 ß-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 ß-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.

Prevalence of virulence and extended-spectrum ß-lactamase (ESBL) genes harbouring Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea.

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 ß-lactamases (ESBLs) and other antibiotic resistance genes along with multidrug resistance signifies the potential threat for consumers.

Antimicrobial resistance of pathogenic Aeromonas spp. isolated from marketed Pacific abalone (Haliotis discus hannai) in Korea.

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 ß = 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.

Phylogenetic relationships, virulence and antimicrobial resistance properties of Klebsiella sp. isolated from pet turtles in Korea.

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 ß-lactamase (ESBL) [blaCTX-M (18·75%)], ß-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.

Species identification, virulence markers and antimicrobial resistance profiles of Aeromonas sp. isolated from marketed hard-shelled mussel (Mytilus coruscus) in Korea.

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, ß-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.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

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 ß-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.

Antibiotic and heavy metal resistance genes in Aeromonas spp. isolated from marketed Manila Clam (Ruditapes philippinarum) in Korea.

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-ß-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.

Incidence of antimicrobial resistance genes and class 1 integron gene cassettes in multidrug-resistant motile Aeromonas sp. isolated from ornamental guppy (Poecilia reticulata).

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.

Multidrug resistant Aeromonas spp. isolated from zebrafish (Danio rerio): antibiogram, antimicrobial resistance genes and class 1 integron gene cassettes.

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.

Aeromonas spp. from marketed Yesso scallop (Patinopecten yessoensis): molecular characterization, phylogenetic analysis, virulence properties and antimicrobial susceptibility.

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.

Incidence of antibiogram, antibiotic resistance genes and class 1 and 2 integrons in tribe Proteeae with IMP27 gene for the first time in Providencia sp. isolated from pet turtles.

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, ß-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 ß-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.

Characterization of virulence properties and multi-drug resistance profiles in motile Aeromonas spp. isolated from zebrafish (Danio rerio).

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 ß-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.