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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.

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.

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.

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.

Pet turtles are considered a source of bacterial infection to humans when handled in captivity. Turtles purchased from 9 pet shops and 8 online markets in Korea were examined to determine whether the turtles and their environment were contaminated with Citrobacter spp. Biochemical tests and morphology revealed that Citrobacter spp. were isolated from 7 fecal and 76 environmental samples. Among the 7 fecal isolates, 5 bacteria were identified as Citrobacter freundii through 16S rRNA gene sequencing. The isolation rate of Citrobacter spp. from soil and water samples increased over time. Each of the isolate's antibiotic resistance was characterized with a disk diffusion test. The strains showed susceptibility against amikacin, ceftriaxone, ciprofloxacin, imipenem, sulfamethoxazole/trimethoprim, and tetracycline, but were resistant to cefoxitin, cephalotin, and chloramphenicol. These results indicate that pet turtles are a potential source of Citrobacter infection in humans in Korea.

The antibacterial activity of essential oil from lemongrass ( Cymbopogon flexuosus ) (LGO) was tested against strains of Edwardsiella tarda (n=4), Photobacterium damselae (n=1), Lactococcus garvieae (n=1), Streptococcus iniae (n=4) and S. parauberis (n=4) isolated from cultured olive flounders in Korea. Disk diffusion assay, MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests showed the strains of Streptococcus spp . were the most sensitive. LGO was bactericidal (MBC/MIC=1-4) for all strains of L. garvieae , S. iniae and the FP5060 strain of E. tarda , and the FP5228, S527 and S1466 strains of S. parauberis suggesting sensitivity to LGO. Gram-negative bacteria were less sensitive than Gram-positive bacteria. Since antibacterial activity of LGO was effective against fish pathogenic bacteria tested in this study, the use of LGO could be helpful in treating such bacterial infections in fish. Key words: lemongrass oil; olive flounders; fish pathogenic bacteria; antimicrobial activity  IN VITRO UAŒINEK OLJA LIMONSKE TRAVE (Cymbopogon flexuosus) PROTI RIBJI PATOGENI BAKTERIJI, IZOLIRANI IZ GOJENEGA MORSKEGA LISTA (Paralichthys olivaceus) Povzetek: Protibakterijsko delovanje eteriAnega olja limonske trave ( Cymbopogon flexuosus ; LGO) je bilo preizkuAieno glede njihovega delovanja proti sevom bakterij Edwardsiella tarda (n = 4), Photobacterium damselae (n = 1), Lactococcus garvieae (n = 1), Streptococcus iniae (n = 4) in S. parauberis (n = 4) pridobljenih iz gojenih morskih listov v Koreji. Difuzijska metoda z diskom ter testi MIC (najmanjAia zaviralna koncentracija) in MBC (najmanjAia baktericidna koncentracija) so pokazali, da so bili sevi Streptococcus spp. najbolj obAutljivi. LGO je imel baktericidno delovanje (MBC / MIC = 1-4) na vse seve L. Garvieae in S. Iniae ter na seva FP5060 in FP5228 E. Tarda in seva S527 in S1466 S. parauberis . Gram-negativne bakterije so bile manj obAutljive od gram-pozitivnih bakterij. Ker je bilo antibakterijsko delovanje LGO uAinkovito proti ribjim patogenim bakterijam, preizkuAienim v tej Aitudiji, bi lahko uporaba LGO pomagala pri zdravljenju tovrstnih bakterijskih okuA¾b pri ribah. KljuAne besede: olje limonske trave; morski list; patogene bakterije rib; protimikrobno delovanje

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.

The antibacterial activity of clove (Syzygium aromaticum) essential oil (CEO) and eugenol was tested against seven Gram-negative and nine Gram-positive fish pathogenic bacteria isolated from cultured olive flounder (Paralichthys olivaceus) in Korea. Eugenol was >99% concentrated and CEO consisted of seven chemical compounds including 83.63% of eugenol. Disk diffusion assay, MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests showed CEO and eugenol inhibited the growth of both Gram-negative and Gram-positive bacteria. CEO (MBC/MIC= 1-2) and eugenol (MBC/MIC= 1-4) were bactericidal for all tested strains. The percentage of relative inhibition zone diameter exhibited high values at higher concentration of both CEO and eugenol. Positive correlation was observed between MAR index values and MIC values of CEO (r =0.61) and eugenol (r =0.15). Since antibacterial activity of CEO and eugenol were effective against fish pathogenic bacteria, the use of CEO and eugenol could be advantageous to treat bacterial infections in aquaculture. Key words: antibacterial activity; clove essential oil (CEO); eugenol; fish pathogenic bacteria; olive flounder PROTIBAKTERIJSKA AKTIVNOST ETERIAŒNEGA OLJA NAGELJNOVIH A½BIC IN EVGENOLA PROTI PATOGENIM BAKTERIJAM RIB, IZOLIRANIH IZ GOJENEGA MORSKEGA LISTA ( Paralichthys olivaceus ) Protibakterijsko delovanje eteriAnega olja nageljnovih A¾bic (Syzygium aromaticum) (CEO) in evgenola je bilo testirano pri sedmih vrstah po Gramu negativnih in devetih vrstah po Gramu pozitivnih patogenih bakterij, ki so bile izolirane iz gojenega morskega lista (Paralichthys olivaceus) v JuA¾ni Koreji. Evgenol je bil 99-odstotno koncentriran, CEO pa je sestavljalo sedem kemiAnih spojin, med njimi je bilo najveA evgenola, ki je predstavljal 83,odstotka eteriAnega olja. Metoda difuzije v trdem gojiAiAu, minimalna zaviralna koncentracija (MIC) in minimalna baktericidna koncentracija (MBC) so pokazale, da sta CEO in evgenol zavirala rast tako po Gramu negativnih kot po Gramu pozitivnih bakterij. CEO (MBC/MIC = 1-2) in evgenol (MBC/MIC = 1-4) sta bila baktericidna za vse testirane seve. Odstotek velikosti relativne zaviralne cone je bil visok pri viAiji koncentraciji CEO in evgenola. Pozitivna soodvisnost je bila opaA¾ena med vrednostmi indeksa MAR in vrednostmi MIC pri CEO (r = 0,61) in evgenolu (r = 0,15). Ker sta CEO in evgenol pokazala uAinkovito delovanje proti patogenim bakterijam rib, bi bila uporaba CEO in evgenola lahko uporabna za zdravljenje bakterijskih okuA¾b v ribogojniAitvu. KljuAne besede: antibakterijsko delovanje; eteriAno olje klinAkov (CEO); evgenol; patogene bakterije rib; morski list

Aeromonas spp. are opportunistic pathogens related to multiple infectious diseases in ornamental fishes. In the present study, the antimicrobial susceptibility, resistance genes, and integrons of 65 goldfish-borne Aeromonas spp. were evaluated. The isolates were identified as A. hydrophila (n = 30), A. veronii (n = 32), and A. punctata (n = 3) by gyrB sequencing. The antimicrobial susceptibility testing of the isolates designated that most of the isolates were resistant to amoxicillin (100.00%), nalidixic acid (100.00%), ampicillin (98.46%), tetracycline (92.31%), rifampicin (86.15%), and cephalothin (61.54%) and each of the isolates showed multiple antimicrobial resistance phenotype (resistant to ≥3 classes of antimicrobials). PCR amplification of antimicrobial resistance genes revealed that the plasmid-mediated quinolone resistance gene, qnrS, was the most prevalent (73.85%) among the isolates. The other antimicrobial resistance genes were detected in the following proportions: qnrB (26.15%), aac(6')-Ib-cr (4.60%), tetA (16.92%), tetE (21.54%), aac(6')-Ib (29.23%), and aphAI-IAB (7.69%). The IntI gene was found in 64.62% isolates, and four class 1 integron gene cassette profiles (incomplete dfrA1, catB3-aadA1, dfrA1-orfC, and qacE2-orfD) were identified. These data suggest that goldfish-borne Aeromonas spp. serve as a reservoir of antimicrobial resistance genes and class 1 integrons.

Pet turtles are well-known to harbor an array of bacterial pathogens which can cause zoonotic infections in humans as well as opportunistic infections in the turtles itself. Essential oils are the natural plant extracts which have been traditionally used for disease treatment. In the present study, the essential oil of lavender (EOL) was examined for its antibacterial activity against thirty-eight strains of turtle-borne pathogenic bacteria belonging to seven species; Aeromonas hydrophila, A. caviae, A. dhakensis, Citrobacter freundii, Proteus mirabilis, Salmonella enterica and Pseudomonas aeruginosa. Antibacterial activity of EOL was tested by means of disk diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests. In addition, the antimicrobial susceptibility pattern of 11 commonly used antimicrobials was examined and the multiple antibiotic resistance (MAR) index was calculated. The results revealed that EOL was active against all tested turtle-borne pathogenic bacteria except P. aeruginosa. The range of MIC and MBC values of EOL against isolates except P. aeruginosa were recorded as 0.5–1% (V/V) and 0.5–2% (V/V), respectively. The MBC/MIC ratio was detected as

Turtle-borne Salmonella enterica owns significance as a leading cause in human salmonellosis. The current study aimed to determine the quinolone susceptibility and the genetic characteristics of 21 strains of S. enterica subsp. enterica isolated from pet turtles. Susceptibility of four antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined in disk diffusion and MIC tests where the majority of the isolates were susceptible to all tested quinolones. In genetic characterization, none of the isolates were positive for qnr or aac(6’)-Ib genes and no any target site mutations could be detected in gyrA, gyrB, and parC quinolone resistance determining regions (QRDR). In addition, neighbor-joining phylogenetic tree derived using gyrA gene sequences exhibited two distinct clads comprising; first, current study isolates, and second, quinolone-resistant isolates of human and animal origin. All results suggest that studied strains of S. enterica subsp. enterica isolated from pet turtles are susceptible to quinolones and genetically more conserved with regards to gyrA gene region.