Comparative analysis unravels genetic recombination events of Vibrio parahaemolyticus recA gene.

Vibrio parahaemolyticus is a gram-negative bacterium capable of causing diseases in humans and aquatic animals. The global relationships among V. parahaemolyticus genomes have been studied using multilocus sequence typing (MLST). Recently, the MLST gene recA has shown difficulties in amplification and/or a larger PCR fragment for some V. parahaemolyticus genomes due to genetic recombination. We aimed to investigate these recombination events of recA gene by analyzing 500 publicly available whole genomes from the NCBI database. The genomes with untypable recA genes were separated using BIGSdb and CGEMLST 2.0 servers, followed by annotation with RAST and NCBI pipelines. Moreover, the variable nature of V. parahaemolyticus was investigated by wgMLST analysis. The hypothetical proteins in recombinant regions were analyzed with VCIMPred tool. In the results, 3 genomes were detected with recA gene recombination, in which 2 were associated with phages and 1 to an AHPND causing strain. All 3 recombinant regions had a G + C content of 39%-40% with 15-30 ORFs, including a newly incorporated recA gene. These acquired recA genes were closely related to 3 different genera namely Aliivibrio, Photobacterium, and Vibrio. The wgMLST analysis indicated genetic recombination events occur independently among V. parahaemolyticus on a global scale. The in silico analysis revealed 4 hypothetical proteins associated with virulence factors in recombinant regions. The present study confirms, recombination events of V. parahaemolyticus recA gene, are diverse and may have an impact on the evolutionary process. Moreover, understanding these genetic recombination events of the recA gene is necessary to determine their STs and, therefore assessing epidemiological relationships.

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.

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.

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.

Outcome of co-infection with opportunistic and multidrug resistant Aeromonas hydrophila and A. veronii in zebrafish: Identification, characterization, pathogenicity and immune responses.

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 ß-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-1ß, 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.

Comparison of virulence genes in Proteus species isolated from human and pet turtle.

The current study was aimed to investigate the prevalence of ureC, rsbA, zapA and mrpA virulence genes using polymerase chain reaction (PCR) in Proteus spp. isolated from 5 commercially popular species of pet turtles and comparison of the mrpA gene sequences of Proteus mirabilis isolates with human clinical isolates. A total of 24 isolates in pet turtles were identified, comprised of P. mirabilis (15), Proteus vulgaris (7) and Proteus hauseri (2). The prevalence of ureC, rsbA, zapA and mrpA genes among all identified Proteus spp. isolates were 91.7%, 50%, 45.8% and 45.8%, respectively. The average percentage similarities of mrpA gene sequence of pet turtle P. mirabilis isolates to human urinary and respiratory isolates were 96.35% and 94.85%, respectively. The prevalence of virulence genes and high similarity of mrpA gene sequences between pet turtles and human P. mirabilis isolates revealed that though pet turtles are healthy, these animals may pose a potential risk of urinary and respiratory infections to humans.

Antibacterial activity of essential oil from lavender (Lavandula angustifolia) against pet turtle-borne pathogenic bacteria.

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 <4, revealing that the tested EOL was bactericidal. Besides, most of the isolates were resistant to different antimicrobials in antimicrobial disk diffusion test. MAR index values of the tested strains were ranging from 0.27 to 0.91. The outcomes indicate that EOL has a potential to be used as an antibacterial agent against pathogenic bacteria isolated from pet turtles.

Quinolone susceptibility and genetic characterization of Salmonella enterica subsp. enterica isolated from pet turtles.

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.

Antimicrobial property of lemongrass (Cymbopogon citratus) oil against pathogenic bacteria isolated from pet turtles.

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.

Prevalence and characterisation of quinolone resistance genes in Aeromonas spp. isolated from pet turtles in South Korea.

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.

Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment.

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.