S.H.M.P. Wimalasena

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.

Prevalence of Citrobacter spp. From Pet Turtles and Their Environment

Abstract 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 isolates 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.

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

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

&NA; 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 &bgr;‐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‐&agr;, il‐1&bgr;, il‐6, il‐12, ifn, ifn‐&ggr;, 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. HighlightsAeromonas hydrophila and A. veronii from wounds of morbid zebrafish showed highly resistant to antibiotics.Naturally co‐infected zebrafish showed highly induced TNF‐&agr;, IL‐1&bgr;, IL‐12 and IFN‐&ggr; transcription.Virulence of A. hydrophila has greater effect than A. veronii during artificial co‐infection.

Antibacterial activity of lime (Citrus aurantifolia) essential oil and limonene against fish pathogenic bacteria isolated from cultured olive flounder (Paralichthys olivaceus)

Abstract 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.

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.

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.