Pseudomonas aeruginosa from pet Chinese stripe-necked turtles (Ocadia sinensis) demonstrating antimicrobial and heavy metal resistance

Leading nosocomial pathogen Pseudomonas aeruginosa has increasingly been reported to be an opportunistic pathogen. In this study, a total of twenty P. aeruginosa isolates were isolated from 40 pet Chinese stripe-necked turtles and examined for their antimicrobial and heavy metal resistance properties. All isolates were multidrug resistance by scoring multiple antimicrobial resistance indices ≥0.2. In the disc distribution test, 100% resistance to ampicillin and oxacillin were detected. In addition to that, 14 (70%) isolate demonstrated amoxicillin resistance. Imipenem, fosfomycin, gentamycin, tobramycin and piperacillin resistance were detected in 40%, 15%, 20%, 10% and 5% of the isolates, respectively. The ESBLs gene that predominated in this study was blaSHV (55%), followed by blaTEM (50%), blaCTX (10%) and blaOXA (5%). The most frequent aminoglycoside resistance gene in this study was aac(6´)-Ib (40%). Class1 integron integrase gene intI1 and class 1 integron gene cassette gene aadA1 were detected in 45% and 35% of the isolates, respectively. All P. aeruginosa isolates demonstrated Cu and Cd resistance. CzcA and CopA genes were detected in 65% and 30% of the isolates, respectively. These findings reveal the presence of pet turtle-born P. aeruginosa can be a potential risk to public health and cannot be excluded as a non-nosocomial source of infections

Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTP-binding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively

A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV

Antimicrobial Activity of Thymol against Pathogenic Gram-negative Bacteria of Fishes

This work investigated the antimicrobial effects of thymol in vitro against seven species of Gram-negative fish pathogenic bacteria namely, Aeromonas salmonicida subsp. masoucida, A. salmonicida subsp. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio vulnificus, V parahaemolyticus and V anguillarum using disk diffusion, minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) tests. In the disk diffusion test, thymol inhibited growth of all bacteria including those known to be resistant to amoxicillin or lincomycin or both. MIC or MBC of thymol against all bacteria were in the range of 0.01 mg/ml to 0.32 mg/ml. The most sensitive was A. salmonicida subsp. salmonicida (0.01 mg/ml for MIC and 0.02 mg/ml for MBC), followed by A. salmonicida subsp. masoucida (0.04 mg/ml for MIC and 0.08 mg/ml for MBC). Based on the present results, thymol has the potential of controlling bacterial pathogens in the aquaculture industry.N

Antimicrobial Activities of Diallyl Disulfide Against Fish Pathogenic Bacteria

As a major pathogen for fish, the antimicrobial activity of Diallyl Disulfide (DADS) was examined for the following bacteria, Aeromonas hydrophila, A. salmonicida ssp. masoucida, A. salmonicida ssp. salmonicida, Edwardsiella tarda, Vibrio vulnificus, V. paraheamolyticus and L. anguillarum. About 10 ug mL(-1) and more of DADS formed a clear inhibitory zone to all pathogenic bacteria in a disk diffusion test. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values were in the ranges of 160-640 and 640-1280 ug mL(-1) of DDS, respectively. The most sensitive pathogen to DDA was V. vulnificus (160 ug mL(-1) for MIC and 640 ug mL(-1) for MBC) followed by E. tarda (320 ug mL(-1) for MIC and 640 ug mL(-1) for MBC). These results suggest bioavailability of DADS for controlling bacterial pathogens in aquaculture.N

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

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

Fate and Survivability of Fish Bacteriophage Inoculated in BALB/c Mice

Understanding of bacteriophage kinetics in mammals or higher organisms is needed, as this determines the potential phage activity in antibacterial treatment. We demonstrated the fate and survivability of fish phage particles in different organs and fluids inoculated into mice. Phage (PPpW-4) specific to Pseudomonas plecoglossicida was intraperitoneally injected to mice with a dose of 108.1 PFU/ml/mouse. Phage titers (103.0~105.2 PFU/g) were detected in blood and other organs within 10 min after injection except in the urine samples. Phage titers in blood (105.1~106.9 PFU/g) were 10~100 times higher than that of the other organs from 10 min until 24 h. It shows that phage was distributed quickly to the different organs by circulatory system via the blood. Phage titers in all organs showed the highest level in 1 h (104.2~106.9 PFU/g) and reduced rapidly until no phage was detected in 72 h. Although phage disappeared in blood within 72 h, phage can still survived in the spleen and kidney until 96 h. Spleen in particular, had an appreciable phage titer of 102.7 PFU/g. There were comparatively high phage titers (103.0~104.7 PFU/g) in urine from 1 h up to 24 h. It means that one possible mechanism of phage elimination is by urinary excretion.Y

Pathogenicity of Streptococcus parauberis to olive flounder Paralichthys olivaceus

Streptococcus parauberis (stain SNUFPC-050803), isolated from diseased olive flounder Paralichthys olivaceus in Jeju Island, Korea, was evaluated for its pathogenicity to healthy juvenile flounder (29.3 g in average body weight). When challenged with the isolate by intraperitoneal injection with tenfold serial dilutions of 4.5 x 10 - 10(6) CFU/fish, the cumulative mortality ranged from 10% to 80% within 14 days except for 4.5 x 10 CFU/fish and control with no mortality. Disease signs were hemorrhage around the mouth, eyes and pectoral fins, pale and friable liver with hepatomegaly and ascitic fluid in the peritoneal cavity. These signs were similar to those of naturally affected fish. S. parauberis was reisolated and identified by PCR method, which confirmed the pathogenicity of the bacterium to olive flounder.Y