Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an important disease in the Ontario fish farming industry and in finfish aquaculture in temperate waters worldwide. The development of antimicrobial resistance by F. psychrophilum is a concern because management of outbreaks of BCWD often requires the use of antibiotics. Seventy-two isolates of F. psychrophilum collected over a 16-year period from farmed salmonids with clinical signs of BCWD were tested for susceptibility to 10 antimicrobial agents using cation-adjusted Mueller-Hinton broth in custom Trek Sensititre susceptibility plates for aquaculture. The minimum inhibitory concentrations (MICs) for the isolates were determined by means of a broth microdilution antimicrobial susceptibility testing method established by the Clinical and Laboratory Standards Institute. Most of the F. psychrophilum isolates had decreased susceptibility to two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim-sulfadimethoxine [> or =0.5/9.5 .tg/mL for 93% of isolates] and trimethoprim-sulfamethoxazole [> or = 0.25/4.8 microg/mL for 89% of isolates]). High MIC values (> or =2 microg/mL) were obtained for florfenicol and oxytetracycline in 53% and 61% of the isolates, respectively, and 83% of the isolates were relatively susceptible (< or =16 microg/mL) to erythromycin. The MIC values were also high for ampicillin, oxolinic acid, and gentamicin.
Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction -- restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type "a", while 21/32 biovar II isolates belonged to either sequence type "c" or "d". More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout (Oncorhynchus mykiss) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.
Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.Bacterial cold water disease (BCWD), caused by Flavobacterium psychrophilum, occurs at low water temperatures and can cause economic losses in the aquaculture industry as a result of direct mortality or vertebral deformities that decrease the market value of fish that survive the infection (31, 35). A number of putative virulence factors of this bacterium have been identified, including extracellular proteases involved in degradation of extracellular matrix components such as elastin, fibrinogen, type IV collagen, actin, and myosin (9,37,47,48). The best studied of these, psychrophilic metalloproteases Fpp1 and Fpp2, are thought to be involved in destruction of host tissues; however, their roles have not yet been demonstrated in vivo (47,48). In a recent study, Sudheesh et al. (53) used two-dimensional acrylamide gel electrophoresis and Western blot analysis to compare a virulent strain and a nonvirulent strain of F. psychrophilum and found a thermolysin that was unique to the virulent strain. A role for the iron uptake-associated gene exbD2 has also been demonstrated. Strains lacking this gene have decreased virulence and confer a high level of protection in rainbow trout fry when they are used for vaccination (4). Other putative virulence factors of F. psychrophilum include a lipo...
The mixture of pickled vegetables with tomato juice, known as winter salad, is one of Iranian traditional fermented foods. The present work aimed to identify the predominant bacterial community in winter salad during fermentation, and to evaluate the antimicrobial activity of its cell-free supernatant (CFS) against Aspergillus niger IBRC-M 30095, Botrytis cinerea IBRC-M 30162, Aspergillus flavus IBRC-M 30092, Pseudomonas aeruginosa ATCC 9027, Salmonella typhi PTCC 1609, Bacillus cereus ATCC 11778, and Staphylococcus aureus ATCC 25923 using disc diffusion agar and microdilution assays. The fermentation dynamics of winter salad, changes in pH, acidity, salt, ash, protein, and fat contents, and bacterial composition were analysed during spontaneous fermentation. A total of 120 bacterial isolates were identified using 16S rDNA sequencing. Results showed that the following genera were dominant in the early stage of fermentation: Lactobacillus (Lb. brevis, Lb. japonicus, Lb. pentosus, Lb. senmaizukei, Lb. plantarum, Lb. acidifarinae, Lb. parabrevis, and Lb. alimentarius) (44%); Leuconostoc (Ln. mesenteroides and Ln. palmae) (13%); Pediococcus (Pc. pentosaceus, Pc. parvulus, Pc. cellicola, Pc. argentinicus, and Pc. stilesii) (7%); Acinetobacter (Ab. johnsonii) (4%); Enterobacter (E. soli) (10%); and unclassified isolates (22%). All isolates were identified successively during fermentation for 40 days; however, the species count changed throughout the fermentation. The CFS of winter salad showed inhibitory activity against all tested fungal species. Ps. aeruginosa and Sa. typhi were the most sensitive bacteria, while the minimum inhibitory percentage (MIP) and minimum bactericidal percentage (MBP) showed that St. aureus (MIPCFS-c 75; MBPCFS-c 75) and Ba. cereus (MIPCFS-c 50; MBPCFS-c 75) were the most resistant bacteria.
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