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PubMed2005

Characterisation of surface blebbing and membrane vesicles produced by Flavobacterium psychrophilum.

The surface of Flavobacterium psychrophilum was examined by electron microscopy to determine if previous findings of haemagglutination positive (HA+) and haemagglutination negative (HA-) abilities could be correlated with expression of pili or of a capsular layer. A thin capsular layer was observed in both HA+ and HA- strains but typical pili were absent. However, long, tubular blebs that released membrane vesicles (MVs) into the supernatant were observed on up to 94% of cells within 1 sample. The surface blebbing was increased for 1 strain following growth on media with restricted iron availability. The MVs had an intact membrane bilayer and were released from blebbing cells of both strains. The protein profiles of MVs, while containing some banding similarity with the profile of outer membrane preparations (OMPs) and of lysed whole cells (WCs), showed several bands that reacted strongly with rabbit anti-whole-cell antisera. Two distinct bands of approximately 62 and 58 kDA were highly expressed in the MVs and not seen in the OMP. MVs contained proteolytic activity towards gelatine but not towards casein and elastin, which were only degraded by live cells. Low molecular weight lipopolysaccharides (LPS) or lipooligosaccharides (LOS) were associated with the MVs. Only the MVs of the HA+ strain possessed haemagglutinin activity. These findings suggest that the F. psychrophilum may, through surface blebbing, release antigenic MVs that contain some proteolytic activity and may aid the bacterium in releasing nutrients from its surrounding environment as well as playing a role in impeding the immune response of its host.

Bacterial Capsules↗
PubMed2005

Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance.

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin.

Base Sequence↗
PubMed2005

Association of Flavobacterium psychrophilum strains with intestinal explants of rainbow trout Oncorhynchus mykiss.

Flavobacterium psychrophilum is an important pathogen in rainbow trout Oncorhynchus mykiss. The portal of entry for F. psychrophilum is not well known. In this study, the role of the intestine as a colonization site for F. psychrophilum was determined. For this purpose, the ability of a high (Dubois) and a low (99/10A) virulence strain of F. psychrophilum to adhere to intestinal explants of rainbow trout was evaluated. After incubation, samples of the gut were examined bacteriologically, histologically and by electron microscopy. The number of gut-associated F. psychrophilum bacteria was significantly higher for the Dubois than for the 99/10A strain. Histological samples clearly showed numerous bacteria of the high virulence strain associated with the intestinal tissue as opposed to only a few bacteria of the low virulence strain. Additionally, extensive exfoliation of intestinal epithelium was noted after incubation with the high virulence strain, but less with the low virulence strain. These findings were confirmed using scanning electron microscopy and suggest that the intestinal epithelium might represent an important site for colonization of the F. psychrophilum strain and may act as a portal of entry for high virulence F. psychrophilum.

Animals↗
PubMed2005

Experimental bath infection with Flavobacterium psychrophilum, inducing typical signs of rainbow trout Oncorhynchus mykiss fry syndrome.

Flavobacterium psychrophilum infection in salmonid fish, known as rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD), is widespread in fish farms and natural waters. Despite many studies in which attempts at infection were made, an adequate method of infection has not yet been established. In this study, we evaluated a bath infection method in which we used bacteria at different stages of growth in the infection of rainbow trout Oncorhynchus mykiss. Rainbow trout with a mean body weight of 1.3 or 5.6 g, respectively, were infected by immersion in a bacterial suspension at different stages of growth (18 to 66 h shaking culture at 15 degrees C). The fish immersed in a logarithmic phase culture showed higher mortality than those in other culture phases. Indeed, 1.3 and 5.6 g fish showed typical clinical signs including ulcerative tissue of the trunk and lack of caudal fin edge. F. psychrophilum was detected by immunohistochemistry (IHC) in these tissue samples. These results indicate that experimental bath infection using a logarithmic phase bacterial solution is the most appropriate method for studies of infectious mechanisms.

Animals↗
PubMed2006

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare.

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.

Animals↗
PubMed2002

Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum.

Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses. It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4). To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-kappaB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2. Flavolipin-induced NF-kappaB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells. Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin. Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-kappaB activation were examined. (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-kappaB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not. In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.

Animals↗
PubMed1998

The role of trace metal ions in aflatoxin B1 degradation by Flavobacterium aurantiacum.

Flavobacterium aurantiacum NRRL B-184 possesses the ability to degrade aflatoxin B1 in solution and in several food items. Aflatoxin B1 is a potent carcinogen that causes significant economic losses to the agricultural and food industry. The role of trace metal ions (Cu2+, Mn2+, Zn2+, and Co2+) were studied in an effort to understand the enzymatic system involved in aflatoxin B1 degradation by F aurantiacum. The effect of divalent chelators (EDTA and 1,10-phenanthroline [OPT]) in the presence of the trace metal ions was studied as well. Aflatoxin B1 (10 microg/ml) was added to 72-h cultures of F aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0). HPLC was used to determine aflatoxin B1 concentration in these cultures. Incubating cells at 30 degrees C with 1 and 10 mM Cu2+, Mn2+, and Zn2+ significantly decreased aflatoxin B degradation after 4 and 24 h (P < 0.05). Decreased degradation was also observed with 1 and 10 mM Cu2+ and Zn2+ after 48 h and with 0.1 mM Cu2+ after 24 and 48 h. Co2+ did not have a significant effect on aflatoxin B1 degradation. EDTA and OPT did not counter the inhibition in the presence of Cu2+. The addition of 1 mM EDTA countered the inhibition by 1 mM Mn2+ after 4 and 24 h, but 1 mM OPT did not counter the inhibition by 10 mM Mn2+ after 4 and 24 h. OPT countered the inhibition by 1 mM Zn2+ after 4 and 48 h. These trace elements inhibit aflatoxin B1 degradation by F aurantiacum. In addition, their presence necessitates higher concentrations (>1 mM) of EDTA and OPT for the removal of their inhibitory effect.

Aflatoxin B1↗
PubMed2000

The influence of divalent cations and chelators on aflatoxin B1 degradation by Flavobacterium aurantiacum.

The influence of divalent cations (Mg2+ and Ca2+) and chelators (EDTA and 1,10-phenanthroline) on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum was determined in an effort to elucidate the possible manner by which this organism degrades AFB1. AFB1 (10 microg/ml) was added to 72-h cultures of F. aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0). High-performance liquid chromatography was used to determine AFB1 concentration in these cultures. Incubating cells with 0.1, 1, and 10 mM Ca2+ for 48 h significantly increased AFB1 degradation by 11.8, 13.5, and 14.0%, respectively, compared with F. aurantiacum cells alone. Likewise, incubation with 0.1, 1, and 10 mM Mg2+ for 48 h significantly increased AFB1 degradation by 13.8, 13.3, and 13.1%, respectively. Incubating the bacterium with either divalent cation for 16 and 24 h did not significantly affect AFB1 degradation (P < or = 0.05). Addition of 0.1, 1, and 10 mM EDTA and 0.1 and 1 mM 1,10-phenanthroline resulted in significant increases in AFB1 degradation after 24 h. Significantly less AFB1 degradation was observed using 10 mM 1,10-phenanthroline after 24-h incubation. These results suggest the involvement of Mg2+ and Ca2+ cations in AFB1 degradation by F. aurantiacum.

Aflatoxin B1↗
PubMed2000

Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic.

The ability of crude protein extracts from Flavobacterium aurantiacum to degrade aflatoxin B1 (AB1) in aqueous solution was evaluated. Crude protein extracts (800 microg of total protein per ml) degraded 74.5% of AB1 in solution. An average of 94.5% of AB1 was recovered after incubation with heat-treated crude protein extracts (800 microg of total protein per ml). DNase I-treated crude protein extracts degraded 80.5% of AB1 in solution, suggesting that removal of aflatoxin by F. aurantiacum is not due to nonspecific binding with the bacterium's genomic DNA. Proteinase K-treated crude protein extracts degraded 34.5% of AB1, providing evidence that degradation of aflatoxin is linked to a protein that is possibly an enzyme. Solution pH affected the amount of AB1 degraded by crude protein extracts after 24 h. Maximum degradation was observed at pH 7 (pH levels tested: 5, 6, 7, and 8), with some AB1 degradation occurring at pH levels as low as 5 and as high as 8. Acidic pH levels were more detrimental to the ability of crude protein extracts to degrade AB1 than was basic pH. The results of this work indicate that the degradation of AB1 by F. aurantiacum may be enzymatic.

Aflatoxin B1↗
PubMed2001

Aflatoxin B1 degradation by flavobacterium aurantiacum in the presence of reducing conditions and seryl and sulfhydryl group inhibitors.

This study was undertaken to determine the effects of reducing conditions (L-cysteine) and seryl (phenylmethylsulfonyl fluoride) and sulfhydryl (divalent cadmium) group inhibitors on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum. High-performance liquid chromatography was used to determine AFB1 concentrations in 72-h cultures of F. aurantiacum. The addition of 0.1, 1, or 10 mM L-cysteine did not have any significant effect on AFB1 degradation by these cultures after incubation for 4, 24, or 48 h (P > 0.05). The addition of 0.1 mM phenylmethylsulfonyl fluoride did not significantly decrease AFB1 degradation (P > 0.05), but 1 mM phenylmethylsulfonyl fluoride significantly decreased AFB1 degradation after 4, 24, and 48 h of incubation (P < or = 0.05). No significant difference in AFB1 degradation was obtained with 0.1 mM Cd2+ after 4, 24, or 48 h of incubation (P > 0.05). The addition of 1 and 10 mM Cd2+ significantly decreased AFB1 degradation compared with the cells containing AFB1 alone after 4 and 24 h (P < or = 0.05). The addition of chelators, 1 mM EDTA and 1 mM o-phenanthroline, did not result in removal of inhibition of AFB1 degradation observed with 1 and 10 mM Cd2+. Higher concentration of chelators (>1 mM) are necessary to overcome the inhibitory effect. Further work on the cellular fractions and/or crude enzyme preparations is necessary to determine if indeed sulfhydryl and seryl groups of the enzymes are involved in AFB1 degradation (by maintaining either the structure or function of the enzyme).

Aflatoxin B1↗
PubMed2001

Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp.

Contaminated surfaces of food processing equipment are believed to be a significant source of Listeria monocytogenes to foods. However, very little is known about the survival of Listeria in processing environments. In a mixed bacterial biofilm of L. monocytogenes and Flavobacterium spp., the number of L. monocytogenes cells attaching to stainless steel increased significantly compared to when L. monocytogenes was in a pure culture. The L. monocytogenes cells in the mixed biofilms were also recoverable for significantly longer exposure periods. On colonized coupons held at 15 degrees C and 75% humidity, decimal reduction times were 1.2 and 18.7 days for L. monocytogenes in pure and mixed biofilms, respectively. With increasing exposure time, the proportion of cells that were sublethally injured (defined as an inability to grow on selective agar) increased from 8.1% of the recoverable cell population at day 0 to 91.4% after 40 days' exposure. At 4 and -20 degrees C, decimal reduction times for L. monocytogenes in pure culture were 2.8 and 1.4 days, respectively, and in mixed culture, 10.5 and 14.4 days, respectively. The enhanced colonization and survival of L. monocytogenes on "unclean" surfaces increase the persistence of this pathogen in food processing environments, while the increase in the percentage of sublethally injured cells in the population with time may decrease the ability of enrichment regimes to detect it.

Bacterial Adhesion↗
PubMedDate not supplied

Flavobacterium breve meningitis in a dog.

An unusual, gram-negative rod was isolated in significant numbers (4+) from the cerebrospinal fluid (CSF) of a dog. This isolate, Flavobacterium breve, has not been identified previously as a pathogen in the dog. The case and the characteristics of the organism are described.

Animals↗
PubMed1979

Colonization of an amputation site by Flavobacterium odoratum after gentamicin therapy.

Flavobacterium odoratum was isolated from the stumps of the amputated toes of a debilitated patient. Gentamicin therapy and the poor state of health of the patient provided suitable conditions for this gentamicin-resistant species to multiply. When the gentamicin therapy was stopped, and as the patient's heal improved, F. odoratum gradually disappeared to be replaced by Staphylococcus aureus. This appears to be the first report of the isolation of F. odoratum in Australia, and it indicates the potential of this resistant species to act as a source of infection for groups at risk.

Amputation Stumps↗
PubMed1988

Properties of a broad spectrum beta-lactamase isolated from Flavobacterium meningosepticum GN14059.

A broad substrate-spectrum beta-lactamase was purified from Flavobacterium meningosepticum GN14059. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be about 30,000 and the isoelectric point was 5.1. The enzyme hydrolyzed various beta-lactam antibiotics including oxyiminocephalosporins and aztreonam. Relative rates, with cephaloridine as 100, were cephalothin 200, cefazolin 48, cefuroxime 153, cefotaxime 51, ceftazidime 20, ampicillin 26, carbenicillin 19, and aztreonam 20. The enzyme activity was inhibited by clavulanic acid, sulbactam, imipenem and cephamycins.

Anti-Bacterial Agents↗
PubMed1999

Flavobacterium meningosepticum bacteremia: an analysis of 16 cases.

BACKGROUND: Flavobacterium meningosepticum is an uncommon pathogen causing nosocomial pneumonia and meningitis in newborns. It is usually resistant to antimicrobial agents used to treat gram-negative bacilli. While the pathogen often results in high mortality and serious sequelae in newborns, it is also found to cause to disease in adults. Therefore, it is necessary to know the full spectrum of the infection in adults and to identify effective antimicrobial agents. METHOD: Microbiology logbooks were reviewed for F meningosepticum isolated from January, 1992, to March, 1996. The medical records of these patients were reviewed. Special attention was paid to clinical manifestations, underlying diseases, risk factors, treatments, and prognosis. Twenty-four antimicrobial agents were tested using antimicrobial susceptibility tests. RESULTS: Eighteen isolates of F meningosepticum were identified from 16 patients. There were 10 men and six women, with a mean age of 63.7 years. The clinical features of infection included fever (> or = 38 degrees C) in 13 patients, chills in seven, shortness of breath in four, rales or rhonchi in four, shock in three and flank pain in two. All except one patient survived without sequelae. Fifteen patients contracted F meningosepticum from nosocomial sources. Of them, seven were suspected to have acquired the pathogen from diagnostic or therapeutic procedures. Bacteremia occurred in these patients within a mean period of 2.2 days. The other eight patients suffered nosocomial bacteremia within a mean period of 33.4 days after admission. The suspected infection route was not identified in only one patient. The organism was resistant to penicillins, cephalosporins, aztreonam, imipenem, aminoglycosides and macrolides. Testing with lomefloxacin, ciprofloxacin and ofloxacin yielded 72.2%, 83.3% and 94.4% susceptibility rates, respectively. Rifampin (61.1%) and trimethoprim-sulfamethoxazole (TMP-SMX) (88.9%) were effective. Vancomycin and minocycline were 100% effective. CONCLUSIONS: F meningosepticum is an opportunistic pathogen of low virulence and rarely causes serious infections in adults. Reducing the use of unnecessary residual devices and invasive procedures may help reduce the incidence of infection. Therapeutic options include vancomycin, TMP-SMX, minocycline, rifampin or fluoroquinolones.

Adult↗
PubMed1999

Identification and management of an outbreak of Flavobacterium meningosepticum infection in a colony of South African clawed frogs (Xenopus laevis).

During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis). Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death. Mortality rate reached 35%, and all infections were fatal. The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine. Treatment with trimethoprim-sulfadiazine was unsuccessful. Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment. Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum. Sanitation and management procedures were effective in controlling the outbreak.

Animals↗
PubMed1998

Serotyping of Flavobacterium meningosepticum by co-agglutination method.

The purpose of this investigation was to evaluate the usefulness of a co-agglutination procedure for the typing of Flavobacterium meningosepticum. The sensitivity and specificity of the co-agglutination test was compared to the slide agglutination test using reference strains of the bacterial species. Antisera were characterized by both technics to determine their titer and working dilution. The specificity of the sera was assessed by performing tests which include strains of other species and serotypes. A collection of 47 strains of F. meningosepticum isolated from clinical specimens were typed by both co-agglutination and slide agglutination methods. Co-agglutination proved to be markedly more specific than the slide procedure although both methods were similar in sensitivity. It was concluded that co-agglutination proved to be an excellent method for the serotyping of F. meningosepticum.

Agglutination Tests↗