Development of Genetic Techniques for the Psychrotrophic Fish Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.     

A proposed serotyping system for Flavobacterium psychrophilum

AIMS: To develop a common serological system for rapid and routine identification of the fish pathogen Flavobacterium psychrophilum. METHODS: Thirty-four isolates of Fl. psychrophilum from different fish species and different geographical areas were typed using a slide agglutination test and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven host-dependent serovars (1: salmon; 2: trout; 3: trout; 4: eel; 5: carp; 6: tench; 7: ayu) were found. Serovar 2 was divided into two antigenic subgroups (2a and 2b). The results achieved by both slide agglutination and ELISA methods were totally consistent with each other. Although both techniques proved to be simple to carry out and useful, only the ELISA allowed identification of Fl. psychrophilum serovars using unabsorbed antiserum and whole-cells as antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proposes a harmonized scheme for serological identification of Fl. psychrophilum to be used for diagnostic and seroepidemiological studies of the diseases it causes.     

An improved growth medium for Flavobacterium psychrophilum

Supplementing cytophaga agar and broth with 0.5 g l-1 each of D(+) galactose, D(+) glucose, L-rhamnose and skimmed milk gave a dramatic improvement in the isolation of the fish pathogen Flavobacterium psychrophilum. By means of spread-plating, approximately double the number of colonies of larger size were obtained on the improved medium compared to cytophaga agar alone. In supplemented cytophaga broth, growth of Fl. psychrophilum was more rapid and generated greater biomass.     

Flavobacterium psychrophilum vaccine development: a difficult task

Bacterial cold water disease (BCWD) is a globally distributed freshwater fish disease caused by the Gram-negative bacterium Flavobacteriumpsychrophilum. It is a particularly devastating infection in fry salmonids and may lead to high levels of mortality. In spite of its economic impact on fish farms, neither the biology of the bacterium nor the bacterium–host interactions are well understood. This review provides a synopsis of the major problems related to critical remaining questions about research into the use of vaccines against F. psychrophilum and the development of a commercial vaccine against this disease. Studies using sera from convalescent rainbow trout have shown the antigenic properties of different proteins such as OmpH, OmpA and FspA, as well as low and high molecular mass lipopolysaccharide of F. psychrophilum, which are potential candidates for subunit vaccines. Inactivated F. psychrophilum bacterins have been successfully tested as vaccines under laboratory conditions by both immersion and intraperitoneal routes. However, the efficacy and the practical usefulness of these preparations still have to be proved. The use of attenuated and wild-type strains to immunize fish showed that these systems offer high levels of protection. Nevertheless, their application clashes with the regulations for environmental protection in many countries. In conclusion, protective vaccines against BCWD are theoretically possible, but substantial efforts still have to be made in order to permit the development of a commercial vaccine.     

Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum

Flavobacterium psychrophilum is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide, and its control mainly relies on antibiotic treatments. To better understand the population structure of this bacterium and its mode of evolution, we have examined the nucleotide polymorphisms at 11 protein-coding loci of the core genome in a set of 50 isolates. These isolates were selected to represent the broadest possible diversity, originating from 10 different host fish species and four continents. The nucleotide diversity between pairs of sequences amounted to fewer than four differences per kilobase on average, corresponding to a particularly low level of diversity, possibly indicative of a small effective-population size. The recombination rate, however, seemed remarkably high, and as a consequence, most of the isolates harbored unique combinations of alleles (33 distinct sequence types were resolved). The analysis also showed the existence of several clonal complexes with worldwide geographic distribution but marked association with particular fish species. Such an association could reflect preferential routes of transmission and/or adaptive niche specialization. The analysis provided no clues that the initial range of the bacterium was originally limited to North America. Instead, the historical record of the expansion of the pathogen may reflect the spread of a few clonal complexes. As a resource for future epidemiological surveys, a multilocus sequence typing website based on seven highly informative loci is available.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis

Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).     

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

We report here the complete genome sequence of the virulent strain JIP02/86 (ATCC 49511) of Flavobacterium psychrophilum, a widely distributed pathogen of wild and cultured salmonid fish. The genome consists of a 2,861,988-base pair (bp) circular chromosome with 2,432 predicted protein-coding genes. Among these predicted proteins, stress response mediators, gliding motility proteins, adhesins and many putative secreted proteases are probably involved in colonization, invasion and destruction of the host tissues. The genome sequence provides the basis for explaining the relationships of the pathogen to the host and opens new perspectives for the development of more efficient disease control strategies. It also allows for a better understanding of the physiology and evolution of a significant representative of the family Flavobacteriaceae, whose members are associated with an interesting diversity of lifestyles and habitats.     

Recovery of Flavobacterium psychrophilum viable cells using a charcoal-based solid medium

AIMS: Flavobacterium psychrophilum is the etiological agent of the cold-water disease in salmonids. This micro-organism is somewhat fastidious being difficult to isolate and culture. The aim of this study was to develop a new solid medium which improves the recovery of viable cells from a sample. METHODS AND RESULTS: Six different media [nutrient agar (NA), NA + charcoal (NAC), enriched Anacker Ordal serum (EAOS), EAOS supplemented with aromatic compounds (EAOSa), EAOS with activated charcoal (EAOC) and EAOC supplemented with aromatic compounds (EAOCa)], three of them containing activated charcoal, were used to recover isolated colonies from a diluted sample of Fl. psychrophilum THC02-90. Pair wise comparisons between different media were carried out using the test of bootstrap to determine the best solid medium and if the presence of activated charcoal increased the number of colonies. The results showed that activated charcoal improved the recovery of viable cells in all the cases and NAC was slightly better than EAOCa but more variable. CONCLUSIONS: Activated charcoal has a great capacity of absorption of toxic compounds and it has no nutritional value, so the problems to culture and isolate Fl. psychrophilum are in part due to an inhibition phenomenon. The use of EAOCa can overcome some of these problems. SIGNIFICANCE AND IMPACT OF THE STUDY: The improvement in Fl. psychrophilum cultivation will facilitate physiological, biochemical and genetic studies with this bacterium.     

Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.     

Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum

AIMS: The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. METHODS AND RESULTS: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugar-modified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. CONCLUSIONS: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum

AIMS: This study developed a new diagnostic method for the bacterium Flavobacterium psychrophilum based on a TaqMan polymerase chain reaction (PCR) assay. METHODS AND RESULTS: Based on reported and newly designed PCR probes, a rapid procedure, that requires no post-PCR processing, was developed for the detection of F. psychrophilum by measuring the fluorescence produced during PCR amplification. Primers were designed to amplify a 971-bp fragment of the 16S rRNA as the target. When different F. psychrophilum strains and other bacterial species, that are taxonomically and ecologically related, were assayed the fluorogenic test was 100% specific in identifying all of the F. psychrophilum strains. The sensitivity of the assay was found to be 1.1 pg DNA and the assay was linear over a range of 0.1 pg-11.2 ng. With pure cultures of F. psychrophilum, the assay was linear over the range 0.4-4.7 x 104 cfu and was able to detect 4.7 cfu per reaction. The analysis was reproducible using either extracted DNA or pure culture. Results using artificially infected fish and diseased fry from natural fish farm outbreaks showed that the assay was useful for diagnosis. CONCLUSIONS: The data showed that the assay was as specific, sensitive, reproducible and rapid but less toxic than the PCR assays described and so very useful for the diagnosis of these micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach permits a rapid, easy and safe routine laboratory diagnosis of F. psychrophilum.     

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.