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.     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.     

Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae.

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.     

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.     

Entericidin Is Required for a Probiotic Treatment (Enterobacter sp. Strain C6-6) To Protect Trout from Cold-Water Disease Challenge

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB<pET101::ecnAB>) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens.     

Relationship between gyrA Mutations and Quinolone Resistance in Flavobacterium psychrophilum Isolates

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.     

Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors

Flavobacterium psychrophilum is an important fish pathogen in salmonid aquaculture worldwide. Due to increased antibiotic resistance, pathogen control using bacteriophages has been explored as a possible alternative treatment. However, the effective use of bacteriophages in pathogen control requires overcoming the selection for phage resistance in the bacterial populations. Here, we analyzed resistance mechanisms in F. psychrophilum after phage exposure using whole-genome sequencing of the ancestral phage-sensitive strain 950106-1/1 and six phage-resistant isolates. The phage-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation, and secretion of extracellular enzymes, which were all impaired in the resistant strains, probably due to superficial structural changes. The clustered regularly interspaced short palindromic repeat (CRISPR) region was unaffected in the resistant isolates and thus did not play a role as a resistance mechanism for F. psychrophilum under the current conditions. All together, the results suggest that resistance in F. psychrophilum was driven by spontaneous mutations, which were associated with a number of derived effects on the physiological properties of the pathogen, including reduced virulence under in vitro conditions. Consequently, phage-driven physiological changes associated with resistance may have implications for the impact of the pathogen in aquaculture, and these effects of phage resistance on host properties are therefore important for the ongoing exploration of phage-based control of F. psychrophilum.     

Genome-informed approach to identify genetic determinants of Flavobacterium psychrophilum phage susceptibility

The fish pathogen Flavobacterium psychrophilum infects farmed salmonids worldwide, and application of bacteriophages has been suggested for controlling disease outbreaks in aquaculture. Successful application of phages requires detailed knowledge about the variability in phage susceptibility of the host communities. In this study, we analysed the genetic diversity of F. psychrophilum hosts and phages from the Baltic Sea area to identify genetic determinants of phage-host interaction patterns. A host range analysis of 103 phages tested against 177 F. psychrophilum strains (18 231 phage–host interactions) identified nine phage clusters, infecting from 10% to 91% of the strain collection. The core genome-based comparison of 35 F. psychrophilum isolates revealed an extremely low overall genomic diversity (>99.5% similarity). However, a small subset of 16 ORFs, including genes involved in the type IX secretion system (T9SS), gliding motility and hypothetical cell-surface related proteins, exhibited a highly elevated genetic diversity. These specific genetic variations were linked to variability in phage infection patterns obtained from experimental studies, indicating that these genes are key determinants of phage susceptibility. These findings provide novel insights on the molecular mechanisms determining phage susceptibility in F. psychrophilum and emphasizes the importance of phages as drivers of core genomic diversity in this pathogen.     

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium (“Pan-Flavo”) and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.     

Isolation and Characterization of Marine Caulobacters and Assessment of Their Potential for Genetic Experimentation

A total of 25 marine caulobacters were isolated from littoral marine sources. Several aspects of their physiology and morphology were examined, as well as their suitability for genetic manipulation in laboratory cultivation. Caulobacters were readily isolated from all sources, including samples from areas containing pollution-related organic compounds. All isolates grew best in media containing seawater, but eight strains grew if sea salts were replaced with NaCl alone, three strains grew at 1/10 the normal sea salt concentration, and one isolate grew, albeit poorly, in freshwater medium. Of the marine isolates, 12 strains grew under anaerobic conditions, indicating that some caulobacters are not obligately aerobic bacteria, as they are currently categorized. Although some freshwater caulobacters are able to oxidize manganese, this capability was not found in these marine caulobacters. Of the marine isolates, 10 strains were resistant to mercury chloride concentrations 10- to 20-fold greater than that tolerated by sensitive bacteria. However, a mercury reductase gene comparable with that found in R100-type plasmids was not detected by gene hybridization. With respect to the potential for genetic experimentation, most strains grew rapidly (3- to 4-h generation time at 30°C), producing colonies on solid media in 2 to 3 days. The isolates were sensitive to antibiotics commonly used in recombinant DNA experiments, and spontaneous drug-resistant mutants were selectable. Conjugal transfer of plasmids from Escherichia coli to several marine caulobacters was demonstrated for four broad-host-range plasmid incompatibility groups, by using both self-transmissible plasmids and cloning-oriented plasmids that require a helper plasmid. Conjugal transfer of broad-host-range plasmids between freshwater and marine caulobacters was also demonstrated in both directions. Native plasmids of approximately 100- to 150-kilobase sizes were found in 2 of the 25 marine Caulobacter strains. The native plasmids were present in relatively high copy number and appeared stable in laboratory culture. In short, the marine caulobacters appeared appropriate as candidates for genetic manipulation and the expression of selected genes in the marine environment.     

Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10³ transformants μg of DNA⁻¹) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.     

A Protective Immune Response Is Generated in Rainbow Trout by an OmpH-Like Surface Antigen (P18) of Flavobacterium psychrophilum

Investigations of the surface characteristics of Flavobacterium psychrophilum, an important pathogen of fish, assisted us in identifying a surface protein termed P18. In the current study, we developed a simple and efficient procedure for the purification of this protein by a two-step method. First, P18 was selectively released from flavobacteria by a heat-HEPES treatment of the cells and then subjected to anion-exchange high-performance liquid chromatography. De novo sequencing was used to generate a fragmented peptide spectrum from purified P18. Comparison of two obtained peptide sequences with a partial genome sequence of F. psychrophilum (INRA, Jouy-en-Josas, France) identified one gene encoding a 166-amino-acid OmpH-like protein that mostly likely undergoes N-terminal cleavage of the 23-residue signal peptide. The susceptibility of the OmpH-like protein to proteinase K treatment and the bacteriostatic/bactericidal activities of anti-OmpH-like protein antibodies indicated that this protein is actually exposed on the surface of F. psychrophilum. Vaccination trials showed that the OmpH-like protein can induce a high titer of anti-OmpH-like protein antibodies which are protective. Taken together, these results suggest that this surface protein produced by F. psychrophilum could be used in future vaccine development as a promising candidate antigen.     

Sequencing of IncX-Plasmids Suggests Ubiquity of Mobile Forms of a Biofilm-Promoting Gene Cassette Recruited from Klebsiella pneumoniae

Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.     

Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (≥10⁸ colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.     

Identification of the Multi-Resistance Gene cfr in Escherichia coli Isolates of Animal Origin

Previous study indicated that the multi-resistance gene cfr was mainly found in gram-positive bacteria, such as Staphylococcus and Enterococcus, and was sporadically detected in Escherichia coli. Little is known about the prevalence and transmission mechanism of cfr in E. coli. In this study, the presence of cfr in E. coli isolates collected during 2010–2012 from food-producing animals in Guangdong Province of China was investigated, and the cfr-positive E. coli isolates were characterized by PFGE, plasmid profiling, and genetic environment analysis. Of the 839 E. coli isolates, 10 isolates from pig were cfr positive. All the cfr-positive isolates presented a multi-resistance phenotype and were genetically divergent as determined by PFGE. In 8 out of the 10 strains, the cfr gene was located on plasmids of ∼30 kb. Restriction digestion of the plasmids with EcoRI and sequence hybridization with a cfr-specific probe revealed that the cfr-harboring fragments ranged from 6 to 23 kb and a ∼18 kb cfr-carrying fragment was common for the plasmids that were ∼30 kb. Four different genetic environments of cfr were detected, in which cfr is flanked by two identical copies of IS26, which may loop out the intervening sequence through homologous recombination. Among the 8 plasmids of ∼30 kb, 7 plasmids shared the same genetic environment. These results demonstrate plasmid-carried cfr in E. coli and suggest that transposition and homologous recombination mediated by IS26 might have played a rule in the transfer of the cfr gene in E. coli.