Figure 1 - available from: BMC Microbiology
This content is subject to copyright. Terms and conditions apply.
Growth curves of F. psychrophilum CSF 259–93 and THC 02–90 parent and passaged strains. Independent triplicates of each strain were grown statically in TYES broth at 16 °C with optical density measurements at 450–580 nm preceded by brief shacking. Abbreviations used: CW - F. psychrophilum CSF 259–93 parent strain, CN and CR – CSF 259–93 passaged 17 times with no and with rifampicin, respectively; B17 – rifampicin attenuated F. psychrophilum CSF 259-93B.17 strain 13 ; TW - THC 02–90 parent strain and TN and TR – THC 02–90 passaged for 17 times with and without rifampicin, respectively
Source publication
Background:
Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent...
Similar publications
Bacterial cold-water disease caused by Flavobacterium psychrophilum is one of the major causes of mortality of salmonids. Three genetic lines of rainbow trout designated as ARS-Fp-R (resistant), ARS-Fp-C (control) and ARS-Fp-S (susceptible) have significant differences in survival rate following F. psychrophilum infection. Previous study identified...
The family Flavobacteriaceae (phylum Bacteroidetes) is a major component of soil, marine and freshwater ecosystems. In this understudied family, Flavobacterium psychrophilum is a freshwater pathogen that infects salmonid fish worldwide, with critical environmental and economic impact. Here, we report an extensive transcriptome analysis that establi...
Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set...
Bacterial Coldwater Disease, an infection caused by Flavobacterium psychrophilum,
causes substantial mortality during the initial feeding of salmonids in
hatcheries around the world. This study evaluated the potential impacts of three
commercial starter diets on Bacterial Coldwater Disease-induced mortality during
rainbow trout (Oncorhynchus mykiss...
Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, in...
Citations
... We investigated CWH conservation in seven highly virulent versus three less virulent strains of Fp. The highly virulent strains were Fp G10, Fp G101, JIP02-86, Fp S-S6, OSU THCO2-90, JIP 08/99, JIP 16/00, 950106-1, and Fp G3 [55,[59][60][61], and the less virulent strains were CR, Fp GIW08, and NCIMB 1947 [59,60,62]. The CWH transcript was conserved in six (highly virulent) strains with 100% sequence identity and three (low virulence strains) (Supplementary File S3). ...
... We investigated CWH conservation in seven highly virulent versus three less virulent strains of Fp. The highly virulent strains were Fp G10, Fp G101, JIP02-86, Fp S-S6, OSU THCO2-90, JIP 08/99, JIP 16/00, 950106-1, and Fp G3 [55,[59][60][61], and the less virulent strains were CR, Fp GIW08, and NCIMB 1947 [59,60,62]. The CWH transcript was conserved in six (highly virulent) strains with 100% sequence identity and three (low virulence strains) (Supplementary File S3). ...
Flavobacterium psychrophilum (Fp), the causative agent of Bacterial Cold-Water disease in salmonids, causes substantial losses in aquaculture. Bacterial outer membrane vesicles (OMVs) contain several virulence factors, enzymes, toxins, and nucleic acids and are expected to play an essential role in host–pathogen interactions. In this study, we used transcriptome sequencing, RNA-seq, to investigate the expression abundance of the protein-coding genes in the Fp OMVs versus the Fp whole cell. RNA-seq identified 2190 transcripts expressed in the whole cell and 2046 transcripts in OMVs. Of them, 168 transcripts were uniquely identified in OMVs, 312 transcripts were expressed only in the whole cell, and 1878 transcripts were shared in the two sets. Functional annotation analysis of the OMV-abundant transcripts showed an association with the bacterial translation machinery and histone-like DNA-binding proteins. RNA-Seq of the pathogen transcriptome on day 5 post-infection of Fp-resistant versus Fp-susceptible rainbow trout genetic lines revealed differential gene expression of OMV-enriched genes, suggesting a role for the OMVs in shaping the host–microbe interaction. Interestingly, a cell wall-associated hydrolase (CWH) gene was the most highly expressed gene in OMVs and among the top upregulated transcripts in susceptible fish. The CWH sequence was conserved in 51 different strains of Fp. The study provides insights into the potential role of OMVs in host–pathogen interactions and explores microbial genes essential for virulence and pathogenesis.
... One of the most common strategies to obtain avirulent (attenuated) mutants is by multiple passages of a wild-type strain on increasing concentrations of an antibiotic, for example, rifampin. This process is effective in generating avirulent mutants, but the mechanism(s) responsible for attenuation has remained vastly uncharacterized [31][32][33][34]. Many vaccines had been obtained using this strategy, including Brucella abortus (RB51) [32], Edwardsiella ictaluri [34], F. psychrophilum [33], and F. columnare [35]. ...
... It is hypothesized that mutations in the rifampin-resistant RNAP affect the activity of this protein to the extent that protein synthesis is affected. This is supported by some rifampin-resistant mutants exhibiting lower growth rates than their parent strains [31]. However, this was not the case for our mutant Fc1723, which displays a similar growth pattern to its parent strain FcB27. ...
Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.
... Moreover, the new conformation of RpoB modifies the polymerase's affinity for certain promoters, causing changes in gene expression [27]. In other bacterial species, such as Escherichia coli or Pseudomonas aeruginosa, mutations in the rpoB gene induce pleiotropic effects that impact processes such as sporulation [28], stringent response [25,29], motility, and virulence [24,30]. ...
Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.
... Our results may also help to explain the lack of virulence of rifampin-resistant F. psychrophilum mutants that have been isolated and examined as vaccine candidates (73). The most attenuated of these strains were deficient in motility (74). Given the link between gliding motility and the T9SS described above, the mutants may have also been compromised for protein secretion, which could have contributed to the attenuated phenotype. ...
Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish, and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare , requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell-surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN . The Δ gldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the Δ gldN mutant was deficient in adhesion, gliding motility, and in extracellular proteolytic and hemolytic activities. The Δ gldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease.
IMPORTANCE: Bacterial cold-water disease, caused by F. psychrophilum , is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors, and targets for the development of control measures.
... Above all, the spontaneous mutation test in this study was carried out on solid medium without antibiotics at 37°C lacking clinically specific environmental pressures, making all mutations possible to survive, which had little to do with their respective fitness. Studies in F. psychrophilum suggested that strains under the rifampin pressure were prone to enrich more single-nucleotide polymorphism, which would be associated with weakened virulence [31]. Therefore, the types of rpoB mutations obtained from spontaneous mutation tests were various, while the types identified in isolates were relatively concentrated. ...
Background
Riemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer.
Results
Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10− 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h.
Conclusions
Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo.
Electronic supplementary material
The online version of this article (10.1186/s12866-019-1478-7) contains supplementary material, which is available to authorized users.
... In fact, the recovery of multiple F. psychrophilum genotypes from a single infected fish has been reported on multiple occasions (9,12,13). In this context, Gliniewicz et al. (28) found no similarities in DNA methylation motifs for two F. psychrophilum strains, which may have been a result of environmental differences from which these strains were recovered, whereby different phage communities contribute to long-term diversification of different F. psychrophilum lineages (i.e., novel genotypes as a by-product of transduction). Even though recombination appears to be a driving force for diversity among F. psychrophilum isolates, the results of our analysis on linkage disequilibrium revealed statistically significant evidence for clonal populations (i.e., I A S value differs significantly from 0). ...
Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.
... This was also true for pNetB transconjugants created with a rifampin-marked chromosome (data not shown). Rifampin resistance and attenuation have been demonstrated in other bacteria, such as Flavobacterium psychrophilum (49). Rifampin marking of strains could therefore also influence other C. perfringens virulence studies performed in the gastrointestinal tract of other animals, such as sheep and cows. ...
Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment.
IMPORTANCE
Clostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.
Multidrug regimes have been considered to constrain selection for resistance compared to monotherapy. However, drug resistance trajectories are influenced by a wide range of conditions which can cause opposing outcomes. Here we employed an in vitro model system to investigate differences in resistance dynamics between mono-, combination and alternating regimes. Across regimes involving three drugs and phage, selection for resistance was decreased in multidrug regimes compared to monotherapy. Surprisingly, across regimes, two out of the four agents used to impose selection had a dominant effect on the overall outcome. Resistance to these agents either caused cross-resistance or obscured the phenotypic effect of other resistance mutations. This indicates that under multidrug therapeutic regimes, a small number of drugs dominate the emergence of antimicrobial resistance.
One Sentence Summary
Resistance evolution in multidrug environments is driven by a small subset of selective agents through pleiotropy and fitness costs.
Flavobacterium species are considered important fish pathogens in wild and cultured fish throughout the world. They can cause acute, subacute, and chronic infections, which are mainly characterized by gill damage, skin lesions, and deep necrotic ulcerations. Primarily, three Flavobacterium species, F. branchiophilum, F. columnare, and F. psychrophilum, have been reported to cause substantial losses to freshwater fish. In this study, we evaluated genomes of 86 Flavobacterium species isolated from aquatic hosts (mainly fish) to identify their unique and shared genome features. Our results showed that F. columnare genomes cluster into four different genetic groups. In silico secretion system analysis identified that all genomes carry type I (T1SS) and type IX (T9SS) secretion systems, but the number of type I secretion system genes shows diversity between species. F. branchiophilum, F. araucananum, F. chilense, F. spartansii, and F. tructae genomes have full type VI secretion system (T6SS). F. columnare, F. hydatis, and F. plurextorum carry partial T6SS with some of the T6SS genes missing. F. columnare, F. araucananum, F. chilense, F. spartansii, F. araucananum, F. tructae, Flavobacterium sp., F. crassostreae, F. succinicans, F. hydatis, and F. plurextorum carry most of the type IV secretion system genes (T4SS). F. columnare genetic groups 1 and 2, Flavobacterium sp., and F. crassostreae encode the least number of antibiotic resistance elements. F. hydatis, F. chilense, and F. plurextorum encode the greatest number of antibiotic resistance genes. Additionally, F. spartansii, F. araucananum, and chilense encode the greatest number of virulence genes while Flavobacterium sp. and F. crassostreae encode the least number of virulence genes. In conclusion, comparative genomics of Flavobacterium species of aquatic origin will help our understanding of Flavobacterium pathogenesis.
