| 1. |
Fernández L,
Secades P,
Lopez JR,
Márquez I,
Guijarro JA,
( 2002 ) Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulence. PMID : 12101310 : DOI : 10.1099/00221287-148-7-2233 Abstract >>
Yersinia ruckeri is a Gram-negative pathogen that causes enteric redmouth disease in salmonids. A gene from Y. ruckeri encoding an extracellular protease termed yrp1 (Yersinia ruckeri protease 1) was cloned from a Sau3AI library constructed in pUC19 and analysed in gelatin-supplemented medium. The nucleotide sequence of the yrp1 gene indicated an ORF encoding a protein of 477 aa. On the basis of the high degree of homology in the amino acid sequence as well as its conservative motifs, this protein was included within the serralysin metalloendopeptidase subfamily (EC 3.4.24.12). The yrp1 N-terminal sequence showed a 14 aa propeptide followed by a 10 aa sequence identical to the one deduced previously from the 47 kDa purified protease. Additional results demonstrated that the yrp1 gene encodes the 47 kDa protein. In contrast to other Yersinia species, the yrp1 protease is secreted by a type I Gram-negative bacterial ABC exporter protein secretion system composed of three genes termed yrpD, yrpE and yrpF, and a protease inhibitor inh. The development of genetic methods for this species has allowed the exploration of the organization and the putative role of the Yrp1 genetic locus. Thus, site-directed insertion mutations into the yrp1 and the yrpE genes were constructed by the integration of the mobilizable suicide vector pIVET8 containing internal portions of both coding sequences. Complementation studies of those mutants with different loci indicated that they are organized as a single operon. The mutant strains lacked protease activity as well as the Yrp1 protein and, although physiologically similar to the parental strain when growing on nutrient broth medium, they were attenuated in virulence when bacteria were injected intraperitoneally into rainbow trout (Oncorhynchus mykiss). This is the first report of defined mutations in Y. ruckeri to show the implication of a factor such as an extracellular protease in pathogenesis.
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2. |
Welch TJ,
Fricke WF,
McDermott PF,
White DG,
Rosso ML,
Rasko DA,
Mammel MK,
Eppinger M,
Rosovitz MJ,
Wagner D,
Rahalison L,
Leclerc JE,
Hinshaw JM,
Lindler LE,
Cebula TA,
Carniel E,
Ravel J,
( 2007 ) Multiple antimicrobial resistance in plague: an emerging public health risk. PMID : 17375195 : DOI : 10.1371/journal.pone.0000309 PMC : PMC1819562 Abstract >>
Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.
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3. |
Fernández L,
Prieto M,
Guijarro JA,
( 2007 ) The iron- and temperature-regulated haemolysin YhlA is a virulence factor of Yersinia ruckeri. PMID : 17259619 : DOI : 10.1099/mic.0.29284-0 Abstract >>
Yersinia ruckeri causes the enteric redmouth disease or yersiniosis, an important systemic fish infection. In an attempt to dissect the virulence mechanisms of this bacterium, a gene encoding a putative protein involved in the secretion/activation of a haemolysin (yhlB), which had been previously identified by in vivo expression technology, was further analysed. The gene yhlB precedes another ORF (yhlA) encoding a Serratia-type haemolysin. Other toxins belonging to this group have been identified in genomic analyses of human-pathogenic yersiniae, although their role and importance in pathogenicity have not been defined yet. In spite of its being an in vivo-induced gene, the expression of yhlA can be induced under certain in vitro conditions similar to those encountered in the host, as deduced from the results obtained by using a yhlB : : lacZY fusion. Thus, higher levels of expression were obtained at 18 degrees C, the temperature of occurrence of disease outbreaks, than at 28 degrees C, the optimal growth temperature. The expression of the haemolysin also increased under iron-starvation conditions. This confirmed the decisive role of iron and temperature as environmental cues that regulate and coordinate the expression of genes encoding extracellular factors involved in the virulence of Y. ruckeri. LD(50) and cell culture experiments, using yhlB and yhlA insertional mutant strains, demonstrated the participation of the haemolysin in the virulence of Y. ruckeri and also its cytolytic properties against the BF-2 fish cell line. Finally, a screening for the production of haemolytic activity and the presence of yhlB and yhlA genes in 12 Y. ruckeri strains proved once more the genetic homogeneity of this species, since all possessed both haemolytic activity and the yhlB and yhlA genes.
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4. |
Delmas J,
Breysse F,
Devulder G,
Flandrois JP,
Chomarat M,
( 2006 ) Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene. PMID : 16626902 : DOI : 10.1016/j.diagmicrobio.2006.02.003 Abstract >>
Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.
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5. |
Mammeri H,
Poirel L,
Nazik H,
Nordmann P,
( 2006 ) Cloning and functional characterization of the ambler class C beta-lactamase of Yersinia ruckeri. PMID : 16553832 : DOI : 10.1111/j.1574-6968.2006.00148.x Abstract >>
Yersinia ruckeri is a gram-negative pathogen causing enteric redmouth disease in salmonids. Previous studies have reported that Y. ruckeri harbors an ampC gene that is expressed at low level. In this present work, the entire ampC gene of Y. ruckeri was cloned and expressed in Escherichia coli. The AmpC enzyme confers resistance to aminopenicillins and narrow-spectrum cephalosporins, which fit well with the kinetic properties of the purified enzyme. Phylogenetic analysis showed that YRC-1 did not share significant sequence identity with known plasmid-mediated or chromosomal AmpC enzymes. This work provides further evidence that fish-pathogenic gram-negative rod species may constitute a reservoir of antibiotic resistance genes.
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6. |
Kotetishvili M,
Kreger A,
Wauters G,
Morris JG,
Sulakvelidze A,
Stine OC,
( 2005 ) Multilocus sequence typing for studying genetic relationships among Yersinia species. PMID : 15956383 : DOI : 10.1128/JCM.43.6.2674-2684.2005 PMC : PMC1151872 Abstract >>
The intra- and interspecies genetic relationships of 58 strains representing all currently known species of the genus Yersinia were examined by multilocus sequence typing (MLST), using sequence data from 16S RNA, glnA, gyrB, recA, and Y-HSP60 loci. Yersinia aldovae, Y. bercovieri, Y. intermedia, Y. pestis, Y. pseudotuberculosis, Y. rohdei, and Y. ruckeri were genetically more homogeneous than were Y. enterocolitica, Y. frederiksenii, Y. kristensenii, and Y. mollaretii. The MLST data concerning the genetic relatedness within and among various species of Yersinia support the idea that Y. pestis and Y. pseudotuberculosis are two lineages within the same species rather than two distinct species. Y. ruckeri is the genetically most distant species within the genus. There was evidence of O-antigen switching and genetic recombination within and among various species of Yersinia. The genetic relatedness data obtained by MLST of the four housekeeping genes and 16S RNA agreed in most, but not all, instances. MLST was better suited for determining genetic relatedness among yersiniae than was 16S RNA analysis. Some strains of Y. frederiksenii and Y. kristensenii are genetically less related to other strains within those species, compared to strains of all other species within the genus. The taxonomic standing of these strains should be further examined because they may represent currently unrecognized Yersinia species.
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7. |
Schiefer AM,
Wiegand I,
Sherwood KJ,
Wiedemann B,
Stock I,
( 2005 ) Biochemical and genetic characterization of the beta-lactamases of Y. aldovae, Y. bercovieri, Y. frederiksenii and "Y. ruckeri" strains. PMID : 15890500 : DOI : 10.1016/j.ijantimicag.2004.08.020 Abstract >>
The beta-lactamases of five strains each of Y. aldovae and "Y. ruckeri", and 10 strains each of Y. bercovieri and Y. frederiksenii were examined phenotypically and genetically. Beta-lactamase activity and induction assays and SDS-PAGE were applied for phenotypic characterization of these enzymes. Genotypically, PCR experiments applying degenerated primer pairs for the detection of AmpC beta-lactamase genes were performed. All yersiniae yielded specific amplification products for ampC and all these strains expressed beta-lactamases. Each species produced its own, species-specific AmpC beta-lactamase. Inducibility of these enzymes was shown for Y. bercovieri, but not for the low-level enzyme producing species Y. aldovae and "Y. ruckeri". In contrast to these species, induction tests for Y. frederiksenii revealed heterogeneous results. Whereas the beta-lactamases of 6 of 10 strains were inducible, the enzyme activities after induction in the remaining four were similar to those measured without an inducer. In addition to the AmpC enzyme, all Y. frederiksenii strains expressed a second beta-lactamase belonging to Ambler class A. The present study enlarges the knowledge about the beta-lactamases of four novel Yersinia species that are likely to be involved in human disease. Beta-lactamases of Y. aldovae and "Y. ruckeri" have been characterized for the first time.
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8. |
Fernández L,
Márquez I,
Guijarro JA,
( 2004 ) Identification of specific in vivo-induced (ivi) genes in Yersinia ruckeri and analysis of ruckerbactin, a catecholate siderophore iron acquisition system. PMID : 15345400 : DOI : 10.1128/AEM.70.9.5199-5207.2004 PMC : PMC520893 Abstract >>
This work reports the utilization of an in vivo expression technology system to identify in vivo-induced (ivi) genes in Yersinia ruckeri after determination of the conditions needed for its selection in fish. Fourteen clones were selected, and the cloned DNA fragments were analyzed after partial sequencing. In addition to sequences with no significant similarity, homology with genes encoding proteins putatively involved in two-component and type IV secretion systems, adherence, specific metabolic functions, and others were found. Among these sequences, four were involved in iron acquisition through a catechol siderophore (ruckerbactin). Thus, unlike other pathogenic yersiniae producing yersiniabactin, Y. ruckeri might be able to produce and utilize only this phenolate. The genetic organization of the ruckerbactin biosynthetic and uptake loci was similar to that of the Escherichia coli enterobactin gene cluster. Genes rucC and rupG, putative counterparts of E. coli entC and fepG, respectively, involved in the biosynthesis and transport of the iron siderophore complex, respectively, were analyzed further. Thus, regulation of expression by iron and temperature and their presence in other Y. ruckeri siderophore-producing strains were confirmed for these two loci. Moreover, 50% lethal dose values 100-fold higher than those of the wild-type strain were obtained with the rucC isogenic mutant, showing the importance of ruckerbactin in the pathogenesis caused by this microorganism.
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9. |
Gibello A,
Porrero MC,
Blanco MM,
Vela AI,
Liébana P,
Moreno MA,
Fernández-Garayzábal JF,
Domínguez L,
( 2004 ) Analysis of the gyrA gene of clinical Yersinia ruckeri isolates with reduced susceptibility to quinolones. PMID : 14711693 : DOI : 10.1128/aem.70.1.599-602.2004 PMC : PMC321256 Abstract >>
Antimicrobial susceptibility of seven clinical strains of Yersinia ruckeri representative of those isolated between 1994 and 2002 from a fish farm with endemic enteric redmouth disease was studied. All isolates displayed indistinguishable pulsed-field gel electrophoresis restriction patterns, indicating that they represented a single strain. However, considering both inhibition zone diameters (IZD) and MICs, the isolates recovered in 2001-2002 formed a separate cluster with lower levels of susceptibility to all the quinolones tested, especially nalidixic acid (NA) and oxolinic acid (OA), compared with the isolates recovered between 1994 and 1998. Analysis of the PCR product of the quinolone resistance-determining region of the gyrA gene from clinical isolates of Y. ruckeri with reduced susceptibility to OA and NA revealed a single amino acid substitution, Ser-83 to Arg-83 (Escherichia coli numbering). Identical substitution was observed in induced OA-resistant mutant strains, which displayed IZD and MICs of quinolones similar to those of the clinical isolates of Y. ruckeri with reduced susceptibility to these antimicrobial agents. These data indicate in that for Y. ruckeri, the substitution of Ser by Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.
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10. |
Welch TJ,
Verner-Jeffreys DW,
Dalsgaard I,
Wiklund T,
Evenhuis JP,
Cabrera JA,
Hinshaw JM,
Drennan JD,
LaPatra SE,
( 2011 ) Independent emergence of Yersinia ruckeri biotype 2 in the United States and Europe. PMID : 21441334 : DOI : 10.1128/AEM.02997-10 PMC : PMC3126439 Abstract >>
Biotype 2 (BT2) variants of the bacterium Yersinia ruckeri are an increasing disease problem in U.S. and European aquaculture and have been characterized as serovar 1 isolates that lack both peritrichous flagella and secreted phospholipase activity. The emergence of this biotype has been associated with an increased frequency of enteric redmouth disease (ERM) outbreaks in previously vaccinated salmonid fish. In this study, four independent specific natural mutations that cause the loss of both motility and secreted lipase activity were identified in BT2 strains from the United States, United Kingdom, and mainland Europe. Each of these was a unique mutation in either fliR, flhA, or flhB, all of which are genes predicted to encode essential components of the flagellar secretion apparatus. Our results demonstrate the existence of independent mutations leading to the BT2 phenotype; thus, this phenotype has emerged separately at least four times. In addition, BT2 strains from the United Kingdom were shown to have the same mutant allele found in U.S. BT2 strains, suggesting a common origin of this BT2 lineage. This differentiation of distinct BT2 lineages is of critical importance for the development and validation of alternative vaccines or other treatment strategies intended for the control of BT2 strains.
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11. |
Méndez J,
Reimundo P,
Pérez-Pascual D,
Navais R,
Gómez E,
Guijarro JA,
( 2011 ) A novel cdsAB operon is involved in the uptake of L-cysteine and participates in the pathogenesis of Yersinia ruckeri. PMID : 21169490 : DOI : 10.1128/JB.01058-10 PMC : PMC3028680 Abstract >>
Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD(50)) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence.
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12. |
Navais R,
Méndez J,
Reimundo P,
Pérez-Pascual D,
Gómez E,
Guijarro JA,
( 2011 ) The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence. PMID : 21131526 : DOI : 10.1128/AEM.01808-10 PMC : PMC3028698 Abstract >>
A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.
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13. |
Murros-Kontiainen A,
Fredriksson-Ahomaa M,
Korkeala H,
Johansson P,
Rahkila R,
Björkroth J,
( 2011 ) Yersinia nurmii sp. nov. PMID : 21037032 : DOI : 10.1099/ijs.0.024836-0 Abstract >>
This study was set up to identify three Gram-negative, rod-shaped strains originating from broiler meat packaged under a modified atmosphere. A polyphasic taxonomic approach, including multilocus sequence analysis (MLSA) of five genes (16S rRNA, glnA, gyrB, recA and HSP60), DNA-DNA reassociation between the closest phylogenetic neighbours and determination of relevant phenotypic properties, was applied. Phylogenetic analysis of the 16S rRNA gene sequences grouped these strains together and within the genus Yersinia. MLSA of the 16S rRNA gene and four housekeeping genes showed that the strains formed a monophyletic group separate from other Yersinia species in all phylogenetic trees constructed. The strains had a phenotypic profile different from those of other representatives of the genus Yersinia, but most similar to that of Yersinia ruckeri. Typical virulence markers for pathogenic Yersinia were not detected. Based on phylogenetic, phenotypic and DNA-DNA reassociation data, a novel species, Yersinia nurmii sp. nov., is proposed for the isolated strains. The type strain is APN3a-c(T) (= DSM 22296(T) = LMG 25213(T)).
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14. |
Dahiya I,
Stevenson RM,
( 2010 ) The UvrY response regulator of the BarA-UvrY two-component system contributes to Yersinia ruckeri infection of rainbow trout (Oncorhynchus mykiss). PMID : 20480360 : DOI : 10.1007/s00203-010-0582-8 Abstract >>
To identify virulence-associated genes of a fish pathogen Yersinia ruckeri, we screened a total of 1056 mini-Tn5-Km2 signature-tagged mutants in rainbow trout by immersion challenge. Of 1056, 25 mutants were found survival-defective as they could not be re-isolated from fish kidney 7 days after infection. Mutated gene in F2-4 mutant, one of the 25 mutants, was homologous to uvrY that encodes UvrY response regulator of BarA-UvrY two-component system (TCS). Mutant F2-4 was significantly more sensitive (P < 0.05) to H2O2-mediated killing and was less able to infect Epithelioma papulosum cyprini cells. However, UvrY mutation did not affect survival of F2-4 mutant in the presence of non-immune fish serum and its ability to grow under iron starvation. In a time-course co-infection, mutant F2-4 had lower bacterial loads on day 1 itself, and by day 5 there was nearly a 1,000-fold difference in infection levels of the parent and mutant strains. The barA homolog of Y. ruckeri was PCR-amplified and sequence analyses identified four domains that were characteristic of hybrid histidine kinases. To conclude, the BarA-UvrY TCS contributes to the pathogenesis of Y. ruckeri in its natural host rainbow trout, possibly by regulating invasion of epithelial cells and sensitivity to oxidative stress induced by immune cells.
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15. |
Souza RA,
Pitondo-Silva A,
Falcão DP,
Falcão JP,
( 2010 ) Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates. PMID : 20493215 : DOI : 10.1016/j.mimet.2010.05.005 Abstract >>
In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.
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16. |
Dahiya I,
Stevenson RM,
( 2010 ) Yersinia ruckeri genes that attenuate survival in rainbow trout (Oncorhynchus mykiss) are identified using signature-tagged mutants. PMID : 20202763 : DOI : 10.1016/j.vetmic.2010.02.003 Abstract >>
To identify genes that enable the enteric redmouth disease bacterium, Yersinia ruckeri, to persist in salmonid fish, 1056 signature-tagged mini-Tn5Km2 transposon mutants of a serotype 1 strain of Y. ruckeri, RS1154, were screened in rainbow trout by immersion infection. Two rounds of screening in fish identified 25 mutants that were not re-isolated from the kidney, 7 days post-infection. Six mutants were tested a third time in fish, in 1:1 competitive challenges with the parent strain; 4 failed to establish in kidney and 2 were present at low levels compared to the parent. Sequence analyses from the single transposon insertion sites in each of the 25 mutants identified genes with sequence homologies to genes for ZnuA, a periplasmic zinc-binding protein of ZnuABC transporter; the UvrY response regulator of BarA-UvrY two-component system; a PtrA protease of the insulin-degrading enzyme family; the RcpA protein of type IV bundle-forming pili; the ParA ATPase of a ParAB DNA-partitioning system; a Wzy polymerase; a polysaccharide deacetylase; a transporter belonging to the major facilitator superfamily and 7 hypothetical proteins of unknown function. The products of 5 of these mutated genes have predicted functions associated with cell surfaces or membranes, which could be important for survival of Y. ruckeri in rainbow trout, while other putative gene products could contribute to infection and invasion processes.
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17. |
Evenhuis JP,
Lapatra SE,
Verner-Jeffreys DW,
Dalsgaard I,
Welch TJ,
( 2009 ) Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis. PMID : 19700548 : DOI : 10.1128/AEM.01415-09 PMC : PMC2765149 Abstract >>
Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.
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18. |
Méndez J,
Fernández L,
Menéndez A,
Reimundo P,
Pérez-Pascual D,
Navais R,
Guijarro JA,
( 2009 ) A chromosomally located traHIJKCLMN operon encoding a putative type IV secretion system is involved in the virulence of Yersinia ruckeri. PMID : 19088314 : DOI : 10.1128/AEM.01377-08 PMC : PMC2643577 Abstract >>
Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes (traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain, as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18 degrees C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.
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19. |
Isabel S,
Leblanc E,
Boissinot M,
Boudreau DK,
Grondin M,
Picard FJ,
Martel EA,
Parham NJ,
Chain PS,
Bader DE,
Mulvey MR,
Bryden L,
Roy PH,
Ouellette M,
Bergeron MG,
( 2008 ) Divergence among genes encoding the elongation factor Tu of Yersinia Species. PMID : 18790860 : DOI : 10.1128/JB.01067-08 PMC : PMC2576667 Abstract >>
Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.
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20. |
Merhej V,
Adékambi T,
Pagnier I,
Raoult D,
Drancourt M,
( 2008 ) Yersinia massiliensis sp. nov., isolated from fresh water. PMID : 18398169 : DOI : 10.1099/ijs.0.65219-0 Abstract >>
Two bacterial organisms, 50640T and 823, were isolated from fresh water in Marseilles, France, and were further identified as members of the genus Yersinia on the basis of their phenotypic characteristics and 16S rRNA gene sequencing. Their unique phenotypic profile differed from that of closely related species of Yersinia bercovieri and Yersinia mollaretii by exhibiting positive indole and inositol tests, and from that of Yersinia frederiksenii by lacking the ability to ferment l-rhamnose. A polyphasic approach, including almost complete 16S rRNA gene sequencing (1461 bp) and partial sequencing of hsp60 (683 bp), gyrB (662 bp), sodA (624 bp) and rpoB (1049 bp) showed that isolates 50640T and 823 exhibited 98.5, 93.5, 90.4, 92.4 and 96.6 % similarity with Y. mollaretii, 98.7, 93.0, 90.1, 89.1 and 96.2 % with Y. bercovieri, and 98.4, 93.2, 89.8, 88.9 and 95.2 % with Y. frederiksenii, respectively. Both isolates exhibited an identical 16S rRNA gene sequence and differed by one to five point mutations in housekeeping gene sequences. Phylogenetic reconstructions based on the combination of these four housekeeping genes indicated that the two isolates formed a unique branch supported by a bootstrap value of 93 %. Their unique phenotypic traits, 16S rRNA gene sequence, together with housekeeping gene sequences exhibiting <97 % similarity with closely related species, and phylogenetic analyses suggested that the two isolates represent a so far undescribed Yersinia species. The name Yersinia massiliensis sp. nov. is proposed for this new taxon (type strain 50640T=CIP 109351T=CCUG 53443T; isolate 823=CIP 109352=CCUG 53444).
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21. |
Navais R,
Méndez J,
Cascales D,
Reimundo P,
Guijarro JA,
( 2014 ) The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence. PMID : 25266819 : DOI : 10.1186/s12866-014-0221-7 PMC : PMC4207315 Abstract >>
The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.
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22. |
Calvez S,
Fournel C,
Douet DG,
Daniel P,
( 2015 ) Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France. PMID : 26104532 : DOI : 10.1186/s13567-015-0200-5 PMC : PMC4477544 Abstract >>
Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.
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23. |
Navais R,
Méndez J,
Pérez-Pascual D,
Cascales D,
Guijarro JA,
( 2014 ) The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions. PMID : 24865652 : DOI : 10.4161/viru.29363 PMC : PMC4105311 Abstract >>
In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.
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24. |
Huang Y,
Michael GB,
Becker R,
Kaspar H,
Mankertz J,
Schwarz S,
Runge M,
Steinhagen D,
( 2014 ) Pheno- and genotypic analysis of antimicrobial resistance properties of Yersinia ruckeri from fish. PMID : 24331744 : DOI : 10.1016/j.vetmic.2013.10.026 Abstract >>
Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 �� 2�XC for 24, 28 and 48 h. Incubation for 24h at 22 �� 2�XC appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 �gg/mL) and nalidixic acid (0.25-0.5 �gg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 �gg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ? 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes.
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25. |
Scott CJ,
Austin B,
Austin DA,
Morris PC,
( 2013 ) Non-adjuvanted flagellin elicits a non-specific protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum) towards bacterial infections. PMID : 23707165 : DOI : 10.1016/j.vaccine.2013.05.025 Abstract >>
Enteric redmouth disease, caused by Yersinia ruckeri, may result in high mortalities in farmed salmonids. Prophylaxis has been achieved with an immersion vaccine comprised of inactivated serovar 1 biotype 1 (motile) Y. ruckeri cultures. However, there has been a growing number of enteric redmouth outbreaks in vaccinated livestock associated with serovar 1 biotype 2 (non-motile) Y. ruckeri strains which do not produce flagellin. It was the aim of this study to evaluate the protective role of flagellin in enteric redmouth vaccines. Results showed that flagellin in the inactivated whole-cell vaccine were not the main immunoprotective molecule in eliciting a protective immune response towards infection. However, use of non-adjuvanted flagellin as a sub-unit vaccine, both in the native and recombinant form, resulted in a potent non-specific protective function towards challenge with biotype 1 (flagellin-producing) and biotype 2 (flagellin-devoid) Y. ruckeri. This vaccine can also protect rainbow trout against other microbial fish pathogens, for example Aeromonas salmonicida. Thus non-adjuvanted flagellin may have potential as a non-specific vaccine for fish towards bacterial pathogens.
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26. |
Stenkova AM,
Isaeva MP,
Shubin FN,
Rasskazov VA,
Rakin AV,
( 2011 ) Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia. PMID : 21655186 : DOI : 10.1371/journal.pone.0020546 PMC : PMC3105102 Abstract >>
OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.
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