Vu TTT, Lu M, Pichpol D, Pham NH; Baumann M, Alter T, Huehn S
Appeared in: Berliner und Münchener tierärztliche Wochenschrift; 129(1) 2016 — S. 48–51; ISSN: 0005-9366; DOI: 10.2376/0005-9366-129-48
Urmersbach S, Aho T, Alter T, Hassan SS, Autio R, Huehn S
Appeared in: BMC Microbiol. 2015 Oct 23;15:229. doi: 10.1186/s12866-015-0565-7; PMID: 26498286; PMCID: PMC4618957
BACKGROUND: Vibrio (V.) parahaemolyticus causes seafood-borne gastro-intestinal bacterial infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Throughout the food chain, V. parahaemolyticus encounters different temperature conditions that might alter metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C to describe the cold and heat shock response. METHODS: Gene expression profiles of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C were investigated via microarray. Gene expression values and RT-qPCR experiments were compared by plotting the log2 values. Moreover, volcano plots of microarray data were calculated to visualize the distribution of differentially expressed genes at individual temperatures and to assess hybridization qualities and comparability of data. Finally, enriched terms were searched in annotations as well as functional-related gene categories using the Database for Annotation, Visualization and Integrated Discovery. RESULTS: Analysis of 37 °C normalised transcriptomics data resulted in differential expression of 19 genes at 20 °C, 193 genes at 4 °C, 625 genes at 42 °C and 638 genes at 15 °C. Thus, the largest number of significantly expressed genes was observed at 15 and 42 °C with 13.3 and 13 %, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS-1, T6SS-2) remained mostly unaffected by heat or cold stress. CONCLUSION: Along with folding and temperature shock depending systems, an overall temperature-dependent regulation of expression could be shown. Particularly the energy metabolism was affected by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts to predict its behaviour under conditions like storage and transport.
Meng Lu, Alter T, Aho T, Huehn S
Appeared in: Letters in Applied Microbiology; 61(3) 2015 — S. 231–237; ISSN: 0266-8254; DOI: 10.1111/lam.12452; Pubmed: 26118852
Vibrio (V.) parahaemolyticus is an aquatic bacterium capable of causing foodborne gastroenteritis. In the environment or the food chain, V. parahaemolyticus cells are usually forced into the stationary phase, the common phase for bacterial survival in the environment. So far, little is known about whole genomic expression of V. parahaemolyticus in the early stationary phase compared with the exponential growth phase. We performed whole transcriptomic profiling of V. parahaemolyticus cells in both phases (exponential and early stationary phase). Our data showed in total that 172 genes were induced in early stationary phase, while 61 genes were repressed in early stationary phase compared with the exponential phase. Three functional categories showed stable gene expression in the early stationary phase. Eleven functional categories showed that up-regulation of genes was dominant over down-regulation in the early stationary phase. Although genes related to endogenous metabolism were repressed in the early stationary phase, massive regulation of gene expression occurred in the early stationary phase, indicating the expressed gene set of V. parahaemolyticus in the early stationary phase impacts environmental survival. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio (V.) parahaemolyticus is one of the main bacterial causes of foodborne intestinal infections. This bacterium usually is forced into stationary phase in the environment, which includes, e.g. seafood. When bacteria are in stationary phase, physiological changes can lead to a resistance to many stresses, including physical and chemical challenges during food processing. To the best of our knowledge, highlighting the whole genome expression changes in the early stationary phase compared with exponential phase, as well as the investigation of physiological changes of V. parahaemolyticus such as the survival mechanism in the stationary phase has been the very first study in this field.
Keywords: Vibrio parahaemolyticus; early stationary phase; exponential phase; microarray; whole genome gene expression
Oeleker K, Alter T, Kleer J, Pund RP,Gölz G, Hildebrandt G, Hühn S.
Appeared in: Journal für Verbraucherschutz und Lebensmittelsicherheit = Journal of consumer protection and food safety; 10(Suppl. 1) 2015 — S. 35–37, ISSN: 1661-5751, DOI: 10.1007/s00003-015-1002-4
Schauer S, Jakwerth S, Bliem R, Baudart J, Lebaron P, Huhulescu S, Kundi M, Herzig A, Farnleitner AH, Sommer R, Kirschner A
Appeared in: Environ Microbiol. 2015, 17; Article first published online: 18 MAY 2015; DOI: 10.1111/1462-2920.12861
In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio cholera-related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid-phase cytometry (CARD-FISH/SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. Vibrio cholerae attached to crustacean zooplankton was quantified via FISH and epifluorescence microscopy. Concentrations obtained by CARD-FISH/SPC were significantly higher than those obtained by culture in 2011, but were mostly of similar magnitude in 2012. Maximum cell numbers were 1.26 × 106 V. cholerae per L in Neusiedler See and 7.59 × 107 V. cholerae per L in the shallow alkaline lakes. Only on a few occasions during summer was the crustacean zooplankton the preferred habitat for V. cholerae. In winter, V. cholerae was not culturable but could be quantified at all sites with CARD-FISH/SPC. Beside temperature, suspended solids, zooplankton and ammonium were the main predictors of V. cholerae abundance in Neusiedler See, while in the shallow alkaline lakes it was organic carbon, conductivity and phosphorus. Based on the obtained concentrations a first estimation of the health risk for visitors of the lake could be performed.
Meng Lu, Alter T, Aho T, Huehn S
Appeared in: FEMS microbiology ecology; 91(5) 2015 — S. 1–12; ISSN: 0168-6496; DOI: 10.1093/femsec/fiv035; Pubmed: 25873464
Viable but non-culturable (VBNC) state is referred to as a dormant state of non-sporulating bacteria enhancing the survival in adverse environments. To our knowledge, only few studies have been conducted on whole genomic expression of Vibrio parahaemolyticus VBNC state. Since a degradation of nucleic acids in V. vulnificus non-culturable state has been detected, we hypothesize that gene regulation of VBNC cells is highly reduced, downregulation of gene expression is dominant and only metabolic functions crucial for survival are kept on a sustained basis. Hence, we performed the whole transcriptomic profiles of V. parahaemolyticus in three phases (exponential, early stationary phase and VBNC state). Compared with exponential and early stationary phase, in V. parahaemolyticus VBNC cells we found 509 induced genes and 309 repressed by more than 4-fold among 4820 investigated genes. Upregulation was dominant in most of non-metabolism functional categories, while five metabolism-related functional categories revealed downregulation in VBNC state. To our knowledge, this is the first study of comprehensive transcriptomic analyses of three phases of V. parahaemolyticus RIMD2210633. Although the mechanism of VBNC state is not yet clear, massive regulation of gene expression occurs in VBNC state compared with expression in other two phases, indicating VBNC cells are active.
Keywords: VBNC; Vibrio parahaemolyticus; early stationary phase; exponential phase; microarray; viable but not culturable state; whole genome gene expression
Rivas AJ, Vences A, Husmann M, Lemos ML, Osorio CR
Appeared in: Infect Immun. 2015 Apr; 83(4): 1246-56. doi: 10.1128/IAI.02608-14
Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyAch secretion. In addition, mutation of epsL in a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating that epsL plays a major role in secretion of the plasmid-encoded HlyApl and Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase gene pilD severely affected hemolysis, which dropped at levels inferior to those of epsL mutants. Promoter expression analyses suggested that impairment of hemolysin secretion in epsL and pilD mutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, single epsL and pilD mutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS and pilD in P. damselae subsp. damselae virulence.
Le Roux F, Wegner KM, Baker-Austin C, Vezzulli L, Osorio CR, Amaro C, Ritchie JM, Defoirdt T, Destoumieux-Garzón D, Blokesch D, Mazel D, Jacq A, Cava F, Gram L, Wendling C.
Appeared in: Frontiers in Microbioly, 2015 Aug 13; 6:830. doi: 10.3389/fmicb.2015.00830. eCollection 2015.; PMID: 26322036
Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.
Keywords: animal model; aquaculture; bacterial disease; european network; genome plasticity; global warming; human health; interactions
Yang Q and Defoirdt T
Appeared in: Environ Microbiol. 2015 Apr; 17(4): 960-8. doi:10.1111/1462-2920.12420
Vibrios belonging to the Harveyi clade are among the major pathogens of aquatic organisms. Quorum sensing (QS) is essential for virulence of V. harveyi towards different hosts. However, most virulence factors reported to be controlled by QS to date are negatively regulated by QS, therefore suggesting that their impact on virulence is limited. In this study, we report that QS positively regulates flagellar motility. We found that autoinducer synthase mutants showed significantly lower swimming motility than the wild type, and the swimming motility could be restored by adding synthetic signal molecules. Further, motility of a luxO mutant with inactive QS (LuxO D47E) was significantly lower than that of the wild type and of a luxO mutant with constitutively maximal QS activity (LuxO D47A). Furthermore, we found that the expression of flagellar genes (both early, middle and late genes) was significantly lower in the luxO mutant with inactive QS when compared with wild type and the luxO mutant with maximal QS activity. Motility assays and gene expression also revealed the involvement of the quorum-sensing master regulator LuxR in the QS regulation of motility. Finally, the motility inhibitor phenamil significantly decreased the virulence of V. harveyi towards gnotobiotic brine shrimp larvae.
Erler R, Wichels A, Heinemeyer EA, Hauk G, Hippelein M, Reyes NT, Gerdts G
Appeared in: Syst Appl Microbiol. 2015 Feb;38(1):16-25. doi:10.1016/j.syapm.2014.10.009
Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments.
Keywords: Database; MALDI-TOF MS; Marine; Pathogenic; Vibrio; Whole-cell fingerprinting
Bliem R, Schauer S, Plicka H, Obwaller A, Sommer R, Steinrigl A, Alam M, Reischer GH, Farnleitner AH, Kirschner A
Appeared in: Applied Environmental Microbiology 81(9) 2015: 3077-3085; doi:10.1128/AEM.03516-14.
Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 102 to 2.3 × 104 cell equivalents liter−1, whereas GR-corrected abundances ranged from 4.7 × 103 to 1.6 × 106 cell equivalents liter−1. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.
Huehn S, Eichhorn C, Urmersbach S, Breidenbach J, Bechlars S, Bier N, Alter T, Bartelt E, Frank C, Oberheitmann B, Gunzer F, Brennholt N, Böer S, Appel B, Dieckmann R, Strauch E
Appeared in: International journal of medical microbiology 2014 Oct;304(7):843-50. doi: 10.1016/j.ijmm.2014.07.010. PMID: 25129553
Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.
Keywords: Baltic Sea; North Sea; Vibrio cholerae non-O1/non-O139; Vibrio parahaemolyticus; Vibrio population; Vibrio vulnificus
Urmersbach S, Alter T, Koralage, MSG, Sperling L , Gerdts G, Messelhäusser U, Huehn S
Appeared in: BMC microbiology 2014 — S. 59–73, ISSN: 1471-2180, DOI: 10.1186/1471-2180-14-59; Pubmed: 24606756
Vibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number. To generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified. Our study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains.
Rivas AJ, Labella AM, Borrego JJ, Lemos ML, Osorio CR
Appeared in: FEMS Microbiol Lett. 2014 Jun; 355(2): 152-62. doi:10.1111/1574-6968.12464
Photobacterium damselae subsp. damselae, a marine bacterium that causes infections in marine animals and in humans, produces up to three different haemolysins involved in virulence, which include the pPHDD1 plasmid-encoded damselysin (Dly) and HlyApl , and the chromosome-encoded HlyAch . We screened 45 isolates from different origins, and found a correlation between their haemolytic phenotypes and the differential haemolysin gene content. All highly and medium haemolytic strains harboured pPHDD1, with amino acid substitutions in HlyApl and HlyAch being the cause of the medium haemolytic phenotypes in some pPHDD1-harbouring strains. Weakly haemolytic strains contained only hlyAch , whereas nonhaemolytic isolates, in addition to lacking pPHDD1, either lacked hlyAch or contained a hlyAch pseudogene. Sequence analysis of the genomic context of hlyAch uncovered an unexpected genetic diversity, suggesting that hlyAch is located in an unstable chromosomal region. Phylogenetic analysis suggested that hlyApl and hlyAch originated by gene duplication within P. damselae subsp. damselae following acquisition by horizontal transfer. These observations together with the differential distribution of pPHDD1 plasmid among strains suggest that horizontal gene transfer has played a main role in shaping the haemolysin gene baggage in this pathogen.
Keywords: Vibrio damsela; damselysin; dly; haemolysin; hlyA; vibriosis
Schirmeister F, Dieckmann R, Bechlars S, Bier N, Faruque SM, Strauch E
Appeared in: Eur J Clin Microbiol Infect Dis. 2014 May;33(5):767-78
Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.
Norouzitallab P, Baruah K, Vandegehuchte M, Van Stappen G, Catania F, Vanden Bussche J, Vanhaecke L, Sorgeloos P and Bossier P
Appeared in: Journal of the Federation of American Societies for Experimental Biology (FASEB ) Journal 28, 2014: 3552-3563.
The notion that phenotypic traits emerging from environmental experiences are heritable remains under debate. However, the recent report of nonmendelian transgenerational epigenetic inheritance, i.e., the inheritance of traits not determined by the DNA sequence, might make such a phenomenon plausible. In our study, by carrying out common garden experiments, we could provide clear evidences that, on exposure to nonlethal heat shocks, a parental population of parthenogenetic (all female) Artemia (originating from one single female) experiences an increase in levels of Hsp70 production, tolerance toward lethal heat stress, and resistance against pathogenic Vibrio campbellii. Interestingly, these acquired phenotypic traits were transmitted to three successive generations, none of which were exposed to the parental stressor. This transgenerational inheritance of the acquired traits was associated with altered levels of global DNA methylation and acetylated histones H3 and H4 in the heat-shocked group compared to the control group, where both the parental and successive generations were reared at standard temperature. These results indicated that epigenetic mechanisms, such as global DNA methylation and histones H3 and H4 acetylation, have particular dynamics that are crucial in the heritability of the acquired adaptive phenotypic traits across generations.—Norouzitallab, P., Baruah, K., Vandegehuchte, M., Van Stappen, G., Catania, F., Vanden Bussche, J., Vanhaecke, L., Sorgeloos, P., Bossier, P. Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model.
Klevanskaa K, Bier N, Stingl K, Strauch E, Hertwig S
Appeared in: Appl Environ Microbiol. 2013 Feb; 80(4):1477-81. doi: 10.1128/AEM.03720-13. Epub 2013 Dec 20
An efficient electroporation procedure for V. vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 x 106 transformants per microgram DNA were achieved. The vector stably replicated in both V. vulnificus and E. coli and was also successfully introduced into V. parahaemolyticus and V. cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.
Sara Urmersbach, Thomas Alter, Tommi Aho, Stephan Huehn
Bechlars S, Wüstenhagen DA, Drägert K, Dieckmann R, Strauch E, Kubick S.
Appeared in: Toxicon, Vol. 76, 15 December 2013, Pages 132–142
Vibrio parahaemolyticus is a recognized enteropathogen causing diarrhea in humans and is one of the major causes of seafoodborne gastroenteritis. An important virulence factor is thermostable direct hemolysin (TDH), a pore-forming toxin, which is able to lyse eukaryotic cells. The active toxin is a tetramer of four identical protein subunits, which is secreted by the pathogen after cleavage of a signal peptide. To establish diagnostic detection systems for TDH we expressed the hemolysin with and without the signal peptide in a prokaryotic cell-free system to obtain pure toxin. In order to purify and to facilitate the isolation from cell lysates we synthesized TDH variants with different tags. Important regulatory sequences for cell-free protein synthesis as well as sequences for N-terminal Strep-tag and C-terminal 6xHis-tag were added by a two-step PCR. For the expression in the cell-free system these linear tdh templates were subjected directly to prokaryotic cell extracts. Protein yields were in the range of 500–600 μg/ml for the preproteins and approx. 300–400 μg/ml for the mature proteins. The identities of expressed proteins were further confirmed by SDS-PAGE, immunological and MALDI-TOF mass spectrometric analyses. The functionality of newly synthesized toxin variants was tested by performing qualitative and semiquantitative hemolysis assays. Cell-free produced mature TDH and its variants were active while the preprotein and its derivatives lacked hemolytic activity. A C-terminal 6xHis-tag showed less influence on functionality compared to the N-terminal Strep-tag.
Keywords: Vibrio parahaemolyticus; Thermostable direct hemolysin (TDH); Cell-free protein synthesis; In vitro translation
Herrfurth D, Oelecker K, Pund R, Strauch E, Schwartz K, Kleer J, Gölz G, Alter T, Huehn S
Appeared in: Journal of Shellfish Research / Dec 2013 / 32(3): 855-859
Species of Vibrio can persist in blue mussels, especially when they are present in high numbers as a result of a large uptake from the aquatic environment. This study investigated the uptake, localization, and persistence of three Vibrio species relevant to human health in blue mussels (Mytilus edulis) after artificial contamination. Mussels M. edulis were kept in tanks of artificial seawater spiked with Vibrio spp. to monitor bioaccumulation of these bacteria in corresponding bivalves. Bacteria accumulated rapidly in the bivalves, reaching high concentrations after 1.5 h. The highest Vibrio sp. counts were detected in the digestive glands, with 6.9 × 108 cfu/g for Vibrio parahaemolyticus, 1.5 × 107 cfu/g for Vibrio cholerae, and 2.2 × 107 cfu/g for Vibrio vulnificus. Among bivalve compartments, the digestive glands showed the fastest enrichment of Vibrio and maintained the highest Vibrio numbers throughout the examination period. After transfer to a tank containing filtered, sterile seawater, the Vibrio load in bivalves showed a continuous reduction. However, even after 7 days of depuration, an average concentration of approximately 103 cfu/g remained in the digestive glands of M. edulis. In clearance assays, a general clearance rate of 0.02 log cfu/g/h was calculated for all three strains. For the first time, in vivo accumulation counts and clearance kinetics of Vibrio within mussel compartments are shown, highlighting a strong concentration of Vibrio in the digestive glands whereas other tissues continued to accumulate significantly less Vibrio.
Keywords: blue mussel, Mytilus edulis, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, food safety, bacterial uptake, bacterial localization
Yu YP, Gong T, Jost G, Liu WH, Ye DZ, Luo ZH
Appeared in: FEMS Microbiol Lett. 2013 Nov; 348(2):112-9. doi: 10.1111/1574-6968.12277
Vibrio owensii is a potential bacterial pathogen in marine aquaculture system. In this study, five lytic phages specific against Vibrio strain B8D, closely related to V. owensii, were isolated from seawater of an abalone farm. The phages were characterized with respect to morphology, genome size, growth phenotype, as well as thermal, and pH stability. All phages were found to belong to the family Siphoviridae with long noncontractile tails and terminal fibers. Restriction analysis indicated that the five phages were dsDNA viruses with molecular weights ranging from c. 30 to 48 kb. One-step growth experiments revealed that the phages were heterogeneous in latent periods (10-70 min), rise periods (40-70 min), and burst sizes [23-331 plaque-forming units (PFU) per infected cell] at the same host strain. All phages were thermal stable and were tolerant to a wide range of pH. The results indicated that these phages could be potential candidates of a phage cocktail for biological control of V. owensii in aquaculture systems
Keywords: Siphoviridae; Vibrio owensii; biological control; lytic bacteriophages
Rivas AJ, Balado M, Lemos ML, Osorio CR
Appeared in: Infect Immun. 2013 Sep; 81(9): 3287-99. doi:10.1128/IAI.00155-13
Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA(pl)). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyAch). Hemolysis was completely abolished in a single hlyAch mutant of a plasmidless strain and in a dly hlyApl hlyAch triple mutant. We found that Dly, HlyA(pl), and HlyAch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyApl and HlyAch, on the one hand, and of the synergistic effect of Dly with HlyApl and HlyAch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie, Atkins, and Munch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyApl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.
Rivas AJ, Lemos ML, Osorio CR
Appeared in: Front Microbiol. 2013 Sep 25; 4: 283. doi:10.3389/fmicb.2013.00283
Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a pathogen of a variety of marine animals including fish, crustaceans, molluscs, and cetaceans. In humans, it can cause opportunistic infections that may evolve into necrotizing fasciitis with fatal outcome. Although the genetic basis of virulence in this bacterium is not completely elucidated, recent findings demonstrate that the phospholipase-D Dly (damselysin) and the pore-forming toxins HlyApl and HlyAch play a main role in virulence for homeotherms and poikilotherms. The acquisition of the virulence plasmid pPHDD1 that encodes Dly and HlyApl has likely constituted a main driving force in the evolution of a highly hemolytic lineage within the subspecies. Interestingly, strains that naturally lack pPHDD1 show a strong pathogenic potential for a variety of fish species, indicating the existence of yet uncharacterized virulence factors. Future and deep analysis of the complete genome sequence of Photobacterium damselae subsp. damselae will surely provide a clearer picture of the virulence factors employed by this bacterium to cause disease in such a varied range of hosts.
Keywords: Photobacterium damselae; damselysin; hemolysin; hlyA; pore-forming toxin
Oeleker K, Alter T, Kleer J, Pund R, Gölz G, Hildebrandt G, Huehn S
Appeared in: Archiv für Lebensmittelhygiene; 64(2) 2013 — S. 50–54; ISSN: 0003-925x; DOI: 10.2376/0003-925X-64-50
Zur Untersuchung der organoleptischen, physikalisch-chemischen und mikrobiologischen Qualität von Kaviar wurden 50 Kaviarproben von verschiedenen Süß- und Salzwasserfischarten aus dem Einzelhandel untersucht. Nur 36 % der Proben waren ohne organoleptische Abweichungen. 46 % der Proben wiesen mesophile aerobe Gesamtkeimzahlen von >2 log10 KBE/g auf. Von diesen zeigte die Mehrzahl (n = 12) Keimzahlen von 3–4 log10 KbE/g. Zwei Proben wiesen dabei eine hohe Keimzahl von >6 log10 KBE. Milchsäurebakterien wurden in 20 % der Proben nach- gewiesen (bis zu 5,8 log10 KbE/g). Hefen wurden in 10 % der Proben detektiert, und Schimmelpilze fanden sich in einer Probe. Coliforme Keime waren in 4 % der Proben nachweisbar mit Keimzahlen von 2,3 log10 KBE/g und 3,4 log10 KBE/g. E. coli war jedoch nicht nachweisbar. Eine Probe enthielt 3,43 log10 KBE/g koagulasepositive Staphylokokken und 5 log10 KBE/g Pseudomonaden. Mycobacterium aubagnense, ein potentieller Zoonoseerreger, wurde in vier Proben nachgewiesen. Weder niedrige pH-Werte, geringe Wasseraktivitäten, hohere Salzkonzentrationen noch eine Pasteurisierung beeinflussten die mikrobiologische Qualität dieser Produkte signifikant. Insgesamt war die mikrobiologische Qualität der meisten Kaviarproben akzeptabel. Dennoch zeigten einzelne Proben hohe mikrobiologische Belastungen. Viele Kaviarproben wiesen dazu organoleptische Abweichungen auf. Um eine Vermehrung der vorhandenen Mikroorganismen in Kaviarprodukten zu begrenzen und daraus folgende Änderungen der organoleptischen Eigenschaften zu vermeiden, ist eine kontinuierliche Kühlung solcher Produkte notwendig. Die Bedeutung von potentiell pathogenen Mycobacterium spp. (inkl. Mycobacterium aubagnense) in Kaviar muss in weiteren Studien evaluiert werden.
To investigate the organoleptic, physicochemical and microbiological quality of caviar, fifty caviar samples from various fresh- and seawater fish species were obtained from retail markets and tested. Only 36 % of the samples were without organoleptic deviations. 46 % of the samples showed cell counts of mesophilic aerobic bacteria of >2 log10 CFU/g. Of these, the majority (n = 12) showed cell counts of 3–4 log10 CFU/g. Two samples exhibited high cell counts of >6 log10 CFU/g. Lactic acid bacteria were found in 20 % of the samples, reaching up to 5.8 log10 CFU/g. Yeasts were detected in 10 % of the samples and moulds were present in one sample. For coliforms, 4 % of the samples showed growth with counts of 2.3 log10 CFU/g and 3.4 log10 CFU/g respectively. However, E. coli was not detectable. One sample contained 3.43 log10 CFU/g coagulase positive staphylococci and 5 log10 CFU/g pseudomonads. Mycobacterium aubagnense, a potential zoonotic pathogen, was detected within four samples. Neither lower pH, lower water activity, higher salt concentrations nor pasteurization influenced the microbiological quality of these products significantly. Taken together, microbiological quality of most caviar samples was acceptable. Nonetheless, single samples showed high microbiological loads. Many caviar samples exhibited organoleptic deviations. To limit microbiological growth and subsequent changes in organoleptic properties, a continuous cooling of these products is necessary. The importance of potentially pathogenic Mycobacterium spp. (incl. Mycobacterium aubagnense) in caviar must be evaluated in future studies.
Keywords: caviar, microbiological quality, organoleptic properties, chemical properties
Böer SI, Heinemeyer EA, Luden K, Erler R, Gerdts G, Janssen F, Brennholt N
Appeared in: Microb Ecol. 2013 May; 65(4): 1052-67
The number of reported Vibrio-related wound infections associated with recreational bathing in Northern Europe has increased within the last decades. In order to study the health risk from potentially pathogenic Vibrio spp. in the central Wadden Sea, the seasonal and spatial distribution of Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae were investigated at ten recreational beaches in this area over a 2-year period. V. alginolyticus and V. parahaemolyticus were found to be omnipresent all year round in the study area, while V. vulnificus occurrence was restricted to summer months in the estuaries of the rivers Ems and Weser. Multiple linear regression models revealed that water temperature is the most important determinant of Vibrio spp. occurrence in the area. Differentiated regression models showed a species-specific response to water temperature and revealed a particularly strong effect of even minor temperature increases on the probability of detecting V. vulnificus in summer. In sediments, Vibrio spp. concentrations were up to three orders of magnitude higher than in water. Also, V. alginolyticus and V. parahaemolyticus were found to be less susceptible towards winter temperatures in the benthic environment than in the water, indicating an important role of sediments for Vibrio ecology. While only a very small percentage of tested V. parahaemolyticus proved to be potentially pathogenic, the presence of V. vulnificus during the summer months should be regarded with care.
Keywords: Vibrio spp.
Bier N, Bechlars S, Diescher S, Klein F, Hauk G, Duty O, Strauch E, Dieckmann R
Appeared in: Appl Environ Microbiol. 2013 Jun; 79 (12)
The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region
Keywords: Vibrio vulnificus
Eichhorn C, Claudia Z, Auerbach C, Goesmann A, Blom J, Alter T, Huehn S, Strauch E, Gunzer F.
Appeared in: International journal of medical microbiology; 303(1) 2013 — S. 112; ISSN: 1438-4221
Pande GSJ, Natrah FMI, Sorgeloos P, Bossier P, Defoirdt T
Appeared in: Veterinary Microbiology, 167 2013: 540-545 ISSN: 0378-1135 DOI: 10.1016/j.vetmic.2013.08.021
Keywords: Macrobrachium-Rosenbergii Larvae, gram-negative Bacteria, Host-pathogen interaction, Host-microbe interaction, Vibriosis, Quorum sensing, Inhibitors, Systems, Pseudomonas Aeruginosa, Artemia-Franciscana, Aeromanas-Hydrophila, Genome Sequence
Defoirdt T, Sorgeloos P
Appeared in: ISME Journal 2012 Dec; 6(12): 2314–2319
Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.
Keywords: quorum sensing, infection, vibriosis, host-microbe interaction
Koralage MS, Alter T, Pichpol D, Strauch E, Zessin KH, Huehn S
Appeared in: J Food Prot. 2012 Oct; 75 (10): 1846-50
This study investigated the prevalence and molecular characteristics of Vibrio spp. in farmed shrimp (Penaeus monodon) in Sri Lanka. A total of 170 shrimp samples (100 g of whole shrimp each) taken from individual ponds from 54 farms were collected 1 week prior to harvest from the North Western Province of Sri Lanka. Overall, 98.1% of the farms and 95.1% of the ponds were positive for Vibrio spp. in shrimp; at the pond level, V. parahaemolyticus (91.2%) was most common, followed by V. alginolyticus (18.8%), V. cholerae non-O1/non-O139 (4.1%), and V. vulnificus (2.4%). Multiple Vibrio spp. were detected in 20.6% of the ponds. None of the V. parahaemolyticus isolates (n = 419) were positive for the virulence-associated tdh (thermostable direct hemolysin) and trh (TDH-related hemolysin) genes. V. cholerae was confirmed by the presence of ompW, and all isolates (n = 8) were negative for the cholera toxin (ctxA) gene. V. cholerae isolates were serogrouped by PCR and identified as V. cholerae non-O1/non-O139. All four V. vulnificus strains, isolated from different ponds of two geographical regions, showed pathogenic potential; they belonged to vcgC sequence type, type B 16S rRNA genotype and contained a pilF polymorphism associated with human pathogenicity. The results of this study revealed the ubiquitous nature of vibrios in farmed shrimp. To minimize the potential risk of Vibrio infections due to handling or consumption of raw or undercooked seafood products, good manufacturing practices as well as proper handling and processing should be addressed.
Janina Breidenbach, Christina Frank
Robert Koch-Institut (RKI) | 2012-05
Keywords: Cholera; Vibrionen
Oberbeckmann S, Fuchs BM, Meiners M, Wichels A, Wiltshire KH, Gerdts G.
Appeared in: Microb Ecol. 2012 Apr; 63 (3): 543-51
Vibrio species are ubiquitously distributed in marine waters all over the world. High genome plasticity due to frequent mutation, recombination, and lateral gene transfer enables Vibrio to adapt rapidly to environmental changes. The genus Vibrio comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions. In recent years, pathogenic Vibrio emerged also in coastal European waters. Little is known about factors driving the proliferation of Vibrio spp. in temperate waters such as the North Sea. In this study a quantification of Vibrio in the North Sea and their response to biotic and abiotic parameters were assessed. Between January and December 2009, Vibrio at Helgoland Roads (North Sea, Germany) were quantified using fluorescence in situ hybridization. Vibrio numbers up to 3.4 × 10(4) cells × mL(-1) (2.2% of total microbial counts) were determined in summer, but their abundance was significantly lower in winter (5 × 10(2) cells × mL(-1)). Correlations between Vibrio and nutrients (SiO(2), PO(4) (3-), DIN), Secchi depth, temperature, salinity, and chlorophyll a were calculated using Spearman rank analysis. Multiple stepwise regression analysis was carried out to analyze the additive influence of multiple factors on Vibrio. Based on these calculations, we found that high water temperature and low salinity best explained the increase of Vibrio cell numbers. Other environmental parameters, especially nutrients and chlorophyll a, also had an influence. All variables were shown to be subject to the overall seasonal dynamics at Helgoland Roads. Multiple regression models could represent an efficient and reliable tool to predict Vibrio abundances in response to the climate change in European waters.
Oeleker K, Huehn S, Pund R, Gölz G, Hildebrandt G, Kleer J, Alter T
Alfeld : Presse Dienstleistungsges. | 2012
Appeared in: Rundschau für Fleischhygiene und Lebensmittelüberwachung; 64(1) 2012 — S. 19–21; ISSN: 0178-2010
Ruwandeepika HAD, Jayaweera TSP, Bhowmick PP, Karunasagar I, Bossier P and Defoirdt T
Blackwell Publishing Asia Pty Ltd | 2012
Appeared in: Reviews in Aquaculture, 4 2015: 59-74
Abstract The Harveyi clade or core group of the Vibrio genus includes both pathogenic and non-pathogenic bacteria. Some of the species belonging to the clade, such as Vibrio harveyi, Vibrio alginolyticus, Vibrio campbellii and Vibrio parahaemolyticus, include serious pathogens of aquatic organisms. Importantly, within these species, some strains are pathogenic, whereas others are not. Other members of the clade, such as Vibrio natriegens and Vibrio mytili are reported to be non-pathogenic. As bacteria belonging to the Harveyi clade show a high diversity with respect to virulence and as the virulent members cause high mortalities in the aquaculture sector, it is important to understand which gene products are responsible for the pathogenicity of the bacteria and how expression of these virulence factors is regulated. This knowledge will ultimately allow for the development of biocontrol measures that aim to reduce the severe losses that are currently faced in the aquaculture sector as a result of vibriosis. In this review, we discuss the pathogenesis of bacteria belonging to the Harveyi clade, their impact on the aquaculture sector, the virulence factors that are involved in pathogenicity and their regulation.
Keywords: Harveyi clade; pathogenesis; vibriosis; virulence factors; virulence regulation
Alter T, Dieckmann R, Hühn S, Strauch E
Behr's Verlag GmbH & Co. KG | 2012
V. parahaemolyticus und V. cholerae spielen als Lebensmittelinfektionserreger weltweit eine zunehmende Rolle. Erfahren Sie in „Pathogene Mikroorganismen: Vibrio“ , welche Bedeutung Vibrio-Bakterien für den gesundheitlichen Verbraucherschutz besitzen, wie sie sich in der Umwelt und der Lebensmittelkette verhalten und welche Präventionsmaßnahmen existieren. - Charakteristika, Pathomechanismen, Nachweisverfahren - Epidemiologie und Bedeutung als Infektionserreger - Industrierelevanz und Präventionsmaßnahmen
Keywords: Pathogene Mikroorganismen, Vibrio, Infektionserreger
Schauer S, R Sommer, AH Farnleitner and AKT Kirschner
Appeared in: Applied and Environmental Microbiology (78) 20 2012: 7369-7375; doi: 10.1128/AEM.02190-12
A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 101 cells ml−1 in the large saline lake Neusiedler See to 5.6 × 104 cells ml−1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.
Rivas AJ, Balado M, Lemos ML, Osorio CR
Appeared in: Infect Immun. 2011 Nov; 79(11): 4617-27. doi:10.1128/IAI.05436-11
Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a marine bacterium that causes infections and fatal disease in a wide range of marine animals and in humans. Highly hemolytic strains produce damselysin (Dly), a cytolysin encoded by the dly gene that is lethal for mice and has hemolytic activity. We found that Dly is encoded in the highly hemolytic strain RM-71 within a 153,429-bp conjugative plasmid that we dubbed pPHDD1. In addition to Dly, pPHDD1 also encodes a homologue of the pore-forming toxin HlyA. We found a direct correlation between presence of pPHDD1 and a strong hemolytic phenotype in a collection of P. damselae subsp. damselae isolates. Hemolysis was strongly reduced in a double dly hlyA mutant, demonstrating the role of the two pPHDD1-encoded genes in hemolysis. Interestingly, although single hlyA and dly mutants showed different levels of hemolysis reduction depending on the erythrocyte source, hemolysis was not abolished in any of the single mutants, suggesting that the hemolytic phenotype is the result of the additive effect of Dly and HlyA. We found that pPHDD1-encoded dly and hlyA genes are necessary for full virulence for mice and fish. Our results suggest that pPHDD1 can be considered as a driving force for the emergence of a highly hemolytic lineage of P. damselae subsp. damselae.
Oberbeckmann S, Wichels A, Wiltshire KH, Gerdts G
Springer Netherlands | 2011-08-01
Appeared in: Antonie van Leeuwenhoek, August 2011, Volume 100, Issue 2, pp 291-307
Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 103 N l−1 and MPNs up to 240 N g−1 were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.
Oberbeckmann S, Wichels A, Maier T, Kostrzewa M, Raffelberg S, Gerdts G
Appeared in: FEMS Microbiol Ecol. 2011 Jan; 75 (1): 145-62
Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.
Randt S, Hühn S, Gölz G, Herrfurth D, Pund R, Strauch E, Alter T
Appeared in: Rundschau für Fleischhygiene und Lebensmittelüberwachung : RFL Heft 3/2011 (Band: 63) S. 93-96
Kirschner AKT, S Schauer, B Steinberger, I Wilhartitz, CJ Grim, A Huq, RR Colwell, A Herzig and R Sommer
Springer-Verlag: Microbial Ecology | 2011
Appeared in: Microbial Ecology April 2011, Volume 61, Issue 3, pp 496-506; DOI: 10.1007/s00248-010-9764-9
Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.
T. Alter, B. Appel, E. Bartelt, R. Dieckmann, C. Eichhorn, R. Erler, C. Frank, G. Gerdt, F. Gunzer,·S. Hühn, J. Neifer, B. Oberheitmann, E. Strauch
Springer-Verlag | 2011
Vibrionen sind salzliebende Bakterien, die in Oberflächen- und Küstengewässern sowie Meerestieren weltweit verbreitet sind. In vielen Ländern stellen pathogene Vibrio-Spezies eine der Hauptursachen von bakteriell verursachten Durchfallerkrankungen dar, die durch den Verzehr von kontaminierten Meeresfrüchten und Fischprodukten und durch verseuchtes Trinkwasser hervorgerufen werden. Vibrio-Infektionen könnten auch in unseren Breiten aufgrund der globalen Klimaerwärmung und der Zunahme des weltweiten Handels mit Meeresfrüchten an Bedeutung gewinnen. Der Forschungsverbund „VibrioNet“ beschäftigt sich mit pathogenen Vibrionen in der marinen Umwelt und in Seafood-Lebensmitteln als potenzielle, neu aufkommende Zoonoseerreger. Durch einen multidisziplinären Forschungsansatz soll eine Bewertung des Risikos ermöglicht werden, das vornehmlich von pathogenen Nicht-Cholera-Vibrionen im zentraleuropäischen Raum ausgeht. Die Verbundforschung wird durch die Zusammenarbeit mit internationalen Partnern aus Ländern, in denen Vibrio-Infektionen eine große Rolle spielen (Bangladesch, Chile, Indien, Thailand und Vietnam), gestärkt.
Vibrio is a genus of bacteria present in surface and coastal waters as well as in marine organisms worldwide. In many countries, pathogenic Vibrio species are a main cause of bacterial diarrhea, which may result from comsumption of contaminated seafood and fish products or from drinking contaminated water. Vibrio infections may also gain in importance in our regions due to global warming and the increase in the world trade of seafood. The research network “VibrioNet” studies pathogenic Vibrios in the marine environment and in seafood consumed by humans as a potential, new emerging zoonotic agent. An assessment of the risk arising from pathogenic non-cholera-vibrios in central Europe is the target of a multidisciplinary research effort. The research network will be strengthened by cooperations with international partners from countries in which Vibrio infections play a major role (Bangladesh, Chile, India, Thailand, and Vietnam).
Keywords: Vibrionen · VibrioNet · Lebensmittel · Meerwasser
Dieckmann R, Strauch E, Alter T
Appeared in: J Appl Microbiol. 2010 Jul; 109 (1): 199-211
AIMS: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI-TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. METHODS AND RESULTS: Starting from sub-colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species-identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. CONCLUSIONS: The MALDI-TOF MS-based method as well as the rpoB sequence-based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI-TOF MS and rpoB sequences revealed a very good congruence of both methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that whole-cell MALDI-TOF MS-based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.
Kirschner AKT, J Schlesinger, AH Farnleitner, R Hornek, B Süß, B Golda, A Herzig and B Reitner
Applied and Environmental Microbiology | 2008
Appeared in: Applied and Environmental Microbiology 74 (7): 2004-2015; doi: 10.1128/AEM.01739-07
Vibrio cholerae non-O1/non-O139 strains have caused several cases of ear, wound, and blood infections, including one lethal case of septicemia in Austria, during recent years. All of these cases had a history of local recreational activities in the large eastern Austrian lake Neusiedler See. Thus, a monitoring program was started to investigate the prevalence of V. cholerae strains in the lake over several years. Genetic analyses of isolated strains revealed the presence of a variety of pathogenic genes, but in no case did we detect the cholera toxin gene or the toxin-coregulated pilus gene, both of which are prerequisites for the pathogen to be able to cause cholera. In addition, experiments were performed to elucidate the preferred ecological niche of this pathogen. As size filtration experiments indicated and laboratory microcosms showed, endemic V. cholerae could rapidly grow in a free-living state in natural lake water at growth rates similar to those of the bulk natural bacterial population. Temperature and the quality of dissolved organic carbon had a highly significant influence on V. cholerae growth. Specific growth rates, growth yield, and enzyme activity decreased markedly with increasing concentrations of high-molecular-weight substances, indicating that the humic substances originating from the extensive reed belt in the lake can inhibit V. cholerae growth.
Huhulescu S, Indra A, Feierl G, Stoeger A, Ruppitsch W, Sarkar B, and Allerberger F
Wien Klin. Wochenschrift | 2007
Appeared in: Wien Klin Wochenschr. 2007; 119 (7-8): 235-41; PMID: 17492351
From 2000 to 2005, 13 infections due to non-O1/non-O139 Vibrio cholerae were documented in Austria. Twelve patients (8 years to 65 years old; 7 male) had symptomatic infections: diarrhea x 5, otitis x 6, septicemia once. All 5 patients who acquired their infections abroad, suffered from diarrhea. The 8 persons without travel history outside of Austria had otitis media (n = 4) or otitis externa (n = 2); the lethal case of septicemia affected a fisherman with underlying malignancy. One isolate was from an asymptomatic child. Detailed data on travel history inside Austria was available for 5 of these 8 patients: all 5 had visited or lived near Austria's largest lake. The concentration of salt in this westernmost steppe lake in Europe is approximately one-twentieth of that of sea water. Why otitis and not diarrhea is the dominating manifestation of non-O1/non-O139 infection acquired in Austria remains to be elucidated. We hypothesize that diarrhea due to Vibrio cholerae serogroups other than O1 and O139 acquired in Austria may simply be unrecognized by the standard operating procedures employed in clinical microbiology laboratories. Testing for Vibrio cholerae is not considered necessary for domestically acquired diarrhea. Only in patients who acquired diarrhea abroad, do physicians sometimes consider cholera as a differential diagnosis, thereby prompting the laboratory to use thiosulfate citrate bile salt sucrose (TCBS) agar plates.