Eckhard Strauch

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

ABSTRACT Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K+ ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei

ABSTRACT A Shiga toxin (Stx)-encoding temperate bacteriophage ofShigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization inShigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producingShigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonneiand laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.

Rapid identification and characterization of Vibrio species using whole-cell MALDI-TOF mass spectrometry

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.

The Obligate PredatoryBdellovibrio bacteriovorusPossesses a Neutral Lipid A Containing α-D-Mannoses That Replace Phosphate Residues: SIMILARITIES AND DIFFERENCES BETWEEN THE LIPID As AND THE LIPOPOLYSACCHARIDES OF THE WILD TYPE STRAINB. BACTERIOVORUSHD100 AND ITS HOST-INDEPENDENT DERIVATIVE HI100

Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a β-(1→6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4′ of the nonreducing end α-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.

Evaluation of Major Types of Shiga Toxin 2e-Producing Escherichia coli Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans

ABSTRACT Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx2e genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx2e genes. DNA sequences specific for the int, Q, dam, and S genes of the stx2e bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx1 or stx2 variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

Taxonomic Studies of Predatory Bdellovibrios Based on 16S rRNA Analysis, Ribotyping and the hit Locus and Characterization of Isolates from the Gut of Animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.

Bacteriophage 2851 Is a Prototype Phage for Dissemination of the Shiga Toxin Variant Gene 2c in Escherichia coli O157:H7

ABSTRACT The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx2cE. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx2c-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli.

Nachweis gentechnischer Veränderungen in Mais mittels PCR

Ein PCR-Nachweis für gentechnisch veränderten Mais «Event 176» der Fa. Ciba-Geigy wurde etabliert. Der Mais enthält Gene, die Selbstschutz gegen den Maiszünsler (Delta-Endotoxin-Gen ausBacillus thuringiensis) und Toleranz gegen das Herbizid Basta (Phosphinothricin-Resistenz-Gen ausStreptomyces hygroscopicus) vermitteln. Zudem enthält der Mais ein Ampicillin-Resistenz-Gen. Für die Amplifikation von Bereichen aus allen drei Genen wurden PCR-Primer entworfen. Mit Hilfe dieser Primer und mit «Event 176»-Mais-DNA als Template konnten die entsprechenden Genbereiche in der PCR amplifiziert werden. Die PCR-Produkte wurden sequenziert, um ihre Identität zu bestätigen. Mit Hilfe der Delta-Endotoxin-PCR wurden, auch in Gegenwart von 104fachem Überschuß nicht gentechnisch veränderter Mais-DNA, fünf haploide Genome der «Event 176»-DNA nachgewiesen. A PCR-test for the genetically modified maize «Event 176» of Ciba-Geigy was established. The maize contains genes conferring resistance to the European corn borer (delta-endotoxin gene fromBacillus thuringiensis) and tolerance to the herbicide Basta (phosphinothricin resistance gene fromStreptomyces hygroscopicus). The maize contains also an ampicillin resistance gene. Primers were designed and using «Event 176»-maize-DNA as template internal regions of the three genes were amplified with PCR. The PCR products were sequenced to confirm their identity. Using the deltaendotoxin primers in PCR down to 5 haploid genomes of «Event 176»-DNA could be detected, even in the presence of a 104fold excess of DNA from non-modified maize.

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis (Paris, 11-12th March 2015).

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.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany

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.