The ability of some Yersinia enterocolitica strains to invade HeLa cells

Many types of Yersinia enterocolitica have been isolated from animal, environmental, food, and human sources but their public health significance remains uncertain. Seventy two strains of Y. enterocolitica were tested for their abilities to invade HeLa cells. The typical clinical strains invade HeLa cells like the other species of invasive pathogens. This characteristic remains even in old stock cultures and can be temperature-sensitive like the motility characteristic. With the use of electron micrographs it was demonstrated that the bacteria were truly intracellular and not merely adhering to the HeLa cell membrane. The esculin-and salicin-positive typical clinical strains did not invade HeLa cells. None of 34 food and water isolates were invasive by this test. The negative Y. enterocolitica strains did not adhere to the cells and cause ambiguous results. The HeLa cell test is simple, inexpensive, rapid, and should prove useful marker for screening the Y. enterocolitica isolates.     

Campylobacter jejuni strains compete for colonization in broiler chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal(r) strain) and one streptomycin-resistant strain (the 02-833L Str(r) strain). In vitro binding assays revealed that the C. jejuni F38011 Nal(r) and 02-833L Str(r) strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str(r) strain relative to that of the C. jejuni F38011 Nal(r) strain competitively inhibited the binding of the C. jejuni F38011 Nal(r) strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str(r) strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal(r) strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Evaluation of the usefulness of selected virulence markers for identification of virulent strains of Yersinia enterocolitica strains. III. Chromosome markers of virulence

It is well known that virulent strains of Y. enterocolitica bear the virulence-associated plasmid pYV. Moreover some authors consider that the pathogenic strains of these bacteria have chromosome encoded phenotypic and genotypic features such as: genes ail and yst which could be used as virulence markers. The virulent strains of Y. enterocolitica do not produce pyrasinamidase and are not able to ferment salicin and cannot hydrolyse esculin. In addition these strains produce thermostable enterotoxin called YST and protein Ail (attachment invasion locus). In contrast to phenotypic virulence makers the biological function of proteins Ail, YST and nucleotide sequence of genes ail and yst is well described. In the presented study one hundred thirty virulence plasmid bearing Y. enterocolitica strains belonging to serogroup O3 were examined for the presence of genes ail, yst, and were tested for their inability to pyrasinamidase production, salicin fermentation and esculin hydrolysis. In addition forty pYV plasmid-cured isogenic strains were included in to the study. Genes ail and yst were detected by polymerase chain reaction (PCR). The obtained results indicate that all tested 130 pYV+ Y. enterocolitica strains as well as 40 plasmid-cured isogenic strains have carried ail and yst genes. All tested strains did not produce pyrasinamidase, hydrolyse esculin and ferment salicin. This generally was in agreement with the observations done by other authors and suggest that the chromosomal virulence markers, especially well described genes ail and yst, could be useful for excluding the potential virulence of Y. enterocolitica strains, which had lost pYV plasmid and have no ail or yst genes. Therefore, in clinical studies, Y. enterocolitica strains isolated directly from patients should be primarily tested for the presence of the virulence plasmid and secondarily, the negative ones could be examined for the presence of the chromosomal virulence markers.     

The polymerase chain reaction: an epidemiological tool to differentiate between two clusters of pathogenic Yersinia enterocolitica strains

A primer set designed to amplify the enterotoxin (yst) gene of pathogenic Yersinia enterocolitica strains generated two different electrophoretic profiles of the target sequence when a collection of strains of worldwide origin was screened. Serovars O:1,3; O:2a,3; O:3; O:5,27 and O:9, known as European strains, produced a 200-bp fragment that matched the size of the target sequence. However, serovars O:4,32; O:8; O:13a,13b; O:20 and O:21, known as American strains, generated two fragments of 1.4 and 1.6 kb. The amplified products of one American strain were sequenced and the presence of the yst gene was confirmed in both fragments. Thus, the potential of the polymerase chain reaction to be used as an epidemiological tool in differentiation between the two clusters of pathogenic strains of Y. enterocolitica could be demonstrated.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

Difference in the extracellular products of two strains of Aeromonas hydrophila virulent and weakly virulent for fish

In this study we first compared the toxigenic profile of Aeromonas hydrophila strains virulent and weakly virulent for rainbow trout. The separation of the toxic factor for fish is also described. Both strains produced hemolytic, enterotoxic, and dermonecrotic activities; the weakly virulent strain produced 20 times more hemolysin. Only the cell-free supernatant of the virulent strain produced a toxic factor for fish. After passage on Sephacryl S-200, the toxic factor for fish was separated from the hemolysin. This toxic factor is heat labile.     

yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq)

Yersinia enterocolitica produces heat-stable enterotoxin (Y-ST) as one of its virulence factors. The yst gene, however, frequently and spontaneously becomes inactive (silent) during storage, which is accompanied by concurrent changes in some biological properties such as colony morphology, growth rate, carbon fermentation and ornithine decarboxylase activity. Northern blot analysis revealed that the level of mRNA for yst was repressed. To investigate the regulatory region, we transformed a yst -silent strain with a chromosomal gene library of Y-ST producing an isogenic counterpart. Out of 3604 clones, one clone resumed the Y-ST production and concurrently other biological properties. An open reading frame in this clone was designated as yrp , y ersinia r egulator for p leiotropic phenotype. Deduced from the nucleotide sequence, Yrp was a small protein of 101 amino acids with no similarity with any regulatory factor described, but showed high homology with an Escherichia coli host factor 1 required for Q_β-replicase, and with an Azorhizobium caulinodans NrfA required for the expression of nifA . The yrp gene mutation caused decreased negative supercoiling of plasmids, as did hfq . The yrp gene could similarly complement Y-ST production in two other silent strains of Y. enterocolitica . In all three silent strains examined, we found various mutations in the yrp region.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.     

Adaptation to abundant low quality food improves the ability to compete for limited rich food in Drosophila melanogaster

The rate of food consumption is a major factor affecting success in scramble competition for a limited amount of easy-to-find food. Accordingly, several studies report positive genetic correlations between larval competitive ability and feeding rate in Drosophila; both become enhanced in populations evolving under larval crowding. Here, we report the experimental evolution of enhanced competitive ability in populations of D. melanogaster previously maintained for 84 generations at low density on an extremely poor larval food. In contrast to previous studies, greater competitive ability was not associated with the evolution of higher feeding rate; if anything, the correlation between the two traits across lines tended to be negative. Thus, enhanced competitive ability may be favored by nutritional stress even when competition is not intense, and competitive ability may be decoupled from the rate of food consumption.     

Genetic variations and in vitro root-colonizing ability for an ericaceous host in Sarcoleotia globosa (Geoglossomycetes)

We discovered that Sarcoleotia globosa (Geoglossomycetes) fruited on the soil of ornamental Erica pot cultures, and its ascospores can germinate on plain agar. These findings prompted us to collect isolates from horticultural and natural environments in Japan and analyze their phylogeny and root colonizing ability. Pure cultures were successfully obtained from ascospores and surface-sterilized ericaceous roots. Phylogenetic analysis based on rRNA internal transcribed spacer sequences revealed that Japanese samples were separated into three strongly supported clades. Individual clade consisted of samples derived from (1) Erica pot cultures, (2) Rhododendron planted in a garden or Vaccinium pot culture, and (3) natural habitats in Hokkaido. Colony characteristics and in vitro root-colonizing morphology observed may correspond to these phylogenetic variations. Irrespective of the clades, all tested strains formed hyphal coils in vital rhizodermal cells of V. virgatum seedlings, which resembled those of ericoid mycorrhizae. Our results represent novel findings that can be the first step in unraveling the currently unknown ecology of geoglossoid fungi.     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

Epizooic ciliates (Vorticella sp.) compete for food with their host Daphnia longispina in a small polyhumic lake

Summary Clearance rates of epizooic ciliates (Vorticella sp.) were measured together with their host, a planktonic cladoceran Daphnia longispina by using fluorescent latex beads as tracers of food. Vorticellans and their host graze on food of same size range (nanoplanktonic algae and bacteria). Individual clearance rates of Vorticella averaged 6.9 and 7.0 l ind^-1 h^-1 and those of Daphnia 463 and 708 l ind^-1 h^-1 for beads with diameter 2.00 and 3.92 m. On the average, epizooic vorticellans together on the carapace of Daphnia cleared particles with rates representing 25–33% of that the host cleared, the maximum rates being 50–80%. In a steeply stratified polyhumic lake vorticellans take advantage of following Daphnia to food patches and they can severely compete for food with their host.     

Comparative study of a DNA hybridization method and two isolation procedures for detection of Yersinia enterocolitica O:3 in naturally contaminated pork products

We compared a DNA-DNA hybridization assay, using a synthetically produced oligonucleotide probe, and two conventional isolation procedures (methods A and B) with regard to their relative efficiency in detecting Yersinia enterocolitica O:3 in naturally contaminated pork products. Method A was as described by Wauters et al. (Appl. Environ. Microbiol. 54:851-854, 1988). Method B has been recommended by the Nordic Committee on Food Analysis (method no. 117, 1987). The genetic probe was used in a colony hybridization assay to detect virulent yersiniae at each of the isolation steps with composed methods A and B. A total of 50 samples of raw pork products obtained from 13 meat-processing factories in Norway were examined. Y. enterocolitica serogroup O:3, biovar 4, was isolated from altogether 9 (18.0%) of the samples by using the two isolation procedures. In contrast, colony hybridization using the genetic probe indicated that 30 (60.0%) of the samples contained virulent yersiniae. All samples which were positive on cultivation were also positive by hybridization. The results indicate that the occurrence of pathogenic Y. enterocolitica in Norwegian pork products is substantially higher than previously demonstrated and, therefore, reinforce our suggestion that pork products represent an important potential source of human infection in Norway. The results also indicate that the use of conventional isolation procedures may lead to considerable underestimation of pathogenic Y. enterocolitica in pork products.     

Intra-individual coexistence of a Wolbachia strain required for host oogenesis with two strains inducing cytoplasmic incompatibility in the wasp Asobara tabida

Cytoplasmically inherited symbiotic Wolbachia bacteria are known to induce a diversity of phenotypes on their numerous arthropod hosts including cytoplasmic incompatibility, male-killing, thelytokous parthenogenesis, and feminization. In the wasp Asobara tabida (Braconidae), in which all individuals harbor three genotypic Wolbachia strains (wAtab1, wAtab2 and wAtab3), the presence of Wolbachia is required for insect oogenesis. To elucidate the phenotype of each Wolbachia strain on host reproduction, especially on oogenesis, we established lines of A. tabida harboring different combinations of these three bacterial strains. We found that wAtab3 is essential for wasp oogenesis, whereas the two other strains, wAtabl and wAtab2, seem incapable to act on this function. Furthermore, interline crosses showed that strains wAtab1 and wAtab2 induce partial (about 78%) cytoplasmic incompatibility of the female mortality type. These results support the idea that bacterial genotype is a major factor determining the phenotype induced by Wolbachia on A. tabida hosts. We discuss the implications of these findings for current hypotheses regarding the evolutionary mechanisms by which females of A. tabida have become dependent on Wolbachia for oogenesis.     

Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations

Growth rates determined by linear regression analysis revealed that Saccharomyces cerevisiae consistently grew more rapidly than Brettanomyces yeasts under a wide array of batch fermentative conditions, including acetic acid stress, in normal gravity (ca. 20°Plato) mashes made from ground corn. Brettanomyces yeasts only grew more rapidly than S. cerevisiae when acetic acid concentrations were elevated to industrially irrelevant levels (>0.45%, w/v). Furthermore, the three Brettanomyces isolates used in this study failed to produce significant quantities of acetic acid under pure culture fermentative conditions. In fact, the small amounts of acetic acid which accumulated in pure culture fermentations of whole corn mash were below the concentration required to inhibit the growth and metabolism of S. cerevisiae. Acetic acid concentrations in pure culture Brettanomyces fermentations exceeded 0.05% (w/v) only in media containing low levels of glucose (<4%, w/v) or when aeration rates were elevated to at least 0.03 vol. air vol.^–1 mash min^–1. Consequently, it was concluded that Brettanomyces yeasts would not be capable of competing with S. cerevisiae in industrial batch fermentations of whole corn mash based solely on growth rates, nor would they be capable of producing inhibitory concentrations of acetic acid in such fermentations.     

Comparative genomic analysis of two Burkholderia glumae strains from different geographic origins reveals a high degree of plasticity in genome structure associated with genomic islands

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, a growing disease problem in global rice production. To better understand its genome-scale characteristics, the genome of the highly virulent B. glumae strain 336gr-1 isolated from Louisiana, USA was sequenced using the Illumina Genome Analyser II system. De novo assembled 336gr-1 contigs were aligned and compared with the previously sequenced genome of B. glumae strain BGR1, which was isolated from an infected rice plant in South Korea. Comparative analysis of the whole genomes of B. glumae 336gr-1 and B. glumae BGR1 revealed numerous unique genomic regions present only in one of the two strains. These unique regions contained accessory genes including mobile elements and phage-related genes, and some of the unique regions in B. glumae BGR1 corresponded to predicted genomic islands. In contrast, little variation was observed in known and potential virulence genes between the two genomes. The considerable amount of plasticity largely based on accessory genes and genome islands observed from the comparison of the genomes of these two strains of B. glumae may explain the versatility of this bacterial species in various environmental conditions and geographic locations.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.