Presence of virulence genes, adhesion and invasion of Arcobacter butzleri.

AIMS: The pathogenic potential of Arcobacter butzleri isolates was investigated by detecting the presence of putative virulence genes and analysing the adhesive and invasive capabilities in cell cultures of human cell lines. METHODS AND RESULTS: The presence of ten putative virulence genes in 52 A. butzleri isolates was determined by PCR. The genes ciaB, mviN, pldA, tlyA, cj1349 and cadF were detected in all, whilst irgA (15%), iroE (60%), hecB (44%) and hecA (13%) were detected only in few A. butzleri isolates. On HT-29 cells, four of six isolates adhered to and three of them were able to invade, whilst all six isolates adhered to and invaded Caco-2 cells with higher degrees. The genes ciaB, cadF and cj1349 of all six isolates were sequenced, but no considerable changes of the amino acids in putative functional domains were observed. CONCLUSION: Selected A. butzleri isolates adhere to and invade HT-29 and Caco-2 cells, which emphasize their human pathogenic potential. The efficiency of invasion depends on the eukaryotic cell line and individual bacterial strain used. We could not show any functional correlation between the amino acid sequence of CadF, CiaB or Cj1349 and the adhesive and invasive phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: We have shown that some A. butzleri strains invade various cell lines. This underlines their pathogenic potential and hints at their relevance in human disease.

[Campylobacteriosis of man : livestock as reservoir for Campylobacter species]. / Die Campylobacteriose des Menschen : Nutztiere als Reservoir für Campylobacter-Arten.

Over the last few years, infections with Campylobacter have significantly increased in Europe and Germany and these bacteria have even surpassed Salmonella as the most prevalent bacteria, causing gastroenteritis. Especially contamination during the handling and consumption of meat products seems to be the most important risk factor which plays a prominent role for transmission to man. In addition, contact with pets and other animals, drinking raw or improperly pasteurized milk, and the tenacity of Campylobacter in different environments, especially water, have also to be considered for an adequate risk assessment. Besides gastroenteritis, arthralgia, and Guillain-Barré syndrome are important clinical complications of Campylobacter infections in man. At the same time, it is mostly unclear why the course of infection in man and in reservoir animals differs significantly, especially as only a few classical bacterial virulence factors have been identified so far. For these reasons, the development of efficient prevention strategies is of utmost importance in order to control campylobacteriosis.

Genomic and phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut.

The ability to colonize the chicken gut was determined for 17 Campylobacter jejuni strains of human and bovine origin. The level of colonization varied according to the strain used for experimental infection. Two Campylobacter isolates from patients suffering from gastroenteritis were found in the group of non-colonizing strains, suggesting that other reservoirs as poultry are also important sources of human Campylobacter infections. Bovine Campylobacter isolates can also effective colonize the chicken intestine and may be a source for poultry infection. The invasion ability of the strains as determined in the cell culture model using Caco-2 cells correlates with their colonization capacity in the chicken gut. The genomic and phenotypic stability of the selected strains were evaluated by analysis of their pulsed-field gel electrophoresis (PFGE) patterns, flaA-typing and in vitro determination of motility, adhesion and invasion abilities after colonizing chickens for up to 21 days. Changes were identified in flaA-types of six isolates and three isolates from chicken showed different patterns by PFGE using SmaI or KpnI as restriction enzymes. One isolate showed phenotypic differences after in vivo passage which were seen in enhancement of adherence to eukaryotic cells, decrease of motility and changes in morphology. These phenotypic changes were not associated with the observed genomic instabilities.

PCR detection of virulence-associated genes in Campylobacter jejuni strains with differential ability to invade Caco-2 cells and to colonize the chick gut.

In this study, the presence of 20 putative virulence genes was examined in 11 Campylobacter jejuni isolates with different colonization and invasion abilities as determined in a chick colonization model and on Caco-2 cells, respectively. The majority of the genes were detected in all strains. Among them, there were genes of the flagellar secretion apparatus like flhA, flhB, flgB, flgE2, the flagellin genes flaA and flaB, invasion-associated genes like ciaB and iamA, the cytotoxin genes cdtA-C, the adhesion related gene cadF, and some genes involved in the colonization process (docA, docB). The plasmid gene virB11 could not be detected in any strain. Specific differences between the isolates were observed only in genes cgtB and wlaN involved in lipo-oligosaccharide (LOS) biosynthesis. The gene cgtB was only detectable in three of five strains with strong colonization and invasion abilities. Probably, wlaN can overcome the lack of cgtB in the two cgtB- isolates.

Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin.

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.

Detection of the induction of Salmonella enterotoxin gene expression by contact with epithelial cells with RT-PCR.

All strains of Salmonella enterica investigated were found to carry the Salmonella enterotoxin gene (stn) as determined by PCR and hybridization studies. However, when using CHO-K1 cells for testing the toxicity of the strains, not all strains showed a toxic effect (cell elongation) on the cells or did so only at a low level. The cultivation of Salmonella in contact with epithelial cells (IEC-6) led to an increase in the production of toxin. The stn gene expression was detectable with the help of the RT-PCR after 3 h of incubation. The RNA of the strains was isolated, transcribed into cDNA (with MMLV-reverse transcriptase) and amplified using PCR. The PCR products were separated electrophoretically using a polyacrylamide gel and detected by silver staining.

Inhibition of Mycoplasma bovis cytadherence by a monoclonal antibody and various carbohydrate substances.

The attachment of Mycoplasma bovis to permanent embryonic bovine lung (EBL) cells was studied in order to identify factors participating in the adhesion process. A monoclonal antibody directed against a 26 kDa protein of M. bovis was shown to reduce cytadherence of strains 120 and 454 by 46% and 70%, respectively. In uninhibited assays, strain 120 which exhibits an intense 26 kDa band in electrophoretic protein patterns adhered more strongly to EBL cell monolayers than strain 454 whose corresponding band is considerably weaker. The findings indicate involvement of the 26 kDa protein in M. bovis adherence. In further inhibition experiments, the ability of N-acetyl-neuraminlactose, glycophorin and dextran sulfate to significantly reduce adherence could be demonstrated. This suggests participation of sialic acid residues and probably also sulfatide groups as binding receptors. The data point to a complex adhesion mechanism with similarities to M. pneumoniae.

Correlation between invasion of Caco-2 eukaryotic cells and colonization ability in the chick gut in Campylobacter jejuni.

In an in vitro cell culture model using Caco-2 cells the adhesion and invasion properties of 11 Campylobacter (C.) jejuni isolates of different origin were studied. Additionally, we investigated the colonization ability of the strains in a chick model. Virtually, all C. jejuni showed cell adherence in the in vitro assay, but there were large differences in the invasion frequencies among the Campylobacter isolates. The colonization ability in the chick gut also differed markedly and enabled the formation of three groups: non-colonizing, weak or delayed colonization and strong colonization ability. On this occasion, we found a putative correlation between invasion of Caco-2 cells and colonization in the chick gut. Non-colonizers are not invasive or only have small invasion indexes. Strains which colonize weakly or exhibit delayed colonization have a medium invasion index and strong colonizers show markedly higher values of this parameter. The characterization of the flagellin gene of the used C. jejuni strains resulted in eight flaA types. There was no association between flaA type and invasion or colonization ability in the chick gut.

Cultivation of Salmonella in contact with epithelial cells.

An in vitro cultivation model for Salmonella having contact to epithelial cells was developed, which led to an increase in the production of toxic substances. The toxin assay on CHO-K1 cells was used for the determination of the toxic activities. Salmonella strains cultivated in contact with a monolayer of the intestinal cell line IEC-6 produced considerably more toxin than Salmonella strains cultivated on VERO cells. The toxin formed was heat-labile.

[The influence of viscosity on adhesion and invasion of Salmonella strains in an in vitro model]. / Untersuchungen zum Einfluss der Viskosität auf Adhäsion und Invasion bei Salmonellen im in-vitro-Modell.

The influence of viscosity on the adhesion and invasion behaviour of Salmonella strains of different origin was investigated using an in-vitro-model. These processes seem to be strain-dependent. Compared to the controls, the number of internalized Salmonella was elevated. This increase was a result of the greater number of Salmonella which adhered to Caco-2-cells and was detected only for strains from organs of calves having died from salmonellosis. The average motility of these strains was determined to be 1.6 +/- 0.5 mm/h. A possible association between adhesion ability and motility was discussed.

[Mixed infections of rotaviruses and Campylobacter jejuni in Caco-2 cells]. / Untersuchungen zu Mischinfektionen von Rotaviren und Campylobacter jejuni an Caco-2-Zellen.

A mixed infection with rotavirus and 3 different Campylobacter jejuni strains was analysed in Caco-2 cells, a cell line highly susceptible to these pathogens. The results obtained showed no influence of the virus preinfection on the Campylobacter jejuni adhesion or internalisation in Caco-2 cells. Confocal laser scanning microscopy of mixed infected cells confirmed these results. The data from the present study indicate that specific rather than nonspecific mechanisms are involved in the interaction between rotavirus, campylobacter and host cells.

Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys.

AIMS: Campylobacter isolates from turkeys were genotyped and characterized by their in vitro virulence properties. Relationships between bacterial genotypes and virulence properties were analysed. METHODS AND RESULTS: Isolates were analysed by pulsed-field gel electrophoresis and fla typing. The toxin production was determined on the phenotypic level using a CHO-K1 cell culture model and on the genotypic level using PCR for detection of the cdtA, cdtB and cdtC genes. Although the cdtB gene was detected from 100% of the Campylobacter jejuni and Campylobacter coli isolates we observed three different morphological pictures on the cells. Cytotoxicity was associated with cell distension or cell rounding. All four Camp. coli strains and one Camp. jejuni strain did not produce any cytotoxic changes on the cells. Adhesion, invasion and survival of Campylobacter isolates were determined in a Caco-2 cell culture model. All isolates adhered to and invaded Caco-2 cells, whereas 64.7% of the strains survived for 48 h in the cells. CONCLUSION: Seventeen Campylobacter isolates from turkeys were classified into four groups with regard to their in vitro abilities. Jackknife analysis revealed a strong association between these groups and genotype clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: Typing methods have generally failed to identify strains with specific virulence properties. This study suggests that a relationship between subgroups of Campylobacter with common in vitro virulence characteristics and genotypes exist.

[In vitro studies of adhesion and invasion by Salmonella strains of bovine epithelial cells]. / In-vitro-Untersuchungen zu Adhäsions- und Invasionseigenschaften von Salmonella-Stämmen bovinen Ursprungs.

24 Salmonella strains were divided into three groups according to the circumstances of isolation. The adhesion and invasion abilities of the strains were determined using two permanent cell lines (IEC-6, VERO) and an epithelial cell line from the small intestine of a calf fetus (pKD). Strains of different groups showed no differences in their ability to adhere to the cells tested. Significant differences were found for the invasion ability. Strains isolated from organs of calves suffering from salmonellosis showed a significant higher invasiveness for permanent cell lines and a considerable higher invasiveness for pKD cells than strains of the other groups.

Formation of cytotoxins by enteric Campylobacter in humans and animals.

Campylobacter (C.) jejuni from persons suffering from diarrhoea, from organs of poultry, C. jejuni and C. fetus ssp. fetus from the gastrointestinal tract of calves and adult cattle as well as a number of reference strains were examined for cytotoxin formation in a CHO-K1 cell culture test. During evaluation, three morphologically different pictures were observed. The first cytotoxin caused a formation of strikingly large, rounded or polymorphic and elongated cells which was associated with reduced growth. The progressive morphological changes corresponded to those described for the Cytolethal Distending Toxin (CLDT) and were assigned to it. The second cytotoxin produced a rounding of cells without a change in their size while at the same time, growth was reduced. In analogy to CLDT, this toxin was termed Cytolethal Rounding Toxin (CLRT). A third morphological picture consisted of cell changes characterized by enlarged polymorphic as well as by small rounded cells. These cell changes were considered as being distinct from the above mentioned ones and referred to as CLTD/CLRT effect. In none of the 39 Campylobacter strains isolated from humans and calves with diarrhoea, a noteworthy cytotonic activity could be detected that would indicate the presence of an enterotoxin.

[Effect of acetate on the formation of the phyoeffector tentoxin by Alternaria alternata (Fr.) Keissler]. / Einfluss von Acetat auf die Bildung des Phytoeffektors Tentoxin durch Alternaria alternata (Fr.) Keissler.

The effect of the cosubstrate acetate on the formation of the phytotoxic substance tentoxin by Alternaria alternata (Fr.) Keissler was investigated. Acetate was taken up and metabolized during growth. The added acetate stimulated considerably the biosynthesis of tentoxin depending on its concentration and time of introduction. There was an increased incorporation of (U-14C)-glucose into the toxin molecule in the presence of acetate in the medium. The results indicate an induction of the biosynthesis of tentoxin by the precursor acetate, probably mediated through the accumulation of the glycolytic intermediates pyruvate and phosphoenolpyruvate.

Studies of the presence of the virulence factors, adhesion, invasion, intracellular multiplication and toxin formation in salmonellas of different origin.

Salmonellas of different origin were classified into two groups (11 strains of common serovars which had been isolated from organs of calves having died from salmonellosis and 18 strains belonging to rare serovars which showed uncommon metabolic characteristics and had been isolated from spices and spiced foods). The strains were examined with regard to different virulence parameters. All salmonellas investigated possessed the genetic information on invasion (invA) and toxin formation (stn). They adhered equally well to epithelial cells, could penetrate into these and survive and multiply inside the cells. The formation of toxic substances could be detected in all strains after co-cultivation with epithelial cells in the CHO-K1 test. Significant differences between the groups of strains could be demonstrated only for the invasion of epithelial cell monolayers. Since adhesion, invasion and the ability of intracellular survival and multiplication as well as toxin formation constitute virulence parameters of salmonellas, it must be assumed that also the Salmonella serotypes studied which have been rarely observed epidemiologically constitute a risk for humans.

[Effect of phosphate and inhibitors of protein synthesis on the growth and tentoxin production of Alternaria alternata (Fr.) Keissler]. / Einfluss von Phosphat und Inhibitoren der Proteinsynthese auf das Wachstum und die Tentoxinbildung von Alternaria alternata (Fr.) Keissler.

The synthesis of a phytotoxic substance, tentoxin, by Alternaria alternata (Fr.) Keissler is negatively controlled by inorganic phosphate. We show here that the influence of phosphate on the production of this secondary metabolite is time-dependent. By using inhibitors of protein synthesis (p-fluorphenylalanine, cycloheximide) together with a resuspension technique, attempts were made to find out when the enzymes responsible for tentoxin synthesis are formed.

Detection and characterization of two cytotoxins produced by Campylobacter jejuni strains.

Campylobacter jejuni strains are able to produce at least two different cytotoxins called "cytolethal distending toxin" (CLDT) and "cytolethal rounding toxin" (CLRT). In this study, we investigated the corresponding changes in CHO-K1 cells using the cell counter and analyzer system CASY 1. Determination of the cell volume after toxin treatment of the cells is a useful criterion for differentiation between the cytotoxic activities produced by Campylobacter strains. Incubation of the cells with crude CLDT resulted in a decrease in the cell count combined with a dramatic increase of the mean cell volume in comparison to the control culture. A decrease in the cell count was also seen as a response to CLRT preparations, while this toxin had no effect on the mean cell volume determined. It was shown that only CLDT caused histone-associated DNA fragments in the cytoplasm of CHO-K1 cells indicating an apoptotic pathway of cell death. In addition, the polymerase chain reaction (PCR) was employed to screen Campylobacter strains for the presence of the cdtB gene sequence, which was detectable in all strains investigated.

[Effect of phosphate on the biosynthesis of tentoxin by Alternaria alternata (Fr.) Keissler]. / Einfluss von Phosphat auf die Bildung von Tentoxin durch Alternaria alternata (Fr.) Keissler.

Alternaria alternata (Fr.) Keissler produces a phytotoxic substance tentoxin. The influence of inorganic phosphate on the formation of this secondary metabolite was analyzed. Distinct phases of growth and metabolite formation can be defined. The first phase shows exponential growth, high QO2, protein and nucleic acid values and a rapid uptake of inorganic phosphate from the medium. The second phase shows linear growth and active tentoxin formation takes place. The highest yields of tentoxin are obtained, when inorganic phosphate in the medium was limited. The phosphate level also influences the ATP-pool of the mycelium. The role of ATP as an effector in phosphate mediated control of tentoxin synthesis was discussed.