Stress Response and Virulence Potential Modulating Effect of Peppermint Essential Oil in Campylobacter jejuni.

Campylobacter jejuni is one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobacter effect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY 0.84x; flhB 0.79x; flgE 0.205x; cadF 0.08x; wlaB 0.89x; porA 0.25x; cbf2 4.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curved C. jejuni cells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEO C. jejuni dominantly exerts a general stress response that elevates the expression of general stress genes like dnaK, groEL, groES (10.41x, 3.63x, and 4.77x). The most important genes dps, sodB, and katA involved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

Curli expression of enterotoxigenic Escherichia coli.

One hundred and four enterotoxin producing Escherichia coli strains of wide geographical origin were tested for the expression of curli fimbriae by transmission electronmicroscopy and by ELISA using curli-specific antibodies, as well as for the presence of curli-specific gene sequences by PCR. All isolates, irrespective of the production of the fimbriae, carried sequences specific for the structure (csgA) and for one of the regulator genes (crl) of curli expression, respectively. Curli fimbriae were detected in 56 strains (53.8 %). Thirty-six strains expressed curli only when growing at 30 degrees C, 4 isolates were weakly curliated at 37 degrees C only, while on 16 strains curli was observed at both temperatures. On isolates carrying curli at both temperatures the expression of the fimbria was significantly stronger at 30 degrees C than at 37 degrees C. Curli proficiency significantly, but not completely, correlated with the binding of the Congo Red dye. The expression of curli did not confer epithelial cell invasiveness to ETEC strains but, once expressed at 30 degrees C, it facilitated the adherence of the bacteria to plastic surfaces. Curli present in more than half of the ETEC strains and expressed preferentially at low temperatures could be a factor facilitating the environmental survival of this food- and water-borne pathogen.

Progesterone as an immunologic blocking factor in human pregnancy serum.

The influence of estriol and progesterone on lymphocyte reactivity was examined by testing the cytotoxic effect on human embryonic fibroblast cells of non-pregnant women's lymphocytes incubated with different concentrations of estriol and progesterone and with complete and progesterone-depleted third trimester pregnancy sera. Progesterone, but not estriol, had a significant inhibitory effect on lymphocyte cytotoxicity at concentrations comparable to those present in pregnancy serum. 95% depletion of progesterone from pregnancy sera caused an 80% loss of inhibitory activity on lymphocyte cytotoxicity. These data suggest that the blood level of progesterone in pregnancy is sufficient to depress lymphocyte reactivity and that progesterone is responsible for the greater part of the serum inhibitory activity in at least the later stages of pregnancy.

Virulence factors of uropathogenic Escherichia coli.

Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.

Characterization of Candida albicans colony morphological mutants and their hybrids by means of RAPD-PCR, isoenzyme analysis and pathogenicity analysis.

A wild-type strain of Candida albicans (S1, ATCC 10261) was used to obtain stable auxotrophic colony morphological mutants (mutant M5 producing only true hyphae and mutant M2 containing 90 % blastospores and 10 % pseudohyphae) by induced mutagenesis. A hybrid was produced by somatic hybridization between these 2 mutants. Out of the isolated 10 clones, 2 stable hybrid clones were chosen and characterized: clone VI. 1M produced rough colonies containing a new, extended cell type (never observed in natural isolates), exhibited unipolar budding, did not form a germ tube, and possessed 12 chromosomal bands. All other features (antifungal and stress sensitivity, adhesion ability, pathogenicity, and isoenzyme and RAPD patterns) were similar to those of mycelial mutant M5. In contrast, the characteristics of clone VI.9S were similar to those of morphological mutant M2.

Spontaneous antibiotic resistance mutation associated pleiotropic changes in Escherichia coli O157:H7.

Besides the well-known O157:H7 clone causing enterohaemorrhagic colitis and haemolytic uraemic syndrome in Europe, Japan and North America, the number of Escherichia coli isolates with non-motile (NM) phenotype has considerably increased. We supposed that spontaneous antibiotic resistance mutation could cause this phenotypic change. To model our hypothesis we isolated rifampicin--(Rif) and ampicillin--(Amp) resistant mutants from E. coli O157:H7 prototype strains 7785 and EDL933. Among Rifr mutants we could isolate strains with no or reduced motility, while the Ampr mutants became hypermotile. The biochemical profile of the mutants had not changed but phage sensitivity and generation time of the mutants were altered. Among the representative strains we did not find polymorphism with Southern blot analysis and no polymorphism was found in the fliC gene of the mutants. The described characteristics have proven to be stable. In a mice virulence assay by intravenous infections the virulence of the derivatives was also found to be changed. In summary, we found that the antibiotic-resistant phenotype in E. coli O157:H7 was coexpressed with several other phenotypic changes including motility and virulence. It can be assumed that expression of the involved phenotypes may be under the influence of a common regulatory cascade. Further work is needed to identify the components and mechanism of this regulatory system.

Genetic diversity in Proteus penneri.

DNA of thirteen Proteus penneri strains derived from four European countries (nine strains from Germany, two strains from United Kingdom, one strain from Turkey, one strain from Hungary) was examined by random amplified polymorphic DNA-PCR (RAPD-PCR) method. RAPD with primer AACGCGCAAC gave different patterns, which suggests a DNA sequence microdiversity within this species. The method provides a fast, economical and reproducible means for typing P. penneri.

Polarization optical analysis of the surface structures of various fungi.

Vicinal hydroxyl groups of the sugar compounds sialic acid and 9-O-acyl sialic acid can be visualised for polarization optical analysis on the surface of different fungi using several topo-optical reactions. We investigated the presence of these molecules in cultures of Cryptococcus neoformans (heterogeneous form), Saccharomyces cerevisiae, Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis by topo-optical reactions. Additionally, we examined brain and stomach tissues of patients with infections by C. neoformans and C. albicans, respectively. The results suggest a highly fashioned orientation of the sugar chains on the fungal surface. Terminal sialic- and O-acyl sialic acid residues are permanently present and orientated in a highly specific way in the cell wall of fungi. Based on the polarization optical analysis after the ABT-r (anisotropic PAS-r), the linear oriented hydroxyl groups of the sugar molecules are localized either perpendicular or parallel to the surface coat, depending on the species. According to the orientation of the vicinal hydroxyl groups, the oligosaccharide chains are orientated vertically. The capsule of the heterogeneous form of C. neoformans presented an especially strong metachromatic reaction and anisotropy. It is especially remarkable that the sterical orientation of sugar chains, and the terminal sialic acid and 9-O-acyl sialic acid molecules, was opposite in the inner and outer layer of the capsule.

Topo-optical investigations on the surface of bacterial cells during the phagocytosis of Klebsiella pneumoniae in mouse.

Polarisation optical methods provide the means to perform sub-microscopic investigations on structures containing spatially highly ordered molecules, for example the cell envelope of prokaryotic cells. Such structures can evoke birefringence, which can be enhanced or modified by different dyes or reagents, thus providing the possibility of a more specific investigation of the composition and structure of bacterial surface compounds. Klebsiella pneumoniae synthesises sterically different carbohydrate-rich structures, including those of the outermost capsular polysaccharide, the polysaccharide somatic antigen of the lipopolysaccharide molecule and the peptidoglycan layer of the cell wall. In the study reported here, the nature and intensity of topo-optical activity of these structures was analysed using the aldehyde-bisulphite-toluidine blue reaction, sialic acid topo-optical reactions and chlorpromazine-eosin charge transfer reactions. Furthermore, a mouse intraperitoneal model was used to analyse alterations in topo-optical characteristics of bacteria during phagocytosis. Both encapsulated and non-encapsulated bacterial cells changed their original pattern and orientation of birefringence after being phagocytosed.

The stability of enterotoxin production in Yersinia enterocolitica and the methanol solubility of heat-stable enterotoxin.

Twenty five strains of Yersinia enterocolitica serogroup O3, were isolated from human enteritis and studied for heat-stable enterotoxin production. Enterotoxin production was found even in the crude supernatant fluid of cultures that had been stored in stock agar for a year. According to the suckling mice and rabbit gut loop tests, after 1 to 5 years storage the filtrate showed heat-stable enterotoxin activity only in a purified and concentrated form. Following more than 5 years storage positive results could be obtained only in rabbit gut loop test. After 9 years the freeze dried strains still showed a full capacity of heat-stable enterotoxin production. Studies with concentrated substances showed that even after more than 9 years, there was no spontaneous loss of heat-stable enterotoxin production, only quantitative changes occurred. The methanol solubility of the heat-stable enterotoxin of Y. enterocolitica is--as distinct from the heat-stable enterotoxin of Escherichia coli--homogeneous and only the methanol soluble fractions showed any activity. The activity of methanol soluble enterotoxin from several years old subcultures could be demonstrated in an isolated rabbit gut loop model even when it failed to show any activity in suckling mice.

Molecular order of carbohydrate components in cell walls of bacteria, fungi and algae according to the topo-optical reaction of the vicinal OH groups.

Selective topo-optical staining of vicinal OH groups with aldehyde-bisulphite-toluidine blue (ABT) has been used for studying the molecular structural order of polysaccharide components in microbial cell walls and capsules. (i) By intensive metachromatic staining in the light microscope, the method demonstrates carbohydrates, containing free vicinal OH or acylaminohydroxyl groups. (ii) The birefringence induced by oriented toluidine blue binding of the ABT reaction, provides information about the linear order of the reacting carbohydrate chains, a possibility not offered by other ultrastructural methods. Furthermore the different optical character of birefringence induced by ABT is indicative of the presence of (a) tangentially oriented polysaccharide chains in the cell wall of the molds and bacterial capsules; or (b) radially oriented OH groups suggesting helical glycan chains of peptidoglucomannane and mucopeptide in the yeasts and bacterial cell walls.

Proteus virulence: involvement of the pore forming alpha-hemolysin (a short review).

The genus Proteus belongs to the tribe of Proteae in the family of Enterobacteriaceae, and consists of five species: P. mirabilis, P. vulgaris, P. morganii, P. penneri and P. myxofaciens. They are distinguished from the rest of Enterobacteriaceae by their ability to deaminate phenylalanine and tryptophane. They hydrolyze urea and gelatin and fail to ferment lactose, mannose, dulcitol and malonate; and do not form lysine and arginine decarboxylase or beta-galactosidase [1]. Colonies produce distinct "burned chocolate" odor and frequently show the characteristics of swarming motility on solid media. P. mirabilis, P. vulgaris and P. morganii are widely recognized human pathogens. They have been isolated from urinary tract infections, wounds, ear, and nosocomial bacteremic infections, often in immuncompromised patients [2-6]. P. myxofaciens has no clinical interest to this time. P. penneri as species nova was nominated by the recommendation of Hickman and co-workers [7]. Formerly it was recognized as P. vulgaris biogroup 1 or indole negative P. vulgaris [8, 9]. Although it has been less commonly isolated from clinical samples than the other three human pathogenic Proteus species, it has nevertheless been connected with infections of the urinary tract, wounds and has been isolated from the feces of both healthy and diarrheic individuals [10-12]. Potential virulence factors responsible for virulence of Proteae are: IgA protease, urease, type3 fimbriae associated with MR/K haemagglutinins of at least two antigenic types, endotoxin, swarming motility and HlyA and/or HpmA type hemolysins [for review see ref. 13]. In the followings we give a survey of accumulated concepts about the position and characteristics of HlyA type alpha-hemolysins both in general and with emphasis on virulence functions in the tribe of Proteae.

New haemagglutinating fimbriae on Escherichia coli strains isolated from urine.

The haemagglutination patterns of 255 urinary Escherichia coli isolates were examined with human (A, Rh+), bovine, chicken and guinea pig erythrocytes in the presence and absence of D-mannose. The strains were divided into four groups according to their haemagglutination properties. About 40% of the isolated agglutinated human red blood cells in the presence of D-mannose. The haemagglutinin of one of these. E. coli O18a, c: K- strain No. 119 was stable, temperature sensitive, did not develop at 18 degrees C and could be isolated by the methods used for the production of fimbriae. Electron microscopy showed fimbriae on the surface of Strain No. 119. An absorbed serum prepared from a derivative cured of haemagglutinating property (No. 119/1) agglutinated all the strains haemagglutinating human erythrocytes in the presence of mannose, but none of those having other haemagglutination patterns. Serologically, the antigen of No. 119 is independent of the K88, K99, "987" and CF I factors and shows some relationship to CF II.

High-affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida.

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a 125I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. 125I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.