Responses of Mytilus galloprovincialis hemocytes to environmental strains of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus.

Marine bivalves are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. In the hemocytes of the mussel Mytilus spp. immune responses to different vibrios have been largely characterized. However, little information is available on the hemocyte responses to human pathogenic vibrios commonly detected in coastal waters and bivalve tissues that are involved in seafood-borne diseases. In this work, functional parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vitro challenge with different vibrios isolated from environmental samples of the Adriatic sea (Italy): V. parahaemolyticus Conero, V. alginolyticus 1513 and V. vulnificus 509. V. parahaemolyticus ATCC 43996 was used for comparison. At the 50:1 bacteria hemocyte ratio, only V. parahaemolyticus strains induced significant lysosomal membrane destabilisation. Stimulation of extracellular lysozyme release, total ROS, O2- and NO production were observed, although to different extents and with distinct time courses for different vibrios, V. vulnificus 509 in particular. Further comparisons between V. parahaemolyticus Conero and V. vulnificus 509 showed that only the latter induced dysregulation of the phosphorylation state of p38 MAP Kinase and apoptotic processes. The results indicate that mussel hemocytes can mount an efficient immune response towards V. parahaemolyticus and V. alginolyticus strains, whereas V. vulnificus 509 may affect the hemocyte function. This is the first report on immune responses of mussels to local environmental isolates of human pathogenic vibrios. These data reinforce the hypothesis that Mytilus hemocytes show specific responses to different vibrio species and strains.

Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

Live and heat-killed Lactobacillus spp. interfere with Streptococcus mutans and Streptococcus oralis during biofilm development on titanium surface.

OBJECTIVES: This research investigates the ability of live and heat-killed (HK) Lactic Acid Bacteria (LAB) to interfere with Streptococcus mutans ATCC 25175 and Streptococcus oralis ATCC 9811 during biofilm formation. DESIGN: Eight Lactobacillus spp. and two oral colonizers, pathogenic Streptococcus mutans and resident Streptococcus oralis, were characterized for their aggregation abilities, cell surface properties and biofilm formation ability on titanium surface. Then, the interference activity of selected live and HK Lactobacillus spp. during S. mutans and S. oralis biofilm development were performed. The cell-free culture supernatants (CFCS) anti-biofilm activity was also determined. RESULTS: LAB possess good abilities of auto-aggregation (from 14.19 to 28.97%) and of co-aggregation with S. oralis. The cell-surfaces characteristics were most pronounced in S. mutans and S. oralis, while the highest affinities to xylene and chloroform were observed in Lactobacillus rhamnosus ATCC 53103 (56.37%) and Lactobacillus paracasei B21060 (43.83%). S. mutans and S. oralis developed a biofilm on titanium surface, while LAB showed a limited or no ability to create biofilm. Live and HK L. rhamnosus ATCC 53103 and L. paracasei B21060 inhibited streptococci biofilm formation by competition and displacement mechanisms with no substantial differences. The CFCSs of both LAB strains, particularly the undiluted one of L. paracasei B21060, decreased S. mutans and S. oralis biofilm formation. CONCLUSIONS: This study evidenced the association of LAB aggregation abilities and cell-surface properties with the LAB-mediated inhibition of S. mutans and S. oralis biofilm formation. Lactobacilli showed different mechanisms of action and peculiar strain-specific characteristics, maintained also in the heat-killed LAB.

Flow cytometric evaluation of Vibrio parahaemolyticus adhesion inhibition to human epithelial cells.

BACKGROUND: The present report demonstrates the usefulness of flow cytometry for a quantitative assessment of adhesion inhibition of a Vibrio parahaemolyticus strain to human epithelial cells to acquire more information about the nature of its adhesins. METHODS: The inhibition of the adhesive process to Hep-2 was assayed by adding several monosaccharides to infected cells monolayers. The quantification of the adherent bacteria, labeled with a specific primary antibody plus a secondary fluorescein isothiocyanate-conjugated antibody, was performed by flow cytometry in comparison with light microscopy. The adherence was quantified in terms of the proportion of cells with adherent V. parahaemolyticus and as the mean of adherent bacteria per cell. RESULTS: The adhesion showed a percentage of 98% with a mean fluorescence channel of 331 comparable to those obtained by light microscopy. The addition of monosaccharides resulted in a D-mannose and N-acetyl-galactosamine sensitive adherence. Even if this environmental strain also showed a mannose-sensitive cell-associated hemoagglutination that could mediate V. parahaemolyticus adherence, our results suggest that different sites for an irreversible adherence to host cell are involved. CONCLUSIONS: Flow cytometry in combination with indirect immunofluorescence is an effective tool to investigate the adhesive process of bacteria to epithelial cells because it is more sensitive and reproducible than visual counting of bacteria performed in light microscopy.

Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products.

In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp. Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V. alginolyticus (81.48%), V. parahaemolyticus (14.8%) and V. cholerae non 0:1 (3.7%). A greater isolation frequency (18.9%) was found for mussels. Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp. isolated. These results confirm that the presence of Vibrio spp. in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable.

Retention of virulence in viable but non-culturable halophilic Vibrio spp.

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

Studies on the development and stability of resistance of Helicobacter pylori to metronidazole and clarithromycin.

Thirty-one strains of Helicobacter pylori, susceptible to metronidazole and clarithromycin, were isolated from 31 biopsy samples from patients with various gastric pathologies. We investigated the possibility of selecting resistant strains and the stability of resistance by exposing isolates to increasing subinhibitory concentrations of metronidazole and clarithromycin using a serial passages technique. Resistance to metronidazole was obtained in 100% of the isolates, while 32.2% displayed resistance to clarithromycin. Resistance to metronidazole was stable in 93.5% (29 of 31) of the isolates, whereas 100% (10 of 10) of the strains were stably resistant to clarithromycin. The stability of the resistance that occurred after three passages on medium containing the two drugs was statistically significant (P <0.001). Thus, the technique of serial passages in vitro could be useful as a first screening in selecting drugs for use in therapeutic protocols for clinical trials.

Comparison of colloidal bismuth subcitrate and metronidazole, both in combination with an H2-antagonist as therapy for Helicobacter pylori.

In this random study, the efficacy of either colloidal bismuth subcitrate (CBS) or metronidazole in combination with an H2-antagonist in the treatment of various gastric pathologies was evaluated, along with the trends in antibody levels. Among the 40 Helicobacter pylori-positive patients with various gastroduodenal pathologies who underwent chemotherapy, 27 were treated with CBS and 13 with metronidazole. H. pylori was eradicated in 48.14% of the patients treated with CBS and 53.8% of those treated with metronidazole. After therapy, no statistically significant or slight decrease in the serum levels of antibodies was found.

Morphological changes and outer membrane protein patterns in Helicobacter pylori during conversion from bacillary to coccoid form.

Conversion from bacillary to fully coccoid form via an intermediate U-and V-shaped form has been described in prolonged cultures of H. pylori. This morphological transformation may be the expression of transitory adaptation to a particular environment and may play an important role in antibiotic resistance and the difficulty to eradicate the pathogen. The aim of this study was to evaluate morphological and outer membrane protein changes in H. pylori during ageing-induced conversion to coccoid morphology. We used two H. pylori strains (the reference NCTC 11639 and a fresh clinical isolate) cultivated in microaerophilic environment at 37 degrees C, monitoring their morphological and biochemical evolutions for 11 days. Microscopic examination revealed the passage from spiral to U- and V-shaped form after 5-8 days of incubation, the conversion to coccoid form and the entry into viable but non-culturable state (VBNC) between days 9 and 11. Protein pattern difference appeared at 97.4 to 45 and 30 kDa molecular weight. Biochemical tests demonstrated not only a modification of outer membrane protein profiles, but also an intra-specific variability by comparison between the two analysed strains. Our findings suggest that structural and outer membrane changes associated with coccoid transformation represent a typical response in H. pylori and may constitute a survival strategy in adverse environmental conditions.

Synthesis of new C-6 alkyliden penicillin derivatives as beta-lactamase inhibitors.

New penicillin, penicillin sulfone and sulfoxide derivatives bearing a C-6-alkyliden substituent were prepared. Their chemical synthesis, in vitro antibacterial activity and inhibition properties against two selected enzymes representing Class A and C beta-lactamases are reported. Compounds 3a-c, 7a-c were able to inhibit either TEM-1 (a Class A enzyme, from Escherichia coli) or P-99 (a Class C enzyme, from E. cloacae), or both enzymes, when tested in competition experiments using nitrocefin as the reporter substrate. However, when tested in combination with amoxicillin, the same compounds did not show synergistic effects against E. coli and E. cloacae strains producing TEM-1 and P99 enzymes, respectively. This finding is most likely related to poor penetration through the bacterial cell wall, as shown by using a more permeable isogenic E. coli strain. Interestingly, a synergistic effect against a strain of S. aureus which produces PC1-enzyme (a Class A beta-lactamase) was observed for compound 3a when used in combination with amoxicillin.

Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems.

AIM: To evaluate the microbial contamination of 91 cosmetics (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use (intact, in-use, ending product) and the protection efficacy of the preservative systems most frequently used in the analysed cosmetic formulations. METHODS AND RESULTS: Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10.6% of tensiolytes (13.5% bath foam, 6.7% shampoo, 10% liquid soaps) were contaminated by Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The efficacy of the preservative systems of two cosmetic products, tested against standard micro-organisms (Staphylococcus aureus ATCC 4338 and Pseudomonas aeruginosa ATCC 9027) and two isolates from cosmetics in this study (S. epidermidis and P. putida), satisfied the Cosmetics, Toiletries, and Fragrance Association and Official Italian Pharmacopeia criteria, while only one tested cosmetic respected the Rapid Challenge Test criterion. CONCLUSIONS: Contaminated cosmetic products are relatively uncommon, but some products, unable to suppress the growth of several micro-organisms, represent a potential health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: The challenge test may be performed not only during the preparation of the preservative system in the intact cosmetics, but also be used to evaluate the protection efficacy during their use.

'In vivo' studies on the pathophysiological mechanism of Vibrio parahaemolyticus TDH(+)-induced secretion.

The thermostable direct haemolysin (TDH) is considered to be the major virulence factors of Vibrio parahaemolyticus; however, poor information is available about its mechanism of action. In our study we examined the capacity of two V. parahaemolyticus TDH-producers (strains 2067 and 3305) to induce fluid secretion in rat ileal loop and to reveal the role of calcium ions (Ca(2+)), calmodulin (CaM), and protein kinase C (PKC) in V. parahaemolyticus TDH(+)-induced fluid secretion. The results show that V. parahaemolyticus TDH(+) strains were able to induce secretion in small intestine; on the contrary, this ability was not evidenced in the V. parahaemolyticus TDH(-) strain used as negative control. The data suggest an enterotoxic activity of haemolysin. Calcium ionophore A23187 and 1-verapamil (calcium channel blocker), when injected alone, induced fluid accumulation in the control loops. A further increase in fluid accumulation (P<0.001) was noted when calcium ionophore was injected along with bacterial suspension of both TDH(+) strains and a significant decrease (P<0.001) in experimental loops when 1-verapamil was inoculated along with bacterial suspension. The other modulating agents increased fluid accumulation in both control and experimental loops, without significant differences with respect to the positive control. Our findings suggest that Ca(2+) appears to be an important messenger involved in the stimulation of intestinal secretion, contrary to PKC and calmodulin which do not appear to have any role.

Detection of Escherichia coli O157:H7 and other intestinal pathogens in patients with diarrhoeal disease.

Six hundred and six faeces specimens from patients with diarrhoea, aged 0->60 and living in the area of Fano (Italy), were examined for Escherichia coli O157:H7, Salmonella, Shigella and thermophilic Campylobacter spp. E. coli O157:H7 was not found, confirming the limited circulation of this bacterium in our country. On the other hand, Salmonellae were the most frequent pathogens (5.6%), followed by Campylobacters (2.3%) and Shigellae (0.3%).

Presence of enteroviruses and reoviruses in the waters of the Italian coast of the Adriatic Sea.

EEC directive 76/160 requires member states to apply microbiological and chemico-physical standards for the quality of recreational waters. In observation of this regulation, in the present study 144 samples of seawater were taken over a 12-month period and tested to determine viral contamination. The samples were collected from the coastal waters of the Italian town of Pesaro, which is located on the Adriatic Sea. Using cell culture techniques, 32.6% of the seawater samples were found to be contaminated with enteroviruses. Isolation of these viruses was most frequent in the summer months. Thus, our results indicate the need to increase the frequency of monitoring of these waters and to eliminate the sources of contamination.

Diffusion of thermophilic Campylobacter in the Pesaro-Urbino area (Italy) from 1985 to 1992.

The results of research on the spreading of campylobacter in the Pesaro-Urbino area carried out from 1985 to 1992 are presented. Materials of different origin were examined: 822 samples of human faeces, 533 animal rectal swabs, 192 samples of domestic sewage, 48 of river water, 96 of sea water and 632 of various types of food. Two hundred and nine strains of campylobacter were isolated (9%), most of which were Campylobacter jejuni (80%), with particular frequency in food products (chicken carcass 45.7%, ground meat and sausage 18.1%) and in river water (31.3%). In contrast, the samples of sea water and dairy cheese products were always negative. It may be concluded that the spreading of campylobacter in the Pesaro-Urbino area is mainly associated with food products of animal origin. Therefore, better controls in the processing of these products may be necessary.

Antipoliomyelitis immunity status in a cohort of young men drafted into military service, residing in the suburban Milan area.

The aim of this study was to verify the antipolio immune status of a cohort of eighteen year-olds residing in the suburban Milan area for whom vaccination histories were available. Seropositivity (titres of neutralizing antibodies > or = 1:8) for the three virus serotypes was detected in 89.2% of the 530 subjects tested, whereas simultaneous seronegativity for all three polioviruses was found for only 0.6% of the subjects.

Prevalence of anti-HCV in subjects of various age groups.

812 serum samples from 382 males and 430 females from various age groups were examined. All the samples were tested for anti-HCV hepatitis C virus, anti-HBc and HBsAg with an enzymeimmunoassay. The total serum prevalence was 2.9% for anti-HVC, 22.2% for anti-HBc and 4.6% for HBsAg. The seropositivity rates of anti-HCV and anti-HBc tended to increase with age, while for HBsAg a more regular pattern was observed for the different subject groups. The fact that anti-HCV are more frequently found together with HBV markers confirms the existence of similar modes of transmission of the two viruses.

Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies.

Previous studies on Tuber borchii fruit bodies in early maturation stages suggested a role of bacteria in sporocarp structural modifications. In order to verify this hypothesis, in the present study we investigated by means of microbial and ultrastructural approaches, the bacterial population of T. borchii sporocarps from intermediate maturation phases to advanced decomposition stages, paying particular attention to chitinolytic and cellulolytic bacteria and to their relationships with ascii and ascospores. We found that Pseudomonas fluorescens and spore-forming Bacillaceae, both able to degrade cellulose and chitin, are present inside the sporocarps in all maturation stages investigated. Moreover, rod-shaped bacteria seem able to erode ascus walls and colonize the interior of ascii containing mature spores. These results suggest a possible role of these bacteria in the process of ascus opening. Moreover, the presence of P. fluorescens and Bacillaceae on isolated mature spores after decontamination suggests an intimate association between these bacteria and the ascospores.

Yersinia enterocolitica and related species isolated in the Pesaro and Urbino area (Italy) from 1981 to 1986.

A total of 23 strains of yersinias, Y. enterocolitica (17), Y. frederiksenii (5) and Y. intermedia (1) characterized according to bio-serogroup and phage type, were isolated from human, animal and environmental samples during a 5-year period. It appears that in the Pesaro-Urbino area Yersinia spp. are infrequent and the strains of Y. enterocolitica belong to environmental and rarely to human pathogenic bioserogroups.

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea.

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae, 46 (22%) as Aer. sobria, 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii. Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.