Tolerance to silver of an Aspergillus fumigatus strain able to grow on cyanide containing wastes.

We studied the strategy of an Aspergillus fumigatus strain able to grow on metal cyanide wastes to cope with silver. The tolerance test revealed that the Minimum Inhibitory Concentration of Ag(I) was 6mM. In 1mM AgNO3 aqueous solution the fungus was able to reduce and sequestrate silver into the cell in the form of nanoparticles as evidenced by the change in color of the biomass and Electron Microscopy observations. Extracellular silver nanoparticle production also occurred in the filtrate solution after previous incubation of the fungus in sterile, double-distilled water for 72h, therefore evidencing that culture conditions may influence nanoparticle formation. The nanoparticles were characterized by UV-vis spectrometry, X-ray diffraction and Energy Dispersion X-ray analysis. Atomic absorption spectrometry revealed that the optimum culture conditions for silver absorption were at pH 8.5.The research is part of a polyphasic study concerning the behavior of the fungal strain in presence of metal cyanides; the results provide better understanding for further research targeted at a rationale use of the microorganism in bioremediation plans, also in view of possible metal recovery. Studies will be performed to verify if the fungus maintains its ability to produce nanoparticles using KAg(CN)2.

Changes in adhesion ability of Aeromonas hydrophila during long exposure to salt stress conditions.

AIMS: Stressful environmental conditions influence both bacterial growth and expression of virulence factors. In the present study, we evaluated the influence of NaCl on Aeromonas hydrophila adhesiveness at two temperatures. This agent is often involved in clinical cases; however, its pathogenic potential is still not fully understood. METHODS AND RESULTS: Bacteria were grown in presence of 1·7%, 3·4%, 6·0% NaCl over a 188 day period and then reinoculated in fresh Nutrient Broth with incubation at 4 and 24°C. Bacterial adhesiveness was tested on Hep-2 cells, and specimens were processed for light, scanning and transmission electron microscopy. Adhesive capacity decreased over time with an increase in reduction percentages depending on NaCl concentrations. At 1·7% NaCl, the reduction was apparently temporary and adhesiveness rapidly recovered in revitalized bacteria, while 3·4%, 6·0% NaCl seemed to be detrimental. Normal, elongated and filamentous bacteria retained adhesiveness capability, although with reduced expression, while in spherical cells, this property seemed to be lost or dramatically reduced. CONCLUSIONS: Our study shows that high osmolarity plays a significant role in adhesion inhibition, therefore having possible implications in the pathogenesis of the infections by Aer. hydrophila. SIGNIFICANCE AND IMPACT OF THE STUDY: This study intends to give a contribution to a better understanding of the pathogenic role of this bacterium whose pathogenicity is still under debate.

Morphological changes of Aeromonas hydrophila in response to osmotic stress.

The adaptive response of bacteria to stressful environmental situations may lead to a modification of physiological and phenotypical characteristics, including morphology. The aim of this study was the analysis of the ultrastructural changes in Aeromonas hydrophila exposed to different NaCl concentrations (1.7%, 3.4%, 6%) at 4 and 24 degrees C for 188 days. Bacterial cultures were processed for scanning and transmission electron microscopy, and specimens were analysed at different times during osmotic stress. SEM reveals the presence of three predominant morphotypes: rod, filamentous and spherical forms, depending on the time and culture conditions. Normal rod cells prevail in 1.7% NaCl growth conditions, maintaining high rates until the end of the trial at 4 degrees C. The most favourable conditions for the elongated morphotype are 3.4% NaCl at 4 degrees C. Spherical forms appear later, increase with time and are the prevalent population at the end of the trial at 24 degrees C, in all culture conditions. TEM reveals the presence of normal, necrotic-like and apoptotic-like forms; these latter forms increase with time according to salt concentration and temperature. Initially, a detachment of the external membrane appears, with cytoplasmic clumping into small, dense masses; as the process continues, both these features become more evident with increasing salt concentrations. This behaviour has been compared to that of eukaryotic cells undergoing growth factor deprivation-induced apoptosis. Occasionally, surface blebs are observed. In conclusion, the study suggests that the exposure of A. hydrophila to stressful conditions (osmolarity, temperature and nutrients) leads to the generation of varying morphotypes, which promote cell survival in adverse conditions and a rapid repopulation in post-stress environments.

Microbial characteristics of marine sediments in bathing area along Pesaro-Gabicce coast (Italy): a preliminary study.

AIMS: This paper evaluates the presence of human pathogen micro-organisms in marine sediments in a coastal area suitable for bathing. In addition, the grain size analysis was performed in order to correlate the characteristics of the sediments and the microbial content. METHODS AND RESULTS: The samples were collected in two small bays along the central Adriatic coast, where breakwaters had been built for the purpose of halting marine erosion. Faecal contamination indicators, Salmonella and Vibrio species, enteric viruses were investigated using standard techniques for isolation and identification. The grain size was determined using calibrated sieves and 'Sedigraph' device. In some samples, the faecal contamination indices overstepped legislative limits. Salmonella was never found. Vibrio and enteric viruses were isolated: the micro-organisms were preferentially abundant in fine sediments. CONCLUSIONS: Marine sediments can represent an important reservoir of allochthonous and marine micro-organisms and the microbial charge correlates with the characteristics of the sediments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that it is important to analyse marine sediments before defining the quality of coastal areas.

Faecal contamination indicators, Salmonella, Vibrio and Aeromonas in water used for the irrigation of agricultural products.

The faecal contamination indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci) and the genera Salmonella, Vibrio, Aeromonas were investigated in water samples used for irrigation. During 4 months, 52 samples were taken. The methods used were: multiple tube fermentation method for faecal contamination indicators and membrane filtration techniques for salmonella, aeromonas and vibrio. Two samples were positive for Salmonella spp., fourteen for Aeromonas spp. and no samples for Vibrio spp. No correlation was found between aeromonas and the indicators of faecal contamination. Regarding Aeromonas spp., 21.6% of the strains were adhesive and 12.6% cytotoxic: this confirms the possible role of aeromonas in human pathologies. These results are important to determine the quality of irrigation water in relation to human health. In fact, the spray or sprinkler irrigation produces bioaerosol, which can contaminate the crops that are likely to be eaten uncooked. In addition, the flood or furrow irrigation represents a risk to field workers.

Immunity to poliomyelitis: situation in northern Italy and in some other European population groups.

The aim of the program to eradicate poliomyelitis is expected to be achieved in the space of very few years and therefore, as they approach this goal, countries find themselves faced with various problems to be tackled. Among these is the decision about how and when to stop polio vaccination, which also depends on seroepidemiological evaluations, necessary because of the possible persistence of circulating strains of poliovirus of vaccine origin which might continue even after the suspension of vaccination. For this reason, the results of seroepidemiological surveys carried out in some European countries in different periods of time and on various samples of the population are discussed. Although the status of immune coverage is satisfactory, a few situations of susceptibility were found; these should be better monitored and, if necessary, corrected to guarantee a homogeneous and lasting resistance of the European population towards polioviruses.

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%).

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.

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.

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.

Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast.

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.

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.

Virulence factors in Aeromonas spp and their association with gastrointestinal disease.

Culture filtrates of eight Aeromonas strains isolated from the feces of 487 subjects (292 diarrhoeic patients and 195 asymptomatic subjects) were tested for toxin production in CHO and McCoy cells, and adhesion and invasive ability in Caco-2 cells. Among these isolates, three Aeromonas sobria and one Aeromonas caviae strains possessed virulence-associated properties. Toxin production was the most common of the three virulence properties. Two A. caviae were associated, in the absence of other diarrhoeagenic agents, with gastroenteritis; however, a virulence marker (cytotoxin) was recognized only in one strain. Two strains of A. sobria isolated from subjects with gastroenteritis were shown to be associated with one (cytotoxicity) or two (adhesion and invasive abilities) virulence factors, respectively. However, a third strain of A. sobria, although cytotoxic and invasive, was isolated from an asymptomatic subject. The results show that Aeromonas spp may act as human enteric pathogens, but also indicate that the significance of several putative virulence factors, such as production of cytotoxin and the capacity to adhere to and invade mammalian cells, remains controversial in explaining the enteropathogenesis of Aeromonads and therefore needs further studies.

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.

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.

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.

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.

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.