Prevalence and genotypic characterization of Clostridium difficile from ruminants in Switzerland.

Clostridium difficile is an emerging enteric pathogen of humans and animals with a known main reservoir in the intestinal tract of various warm-blooded animals. This study was carried out to evaluate the prevalence of C. difficile in 150 rectal swab samples collected from cattle and goats in Switzerland. The overall prevalence of C. difficile was 6.6%. The isolates belonged to the PCR ribotypes 033, 066, 070, 003, 001 and 137. In addition, the occurrence of C. difficile in faecal samples collected from farm ground was also evaluated and C. difficile was detected in 21% (7/30) of these samples. These isolates belonged to ribotypes 033, 066, 014 and 137. Several isolates pertained to ribotypes known to cause Clostridium difficile infections in humans.

Occurrence of toxigenic Clostridium difficile in edible bivalve molluscs.

Clostridium difficile is an anaerobic bacterium commonly considered to be responsible for antibiotic-associated gastrointestinal diseases, ranging from diarrhea of varying severity to pseudomembranous colitis. The aim of this study was to assess the occurrence of C. difficile in marine edible bivalve molluscs, which, as filter feeding organisms, are able to accumulate particles suspended in water, including microorganisms. Samples of Mytilus galloprovincialis, Tapes philippinarum, and Venus verrucosa were collected from mussel farms and fishmongers in the province of Naples (Southern Italy). C. difficile was found in 49% of the 53 samples investigated. Sixteen isolates were grouped in 12 known different PCR ribotypes (001, 002, 003, 010, 012, 014/020, 018, 045, 070, 078, 106, and 126), whereas 10 additional isolates were grouped in 8 new PCR riboprofiles. Two toxinotypes (0 and V) were found. Fifty eight percent of the isolates were toxigenic. These findings indicate that toxigenic C. difficile strains can be isolated in bivalve molluscs. Marine filter feeding organisms, therefore, may be considered as reservoir of toxigenic strains of C. difficile. The ingestion of raw or poorly cooked contaminated seafood and the high temperature resistance of the spore-forming C. difficile could represent an important source of exposure and pose human health concern.

Phenotypic characterization and putative virulence factors of human, animal and environmental isolates of Plesiomonas shigelloides.

Plesiomonas shigelloides (a bacterium widely distributed in aquatic ecosystems causing both intestinal and extra-intestinal diseases) shows a host of putative virulence markers, such as hemolysins, cytotoxins, production of exoenzymes associated with pathogenicity, adhesive ability and vacuolation of cell lines in vitro. Technical difficulties in detecting some of these virulence factors together with scantiness of epidemiological information, due to the lack of routine analysis for P. shigelloides as etiological agent of gastroenteritis, lead to sporadic and occasional finding of these bacteria. All this casts doubt on the real virulence potential of P. shigelloides and fuels a debate about assignment of these bacteria to the list of human pathogens. Here we demonstrated the phenotypic diversity and the putative virulence markers by examining serotype biochemical and virulence properties of 60 strains of P. shigelloides isolated from human, animal and environmental samples in different countries, which showed the unpredictable occurrence of the above properties depending on various locations and diverse sources.

Inhibition of mammalian cathepsins by Plesiomonas shigelloides.

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods.

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.

Potential virulence-associated properties of Plesiomonas shigelloides strains.

Serotyping and some potential virulence-associated markers were investigated in Plesiomonas shigelloides strains isolated from humans, animals and aquatic environments. Surface properties of these strains were evaluated using Congo red binding, salt-aggregation test, bacterial adherence to xylene and motility. Production of pancreatic elastase, proteinase (consistent with subtilisin Carlsberg), triacylglycerol lipase, histidine decarboxylase and beta-hemolysin was also determined. In addition, detection of signal molecules such as C4-C8 unsubstituted N-acylhomoserine lactones (AHLs) was performed. The serological typing of the P. shigelloides strains showed that the isolates belonged to 13 different serovars. The majority of the strains were hydrophobic and motile. The strains produced low levels of elastase, proteinase and histidine decarboxylase whereas triacylglycerol lipase activity was relatively high. Only 23.3 % of the strains produced hemolysin. The AHLs signal molecules were not detected. P. shigelloides strains were able to produce a variety of potential virulence markers which may be involved in the pathogenesis of Plesiomonas-associated infections.

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand.

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.

The effect of disinfectants on Plesiomonas shigelloides.

The effects of ten commercially available disinfectants on virulence associated properties of Plesiomonas shigelloides were tested. All the disinfectants tested contained quaternary ammonium salts. The majority of the disinfectants when used at subinhibitory concentrations increased surface hydrophobicity as evaluated by bacterial adherence to xylene and decreased bacterial motility in a concentration dependent manner. Disinfectants did not significantly affect lipase activity. However, more than half of the antimicrobials tested increased the resistance of bacteria to hydrogen peroxide. The disinfectants, in a similar manner to antibiotics at concentrations below MIC, interfered with potential virulence factors of Plesiomonas shigelloides.

Incidence and characterisation of Aeromonas spp. in environmental and human samples in southern Italy.

The incidence and characterisation of Aeromonas species in human and environmental samples in southern Italy were investigated. The results emphasize that 12.3% of the 210 examined patients carried Aeromonas spp. in their faeces. These results underline the need to include Aeromonas spp. in the list of routinely analysed enteropathogens in all diarrhoeal stool samples, especially in children below 10 years of age, elderly persons and immunocompromised patients.

RAPD-PCR and PFGE as tools in the investigation of an outbreak of beta-haemolytic Streptococcus group A in a Swedish hospital.

We evaluated the random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques for studying an outbreak of beta-haemolytic streptococci group A (GAS) occurred at two maternity wards at Danderyd hospital, Stockholm, Sweden. All the isolates were of T-type 8,25. The RAPD technique revealed that all RAPD-PCR profiles were identical. PFGE showed that all the patterns but one were identical. These patterns were compared with 10 different T-type GAS from the strain collection of the Swedish Institute for Infectious Disease Control (SMI) and T-type 8,25 from different years and locations. The SMI strains exhibited patterns different from each other and all different from the isolates from Danderyd hospital. Moreover, RAPD could not differentiate among the T-type 8,25 isolates from different years and locations but PFGE showed differences among the amplicons. Our results indicated that the RAPD and PFGE techniques could be efficient tools in epidemiological studies of GAS.

The importance of pathogenic organisms in sewage and sewage sludge.

Deficient sanitation poses a serious threat to human and animal health, involving complex relationships between environments, animals, refuse, food, pathogens, parasites, and man. However, by sanitizing and stabilizing the organic matter of sewage sludge, agriculture can utilize it to maintain soil, water, and air quality. As ingredients in soil amendments, such bioresidues are a source of nutrients for plants. Stabilization and sanitation of sewage sludge safely couple its recycling and disposal. This coupling becomes increasingly important as economic and environmental constraints make strategies for waste disposal more difficult to apply. The occurrence of viruses, bacteria, yeasts, fungi, and zooparasites in sewage sludge is reviewed in this article, and consequential epidemiologic concerns that arise from sewage sludge recycling is also addressed.

Enteropathogenicity of Plesiomonas shigelloides and Aeromonas spp. in experimental mono- and coinfection with Cryptosporidium parvum in the intestine of neonatal BALB/c mice.

Enteropathogenicity of Plesiomonas shigelloides, Aeromonas hydrophila, A. caviae and A. sobria was studied both in monoinfections and in coinfections with coccidium Cryptosporidium parvum in neonatal BALB/c mice. In monoinfection experiments, neonatal BALB/c mice were orally infected with 7 x 10(7) or 7 x 10(8) CFU, respectively, of a strain of P. shigelloides or a strain of an Aeromonas spp. In coinfection experiments, the neonatal mice were, in addition to being orally infected with one of the four bacterial species, orally infected with an inoculum containing 10(5) oocysts of C. parvum. Results from monoinfections with P. shigelloides revealed long-term colonisation of the neonatal mouse intestine by this pathogen, along with associated pathological lesions. The lesions varied in severity from atrophy to necrosis of the mucosal inner surface of the ileum and colon, with predilection to the colon and brush border of colonic enterocytes. The effects of coinfection of P. shigelloides with C. parvum were characterised by bacteremia and heavy colonisation of the intestine by P. shigelloides. In addition, extensive necrotising inflammatory changes in the ileum and colon were accompanied by diarrhoea and deaths of coinfected mice. In contrast, the results from monoinfections of neonatal mice with Aeromonas spp. showed only a short-term colonisation of the intestine by the pathogen. However, when mice were coinfected with A. hydrophila and C. parvum, then the growth of the bacterial species was prolonged, and occurred in both the spleen and intestine. However, no substantial clinical or histopathological changes were observed in mice, whether monoinfected with Aeromonas spp. or coinfected with C. parvum. Our study suggests that experimental monoinfections of neonatal BALB/c mice with P. shigellodes, Aeromonas spp. and C. parvum, together with coinfections (each bacterial species with the protozoan C. parvum), may serve as a useful model to study the initial steps of gastrointestinal colonisation and diarrhoeal disease syndromes caused by enteropathogenic bacteria and protozoa, individually and in combination.

Specific detection of Plesiomonas shigelloides isolated from aquatic environments, animals and human diarrhoeal cases by PCR based on 23S rRNA gene.

Twenty-five strains of Plesiomonas shigelloides isolated from aquatic environment, 10 strains from human cases of diarrhoea and five strains from animals were identified by the polymerase chain reaction technique based on 23S rRNA gene. For this purpose, two primers targeted against part of the 5' half of the 23S rRNA gene of P. shigelloides (Escherichia coli number C-912, G-1195; Plesiomonas number C-906, G-1189) were designed. Results from our study indicated that this method might serve as a tool for a rapid and sensitive identification of P. shigelloides from different environmental and clinical sources.

Isolation, biochemical and serological characterisation of Plesiomonas shigelloides from freshwater in Northern Europe.

Isolation and characterisation of Plesiomonas shigelloides from fresh water in Northern Europe is reported in this study. The organisms were isolated from two lakes and a river in Sweden. All isolates of P. shigelloides showed an identical biochemical profile and belonged to different serotypes, namely, O18, O23, O26, O58 and O60. The study indicates that P. shigelloides may occur in the aquatic environment of cold climates and as a result, it is likely to be associated with human infections caused by waterborne pathogens in geographical areas with similar climatic conditions.

Prevalence and diversity of Aeromonas and Vibrio spp. in coastal waters of Southern Italy.

A survey was undertaken to examine sea water and sediment for the presence of Vibrio and Aeromonas spp. along approximately 900 km of coast in Southern Italy during early and late summer. A quantitative analysis was also done to evaluate the water fecal contamination at the stations examined. The results indicate that all the investigated areas were submitted to a wide spatial fluctuation of fecal contamination and that Vibrio and Aeromonas spp. were present in both high and low fecal-contaminated stations. Sixty two percent of the investigated samples were positive for Aeromonas spp., while 42% of samples were positive for Vibrio spp. It was interesting to note that 38% of the positive stations for both Aeromonas and Vibrio spp. showed a fecal coliform contamination of water at < 10(2) cells 100 ml(-1). Thus, these findings support the hypothesis that the bacterial indicators (such as fecal coliforms) do not always satisfactorily reflect the hygienic quality of water. The presence of Vibrionaceae on copepods was also investigated. Copepods were sampled at a station located inside the harbour of the city of Naples and were found contaminated by V. cholerae non-O1, V. alginolyticus, V. fluvialis and A. caviae. Furthermore, the antibiotic resistance patterns of isolated bacteria showed the presence of a number of resistant strains among the isolates. In order to discriminate the isolates on the basis of their biochemical profiles and/or antibiotic resistance patterns, cluster analysis was carried out which showed that no unique assay could fully discern these isolates. However, the best discrimination resulted from complete pattern profile based on both biochemical profiles and antibiotic resistance patterns.

Identification of Escherichia coli recovered from milk of sows with coliform mastitis by random amplified polymorphic DNA (RAPD) using standardized reagents.

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.

Neurotoxic effect on two fish species and a PC12 cell line of the supernate of Vibrio alginolyticus and Vibrio anguillarum.

The biological effects of supernates obtained from different strains of Vibrio alginolyticus and Vibrio anguillarum isolated from diseased fish have been studied by inoculation on two fish species, eel and rainbow trout, and two fish cell lines. These supernates possess neuroexcitatory properties, and so, when they are injected into both fish species, they trigger convulsions, wriggling, contortive swimming and respiratory arrest coupled with increased respiratory reflex. Furthermore, after the application of the supernates on cultures of noradrenergic pheochromocytoma PC12 cells, an increase of acetylcholine, released from the cells was obtained. The amount of released acetylcholine depends on the source of assayed supernates and on the dose applied to the cells. On the basis of the results obtained with PC12 cells, we suggest that the supernates from pathogenic Vibrio strains injected into fish may elicit an increased release of acetylcholine in the motor endplate of some muscles related to locomotion and ventilation of the inoculated fish.

Virulence determinants of Escherichia coli isolated from milk of sows with coliform mastitis.

Escherichia coli isolates recovered from the milk of sows with coliform mastitis were examined to determine their biochemical and serologic characteristics, serum resistance and ability to adhere to fibronectin and bovine fetal fibroblasts. No common biotype was identified, and a variety of serovars were detected. Ninety-five per cent of the investigated strains were serum resistant in swine serum. Binding to fibronectin was demonstrated in most of the strains. At a binding level above 13% of the added fibronectin the strains also adhered to fibroblasts. The results of the study indicate a galactogenous route of infection. The importance of the identified virulence factors need to further elucidated.

Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Baltic Sea: a case study.

Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Swedish part of the Baltic Sea is reported. The pathogen was isolated from both lung and spleen specimens. All of the A. hydrophila isolates produced haemolysin and Vero active cytotoxin. The aerolysin gene was found in all tested isolates as evidenced by the polymerase chain reaction (PCR) technique. Also, all isolates tested showed identical patterns of biochemical and antibiotic resistance. As Aeromonas spp. commonly occur in aquatic environments, we suggest that organisms from this genus may also play an important role as opportunistic pathogens in morbillivirus infected seals.