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.

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.

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.

Cytotonic enterotoxins and cytotoxic factors produced by Salmonella enteritidis and Salmonella typhimurium.

Strains of Salmonella enteritidis and Salmonella typhimurium isolated from human diarrheal cases produced heat-labile enterotoxin(s) and cytotoxic factor(s) which elongated, lysed or deformed Chinese hamster ovary cells in tissue culture. The toxin(s) caused fluid accumulation in ligated rabbit gut loops and produced increased skin permeability. Salmonella toxin produced by these strains does not cross-react immunologically with high titer Vibrio cholerae toxin antisera or heat-labile Escherichia coli enterotoxin antisera used in this study and does not bind to galactose--Sepharose gel. The activity of the toxin was not inhibited by GM1-ganglioside.

Growth of and toxin production by Aeromonas hydrophila and Aeromonas sobria at low temperatures.

The effects of different temperatures on the growth and toxin production of Aeromonas hydrophila and Aeromonas sobria were studied. The results showed that these Aeromonas species are not only able to grow at low temperatures (e.g. at 4 and 10 degrees C) but may also produce cytotoxin, hemolysin and enterotoxin under suitable growth conditions.

Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study.

A large number of Aeromonas spp. have been found in drinking water from a drilled well in Sweden. Isolates identified as A. hydrophila were tested for production of enterotoxin, hemolysin, enzymes and for resistant patterns to different antibiotics. The enterotoxin-producing A. hydrophila could be responsible for the long-term diarrhoeal case of a 1 1/2 year old child who consumed the contaminated water.

Binding of fibronectin to Escherichia coli isolated from bovine mastitis from different geographical regions.

Seventy strains of Escherichia coli, isolated from bovine mastitis in Australia, Denmark, Norway and the U.S.A., were tested for their ability to bind fibronectin. Fifty-three strains (76%) interacted with iodinated fibronectin at a level exceeding 5% of the total radioactivity added. Binding of the amino-terminal (29 kD) fragment of fibronectin was tested for 15 strains, and 6 strains (40%) bound greater than 5%. Bacteria binding the 29 kD fragment at greater than or equal to 19% of the added protein, consistently showed "high" attachment to bovine skin fibroblasts. These cells were shown by immunofluorescence to produce extracellular matrix containing fibronectin. Strains binding lower amounts of fibronectin or 29 kD fragment adhered poorly to these fibroblasts.

Enterotoxigenic Escherichia coli (ETEC) and Klebsiella pneumoniae isolated from dogs with diarrhoea.

Faecal samples from 148 dogs with diarrhoea and from 15 healthy dogs were cultured for bacterial pathogens with enterotoxigenic properties. The aim of the study was to define the toxin profile (production of heat-labile [LT] and heat-stable [ST] toxins) and possible surface fimbrial antigens. Enterotoxigenic bacteria were isolated from 6 (4.1%) dogs with diarrhoea, four of these were Escherichia coli and two were Klebsiella pneumoniae. The E. coli strains and K. pneumoniae strains were producing both LT and ST toxins. The LT toxin from these strains was not neutralized by human anti-LT serum or anti-choleragen and did not cause coagglutination with Staphylococcus aureus coated with anti-human-LT. This suggests that the LT toxin produced by these canine isolates is non-identical to LT toxin from human strains. Three of the ETEC strains were haemagglutinating and showed surface hydrophobic properties. Electron microscopy showed that canine ETEC isolates possessed fimbriae of two different types: thick (5-5.5 nm) and thin (2-3 nm).

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.

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.

Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction.

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

Detection of potential virulence markers of Vibrio vulnificus strains isolated from fish in Sweden.

A variety of potential virulence markers such as the production of cytotoxin, haemolysin, exoenzymes, bactericidal action of sera, presence of capsule and adhesion to human intestinal cells were investigated on Vibrio vulnificus strains isolated from eels in Sweden. The strains had the capacity of producing all or some of the above-mentioned virulence markers, to varying degrees though none of the strains produced any capsule. The strains also bound specifically to human intestinal cells in vitro with maximum adhesion levels of 30 bacteria/cell. The results on binding of V. vulnificus cytotoxin to HeLa cells, showed that a very short exposure time (30 min) was required for inducing the cytotoxic effects. V. vulnificus is a relatively new addition to the list of bacteria pathogenic for humans, and since there are increasing reports on its isolation from aquatic environments and seafood (e.g. raw oysters, crabs and shellfish), the results on virulence profiles of V. vulnificus strains presented above emphasize the importance of these organisms in public health and epidemiological studies.

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.

Fatal septicaemia caused by Aeromonas hydrophila in a patient with cirrhosis.

In this case report from Italy we describe a fatal infection caused by A. hydrophila in a 39 yr old cirrhotic patient. This pathogen was isolated as a pure single culture from the patient's blood sample. The patient died on the second day of hospitalization from overwhelming sepsis. The A. hydrophila isolate was tested for different potential virulence properties, such as invasiveness, adherence, exotoxins production, presence of fimbriae and for the patterns of resistance to a variety of antimicrobial agents. Although, the Aeromonas species are infrequently reported as a cause of human infections, the present case study confirms the capability of these pathogens to induce serious human infections.

Ecological relationship between Aeromonas and Vibrio spp. and planktonic copepods in the coastal marine environment in southern Italy.

The colonisation of planktonic copepod integument by bacteria belonging to the family of Vibrionaceae is a well described phenomenon. In this study, besides reporting on the occurrence of Vibrionaceae and other enteropathogens, we further report on the bacterial attachment to the Estuarine copepod Acartia margalefi in a faecal polluted coastal lagoon near Naples, Southern Italy. In addition, we also performed a laboratory experiment to study the ability of 7 bacterial strains (Vibrio cholerae non-Ol, V. mimicus, V. parahaemolyticus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli and Pseudomonas sp.) to colonise the copepod integument. For this laboratory study, 4 different species of copepods, namely Temora stylifera, A. clausi, Centropages typicus and Paracalanus parvus sampled from the Gulf of Naples (Southern Italy) were used. Scanning electron microscope (SEM) studies on the copepods sampled from the lagoon indicated that the bacterial attachment on the integument of Acartia margalefi was mainly on the ventro-lateral body region of the host and in the joints of the thoracic segments, as well as on the swimming and feeding appendages. This infestation, made by rod-like bacteria, was absent in winter but reached peak values of 70% frequency in June. The laboratory studies showed that while V. cholerae non-O1 and A. hydrophila attached on live and dead copepods, respectively, the V. parahaemolyticus, V. alginolyticus, V. mimicus, E. coli and Pseudomonas sp. failed to colonise either live or dead copepods. Thus, this study provides further valuable information about the ecological relationship between different microorganisms (pathogens) and copepods in the coastal marine environment in Southern Italy.

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.

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.

Infection of rabbit mammary glands with ovine mastitis bacterial strains.

An experimental model was developed in rabbits to study ovine mastitis. A total of 19 ovine mastitis bacterial strains (seven Staphylococcus aureus, four Staph. chromogenes, four Staph. hyicus and four Escherichia coli) were used for mammary gland infections. The histopathological results showed that the ovine mastitis types corresponded to experimental infections produced in the rabbit with the ovine strains. These results helped the grading of the bacterial species tested according to the severity of their effects on the mammary gland. The most pathogenic species was Staph. aureus, followed by E. coli, Staph. hyicus and Staph. chromogenes, in that order. There was, however, variation among strains within a given species (e.g. one out of seven Staph. aureus strains gave rise to a mild infection in sheep and rabbits). The procedure was simple and consisted of introducing bacterial suspensions through alternate teat ducts of does with the help of a cannula. It helped minimize the number of animals required in the experiments.

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.