Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources.

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks.

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.

Effects of medium composition, calcium, iron and oxygen on haemolysin production by Plesiomonas shigelloides isolated from water.

AIMS: The effects of medium composition, calcium, iron and oxygen tension on the haemolytic activity of Plesiomonas shigelloides were investigated. METHODS AND RESULTS: The haemolytic activity of seven strains of Ple. shigelloides was tested on the surface of Luria Agar (LA), Brain Heart Infusion Agar (BHIA) and Trypitic Soy Agar (TSA) containing 5% (v/v) sheep blood, and in the Agar Overlay (AO) assay. All strains produced beta-haemolysis in the AO assay in three media, and on the surface of LA. The kinetics of growth and haemolytic activity of Ple. shigelloides 9P3-1 were evaluated in six different media, and the highest production of haemolysin occurred in Luria Broth (LB). The haemolytic activity of 9P3-1 was stimulated by Ca2+ and inhibited by EDTA. Addition of iron to the culture medium did not affect bacterial growth, although it reduced bacterial haemolytic activity. In the presence of an iron chelator, growth of the 9P3-1 was inhibited, but its haemolytic activity was enhanced. CONCLUSION: The haemolytic activity of Ple. shigelloides depends on medium composition, and that it is regulated by iron and is calcium-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the importance of optimization of media composition and oxygen tension for detection of Ple. shigelloides haemolytic activity.

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil.

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambé Stream (State of Paraná, Brazil).

The incidence of Vibrio cholerae, Aeromonas spp. and Plesiomonas shigelloides was determined in water samples from Cambé Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve Aer. hydrophila, 12Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp. and V. cholerae, while none of the Ple. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp. and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.

Pre-treatment with Concanavalin-A increases resistance of mice to peritoneal infection by Serratia marcescens.

Mice pre-treated with Concanavalin-A largely survived an intra-peritoneal inoculum of 2 x 10(7) Serratia marcescens, whereas all control mice died within 15 h of inoculation. A subpopulation of peritoneal macrophages from Con-A pre-treated mice was able to phagocytose the bacteria in vitro (6.7 SEM 1.2% phagocytosing cells) and in vivo (16.9 SEM 2.1%), whereas control phagocytes did not phagocytose S. marcescens. The survival of Con-A pre-treated mice allowed their immunisation with living bacteria, and the antiserum thus produced increased the phagocytosis of S. marcescens in vitro. Control mice largely survived an inoculum of S. marcescens suspended in 50% immune serum, although the bacteria were resistant to the bactericidal activity of that serum. These results suggest that, in contrast to the delayed humoral protection afforded by immunisation, phagocytosis by phagocytes activated by Con-A conferred early protection to mice against experimental infection by S. marcescens.

Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea.

Nineteen Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups were isolated from calves with diarrhea in Paraná State. Brazil, and studied for virulence markers associated with EPEC or enterohemorrhagic E. coli (EHEC). The 19 isolates belonged to 12 serotypes with isolates of O26:H11, O119:H25 and O114:H- being the most prevalent Localized adherence (LA) was demonstrated for 37% of the isolates, consisting of all four O26:H11, both O114:H- and one O114:H40 isolates. All the LA strains were positive in the fluorescent-actin staining (FAS) test and possessed attaching-effacing E. coli (eae) sequences, but only O114 strains hybridized with the EPEC adherence factor (EAF) probe. None of the strains produced Shiga-like toxins (Verotoxin). Only the O26:H11 strains hybridized with the EHEC plasmid specific (CVD419) probe and were enterohemolytic, properties associated with EHEC strains. This investigation demonstrates that among the bovine strains isolated only those of serogroup O114 behaved as typical EPEC.

Microbiology of frozen goat meat and toxin production by Bacillus cereus isolated therefrom.

Microbial analysis of commercial samples of freshly frozen goat meat and those stored at - 12 degrees C for one week revealed high counts of aerobic bacteria, Bacillus cereus, Staphylococcus aureus and Streptococci. These counts increased with storage. Psychrophilic bacteria were higher in number than mesophiles. The counts of Salmonella were zero CFU/g in both freshly frozen and stock frozen meat. No yeast or moulds were encountered. Mouse tests of crude culture filtrate of B. cereus and preparations from meat samples confirmed the production of toxin.