ABSTRACT
ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.
Subject(s)
Animals , Male , Rats , Cell Survival/drug effects , Plesiomonas/metabolism , Cytoplasmic Vesicles , Virulence Factors , Rivers/microbiology , Enterotoxins/pharmacology , Vero Cells , Neutralization Tests , Microscopy, Electron, Scanning , Chlorocebus aethiops , Plesiomonas/pathogenicity , Plesiomonas/ultrastructure , Lethal Dose 50ABSTRACT
Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.
Subject(s)
Cell Survival/drug effects , Cytoplasmic Vesicles , Enterotoxins/pharmacology , Plesiomonas/metabolism , Rivers/microbiology , Virulence Factors , Animals , Chlorocebus aethiops , Lethal Dose 50 , Male , Microscopy, Electron, Scanning , Neutralization Tests , Plesiomonas/pathogenicity , Plesiomonas/ultrastructure , Rabbits , Vero CellsABSTRACT
OBJECTIVES: Resistance of Plesiomonas shigelloides to cephalosporins at higher cell densities has been reported. We investigated whether these inoculum effects are due to the production of beta-lactamases. METHODS: beta-Lactamase production of five P. shigelloides strains was characterized by activity tests, SDS-PAGE and isoelectric focusing. For all strains, MIC values of different cephalosporins were determined by microdilution methodology using inocula of 1 x 10(5) cfu/mL and 1 x 10(6) cfu/mL. Subsequently, the morphology of cells was determined by light microscopy. For one isolate, kill kinetics of cefpodoxime were determined using batch cultures with the lower and higher inocula. RESULTS: Four of five P. shigelloides strains were shown to be beta-lactamase-positive, producing different amounts of constitutively expressed non-inducible enzymes. Inoculum effects for cephalosporin susceptibility were observed for all strains. Examination of cells revealed a very strong filamentation, with filament sizes ranging from 100 microm up to 2 mm. The kill kinetics with cefpodoxime showed similar killing capacities of the antibiotic at both inoculum sizes. CONCLUSIONS: The reported resistance of P. shigelloides to cephalosporins at higher cell densities is not due to an inoculum-dependent regulation of beta-lactamases, but can be explained by the formation of extensive filaments.
Subject(s)
Cephalosporins/pharmacology , Plesiomonas/drug effects , Colony Count, Microbial , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Isoelectric Focusing , Kinetics , Microbial Sensitivity Tests , Plesiomonas/enzymology , Plesiomonas/ultrastructure , beta-Lactamases/biosynthesis , beta-Lactamases/metabolismABSTRACT
In total, 131 strains of Plesiomonas shigelloides isolated from various sources were tested for peritrichous flagella by a flagella staining method. When incubated on a solid medium for 18 hr at 25 C, peritrichous flagella were demonstrated in 89 (68%) of them. With an electron microscope, the peritrichous flagella were clearly distinguished from the lophotrichous ones by their wavelength.
