Vibrio cholerae O139 requires neither capsule nor LPS O side chain to grow inside Acanthamoeba castellanii.

Vibrio cholerae, the causative agent of cholera, has the ability to grow and survive in the aquatic free-living amoeba Acanthamoeba castellanii. The aim of the present study was to examine the ability of the clinical isolate V. cholerae O139 MO10 to grow in A. castellanii and to determine the effect of the bacterial capsule and LPS O side chain on intracellular growth. Results from co-cultivation, viable counts, a gentamicin assay, electron microscopy and statistical analysis showed that the association of V. cholerae O139 MO10 with A. castellanii did not inhibit growth of the amoeba, and enhanced growth and survival of V. cholerae O139 MO10 occurred. The wild-type V. cholerae O139 MO10 and a capsule mutant or capsule/LPS double mutant grew inside A. castellanii. Neither the capsule nor the LPS O side chain of V. cholerae O139 was found to play an important role in the interaction with A. castellanii, disclosing the ability of V. cholerae to multiply and survive inside A. castellanii, as well as the role of A. castellanii as an environmental host for V. cholerae.

Ultrastructure of coccoid viable but non-culturable Vibrio cholerae.

Morphology of viable but non-culturable Vibrio cholerae was monitored for 2 years by scanning and transmission electron microscopy. Morphological changes included very small coccoid forms, after extended incubation at 4 degrees C and room temperature, and sequential transformation from curved rods to irregular (approximately 1 microm) rods to approximately 0.8 microm coccoid cells and, ultimately, to tiny coccoid forms (0.07-0.4 microm). Irregular rod-shaped and coccoid cells were equally distributed in microcosms during the first 30-60 days of incubation at both temperatures, but only coccoid cells were observed after incubation for 60 days at 4 degrees C. When V. cholerae O1 and O139, maintained for 30-60 days at both temperatures, were heated to 45 degrees C for 60 s, after serial passage through 0.45 microm and 0.1 microm filters, and plating on Luria-Bertania (LB) agar, only cells larger than 1 microm yielded colonies on LB agar. Approximately 0.1% of heat-treated cultures were culturable. Cell division in the smallest coccoid cells was observed, yielding daughter cells of equal size, whereas other coccoid cells revealed bleb-like, cell wall evagination, followed by transfer of nuclear material. Coccoid cells of V. cholerae O1 and O139 incubated at 4 degrees C for more than 1 year remained substrate responsive and antigenic.

Reduction in capsular content and enhanced bacterial susceptibility to serum killing of Vibrio cholerae O139 associated with the 2002 cholera epidemic in Bangladesh.

Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.

Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae.

Vibrio cholerae is a highly infectious bacterium responsible for large outbreaks of cholera among humans at regular intervals. A seasonal distribution of epidemics is known but the role of naturally occurring habitats are virtually unknown. Plankton has been suggested to play a role, because bacteria can attach to such organisms forming a biofilm. Acanthamoebea castellanii is an environmental amoeba that has been shown to be able to ingest and promote growth of several bacteria of different origin. The aim of the present study was to determine whether or not an intra-amoebic behaviour of V. cholerae O139 exists. Interaction between these microorganisms in co-culture was studied by culturable counts, gentamicin assay, electron microscopy, and polymerase chain reaction. The interaction resulted in intra-amoebic growth and survival of V. cholerae in the cytoplasm of trophozoites as well as in the cysts of A. castellanii. These data show symbiosis between these microorganisms, a facultative intracellular behaviour of V. cholerae contradicting the generally held view, and a role of free-living amoebae as hosts for V. cholerae O139. Taken together, this opens new doors to study the ecology, immunity, epidemiology, and treatment of cholera.

[Survival and growth of Vibrio cholerae O139 inside Acanthamoeba].

OBJECTIVE: To study the survival and growth of Vibrio cholerae inside the Acanthamoeba polyphage. METHODS: Survival and growth of Vibro cholerae O139, co-cultured with Acanthamoeba polyphaga, was observed inside the trophozoites and cysts, using Gram stain and electron microscope. RESULTS: Viable O139 was observed inside the amoebal vacuoles in 24 hours. Vacuoles were filled with more bacteria along with the longer period of co-culture. The process of O139 infection with Amoebae would include uptake, formation of O139 vacuole, multiplication, trophozoites lysed and expel under electron microscopy. Some infected trophozoites could subsequently encyst and the surviving O139 could locate in the vesicles inside the cysts. CONCLUSION: O139 might survive and multiply in the trophozoites and reside inside the cysts of Amoebae, suggesting that Acanthamoebae might serve as one of the environmental hosts of Vibro cholerae.