Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment

Abstract

The factors that enhance the waterborne spread of bacterial epidemics and sustain the epidemic strain in nature are unclear. Although the epidemic diarrheal disease cholera is known to be transmitted by water contaminated with pathogenic Vibrio cholerae, routine isolation of pathogenic strains from aquatic environments is challenging. Here, we show that conditionally viable environmental cells (CVEC) of pathogenic V. cholerae that resist cultivation by conventional techniques exist in surface water as aggregates (biofilms) of partially dormant cells. Such CVEC can be recovered as fully virulent bacteria by inoculating the water into rabbit intestines. Furthermore, when V. cholerae shed in stools of cholera patients are inoculated in environmental water samples in the laboratory, the cells exhibit characteristics similar to CVEC, suggesting that CVEC are the infectious form of V. cholerae in water and that CVEC in nature may have been derived from human cholera stools. We also observed that stools from cholera patients contain a heterogeneous mixture of biofilm-like aggregates and free-swimming planktonic cells of V. cholerae. Estimation of the relative infectivity of these different forms of V. cholerae cells suggested that the enhanced infectivity of V. cholerae shed in human stools is largely due to the presence of clumps of cells that disperse in vivo, providing a high dose of the pathogen. The results of this study support a model of cholera transmission in which in vivo-formed biofilms contribute to enhanced infectivity and environmental persistence of pathogenic V. cholerae.

Fig. 1.

Fluorescent antibody-based detection of V. cholerae O1 CVEC in water samples. Shown are aggregates of CVEC reacting with V. cholerae O1-specific monoclonal antibody (A–C), as well as planktonic cells of V. cholerae O1 (B and D). C shows high magnification of a CVEC clump that appears to contain both coccoid (starved) and rod-shaped V. cholerae O1 cells. The corresponding water samples produced viable V. cholerae O1 in the AST assay, as well as in the ileal loop assay in rabbits, and further analyses confirmed that these isolates were identical to the strain responsible for recent epidemics of cholera in Bangladesh.

Fig. 2.

Micrographs of different fractions of human cholera stools. Both planktonic cells and biofilm-like clumps of V. cholerae cells are present in unfractionated stool (A). The planktonic cells (B) were separated from the clumps (C) by differential centrifugation, and the precipitated clumps were further washed to remove free cells. Numerous V. cholerae cells in multiple layers were found tightly attached to debris in stools, forming biofilms (C).

Fig. 3.

Infectivity of V. cholerae shed in human cholera stools. Shown are mean competition indices of V. cholerae O1 present in different fractions of stools from 10 different cholera patients, against a reference strain grown to logarithmic phase. Each mixture of test and reference strains was assayed in 5–10 different mice. Results shown here are for stools that were negative for the presence of any lytic vibriophage.