Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina

Abstract

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Rio de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.

FIG. 1.

Map showing the sampling stations for VNC V. cholerae O1.

FIG.2.

Fluorescent photomicrographs of V. cholerae O1 attached to microplankton (A), in a water sample (B), and attached to zooplankton (C).

FIG. 3.

Heminested PCR for ctxAB detection. Lane M, 100-bp ladder; lane 1, negative control (PCR master mixture); lanes 2, 4, 5, 6, 8, and 9, negative samples; lanes 3, 7, 10, and 11, positive samples; lane 12 positive control (V. cholerae O1 strain O425).

FIG. 4.

Multiplex PCR for the detection of O1/O139 genes. Lane M, 100-bp ladder; lane 1, negative control (PCR master mixture); lanes 2 to 5, positive samples; lane 6, O1 positive control strain ATCC 14035; lane 7, O139 positive control strain MO4045.

FIG. 5.

Multiplex PCR for the detection of ctxA/tcpA. Lane M, 100-bp ladder; lanes 1 to 8, positive samples; lane 9, positive control V. cholerae O1 strain O425; lane 10, negative control (PCR master mixture).