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 Río 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.Cholera reemerged in Latin America in 1991 after being absent from the continent for nearly a century. In Argentina, there have been seven epidemics since 1992, all of which were caused by El Tor biotype Vibrio cholerae O1. These cholera outbreaks occurred mainly during the summer months, and, consistent with reports from other geographic regions of the world, strains of V. cholerae O1 were isolated from water samples collected from rivers during epidemic periods. However, the microorganism could not be recovered from the environment during interepidemic periods (12).V. cholerae is a natural inhabitant of aquatic environments, and it has been found to survive for extended periods in estuarine and brackish waters (12,13,15). Furthermore, in response to extreme environmental conditions, the bacteria may enter a dormant state (7, 30), which has been designated the viable but nonculturable (VNC) state, since the organism cannot be recovered by using traditional culture media. It has been hypothesized that it is in the VNC state that V. cholerae survives in the environment during interepidemic periods. Also, interaction with plankton appears to play an important role in the ecology of the microorganism and to facilitate persistence, mainly in response to low temperatures and reduced nutrient concentrations (12,13,14,19,21).In Argentina, V. cholerae non-O1 was isolated from samples collected from the Río de la Plata in previous studies (5). Several facts, such as the seasonality of the outbreaks most likely associated with temperature shifts and plankton blooms, suggested that there are environmental reservoirs of V. cholerae. Additionally, in a recent study in which the genetic diversity of V. cholerae O1 strains isolated during the seven outbreaks that occurred in this country was analyzed, most of the cholera toxin (CT)-producing strains were found to belong to a single clonal group already recognized as the Latin American epidemic stra...

Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage

“…The hypothesis that cholera epidemics are triggered by environmental factors has been embraced by many environmental microbiologists, climatologists, and ecologists during the last decade (18)(19)(20). The fact that cholera epidemics seem to occur in several different foci simultaneously (21, 22) is often cited as evidence for an environmental component to its epidemiology.…”

“…However, because the nonculturable state by definition is a condition when cells are incapable of forming a colony on commonly used media, it has been impossible to isolate the strains responsible for generating such cells in the environment. Thus, reported demonstrations of the existence of viable but nonculturable (VBNC) V. cholerae cells in the environment relied on fluorescent antibody techniques (19,20). Some reports suggest that V. cholerae can be converted to a VBNC state under defined laboratory conditions, and in one such case, nonculturable cells were putatively converted to viable cells by passage through the guts of human subjects (28).…”

Growth of Vibrio cholerae O1 Ogawa Eltor in freshwater

“…Accordingly, V. cholerae O1 was frequently detected in estuary and coastal waters, and growth in sterile-filtered natural seawater under defined laboratory conditions has been reported (Binsztein et al, 2004;Louis et al, 2003;Mourino-Perez et al, 2003;Worden et al, 2006). In addition, laboratory studies using artificial seawater demonstrated growth of V. cholerae O1 down to a salinity of 5 g l 21 (Singleton et al, 1982a, b).…”

Antagonistic effect of Anabaena fertilissima CCC597 on pathogenic Vibrio cholerae propagating in association with cyanobacterial community in freshwater bodies of Eastern Madhya Pradesh

“…Their purity was checked by taking the ratio of their absorbance at A260/A280 nm. The yield of each sample was also calculated by using the following formula: A260 × dilution factor × 50 µg ml -1 PCR amplification reaction for detecting VCO1/ VCO139 genes of V. cholera Amplification reaction was carried out for V. cholerae O1 and O139 strains associated with bloom/scum materials using above DNA preparations and primer pairs specific for VCO1 and VCO139 genes as detailed subsequently (Binsztein et al, 2004), procured from Imperial Life Sciences, India. Reaction mixture was prepared according to Jungblut and Neilan (2006) and Kumar et al (2011) and thermal cycling was performed according to the Binsztein et al (2004) with an initial denaturation step at 94°C for 5 min, followed …”

“…Reaction mixture was prepared according to Jungblut and Neilan (2006) and Kumar et al (2011). Thermal cycling and subsequent electrophoresis of amplified products was carried out according to the Binsztein et al (2004) using the primer pairs selective for VCO1 and VCO139 genes.…”