The effect of shrimp chitin on the life cycle of Vibrio cholerae and the subsequent transmission of cholera

The bacterium Vibrio cholerae O1 causes epidemics of the disease cholera, and incidences have been well-documented in areas such as Bangladesh. This pattern is particularly clear at two main seasonal peaks each year, which coincides with plankton blooms in the spring and in the autumn. Chitin, a highly abundant substrate in the marine environment, is thought to affect the population dynamics of V. cholerae, and it is mainly found in the exoskeletons of crustaceans. These invertebrates feed on the highly abundant zooplankton during the blooms, which act as a reservoir for the bacterium. Chitin is heavily colonised by the chinolytic V. cholerae, which breaks it down into soluble constituents that can be further colonised by these bacteria.

                   

 There are three stages to the life cycle of V. cholerae; the non-culturable state, the reservoir, and the culturable, toxigenic stage which results in cholera transmission. However, the mechanisms and the capacity of the bacteria to regain their culturability are not well understood. Shrimp chitin from the estuarine of Bangladesh was used as the sole nutrient in two different types of microcosms (artificial ecosystems created for this experiment), the Mathbaria water (MW) and Mathbaria water supplemented with chitin chips (MW-CC), to determine how chitin influences both the natural cycle of V. cholerae and seasonal occurrence of cholera in this area of Bangladesh.

DFA Counts

In the initial counts for Direct Fluorescent Antibodies (DFA), there were similar numbers of V. cholerae in the MW and MW-CC microcosms. However, a gradual reduction in counts was found in both microcosms in the subsequent weekly intervals, with higher cell count for a longer duration in MW-CC (Cells in MW water were only counted for 49 days). The cells in the MW-CC microcosm remained culturable for longer than 174 days (when growing on LB agar), but the counts declined to <10 before the next interval at day 189.

Multiplex-PCR

Two particular toxigenic genes in V. cholerae, wbe and ctxA, could still be amplified using Multiplex-PCR (M-PCR) up to day 174 for MW-CC, but only for 49 days in the MW microcosm. However, the M-PCR only shows the presence of multiple genes, and do not mention whether the genes are expressed or not. It may be more appropriate to use reverse-transcriptase-PCR (RT-PCR) to give a more accurate length of time that the toxigenic genes are expressed for and hence how long V. cholerae remains in this life stage.

MW with Added Shrimp Chitin Chips

Chitin degradation was recorded for six months, and it was observed that chitin became degraded after the chips were initially colonised. During the experiment, it was noted that large numbers of cells formed clusters of biofilm, and after further degradation, the majority of the V. cholerae bacteria were embedded within the biofilm up to day 189, although there was still some further colonisation of the degraded residue. V. cholerae remained culturable up to 174 days, but no longer showed active growth by day 189.

Addition of HCl

The chitin biofilm formed by the bacterium was thought to act as a shelter from harmful environmental conditions, as it is suggested that V. cholerae can survive the stomach acid in humans when they consume drinking water from the Mathbaria waters containing chitinous material. When concentrated HCl was added, it was found that the homogenate of the chitin biofilm still had 10⁴ cfu/ml of toxigenic cells present. These results could be supported further by repeating the experiment in the control MW microcosm as a comparison.

The study gives support for the ability of V. cholerae O1 to persist in the plankton reservoir between epidemics using chitin in estuarine water, which shows that this biopolymer keeps the cells in the exponential active growth phase for a longer duration. The V. cholerae that are normally found in the environment are typically toxigenic, but the chitin acts as a food source to activate the toxigenic stage. However, this paper seems to assume that chitin degradation is the single cause for the activation of toxigenic V. cholerae, even though there could be a multitude of interacting factors involved, and the authors make many inferences biased on their experiments. 

Ellie Vaughan & Dave Watt

Nahar, S., Sultana, M., Naser, M.N., Nair, G.B., Watanabe, H., Ohnishi, M., Yamamoto, S., Endtz, H., Cravioto, A., Sack, R.B., Hasan, N.A., Sadique, A., Huq, A., Colwell, R.R., and Alam, M. (2012) Role of shrimp chitin in the ecology of toxigenic Vibrio cholerae and cholera transmission. Frontiers in Microbiology, 2(260). doi: 10.3389/fmicb.2011.00260