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American Journal of Applied Sciences
Year: 2009  |  Volume: 6  |  Issue: 3  |  Page No.: 439 - 444

Survival of Vibrio cholerae O139 in Association with Anabaena variabilis in Four Different Microcosoms

Zannatul Ferdous    

Abstract: Investigation was carried out to see the association of Vibrio cholerae O139 with Anabaena variabilis, in four different types of water. Conventional media were used to count culturable bacteria while epi fluorescence microscopy to count the viable but non culturable (VBNC) V. cholerae O139. It was found that the culturable form of V. cholerae O139 found 9 days in the control artificial sea water and 4 days in river water and BOD dilution water. However, in association with A. variabilis, culturable form was found for 6 days in artificial sea water, 4 days in distilled water, 1 day in BOD dilution water, 4 days in river water. The bacterial count of surrounding water decreased inversely with the bacterial count in association with algae. Among these four microcosm water, associations of V. cholerae O139 with algae was higher in artificial sea water. In control water of four microcosms, direct viable count (DVC) gradually decreased with time. In association with algae the DVC increased after some time then reached a peak in 4-8 days depending on the types of water. The epi fluorescence microscopy indicated that V. cholerae O139 survived for more than one month in association with algae. Present study demonstrated that survival of V. cholerae O139 appears to get advantages in association with A. variabilis in different types of water.

Fig. 1 showed the survival pattern of V. cholerae O139 with Anabaena sp. in artificial sea water. In control flask water the culturable count increased 1.038 log10 CFU mL-1 from 0 h-24 h Then culturable count slightly decreased throughout 2nd day, 4th day and 6th day reading and reduced to 3.374 log at 9th day. After that no culturable V. cholerae O139 could be detected even after enrichment in APW. Incase of water where Anabaena sp. was floating (supernatant) the culturable count increased 1.113 log from 0-4 h and then the culturable count of V. cholerae O139 decreased to 2.225 log at 6th day. In association with algae the culturable count increased 0.961 log at 4th day of sampling.

In the control flask of distilled water (Fig. 2) the culturable count increased 0.873 log from 0 h to day 3. This count slightly decreased an amount of 0.382 log on day 9. In case of water where Anabaena sp. was floating the culturable count decreased an amount of 3.973 log from 0 h to day 3. In association with algae the culturable count increased 0.312 log from 0 h-4 h. This culturable count deceased an amount of 2.523 log at 3rd day reading.

In control flask of river water (Fig. 3) the culturable count increased 0.056 log from 0-6 h. Then culturable count gradually decreased 3.398 log at day 4. In case of water where Anabaena sp. was floating the culturable count gradually decreased 0.306 log from 0 h to day 2. In association with algae the culturable count gradually increased 0.286 log from 0 h to day 2. This culturable count deceased 1.136 log at day 4.

Figure 4 showed the survival and culturable pattern of V. cholerae O139 with Anabaena sp. in BOD dilution water. In contol flask water, the culturable count increased 0.325 log from 0 h to day 4. Incase of control water, V. cholerae O139 survived upto 4 days. In case of water where Anabaena sp. was floating the culturable count decreased to 1.19 log from 0 h of sampling to 24 h.

Fig. 1: Culurable count of V. cholerae O139 in artificial sea water microcosm (ph 8.0)

Fig. 2: Culurable count of V. cholerae O139 in distilled water microcosm (ph 8.0)

Fig. 3: Culturable count of V. cholerae in river water microcosm (pH 7.94)

In association with algae the culturable count increased 0.488 log from 0-2 h of sampling but a decrease of 0.322 log was observed at 24 h reading.

In artificial sea water the multiplication of V. cholerae as VBNC state was observed in association with Anabaena sp. (Fig. 5). The initial count of VBNC cell was 6.68 log 10 cells/gm which increases to 7.28 log10 cells/gm on day 4 and at the end of the experiment, the count was 6.431 log10 cells/gm.

Fig. 4: Culturable count of V. cholerae in BOD dilution water (pH 7.70)

Fig. 5: Flurescent antibody Direct Viable Count (DVC) of V. cholerae O139 in artificial sea water microcosm ( pH 8.0)

The count in algal water at 0 h was 5.57 log10 cells/mL, decrease to 3.69 log10 cells/mL on days 14. At 30th day the count was 3.75 log10 cells/mL. No significant increase or decrease of VBNC count was observed in control water.

In river water the VBNC count of V. cholerae in Anabaena sp. was increase to 7.1 log10 cells/gm from the initial 6.11 log10 cells/gm at 8 day while at 14th day, the count decrease to 4.41 log10 cells/mL algal water (Fig. 6). Insignificant changes of VBNC count was found in control water.

In distilled water, the VBNC count of V. cholerae either inside or surface of the Anabaena sp. gradually increased at day 8 (from initial 6.15 log10 cells/gm to 8.07 log10 cells/gm) and then gradually decreased to 7.17 log10 cells/gm at day 30 (Fig. 7). In Anabaena water VBNC of V. cholerae maintained a steady state with initial count and the end count (5.87 log10 cells/mL at 0 h, 5.44 log10 cells/mL at day 30). A decrease of 1.01 log10 cells/mL was observed in control water from 0 h-day 30.

Fig. 6: Flurescent antibody Direct Viable Count (DVC) of V. cholerae O139 in distilled water microcosm (pH 8.0)

Fig. 7: Flurescent Antibody Direct Viable Count (DVC) of V. cholera O139 in River water microcosm (pH 7.94)

Fig. 8: Flurescent antibody Direct Viable Count (DVC) of V. cholerae O139 in BOD dilution water microcosm (pH 7.70)

V. cholerae O139 of control water of BOD dilution water, did not show any significant changes in DVC count at 0 h for DVC was log10 5.252 cells/mL ( Fig. 8). In association with Anabaena sp. the highest count of DVC was found at day 8 (log10 7.279 cells/mL). After day 8 the DVC counts gradually decreased to log10 5.960 cells/mL at day 30. In case of water where Anabaena sp. was floating, DVC did not differ very much from 0 h to day 8 (log10 4.954 cells/mL, log10 5.176 cells/mL respectively). The DVC could be observed at day 14 sample and onwards.

Artificial sea water provided the best environment for the V. cholerae O139 for more attachment with algae in this experiment. On the other hand, BOD dilution water seemed less friendly for the growth of V. cholerae O139. This suggests that water quality has role in the survival of V. cholerae in association with Anabaena sp. This finding can be supported by previous studies. Water quality plays important role in bateria-algal association and transmission of cholera[23,24]. Here the culturable count in algal water was inversely proportionate to the count of bacteria-algal association. Higher count found in algal water at initial stage which gradually decreased within 2-4 days. This trend was completely opposite in bateria-algal association. It inferred that V. cholerae O139 entered into the mucilaginous sheath of algae for shelter and nutrition. This finding is also supported by the study of Schneider and Parker[25] where they found that V. cholerae can secrete an enzyme mucinase which degrade the mucin and mucin like substances present in the plant cells, helps to penetrate bacteria into algae. More studies have demonstrated that blue green algae, Anabaena sp. may act as a reservoir of V. cholerae[26]. Islam[27] also demonstrated that V. cholerae O1 can survive and attach in association with four marine algae longer period than water surrounding marine algae. This also infers that V. cholerae can make association with algae. Strain V. cholerae O139 remained in VBNC in blue-green algae in large number than in control water or in algal water. This result is concordance with another study by Munro[28] where they found that in sea water VBNC V. cholerae O1 may survive for some times.

In brief this research leads to following findings: Survival rate of the bacteria with the algae depends on the quality of the water, V. cholerae O139 survives long time in control water because of the absence of nutrient intake by algae and interference of other bacteria and A. variabilis can act as a reservoir of V. cholerae O139 where they can hide for long time as VBNC form.

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