Isolation and Identification of Total Heterotrophic Bacteria and Human Pathogens in Water and Sediment from Cuddalore Fishing Harbour after the Tsunami

MATERIALS AND METHODS

Study area: The Uppanar estuary is located at Cuddalore (Lat. 11 43’ N; Long 79 49’ E) on the South East coast of the peninsular India (Harbor). Field collections were carried out during July-December, 2008 at two seasons viz, pre monsoon and monsoon (Fig. 1).

Sample collection: Water quality parameters such as temperature, salinity and pH were monitored. Atmospheric and surface water temperatures were measured using standard mercury filled centigrade thermometer, salinity was estimated with the help of a hand Refractometer (Atago, Japan) and pH was measured using Elico pH meter (Model LC-120). The surface water sample was collected from sterile screw capped bottles for bacteriological assessment and the sediment sample was collected by employing an alcohol rinsed and air-dried small Peterson’s grab, which were aseptically transferred into new polyethylene bags using a sterile spatula. All samples were brought to the field laboratory in portable icebox with in 2 h.

Bacteriological analysis: One milliliter of water sample and 1 g of sediment sample was added in 99 mL of 50% of aged sea water separately and then serially diluted using the same diluents. A 0.1 mL of the serially diluted sample was inoculated in to the Zobell marine agar and some selective media (TCBS, EMB) to enumerate the THB and also to isolate the specific pathogens. After inoculation the plates were incubated in an inverted position at a temperature of 28±2^oC for 24 to 48 h.

Fig. 1:	Map showing the location of sampling site in Cuddalore Fishing Harbour

After incubation the colony was enumerated. Heterotrophic bacterial population was expressed as colony forming units in water (cuf mL^-1) and sediments (cfu g^-1).

RESULTS

Seasonal variation in water quality parameters: Temperature is considered one of the most important environmental factors affecting growth and survival of microorganism which ranged from (27-36.5°C) (Fig. 2). Another important factor is salinity which profoundly influences the abundance and distribution of the microorganism in the estuarine environment which ranged from (20-30.4 ppt) (Fig. 3). The hydrogen ion concentration was recorded minimum in the month of July and maximum in the month of August and November which ranged from (8-8.3) (Fig. 4). Thus the seasonal variations of water quality have been change depends upon the temperature, salinity and pH of overlying water and nature of effluent discharges from industries.

Total heterotrophic bacterial population: THB load in sediments were ranged from 11.3x10³ cfu g^-1 as maximum and 3.0x10⁶ cfu g^-1 as minimum and in water 10.5 x10³ cfu mL^-1 as maximum and 3.2x10⁶ cfu mL^-1 as minimum in monsoon season. A total of 10 strains were isolated from water and sediment samples from Cuddalore fishing harbour (Fig. 5).

Fig. 2:	Water temperature of the Uppanar estuary from the July to December 2008

Fig. 3:	Water Salinity of the Uppanar estuary from the July to December 2008

Fig. 4:	pH of the Uppanar estuary from the July to December 2008

Based on colony morphology 3 genera were identified (Table 1) viz., V. cholerae, V. parahaemolyticus and E. coli (Fig. 6, 7).

Population density of V cholerae in the water sample was higher (5.1x10⁴ cfu mL^-1) at monsoon and lower (2.4x10⁴ cfu mL^-1) in premonsoon whereas in case of sediment (5.9x10³ cfu g^-1) in monsoon and minimum (2.1x10³ cfu g^-1) in pre monsoon were recorded V. parahaemolyticus the population density was higher in water sample (3.3x10³ cfu mL^-1) at monsoon and lower (2.1x10³ cfu mL^-1) in pre monsoon but in case of sediment higher density (4.1x10³ cfu g^-1) in monsoon and the lower density (2.7x10³ Cfu g^-1) in pre monsoon were recorded.

Fig. 5:	Population density of V. cholerae in water and sediment samples collected from Cuddalore fishing harbour

Fig. 6:	Population density of V. parahaemolyticus in water and sediment samples collected from Cuddalore fishing harbour

Fig. 7:	Population density of E. coli in water and sediment samples collected from Cuddalore fishing harbour

Population density of E coli was higher in water (3.4x10⁵ cfu mL^-1) in monsoon and lower in (1.9x10⁵ cfu mL^-1) in premonsoon and in sediment sample higher density (4.9x10³ cfu g^-1) in monsoon season and lower (1.7x10³ cfu g^-1) in pre monsoon (Fig. 8-12). Identification of pathogenic bacteria followed by Bergey’s manual (Table 1).

Fig. 8:	The THB in Zobell’s Marine Agar (Water sample)

Fig. 9:	The THB in Zobell’s Marine Agar (Sediment sample)

Table 1:	Identification of pathogenic bacteria followed by Bergey’s manual

Fig. 10:	The quadrant streaking of V. cholera (TCBS-Sediment)

Fig. 11:	The quadrant streaking V. parahaemolyticus (TCBS -Sediment)

Fig. 12:	The quadrant streaking of E. coli (EMB-Sediment)

DISCUSSION

A change in physico chemical parameters alters the microbial environment leading to alteration in microbial community. In the present study the Cuddalore fishing harbor area was affected by tsunami waves. This alteration of the environment and the microbial population of this area is identifiable from the study. Water sampled immediately after the Tsunami in 2005 showed a reduction in TVC and in the counts of E. coli and Vibrio sp. However in the later month, the water and sediment sample showed a high in the bacterial flora (Balasubramanian, 2005). The environmental factors such as pH has low influence on the survival and proliferation of this bacterium particularly when nutrient loading is heavy in the coastal water due to discharge of sewage and industrial effluents (Velammal, 1993). Thus, the present study revealed the load of Vibrio was more in alkaline pH.

Present investigation highlights the ubiquitous distribution of the THB and pathogenic bacteria in the water and sediment samples collected from the Uppanar estuary. THB and pathogenic bacterial densities were higher in sediments than in water samples. Wollast (1991) reported that the coastal and shelf sediments play a significant role in the demineralization of organic matter which supports the growth of microbes. Anon (1997) also reported the higher bacterial population density in the sediments than water in generally due to the rich organic content of the former and the lesser residence time of the microorganisms in the water column than the sediments. Total heterotrophic bacterial population various from seasons to season. The high bacterial population during monsoon may be due to the rain water flow which brings huge quantities of nutrients (Martin, 1981; Sathiyamurthy et al.,1992). During summer the population level was maintained at low in water (Velammal, 1993; Natarajan et al., 1980) also observed very low levels of pathogens in estuarine and marine waters during summer season. In addition to temperature and salinity, nutrients availability also plays a major role in the distribution of marine bacteria (Carlucci, 1974). Pathogenic bacteria such as V. cholerae, V. parahaemolyticus and E. coli have been recorded. Population density of Vibrio sp. in the marine environment is usually more because Vibrios can occur in a wide range of aquatic environments including estuaries, marine and coastal waters and sediments (Urakawa et al., 2000; Thompson et al., 2004). In the present investigation Vibrios contributed only 72% and E. coli contribution only 15% contribution in the coastal areas of the Uppanar estuary, which incase higher microbial population due to Vibrio in the coastal environment of the Uppanar estuary.

The greatest enhancement of total heterotrophic bacteria was seen in the present study when compared with the counts of total heterotrophic bacteria like Vibrio spp. and E coli in Uppanar and Vellar estuary. Jayalakshmi (1992) recorded 3.6x10⁹ cfu mL^-1 in water and 1.5x10⁷ cfu g^-1 in sediment during monsoon and also reported Vibrio sp. in the month of monsoon 6.4x10¹ cfu mL^-1 in water and 8.3x10¹ cfu g^-1 in sediment. E. coli also isolated in monsoon season like 8.7x10⁴ cfu mL^-1 in water and 9.6x10⁴ cfu g^-1 in sediment. Premalatha (2001) recorded Vibrio sp. 3.8x10⁴ cfu mL^-1 in water and 3.4x10⁵ cfu g^-1 in sediment, during monsoon season and distribution of THB in Uppanar estuary during the month of monsoon season, the higher population was observed like 8.7x10⁶ cfu mL^-1 in water and 9.4x10⁷ cfu g^-1 m in sediment. The early reports supports to the result obtained as and in the sediments showed it’s lower of 7x10³ cfu g^-1 in (summer) and higher of 46.5x10³ cfu g^-1 in (Monsoon). This trend could be the rescan of Tsunami water (Swaranakumar et al., 2008). In areas where there is not a lot of sunlight, bacteria thrive and maintain a good population. In the presence of sunlight the bacteria become inactive and eventually die. Visual light may be the cause rather than the ultra violet for the effect and decrease in the bacteria population (Fujioka et al., 1981).

Whereas, the present study shows similar values of V. cholerae, V. paraheamolyticus and less values of E. coli when compared to them. However, the sediment was found to record higher values in the investigated areas compared to the water column. The microbial population was found to have only marginal differences between sediment and water column after tsunami (Table 2).

Table 2:	Comparison of bacterial load with the earlier studies

CONCLUSION

The present observation on the THB of Uppanar estuary revealed higher level of population in the sediment and the water column. In the present study, an increase in population density was noticed during the monsoon season (11.3x10⁴ cfu g^-1) this was due to the addition of terrigenous material through land run off carrying high bacterial population. This could have been achieved by eluting a large number of soil bound bacteria and transporting them into the study area.

ACKNOWLEDGMENT

Authors thanks Prof. T. Balasubramanian, Director Faculty of Marine Science, Centre of Advanced Study in Marine Biology, Annamalai University for providing permission and facilities in the study period.