Estuaries and coastal regions are continuously subjected to natural as well as various forms of anthropogenic activities. These anthropogenic activities can change the quality of coastal water rendering it unsafe for recreation or human use1,2. The sources of water pollution include point (sewage discharge, domestic wastes and industrial effluents) and non-point sources (agricultural and urban runoff) which brings pathogenic bacteria into the coastal and estuarine waters3. The presence of various pathogens (e.g. Escherichia coli, Salmonella spp., Shigella spp., Vibrio cholerae, V. parahaemolyticus, V. alginolyticus, Pseudomonas spp., Enterococcus faecalis, total coliforms etc.) have been reported in the estuarine and coastal waters of India4-10. Most of these pathogens are responsible for waterborne diseases (gastroenteritis, diarrhea, dysentery, typhoid, cholera, food poisoning and wound infection) in the humans3, either due to consumption of contaminated sea food or contact with water7. The attachment of allochthonous pathogenic bacteria to the particulate matter in the water column protect themselves from other environmental and biotic factors and are found in high numbers in the sediments rich in organic matter, silt and clay11,12. Sediment acts as a reservoir of pathogenic bacteria in the estuarine systems12,13. There are reports on the re-suspension of coliforms and faecal indicators from sediment bed due to recreational activities, tidal currents and storms14,15. The re-suspension of sediment could be another source in adding pathogenic bacteria to the overlying water column other than anthropogenic activities. The transport of re-suspended sediment particles is land-ward during flood tide and bay-ward during ebb tide16.
Vibrio cholerae, V. parahaemolyticus and V. vulnificus are generally found in the marine and brackish environments and are serious human pathogens17. Kanungo et al.18 reported that high numbers of cholera outbreaks in India were found in West Bengal, Orissa, Maharashtra and Kerala from 1997-2006. According to World Health Organization (WHO), a total of 589,854 cholera cases were found in 58 countries worldwide during 201119. As sediment acts as reservoir of pathogenic bacteria, monitoring their load in the sediment can provide stable indicator of long-term bacterial abundance in the water column20.
Zuari estuary is located along the central west coast of India (15ο27.5′ N, 73ο48′ E) and is influenced by the south west (SW) monsoon21, hereafter referred to as the monsoon. The seasonality in this estuary can be categorized into three seasons: Pre monsoon (February-May), Monsoon (June-September) and Post monsoon (October-January)22. During the monsoon, the run-off is reported to range from 100-400 m3 s-1 in the Zuari estuary23. The amount of sewage discharged into the Mandovi-Zuari estuaries is estimated to be about 30 million litres per day24. Zuari estuary is used for navigation purpose, fishing, shellfish harvesting, transport of ores, discharge of wastes from transport ships, port and tourist related activities. There are few studies on the abundance of pathogens in the sediments in the midstream of the channel and the bay of the Zuari estuary6,25-27. The present study assessed the distribution of pathogenic bacteria in the surface sediments along the estuarine banks which are the active sites of anthropogenic activities. Fortnightly sampling was carried out in the Zuari estuary for a period of 17 months from November 2010-May 2012 covering all three seasons (pre monsoon, monsoon and post monsoon) of the year. The present study also addressed the influence of tidal conditions observed during the sampling period on the distribution of pathogenic bacteria in the sediments.
Sampling was carried out in the Zuari estuary which is located along the central west coast (Goa) of India (Fig. 1). Zuari river has length of 50 km and average depth of 5 m. The mouth of the estuary is funnel-shaped with ~5 km width and narrows down towards the head of the estuary28. The estuary remains well mixed during pre monsoon season. However, it is stratified during monsoon season with less dense fresh water on the surface and more dense saline water at the bottom. The tides are semi-diurnal, with a height of ~2.3 m during spring tide and ~1.5 m during neap tide21. The surface sediment samples were collected from seven selected sites lining the banks of the Zuari estuary and divided into 5 categories (Fig. 1). The station 1 (Odxel Beach – OB) and station 2 (Bambolim Beach – BB) are categorized as beach influenced stations. Stations 3 (Agacaim – AG) and 4 (Cortalim – CR) are located towards upstream and influenced by movement of ferry boats and fishing trawlers. The anthropogenic and industrial influence is prominent at station 5 (Chicalim – CH). The station 6 (Vasco – VS) is close to Marmugao port and is situated towards the mouth of the estuary. Tidal influence is high at station 7 (Marmugao Bay – MB) which is located at the mouth of the estuary. The details of the sampling locations are given in Table 1.
Collection of samples
Fortnightly sampling was carried out during post monsoon-I (November-December, 2010 and January, 2011; POM-I), pre monsoon-I (February-May, 2011; PreM-I), monsoon (June-September, 2011; MON), post monsoon-II (December-January, 2011/2012; POM-II) and pre monsoon-II (February-May, 2012; PreM-II) seasons at the Zuari estuary. The sampling was initiated at station 1 at ~7:00 am and concluded at station 7 at ~11:00 am. The different phases of tides were noticed during the sampling period in the tide table. A slack period before ebb tide was observed during POM-I sampling and ebb tide during POM-II. Flood tide was observed during PreM-I season and ebb tide during PreM-II. Monsoon sampling was done during the slack period before flood tide. Thus, except, PreM-I season, all other samplings were carried out either during slack period before ebb or flood tide or during ebb tide. Surface sediment samples were collected by using a van Veen grab and top ~2 cm of sediment was used for assessing the Total Viable Count (TVC), pathogenic bacteria, Total Organic Matter (TOM), Total Organic Carbon (TOC), Total Nitrogen (TN), proteins and carbohydrates. Sediment samples to be analysed for total bacterial count (TBC) were fixed with formaldehyde (final concentration 1-2 % v/v). The samples were transported to the laboratory in an ice box. The near bottom water samples were also collected by using Niskin sampler for analysis of dissolved oxygen (DO), salinity and temperature. The Winkler titrimetric method was used for estimation DO29. Digital Thermometer (EUROLAB) and Master Refractometer (ATAGO, Japan) were used to measure the temperature and salinity of sea water respectively.
Enumeration of Total Viable Count (TVC) and pathogenic bacteria in the surface sediments
Enumeration of bacteria in the sediment samples was carried out by using standard spread plate technique. The different selective media were purchased from HiMedia (Mumbai) and prepared in distilled water following manufacturer’s instructions. One gram of wet sediment was mixed in 10 ml of filtered, autoclaved sea water followed by vigorous shaking to dissociate the bacteria from the sediment and left for 5-10 min for sediment particles to settle. Subsequently, 1 ml of supernatant was serially diluted in 9 ml of filtered, autoclaved sea water to get 101, 102, 103 dilutions and 0.1 ml from each of the dilutions were plated on Zobell Marine Agar (ZMA) for total viable (culturable) bacteria and incubated at room temperature (28±2ºC) for 24 hrs. Pathogenic bacteria were enumerated by spreading samples on different selective media like MacConkey agar which differentiates E. coli (pink/red coloured colonies) from Shigella/Salmonella species (transparent, colourless colonies). All colonies on MacConkey agar are reported as total coliforms (TC = E. coli and Shigella/Salmonella spp.). Thiosulphate-citrate-bile-salts sucrose (TCBS) agar was used to differentiate Vibrio spp. depending on size and colour. V. cholerae grow as raised, yellow coloured colonies having diameter <2 mm, V. alginolyticus are bigger (>2 mm diameter) in size and produces yellow coloured colonies. However, V. parahaemolyticus develop as green coloured colonies on TCBS agar. Xylose-Lysine Deoxycholate (XLD) agar differentiates Salmonella (pink coloured colonies with black centre) from Shigella (pink colonies) species. Enterococcus confirmatory agar was used for Enterococcus faecalis (blue coloured colonies) and General Streptococci species (white colonies). Hichrome EC O157:H7 selective agar was used for E. coli O157:H7 (purple coloured colonies) in which Hichrome EC O157:H7 selective supplement was added aseptically. All selective media for specific pathogens were incubated at 37ºC for 24 hrs and the counts are expressed as Colony Forming Unit (CFU) g-1. The abundance of Shigella and Salmonella spp. were determined later to POM-I season (November-December, 2010).
Enumeration of Total Bacterial Count (TBC) in the surface sediments by using Flow Cytometry
Flow Cytometry (BD-Biosciences, USA) was used to analyse formaldehyde fixed sediment samples for TBC. Sediment samples (1 g) suspended in 10 ml of autoclaved sea water were sonicated at 50% power in the water bath sonicator (Ultrasonic cleaner, Equitron) for 1 min and three times to separate the cells from sediment particles30 and further centrifuged at 3000 rpm for 1 min and the supernatants were recovered. One ml of supernatant was passed through BD cell strainer cap to remove larger particles and subsequently stained with SYBR Green I (Molecular Probes, USA) at 1:10,000 final concentration and incubated for 15 min at room temperature in the dark31. After incubation, the samples were analyzed using a BD FACSAriaTM II (BD-Biosciences, USA) flow cytometer equipped with a nuclear blue laser 488 nm which can differentiate green fluorescence excited by blue laser. Emitted light was collected through the filter sets of 488/10 band pass (BP) for right angle light scatter (SSC) and 530/30 BP for green fluorescence. Fluorescent beads (1μm, Polysciences) were used as internal standards for calibration. Gating was done against SSC versus green fluorescence (FITC) using BD FACS Diva software and results are expressed as cells g-1 of sediment.
Analysis of biogeochemical parameters of surface sediments
Before analysis, the sediment samples stored at -20ºC were thawed, dried at 50ºC and ground to fine powder using mortar and pestle. The sedimentary TOC content was determined by wet oxidation with chromic acid as described by El Wakeel and Riley32 and converted into TOM content by using a factor of 1.724 as described by Bhosle and Dhople33 and is expressed as dry weight percentage (%). Total nitrogen (TN) was determined using CHNS analyzer (Vario MICRO Select Elementar) using sulfanilamide as standard and expressed as dry weight percentage (%). The carbohydrate (CHO) content was estimated according to Dubois et al.34 method by using spectrophotometer (UV-1800 Spectrophotometer, Shimadzu) using glucose as standard and expressed as mg g-1 of sediment dry weight. The protein (PRT) content was estimated according to Hartree35 method using bovine serum albumin as calibration standard and results are reported as mg g-1 of sediment dry weight. Sediment samples previously combusted in a muffle furnace at 450ºC for 4 hours were used as the blanks for biochemical analysis.
Data analysis
A correlation analysis was performed in order to understand relationship between different bacterial species (log transformed) and near bottom water salinity, dissolved oxygen and tide. This analysis was done using Statistica 8.0 statistical package.
Results
Physico-chemical parameters of the near bottom water
The near bottom water temperature during the study period varied from 25.7ºC-31.1ºC, with maximum temperature during the PreM-I. The average salinity fluctuated between 22 to 34. Salinity was low (4) towards the upstream at CR during MON while, it was high (37) at the mouth (MB) during the PreM-I season. DO in the near bottom water was high during PreM-I and low during POM-II and ranged from 1.55-7.83 mg L-1. A strong correlation between DO and tide (r = 0.67; p ≤ 0.001) indicates the influence of tide on DO in the near bottom water.
Spatio-temporal variations of the bacterial populations (TBC, TVC and pathogenic bacteria) in the surface sediments
The average abundance of TBC in the sediment was high (1.77×108 cells g-1) at CH, a lower middle estuarine station and minimum (6.54×107 cells g-1) at the mouth of the estuary (MB) (Fig. 2a). The abundance of TBC was high during MON, low during POM-I (Fig. 3a) and was positively influenced by near bottom water DO (r = 0.46; p ≤ 0.001) and negatively by salinity (r = -0.26; p ≤ 0.006). Tide showed a significant influence (r = 0.38; p ≤ 0.001) on the distribution of TBC (Table 2). The TVC ranged from 3.64×105-1.48×106 CFU g-1 with maximum abundance towards the upstream at CR during POM-I season (Fig. 2b & 3b).
The average abundance of TC (3.34×105 CFU g-1) and general Streptococci spp. (GS) (1.22×103 CFU g-1) was high during POM-I at CR (Fig. 2c). TC were positively influenced by bottom water DO (r = 0.26; p ≤ 0.006), tide (r = 0.25; p ≤ 0.009) and negatively by the salinity (r = -0.21; p ≤ 0.030) (Table 2). The average abundance of TC decreased from POM-I to POM-II season (POM-I>PreM-I>MON>POM-II) (Fig. 3c). E. coli O157:H7 was found towards upstream stations only during MON (Supplementary Table 1) and were high (3.90×103 CFU g-1) at CR. The Shigella spp. and VA were high towards the mouth of estuary during PreM-II, whereas, Salmonella spp. were high towards the upstream during PreM-I. The abundance of VC ranged from 3.02×104 to 8.48×105 CFU g-1 and was high at MB, the mouth of the estuary during POM-I (Fig. 2d) and positively influenced by near bottom water salinity (r = 0.23; p ≤ 0.015). However, VP were high (9.85×104 CFU g-1) towards upstream at AG (Fig. 2f) and influenced by tide (r = 0.25; p ≤ 0.009) (Table 2). Seasonal variation showed high abundance of VC and VP during POM-I (Supplementary Table 1). The percentage occurrence of pathogenic bacteria in the surface sediments showed dominance of VP (88.8%) and VA (80.9%) among autochthonous Vibrio spp. and TC (79.3%) and Shigella spp. (69.6%) among allochthonous pathogens (Table 3).
Biogeochemical composition of the surface sediments
The upstream stations (AG and CR) and anthropogenically influenced lower estuarine station (CH) were characterised by relatively high content of TOM, TOC and TN as compared to stations towards the mouth of the estuary (Table 4a). TOM, TOC and TN in the surface sediments ranged from 0.08 to 6.98%, 0.05 to 4.05% and 0.00 to 0.35% respectively at the Zuari estuary (Table 4b). High amount of TOM and TOC was observed during MON. The spatial and seasonal distribution indicated that the concentration of CHO was more than PRT in the sediments. The concentration of PRT and CHO varied from 0.13 to 16.1 mg g-1 and 0.45 to 18.6 mg g-1 dry weight of sediment respectively (Table 4b).
Discussion
Bacteria in the estuarine and marine environment play a significant role in the biogeo-chemical processes, nutrient cycling and organic matter degradation36. The present study showed high abundance of TBC in the sediments of CH, a lower middle estuarine station which is influenced by anthropogenic activities. This site is actively used for shipbuilding workshops, yards and subjected to various land-based anthropogenic activities37. A recent study on microbial community structure in the surface sediment of CH in the Zuari estuary reported dominance of Gammaproteobacteria which includes members of potential pathogens such as Enterobacteriaceae, Vibrionaceae and Pseudomonadaceae38. In general, high abundance of TBC was observed during MON. Large amount of fresh water discharge from catchment area during MON could be one of the sources bringing in land run-off rich in bacterial load and anthropogenic input. Zuari estuary receives maximum sediment discharge during peak rainfall22. Recently, Shynu et al.39 studied the ɗ13Corg in the sediment and reported terrestrial organic matter to be the dominant component in the lower region of the Zuari estuary during MON. The ratio of TOC/TN in the sediment has been used to distinguish the autochthonous or marine organic matter from allochthonous or terrestrial input. The protein rich algae and plankton are characterised by low C/N ratio (4-10) than terrestrial land plants (>20) which are rich in cellulose40. TOC/TN ratio in the present study was 15.44 during MON which indicates mixed input of terrestrial and marine derived organic matter in the surface sediments and could be another possibility for high abundance of TBC. Mahalakshmi et al.41 also observed high density of heterotrophic bacteria in the sediment and water column during the MON at the Uppanar estuary located on the south east coast of India.
The TVC, TC and general Streptococci spp. were high at CR which is located at the entrance of channel near the junction between bay and upstream region of Zuari river and is influenced by tidal currents, waves, movement of fishing trawlers, ferry boats and shallow in depth. Previous study by Dessai and Nayak42 reported that the sediments in this region are rich in silt and clay content due to deposition of fine particles during low energy condition and colloidal aggregates during estuarine mixing, whereas, the mouth region is rich in sand owing to intense tidal currents which causes the transfer of fine grain particles away from the mouth. This station was also characterized by high TOM, TOC, TN, proteins and carbohydrates which possibly favoured high abundance of TVC, TC and general Streptococci spp. A parallel study on the sediment texture reported that the stations towards the upstream are rich in silt and clay (54.6%) content as compared to those located at the mouth (19.3%) of the estuary (Desai and Atchuthan; personal observation). Perkins et al.11 also reported high abundance of pathogenic bacteria in organic matter, silt and clay rich sediments at the Conwy estuary, UK. The attachment of fecal coliforms to the fine particles increases their viability and transport to the sediment bed43. The fine grained sediments with large surface area have more adsorptive capacity for organic matter44 and can influence the accumulation of organic matter and bacteria. TOC/TN ratio indicated input of both autochthonous and terrestrial organic matter at CR which supported high abundance of allochthonous coliforms. The dominance of carbohydrates over proteins and low PRT/CHO ratio (<1) indicates detrital heterotrophic nature of the environment45. The abundance of TC in the present study ranged from None Detected (ND) – 88.7×105 CFU g-1 and was almost similar to the previous study (ND – 47.7×105 CFU g-1) by Ramaiah et al.25 but the abundance of E. coli was 2 folds lower (ND – 3.90×103 CFU g-1) than the previous study (ND – 6.4×105 CFU g-1) by Ramaiah et al.25. However, their study did not include the influence of tides, as the flood tide and ebb tide to certain extent causes decrease in the abundance of pathogenic bacteria from the sediment. A recent study by Khandeparker et al.27 reported that depending on the riverine discharge and tidal amplitude interplay sediment re-suspension mediated increase in SPM significantly regulates bacterial population in the Zuari estuary. The re-suspension of sediment will affect the quality of overlying water by adding SPM, organic matter and pathogens which could be hazardous to human health. The assessment of pathogenic bacteria in the sediment can improve our understanding of the behaviour, fate and mitigation of potential pathogens in the sediment as well as water body46.
The abundance of TVC, TC, general Streptococci spp. and VP was significantly higher during slack period before ebb tide of POM-I than ebb tide of POM-II. This variation in the TVC and pathogenic bacterial abundance can be attributed to the nature of sediment as well as tidal conditions observed during POM. It is well known that the re-suspended particles settle to the sediment bed during slack period before starting of ebb tide and get re-suspended from the sediment with ebb currents, remain suspended for a certain degree and then settle to the bottom47. The pathogenic bacteria attached to the suspended particles increase their downward flux to the bottom sediment11,48. This could be possible reason for high abundance of coliforms, VP and general Streptococci spp. in the sediments towards the upstream station during POM. The negative influence of near bottom water salinity on the abundance of total coliforms in the sediment indicates their extraneous input into the sediment. The presence of pathogenic strain, E. coli O157:H7 in the sediment towards the upstream stations only during MON season can be attributed to land run-off and sewage input. Nagvenkar and Ramaiah26 also reported the influence of land run-off and domestic sewage input on the abundance of fecal coliforms in the Zuari-Mandovi estuaries.
High abundance of V. cholerae was observed at the mouth of the estuary during slack period prior to ebb tide of POM. A recent study by Khandeparker et al.27 also reported high numbers of VC in the water column at the mouth of the Zuari estuary and their abundance was influenced by suspended particulate matter (SPM), nutrients and salinity. The ratio of TOC/TN was low (8.71) at the mouth of the estuary indicating contribution of phytoplankton which could be a possible reason for high abundance of VC, as Vibrio spp. are associated with plankton population49. The abundance of Shigella spp. and VA was high towards the mouth of the estuary during ebb tide of the PreM-II season indicating their deposition during ebb tide. However, Salmonella spp. were high towards the upstream during flood tide of the PreM-I. The flood tide currents causes re-suspension of sediment bed and transfer particles towards land whereas, during ebb currents particles move towards sea and settle to the bottom sediment47,50. An earlier study in this estuary has also pointed out that the effluents containing high numbers of pathogenic bacteria accumulate towards downstream during low tide-period6.
Overall, the present study showed that the percentage (%) occurrence of VP and VA was high in the sediments and is not surprising, as they are autochthonous to the estuarine and marine ecosystems17. High prevalence of coliforms and Shigella spp. among allochthonous pathogens can be attributed to their adaptability and survival in the sediment. Pathogenic bacteria adsorbed to sediment particles protect themselves from UV light, phage attack and protozoan grazing and survive for longer time in the sediment11-13,46. The fecal indicator bacteria can even proliferate and re-grow in the organic matter and fine particle rich sediments51. Moreover, VP emerged as a dominant bacterium among all the pathogens in the sediment during the study period (Table 4). VP can tolerate wide range of salinities and is able to use large numbers of substrates for the growth52,53. VP are found to be associated with copepods and play a significant role in nutrient cycling by mineralization of chitinous material with the help of chitinase enzymes53. Also, it has been reported that VP are associated with phytoplankton and their growth is influenced by decaying planktonic cells54. Zuari estuary is productive during non-monsoon seasons and subjected to heterotrophy during monsoon due to large input of terrestrial organic matter and nutrients through land run-off22,55. Estuaries play a significant role in trapping and modification of autochthonous and terrestrial organic matter56. The present study showed mixed input of marine and terrestrial organic matter in the surface sediments and low PRT/CHO ratio (<1) indicates the presence of detrital organic matter. High prevalence of VP in the surface sediments suggests their role in the mineralization of detrital organic matter derived from marine and terrestrial inputs.
Conclusions
The present study revealed a significant role of organic matter content, seasons and tide on the distribution of pathogenic bacteria in the surface sediments along the banks of the Zuari estuary. The organic matter was of mixed origin (autochthonous and terrestrial) towards upstream and autochthonous at the mouth of the estuary and had profound influence on the distribution of pathogenic bacteria in the surface sediments. The low (<1) PRT/CHO ratio suggests utilization of proteins and a heterotrophic environment in the sediment. High abundance of pathogenic bacteria in the sediment during slack period before ebb tide indicates their deposition in the sediment. VP and VA were the more frequently occurring bacteria among autochthonous whereas, TC and Shigella spp. were dominant among allochthonous pathogens in the sediment. Future studies should address the interaction of pathogenic bacteria with suspended particles, their transport and survival in the sediment which will be helpful in understanding their ecology.