Pathogenic microbial contamination status and antibiotic resistance of isolated Salmonella spp. in well water of Jaffna Peninsula

Background: The incidences of typhoid fever which is caused by Salmonella spp. has markedly increased in Sri Lanka in the recent past. Out of 12, 823 typhoid cases reported during 2005-2014, 2588 cases were reported from Jaffna Peninsula. Health ministry statistics also showed that, occurrence of significant intestinal diseases in Jaffna during 2005-2014. The only way of obtaining water for drinking and domestic consumption of people in Jaffna Peninsula is from dug wells therefore concern on quality of groundwater is important, as these diseases are water borne.Objectives: To evaluate the microbial contamination status with a special emphasis on Salmonella spp. and Shigella spp. and their antibiotic resistance.Methods: Forty sampling points were randomly selected covering the whole peninsula. Total coliform, and fecal coliform, Salmonella spp. and Shigella spp. were enumerated and identified according to the methods given by WHO. Antibiotic resistance of Salmonella sp. and Shigella sp. were carried out using antibiotic susceptibility tests according to CLSI guidelines. AST was carried out using Agar disk diffusion method using a commercially available susceptibility test disk.Results: The results of the study revealed that the 100% of the samples were contaminated with total and fecal coliform and they were not within the range given by the WHO and SLS drinking water quality standards. Further, it was found that 8% of sampling locations were contaminated with Salmonella spp. and all the positive wells are being used to extract water for drinking. However, no Shigella spp. was recorded during the time of sampling. Serovar identification revealed that the isolated Salmonella strains belong to Salmonella weltevreden, a human pathogen which causes water borne intestinal diseases. Further, the isolated Salmonella spp. were tested for their antibiotic susceptibility. Six different commonly used antibiotics for typhoid and intestinal diseases were used. Out of them one strain of Salmonella weltevreden showed resistance to Gentamicin and intermediate resistance to Amoxicillin.Conclusions: Thus, the results of the study is alarming, it relives that almost all the parts of Jaffna Peninsula are contaminated with coliforms. The contamination of ground water sources with Salmonella spp. is also posing a threat. The results also showed that there’s a development of resistance to commonly used antibiotics. Therefore, the importance of proper strategic plan to monitor microbial contamination of ground water along with proper disposal practices of antibiotics in order to avoid development of antibiotic resistance in the environment are much needed

Determination of the Insecticide Azamethiphos in Water and Soil using High Performance Liquid Chromatography

Azamethiphos (AZA) is an organophosphorus insecticide used to control flies and cockroaches. Most of open dump sites in Sri Lanka AZA is applied as insecticide to control fly and studies on the occurrence of the insecticide in the environment after application was not recorded. Thus, the aim of this study was to optimise an analytical method to detect AZA in the environmental samples using High Performance Liquid Chromatography (HPLC). AZA in water samples were extracted using the Solid Phase Extraction (SPE) method following the methanol elute, rotary evaporation at 44.4o C and the final residue was re-dissolved in 2.00 mL of HPLC grade Acetonitrile–water (20:80 v/v) for HPLC analysis. AZA residues in soil samples collected from dump site at Karadiyana was subjected to extract AZAinto Acetonitrile and then the solid phase extraction followingrotary evaporation andre-dissolved in 1.00 mL of acetonitrile-water (20:80, v/v) prior to analysis. Quantification of AZA was carried out using the HPLC system consisting of Agilent 1200 series. Two mobile phases were optimised for the isocratic run (Acetonitrile: water=70:30). About 25 μL sample was injected into at a flow rate of 1 mLmin-1. Concentration of AZA were determined using the peak area (at 294 nm) of following the calibration curve and the detection limit for the method was 1 ppm. Soil and water recovery of the method were 94.65±0.13 μgL-1 and 94.02±0.09 μgL-1 respectively. Levels of AZA in collected water samples from dumping site area ranged from 895.35 μgL-1 to 924.81 μgL-1 and levels of AZA in soil samples ranged around 12,000 μgL-1. Thus the optimise method is to be found more reliable and validate to detect and quantification of Azamethiphos in the environmental samples.Keywords: Azamethiphos, High performance liquid chromatography (HPLC), Solid phase extraction (SPE), Environmental sample

Community-level analysis of anthropogenic impacts on rocky shore communities in Sri Lanka

Human activities threat seashore communities in many areas of the world and their impacts on coastal ecosystem are a matter of increasing concern. Present study describes the anthropogenic disturbances on the rocky shore community structure by comparing the benthic communities of disturbed and non-disturbed areas in Sri Lanka where rocky shores remain virtually untouched so far by experimental studies. Impacts of human disturbances; trampling, handling and exploitation on the community structure of rocky macro benthic assemblages were tested in high-, mid- and low-intertidal area by stratified sampling method at Rumassala marine sanctuary and adjacent two localities at either side of the marine sanctuary, Galle and Unawatuna. Univariate measures, log series model and multivariate techniques were used to discriminate the communities with respect to the disturbances. Proximity of study localities and indistinguishable physicochemical parameters of sea surface water (temperature, Salinity, conductivity, dissolved oxygen and pH) indicated that overall environment of the study area was relatively uniform and changes in community structure was due to human disturbances. Human disturbances documented as visitor censuses by transect walk method lead to categorize the Rumassala as non-disturbed, Unawatuna as disturbed with Galle being of moderately disturbance. Community abundance showed a decreasing trend along with increasing disturbances, albeit community biomass, due to opportunistic algae species, increased in parallel to disturbances. Computed Index of disturbance revealed that macrofauna was very sensitive to the disturbances and increased disturbances reduced their competitive pressure on macroalgae, leading to outstanding macroalgal growth. Macroalgal domination in disturbed communities contributed to lessen the heterotrophic dominance in Galle and Unawatuna. 96 macrobenthic taxa were encountered from the study and Species richness, Shannon and Fisher’s diversity index showed decreasing trend with increasing disturbances. Moreover, initial rising slope of species accumulation curves for three communities indicated that community evenness was low in disturbed communities. The community compositions from dominant conservative species to fast growing opportunistic species were well described by the log series model, showing left skewed distribution. The SIMPER analysis further confirmed that most biomass dominant species in stress status were the opportunistic species such as Valoniopsis pachynema, Gracilaria cassa and Padina boerggesenii. Providing strong support to the results highlighted from univariate measures and log series model, multivariate cluster analysis and nMDS plots completely separated three communities at ordinal scale. This separation was further confirmed by one-way ANOSIM test with significantly higher (2.9%) similarity of study sites within each community. Present study indicated that, allowing less time for recovery, human activities created press-type disturbance on macro-benthic assemblages, and subsequent changes in community structure could be attributed to less suitable substrata. Study proposed that ecological stress is the best measured by multiple methods, and results from different approaches provide the robustness necessary to judge the reliability of the conclusion. Present study strongly supports the growing concern that human activities impact on intertidal assemblages all over the world by changing community composition. Present finding is a contribution to the published literature that is scarcer from tropical rocky shores

In-vitro Toxicity Study of Cylindrospermopsin on Wistar Rats

Naturally derived cyanotoxin, Cylindrospermopsin (CYN) found in fresh water systems poses a threat to human health worldwide. Cylindrospermopsin (CYN) is most potent cyanotoxin which affects the functions of kidney and liver. The present study was carried out to determine the possible effects of Cylindrospermopsin on mammals using male Wistar rats as an animal model. All experimental protocols were approved by the ethics committee of Faculty of Medical Science, University of Sri Jayewardenepura (No. 17/18). Thirty-five Wistar rats were divided into five groups (n=7) and rats were orally treated with different concentrations of cyanotoxin; Cylindrospermopsin (2.5 μg/L, 2 μg/L, 1.5 μg/L) and control group was treated with distilled water for 90 days. Fifth group received environmental water sample contaminated with Cylindrospermopsin (2.3 μg/L) obtained from randomly selected well in Padaviya. Blood and urine samples from each individual were collected at 0, 7, 14, 28, 42, 60, 90 days intervals and the collected samples were subjected to serum creatinine and urine creatinine analysis using creatinine assay kits. Cylindrospermopsin in urine samples were quantified by the ELISA methods. Aspartate aminotransferase (AST), Aspartate alaninetransferase (ALT) and Full Blood Count (FBC) were analysed. The mean body weight of treated (200 to 310 g) and control groups (200-335 g) of the experiment gradually increased until fourteenth week. There was no significant difference of body weights between treated and control groups (p=0.08). The absolute and relative (% body weight) weights of liver and kidneys of the treated groups were less than control group. Cylindrospermopsin dose 2.5 μg/L, 2 μg/L , 1.5 μg/L, 2.3 μg/L and control showed increased serum creatinine levels from 0.62 to 0.87 mg/dL, 0.64 to 0.86 mg/dL, 0.64 to 0.86 mg/dL, 0.61 to 0.83 mg/dL and 0.6 to 0.79 mg/dL respectively after 90 days treatment. The control group did not show a significant cause. Rat treated with different concentrations of Cylindrospermopsin, 2.5 μg/L, 2 μg/L, 1.5 μg/L, 2.3 μg/L and control showed gradually decreased of urine creatinine level from 34 to 55 mg/dL, 40 to 54 mg/dL, 38 to 54 mg/dL, 32 to 53 mg/dL and 43 to 54 mg/dL respectively after 90 days treatment and statistically significant (p<0.05) difference was found between treated and control groups. The highest Aspartate Aminotransferase (AST) and Aspartate Alaninetransferase (ALT) levels were obtained from Cylindrospermopsin dose 2.5 μg/L at 90 days exposure. Cylindrospermopsin concentration in urine gradually increased from 1.5 to 2.32 μg/L, 0.9 to 1.7 μg/L, 0.8 to 1.1 μg/L and 0.4 to 1.5 μg/L when animal exposed to 2.5 μg/L, 2 μg/L, 1.5 μg/L and 2.3 μg/L concentrations of Cylindrospermopsin where control did not show. Thus, the result of present study show consumption of Cylindrospermopsin contaminated water may lead to liver and kidney injuries.Keywords: Cylindrospermopsin (CYN), Wistar rats, ELISA, AST, AL

Microbial Fuel Cell: A New Technology to Generate Power Using Cyanobacteria and Wastewater

Microbial fuel cells make it possible to generate electricity using wastewater and cyanobacteria. Microorganisms in wastewater actively catabolise substrate, and bioelectricities are generated. This study was directed to investigate the effect of cyanobacteria to electricity generation. Two cyanobacteria species, Microcystis sp. and Chroococcus sp. were sampled from Beira Lake and cultured in BG11 and Zarrouk medium respectively without aeration under 2500 lux light. Except cyanobacteria species all other conditions were same in both setups. MFC container made with poly acrylic sheet contain two chambers and silicone was used to seal the chambers except the lids. Two chambers were separated using cation exchange membrane (CMI-7000S) which allows only H+ ions to transfer from anode to cathode. Both electrodes were connected to an external resistor (0.33 Ω) and internal resistance was neglected. Carbon cloth (2×4) cm2 were used as electrodes. Rice washed water with neutral red as a mediator was used as anolyte and 1 L of each cyanobacteria culture was used as catholyte in two setups separately. Control set up was maintained by adding sterilised water as catholyte. The voltage generated at each sampling time was recorded in both setups and current, power were calculated. Setup 01 with Microcystis sp. presented highest values such as voltage 1,184 mV, current 3,587.88 mA and power 4.24 mW. Subsequently setup 02 with Chroococcus sp. represented voltage as 1111 mV with current 3,366.67 mA and power 3.74 mW. Water treatment efficiencies were recorded as reduction in COD, Nitrate, Nitrite and Orthophosphate by 88.96%, 83.45%, 60.36%, 59.59% in setup 01 and 61.94%, 32.05%, 29.05%, 26.01% in setup 02 respectively. The findings of the present study reveal that single cell MFC with Microcystis sp. as cathode is a promising source of bioelectricity generation and wastewater treatment under laboratory conditions.Keywords: Microbial fuel cell, Rice washed water, Wastewater treatment, Electricity generatio

Detection of Microbial Degradation of Natural Rubber Latex by Isolated Native Bacteria in Sri Lanka

Rubber waste management plays an important role to deal with high production andconsumption of rubber. The main constituents of natural rubber is cis 1,4-polyisoprene. It isrelatively resistant to microbial decomposition compared with many other natural polymers.The aim of study was to evaluate the potential effects of the Bacillus cereus, Enterobacter sp.and Enterobacter ludwigii on natural rubber latex degradation. Aliquots of 100 ml mineralsalt medium were prepared and latex was spiked at different concentrations to make finaldilutions at 1:10,000, 1:15,000. Each isolates were separately inoculated (3% v/v) to eachdilution and incubated at 120 rpm for 12 days. Pour plate method was performed to confirmthe resistance of bacteria against to latex. The amount of CO2 liberation was determinedduring the cultivation of cells in mineral salt medium and the released CO2 was trapped by1M NaOH. Quantification of CO2 was performed by titrimetric method. The liberation ofCO2 was gradually increased and reached at 8.67, 9.78 and 10.31 ppmfor E.ludwigii, B.cereus and Enterobacter sp. respectively. CO2 liberation was not detected incontrol. Based on the calculation adapted to the present study, 100% of latex degradation oflatex by released CO2 value should equal to 12.5 ppm. The present study showed thatB.cereus, E. ludwigii and Enterobacter sp. have harber gene which can produce enzyme todegrade latex and future studies are needed to elucidate the degradation mechanism.Keywords: Natural Rubber Latex, E.ludwigii, B. cereus, Enterobacter sp

Contamination of Tributyltin on Selected Marine Invertebrates Collected from Selected Fishery Harbors in Sri Lanka

Tributyltin (TBT) included in the organotin group is considered some of the most toxic chemicals introduced into aquatic environments by anthropogenic activities. Because of it broad range of biocidal activities, they are used for different agricultural and industrial purposes. However, TBT deleterious effects on aquatic biota are well reported and included endocrine distruption, immunotoxicity, neurotoxicity, genotoxicity and metabolic dysfunction. Aquatic invertebrates are key members of zooplankton and have vital roles in food chains. Therefore, the endocrine- disrupting effects of TBT on invertebrates can be transferred to other organisms including human being. This study aims to find the contamination status of TBT in invertebrate samples collected from selected fishery harbors where boat activities are high. Perna viridis, P. perna, Crassostrea madrasensis and Thais sp. in nearly same size were collected from Galle, Kirinda, Mirissa and Trincomalee fishery harbors where the highest water dissolved TBT concentrations were recorded in preliminary screening. Composite sample of each species (10 g) were subjected to TBT extraction using Solid Phase Micro Extraction (SPME) fiber and Potassium Borohydride was used as a deriving agent of TBT chloride into TBT hydride. Quantifications were done in Gas Chromatography Mass Spectrometry (GC-MS) using HP5 column and followed the splitless mode. The results showed that the highest TBT concentration was recorded in P. viridis (53±1.7 ngL-1) following P. perna, C. madrasensis and Thais sp. were 41±2.5 ngL-1, 43±2.4 ngL-1 and 28±1.4 ngL-1 respectively. The concentrations of TBT in these marine invertebrate samples exceeded the concentration which cause severe adverse effects (1 ngL-1). Hence, fishes and marine mammals can be contaminated by ingesting TBT contaminated invertebrates. Therefore, the consumption of contaminated seafood could be risks to human health.Keywords: Tributyltin (TBT), Marine invertebrates, Imposex, Solid Phase Micro Extraction (SPME), Gas Chromatography Mass Spectrometry (GC-MS

Effect of Total Phosphorous, pH and Electric Conductivity on the Seasonal Occurrence of Geosmin in Some Water Bodies Sri Lanka

Cyanobacteria and actinomycetes are well known for their capability to produce taste and odour (T and O) causing compounds in water as their secondary metabolites. Geosmin (trans-1, 10-dimethyl-trans-9-decalol)) is one of the major T and O causing compounds producing earthy T and O and has become a major issue in worldwide water sector. Despite of not detecting as a human health hazard, presence of this odorant directly causes drinking water aesthetically unacceptable and leads to consumer rejection of treated drinking water due to conventional treatment processes do not remove Geosmin. Geosmin is prevailing in some Sri Lankan raw water bodies where water is taken for treatment purposes by National Water Supply and Drainage Board. The objective of the present study was to find the seasonal variation of Geosmin in Sri Lankan water bodies with respect to some physico-chemical aspects aiming to manage the T and O issue in Sri Lanka. Sampling was carried out from June 2016 to June 2018 covering both dry and wet seasons. Geosmin contamination level in 20 water bodies in Sri Lanka were analysed by Solid Phase Micro Extraction (SPME) coupled with Gas Chromatography Mass Spectrometry (GC/MS). The physico-chemical parameters of the water body were measured using standard spectrometric and titrimetric methods. Quantification and enumeration of phytoplankton were carried out with standard method using Acidified Lugol’s solution at final concentration of 1% following natural sedimentation. Geosmin concentration of the analysed samples varied between 7.8±3.27 to 34.6±1.32 ng/L throughout the dry season and from 0 to 18.3±1.22 ng/L during the wet season. Among the selected raw water bodies; the highest level of Geosmin was recorded in Sagama tank (34.6±1.32 ng/L) while the lowest was detected in Nallachchiya tank (7.8±3.27) during the dry season. During the wet season, the highest level of Geosmin was recorded in Sagama tank (18.3±1.22 ng/L) where not detected levels were recorded in Kondawatuwana tank, Jayanthi tank, Kanthale tank and Unnichchi tanks. Anabaena sp., Microcystis sp., Cylindrospermopsis sp., Oscillatoria sp., Cyclotella sp. Volvox sp., Gloeocystis sp., and Uroglenopsis sp. are the major taste and odour forming cyanobacteria recorded from the reservoirs during the study. Total cyanobacteria cell density varied between 102,560±2.28 cells/ml (Thuruwila tank) to 3,649±0.34 cells/ml (Ridiyagama tank) in dry season whereas from 98,235±4.73 (Beire lake) cells/ml to 135±3.11 cells/ml (Kanthale tank) during wet season. Detected Geosmin level in dry season was greater than the wet season. The Pearson’s correlation coefficient analysis revealed a significantly strong positive correlation between Geosmin level and the total phosphorus concentration (r=0.850, (p<0.05), water pH (r=0.788 (p<0.05)) and the Electrical Conductivity level (r=0.612, (p<0.05). Further, it was found that cyanobacteria cell density (r=0.691, (p<0.05)) had a significant positive correlation with Geosmin level in water bodies. These findings are crucial in understanding and managing natural reservoirs to eliminate T and O issue to provide a better quality drinking water to Sri Lankan general public.Keywords: Geosmin, SPME, GC/MS, Physico-chemical parameters, Seasonal variatio