Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification.

Tourists visiting to endemic zones may acquire Enterotoxigenic Escherichia coli (ETEC) infection resulting into diarrhea due to consumption of contaminated potable waters. In this study, a qPCR assay (SYBR Green), targeting LT1 and ST1 genes was designed to quantify ETEC in potable waters derived from civic water supply. The assay could detect lowest 1CFU/PCR targeting LT1/ST1 gene from ten-fold diluted culture of the reference strain (E. coli MTCC 723) and is ten-fold more sensitive than the conventional PCR. The quantification of the ETEC in potable waters collected from civic supply of a major city of the northern India exhibiting high flow of tourists reveals that all the sites that ran along sewage line were contaminated by the ETEC. Contamination was due to percolation of sewage. The assay could be used for the regular monitoring of potable water in places exhibiting heavy flow of tourists to prevent ETEC induced diarrhea.

Culture-free detection and enumeration of STEC in water.

Shiga toxin-producing Escherichia coli (STEC) causes worldwide outbreaks of food and waterborne diseases. Rapid identification of causative agents is critical for early intervention in the case of widespread diarrheal epidemics to prevent mortality. In this study, a Molecular-Beacon targeting stx2 gene (highly associated with human illness) was designed to develop a culture-independent real-time PCR assay for detection and quantification of STEC in water samples. The assay could detect lowest 10 genomic equivalent (GE) of the reference strain (E. coli I.T.R.C.-18) per PCR or 100 GE/mL. The presence of 10(6)CFU/mL of non-pathogenic E. coli DH5α has no impact on sensitivity of the assay. The assay could successfully enumerate STEC in surface water (collected from a sewage impacted river) and potable water samples collected from Lucknow city without prior enrichment. The assay will be useful in pre-emptive monitoring of surface/potable waters to prevent waterborne outbreaks caused by STEC.

Enterotoxigenic Escherichia coli in sewage-impacted waters and aquatic weeds: quantitative PCR for culture-independent enumeration.

AIM: To develop quantitative PCR for culture-independent enumeration of enterotoxigenic Escherichia coli (ETEC) in sewage-impacted waters and aquatic weeds. METHODS AND RESULTS: Two fluorescent probes (TaqMan and FRET) based on two different real-time PCR chemistries were designed in highly conserved region of LT1 gene encoding heat labile enterotoxin. Both the assays could detect 2 CFU ml(-1) from serially diluted (two-fold and ten-fold) culture of reference strain (E. coli MTCC 723). FRET performed better in terms of CT value and PCR efficiency than TaqMan. The presence of 10(6) CFU ml(-1) of nonpathogenic E. coli reduced the detection limit two-fold with both the probes. However, the performance for two chemistries in various environmental samples was significantly (student's t-test, P<0.05) different. CONCLUSION: It could be inferred from this study that real-time PCR chemistries (TaqMan and FRET) could detect very few copies of target DNA in pure cultures, but may give varied response in the presence of nonspecific DNA and natural inhibitors present in environmental sample matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The assays can be used for pre-emptive monitoring of aquatic weeds (a potential nonpoint source), surface and potable waters to prevent waterborne outbreaks caused by ETEC.

Determination of antimicrobial resistance and virulence gene signatures in surface water isolates of Escherichia coli.

AIMS: To determine the occurrence of Escherichia coli harbouring virulence markers of shiga- or entero-toxins and resistance to antimicrobials in surface waters. METHODS AND RESULTS: Surface water samples were collected at six locations of the river Gomti. E. coli isolates (n = 90) were characterized for their pathogenic potential using polymerase chain reaction to detect virulence genes as well as their sensitivity to antimicrobial agents using disc diffusion methods. In this study, 57.8% of E. coli isolates exhibited resistance to three or more antimicrobial agents. Sensitivity to cephotaxime, gentamicin and norfloxacin was observed in 7.8%, 48.9% and 77.8% of isolates, respectively. Both stx1 and stx2 genes were present in 15.6% of isolates while remaining isolates had either stx1 (17.8%) or stx2 (6.7%). The stx1 gene (33.3%) was more prevalent than stx2 (22.2%). The results indicate that the LT1 and ST1 genes were positive in 21.2% of isolates. CONCLUSIONS: The presence of multi-drug resistance and virulence genes in E. coli isolated from surface water being used for domestic and recreational purposes may result in waterborne outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: The data will be useful in monitoring surface waters for forecasting and management of waterborne outbreaks.

Efficacy of various amendments for amelioration of fly-ash toxicity: growth performance and metal composition of Cassia siamea Lamk.

Plants of Cassia siamea Lamk were grown in garden soil (control), fly-ash (100%) and fly-ash amended by various ameliorants (cowdung manure, press-mud, garden soil; 1:1, w/w). The plants survived in fly-ash (100%) though their growth was less in comparison to the treatments. Fly-ash+press-mud (1:1, w/w) proved to be the best combination as growth (total biomass, leaf number, photosynthetic area, total chlorophyll and protein) was significantly high in this treatment followed by cowdung manure and garden soil. Leaves and roots accumulated significant amount of Cu, Zn, Ni and and Fe. However, the concentration of all the metals was more in roots than leaves except Ni. Although, fly-ash contains high amount of metals but the metal uptake was more in the plants grown in fly-ash+press-mud mixture. Inspite of high metal availability in fly-ash and press-mud mixture, plant growth was good. This might be attributed to the some metal detoxification mechanism active in this treatment. The present study concluded that C. siamea seems to be a suitable plant for developing a vegetation cover on fly-ash dumps.

Bioaccumulation of toxic metals (Cr, Cd, Pb and Cu) by seeds of Euryale ferox Salisb. (Makhana) W.

The level of toxic metals Cr, Cd, Pb and Cu was determined in seeds, water and sediments collected from nine closed waterbodies of Darbhanga, north Bihar, used for cultivation of the edible aquatic macrophyte Euryaleferox Salisb. during harvesting season of the crop for two successive years (1996 and 1997). Seeds bioconcentrated appreciable amount of these toxic metals in the order Pb > Cr > Cu > Cd. The increased load of metal pollution due to domestic and municipal discharges threatened the habitats of the plant. The toxic metal contents in seeds were found positively correlated with the ambient concentration of metals in water and sediments. The importance of these findings has been discussed for national water resource economy of the country and human health perspectives.

Chromium (VI) accumulation reduces chlorophyll biosynthesis, nitrate reductase activity and protein content in Nymphaea alba L.

Plants of Nymphaea alba L. grown at various levels of chromium (VI) ranging from 1 to 200 microM accumulated chromium in concentration and duration-dependent manner. At all Cr levels, chromium accumulation by various plant tissues followed the order roots > leaves > rhizomes. Approximately 93% of total chromium present in the medium was accumulated by plants at lowest conentration (1 microM) used in the experiment. Chromium-induced toxicity appears at 1 microM chromium resulting in the build-up of delta-aminolevulinic acid (ALA) and reduced activities of delta-aminolevulinic acid dehydratase (ALAD) and nitrate reductase (NR), total chlorophyll (Chl) and protein contents. Ch1a was more sensitive than Ch1b to chromium toxicity. It could be inferred that chromium toxicity is not located at the level of ALA synthesis, but, probably at the ALAD activity which was more severely affected during chlorophyll biosynthesis. Finally, impaired chlorophyll biosynthesis resulted in reduced total chlorophyll content.