Exploring the potential reservoirs of non specific TEM beta lactamase (bla(TEM)) gene in the Indo-Gangetic region: A risk assessment approach to predict health hazards.

The emergence of antimicrobial resistant bacteria is an important public health and environmental contamination issue. Antimicrobials of ß-lactam group accounts for approximately two thirds, by weight, of all antimicrobials administered to humans due to high clinical efficacy and low toxicity. This study explores ß-lactam resistance determinant gene (blaTEM) as emerging contaminant in Indo-Gangetic region using qPCR in molecular beacon format. Quantitative Microbial Risk Assessment (QMRA) approach was adopted to predict risk to human health associated with consumption/exposure of surface water, potable water and street foods contaminated with bacteria having blaTEM gene. It was observed that surface water and sediments of the river Ganga and Gomti showed high numbers of blaTEM gene copies and varied significantly (p<0.05) among the sampling locations. The potable water collected from drinking water facility and clinical settings exhibit significant number of blaTEM gene copies (13±0.44-10200±316 gene copies/100mL). It was observed that E.crassipes among aquatic flora encountered in both the rivers had high load of blaTEM gene copies. The information on prevalence of environmental reservoirs of blaTEM gene containing bacteria in Indo-Gangetic region and risk associated will be useful for formulating strategies to protect public from menace of clinical risks linked with antimicrobial resistant bacteria.

Quantification of Salmonella Typhi in water and sediments by molecular-beacon based qPCR.

A molecular-beacon based qPCR assay targeting staG gene was designed for specific detection and quantification of S. Typhi and validated against water and sediment samples collected from the river Ganga, Yamuna and their confluence on two days during Mahakumbha mela 2012-2013 (a) 18 December, 2012: before six major religious holy dips (Makar Sankranti, Paush Poornima, Mauni Amavasya, Basant Panchami, Maghi Poornima and Mahashivratri) (b) 10 February, 2013: after the holy dip was taken by over 3,00,00,000 devotees led by ascetics of Hindu sects at Sangam on 'Mauni Amavasya' (the most auspicious day of ritualistic mass bathing). The assay could detect linearly lowest 1 genomic equivalent per qPCR and is highly sensitive and selective for S. Typhi detection in presence of non specific DNA from other bacterial strains including S. Paratyphi A and S. Typhimurium. It has been observed that water and sediment samples exhibit S. Typhi. The mass holy dip by devotees significantly affected the water and sediment quality by enhancing the number of S. Typhi in the study area. The qPCR developed in the study might be helpful in planning the intervention and prevention strategies for control of enteric fever outbreaks in endemic regions.

Bio-capture of S. Typhimurium from surface water by aptamer for culture-free quantification.

In this study, a DNA aptamer was used to bio-capture Salmonella enterica serovar Typhimurium from surface water collected from highly endemic zone prior to culture-free detection through Molecular-Beacon based real-time PCR assay targeting invA gene. The assay could detect S. Typhimurium cells (1 CFU/PCR or 100 CFU/ml) selectively captured by serovar specific DNA aptamer. The observations indicate that all the water samples (n=40) collected from the river Gomti were contaminated by S. Typhimurium (31400-1 × 10(7) CFU/100 ml). The pre-analytical step in the form of serovar specific DNA aptamer based bio-capture of the bacterial cell was found to enhance the sensitivity of the florescent probe based real-time PCR assay during detection of S. Typhimurium in environmental samples exhibiting natural PCR inhibitors and high background bacterial flora. The assay could be used for the regular monitoring of surface waters for forecasting and management of non-typhoidal Salmonellosis in south Asia.