Q-PCR Based Culture-Independent Enumeration and Detection of Enterobacter: An Emerging Environmental Human Pathogen in Riverine Systems and Potable Water.

Chandra B Patel,Ram S Upadhyay,Rishi Shanker,Vijai K Gupta

doi:10.3389/fmicb.2016.00172

Abstract

The availability of safe and pristine water is a global challenge when large numbers of natural and anthropogenic water resources are being depleted with faster rate. The remaining water resources are severely contaminated with various kinds of contaminants including microorganisms. Enterobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. Enterobacter was earlier used as an indicator bacterium along with other fecal Coliforms namely Escherichia coli, Citrobacter, and Klebsiella, but it is now known to cause various diseases in human beings. In this study, we have collected 55 samples from potable water and riverine system and proved their presence using their conserved sequences of 16S rRNA and 23S rRNA genes with the help of SYBR green real-time PCR, which showed very high specificity for the detection of Enterobacter. The Enterobacter counts in potable water were found to 1290 ± 32.89 to 1460 ± 39.42 cfu/100 ml. The Enterobacter levels in surface water were 1.76 × 104 ± 492, 1.33 × 104 ± 334, 1.15 × 104 ± 308, 2.56 × 104 ± 802, 2.89 × 104 ± 962, 8.16 × 104 ± 3443 cfu/100 ml; the levels of Enterobacter contamination associated with hydrophytes were 4.80 × 104 ± 1804, 3.48 × 104 ± 856, 8.50 × 104 ± 2074, 8.09 × 104 ± 1724, 6.30 × 104 ± 1738, 3.68 × 104 ± 949 cfu/10 g and the Enterobacter counts in sediments of the river, were 2.36 × 104 ± 703, 1.98 × 104 ± 530, 9.92 × 104 ± 3839, 6.80 × 104 ± 2230, 8.76 × 104 ± 3066 and 2.34 × 104 ± 732 cfu/10 g at the sampling Site #1, Site #2, Site #3, Site #4, Site #5, and Site #6, respectively. The assay could be used for the regular monitoring of potable water and other water reservoirs to check waterborne outbreaks.

Highlights

The change in the global environment has led to the inappropriate distribution of microbial species, which resulted into emergence and re-emergence of microbial pathogens
The SYBR Green based quantitative PCR (qPCR) assays targeting 16S rRNA, 23S rRNA genes were used to detect Enterobacter quantitatively, in the potable water samples collected from urban boundaries of a city Lucknow
The Enterobacter levels at sites: A–J targeting 16S rRNA, 23S rRNA genes were 13320 ± 428.90 CFU/100 ml, ND, ND, ND, 1304 ± 34.94, 1290 ± 32.89, 1460 ± 39.42, ND, ND, and ND, respectively (Table 2). These findings showed that the Enterobacter were present at the sampling site: A and site: E, site: F site: G only

Summary

Introduction

The change in the global environment has led to the inappropriate distribution of microbial species, which resulted into emergence and re-emergence of microbial pathogens. Surface and potable waters are heavily contaminated with microorganisms leading to various waterborne diseases and outbreaks. Some indicator microorganisms are required to trace the presence of possible pathogens in the aquatic environment. The guidelines and regulations for safe recreational and potable waters require determination of the absence of ‘indicator’ microorganisms. Coliform bacteria normally exist in the intestinal tract of warm-blooded animals and humans, and are discharged through fecal matter. Most coliform bacteria can exist widely in the natural environment, including soil, surface water, and to some extent in groundwater (Francy et al, 2000). Non-pathogenic coliforms can survive in water in adverse conditions as compared to other pathogenic bacteria. There are four genera of fecal coliforms namely Escherichia, Klebsiella, Enterobacter, and Citrobacter

Methods

Results

Conclusion