Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Abstract

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.

Introduction

Wastewater is the source of many human enteric pathogens (Nayak and Rose, 2007, Lee et al., 2006, Kamel et al., 2010, Robertson et al., 2006) and often associated with swimming-acquired illnesses in natural waters (Wade et al., 2006). Adequate wastewater treatment prior to effluent discharge plays a critical role in minimizing public health risks. On-site wastewater disposal using septic tanks has also been an issue regarding pathogen entry into and transmission through water particularly groundwater (Fong et al., 2007) and septage treatment and application on land has received little attention in regard to microbial quality. In most states, fecal coliform bacteria are still used to address wastewater treatment using the National Pollutant Discharge Elimination System permit programs and are disconnected from ambient water quality monitoring, in which coastal states are moving toward Escherichia coli (E. coli) and enterococci.

E. coli and enterococci have long been used as indicators of fecal pollution for recreational and drinking waters (USEPA, 2002, USEPA, 2005a); and cultivation methods are used as the gold standard for the enumeration of these bacteria in water (Messer and Dufour, 1998). Recent advances in molecular biology such as polymerase chain reaction (PCR) and particularly quantitative PCR (qPCR) have revolutionized microbiology. Quantitative PCR has many advantages over standard cultivation methods, such as producing results rapidly (30 min–2 h), the ability to detect viable but non-cultivatable (VNBC) pathogens, and providing quantitative results with a wide detection range (10⁰–10⁸ copies/reaction). However, it is still recognized that both inactivated and live microbes will be detected, which is a disadvantage when evaluating disinfection processes. Never-the-less, evaluation and application of these qPCR methods for routine monitoring of fecal contamination in recreational waters is ongoing in the US (Haugland et al., 2005, Noble et al., 2006, Wade et al., 2006, Lavender and Kinzelman, 2009) as well as in wastewaters (Silkie and Nelson, 2009, Varma et al., 2009, Wery et al., 2008, Frahm and Obst, 2003).

One of the other disadvantages of using routine bacterial indicators is that the source cannot be identified while the specificity of molecular methods has lead to development of a field known as Microbial source tracking (MST) that has enabled the identification of animal or human sources of fecal contamination (Scott et al., 2002, Simpson et al., 2002, USEPA, 2005b). Many human fecal specific assays to address sewage discharges impacting water quality have targeted species in the Bacteroides–Prevotella group directed toward detection of 16S rRNA genes (Seurinck et al., 2005, Layton et al., 2006, Okabe et al., 2007). Bacteroides spp. are obligately anerobic, Gram negative, rod shaped, and non-endospore forming bacteria and are normally commensals that constitute the most numerous members of the intestinal flora of all warm blooded animals (Wexler, 2007). There are still concerns regarding cross reactivity of some of these genetic markers with feces from humans and other animals including cats and dogs (Sadowsky et al., 2007, Kildare et al., 2007). Some qPCR assays based on 16S rRNA genes have been reported to cross react with fish DNA (McLain et al., 2009). Moreover, the exact copy number of these 16S rRNA genes present in one cell is not known which makes conversion of qPCR copy number to cell equivalents rather difficult. Recently, Yampara-Iquise et al. (2008) examined a single copy putative mannanase 1-6 gene of Bacteroides thetaiotaomicron as a human fecal source tracking marker with good specificity.

The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility in contrast to E. coli and enterococci as measure by qPCR and cultivation. In this study, a qPCR assay targeting uidA gene for E. coli was developed and used, modified from Frahm and Obst (2003). Enterococci qPCR assay focused on the use of primers and probes designed for detection of the 23S rRNA gene sequences (Haugland et al., 2005, Silkie and Nelson, 2009). Samples were collected throughout the wastewater treatment processes as well as from septage before and after treatment. Cultivable counts of E. coli and enterococci were compared to the qPCR data generated and an automated DNA extractor was compared to a commercially available QIAmp DNA mini kit (Qiagen, Valencia, CA, USA).