A real‐time polymerase chain reaction assay for quantitative detection of the human‐specific enterococci surface protein marker in sewage and environmental waters

Abstract

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.

Role of House Flies in the Ecology of Enterococcus faecalis from Wastewater Treatment Facilities

“…This prevalence and concentration is comparable to other studies that have screened sewage for the enterococci [2,9,19,47,51]. Sixty six percent of house flies were positive for enterococci at a concentration ~10 3 CFU/fly.…”

“…Waste received from environments under significant antibiotic pressure, such as hospitals, consistently contains a greater proportion of singly and multiple drug-resistant bacteria [9,22,26,28,38,60] and AR enterococci are commonly recovered from sewage [2,8,9,28,38,47,51,55]. House flies and other filth flies often have direct and unhindered access to many steps in the waste processing flow and therefore may acquire bacteria associated with the waste.…”

Enterococci in the Environment

“…The esp gene of E. faecium (esp fm ) is strongly human associated (5,287,357), although a low frequency of cross-reactivity with nonhuman feces has been noted, and it is not readily detected in some sewage sources, such as on-site (septic) systems (4,357). The occurrence of esp fm was correlated with human polyomaviruses in polluted surface waters in Florida (227) and with fecal coliforms in another study (197).…”

“…It has also been used in field studies in Florida, the Great Lakes, and Australia (5,39,196,209). A quantitative PCR (qPCR) method for esp fm has been developed and used in field studies in Australia (4). Interestingly, the presence of the esp gene was found to affect the transport of E. faecium in saturated quartz sands by lowering bacterial mobility through increased attachment to sand particles (184).…”

Assessing the impact of socio-economic development and climate change on faecal indicator bacteria in the Betna River, Bangladesh

“…An average E. coli concentration of 1.5×10 6 cfu/100ml in untreated wastewaters was assigned as input based on the concentrations reported by Payment et al (2001), Carlos et al (2013) and Sokolova [62] et al (2013). Enterococci concentration in untreated wastewaters was set to1.0×10 6 cfu/100 ml, which is comparable to the value reported by Ahmed et al (2008).…”