Decay of Bacterial Pathogens, Fecal Indicators, and Real-Time Quantitative PCR Genetic Markers in Manure-Amended Soils

Abstract

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manureamended agricultural soils. Known concentrations of transformed green fluorescent protein-expressing Escherichia coli O157:H7/pZs and red fluorescent protein-expressing Salmonella enterica serovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of ؊20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (Enterococcus spp., E. coli, and Bacteroidales) exhibited decay rate coefficients similar to that of E. coli O157:H7/pZs but not of S. enterica serovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (stx1 and ttrRSBCA, respectively), and FIB genetic markers were also correlated (r ‫؍‬ 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Hostassociated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB, Salmonella enterica serovar Typhimurium/pDs, and E. coli O157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.Cultivation-based methods for fecal indicator bacteria (FIB) such as Escherichia coli and Enterococcus spp. have long been used to indicate potential public health risks associated with water impacted by human and other animal feces (53). FIB cultivation methods are simple to perform and inexpensive. However, these methods require 18 to 24 h following sampling to generate test results; this allows potential exposure of the public to fecal pathogens in the interim. Regulatory agencies, business owners, and other stakeholders have expressed interest in more rapid and specific methods to identify water quality impairment.Emerging real-time quantitative PCR (qPCR) methods designed to estimate the concentration of fecal pollution by targeting genomic DNA (gDNA) from FIB such as Bacteroidales, Enterococcus spp., and E. coli are now available and can generate test results in just a few hours after sampling (10,16,48). Some of these genetic markers can be correlated to public health risk and may soon be incorporated by the U.S. Environmental Protection Agency into water quality standards in the United States (16,59). These genetic markers may also detect viable but nonculturable (VBNC) cells tha...

Depth-Dependent Survival of Escherichia coli and Enterococci in Soil after Manure Application and Simulated Rainfall

“…It has been found that both E. coli and enterococci can survive in the soil for several weeks after deposition, if not longer (7,8). Some studies have found that populations of these organisms can experience initial growth in the soil that can extend their prevalence for longer than previously thought.…”

“…Contamination of soil and water leading to foodborne illness can originate from numerous sources, such as direct deposition by animals, overflow or leakage of faulty septic and sewage systems, or manure application to agricultural fields and pastures (3-5). The potential presence of pathogens that cause foodborne illness can be inferred via the monitoring of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci (6).It has been found that both E. coli and enterococci can survive in the soil for several weeks after deposition, if not longer (7,8). Some studies have found that populations of these organisms can experience initial growth in the soil that can extend their prevalence for longer than previously thought.…”

Effects of tillage and poultry manure application rates on Salmonella and fecal indicator bacteria concentrations in tiles draining Des Moines Lobe soils

“…In addition to pathogens, PM commonly contains nonpathogenic bacteria, including Escherichia coli (EC) and enterococci (ENT) (Terzich et al, 2000;Rogers et al, 2011). Studies of human exposures have confirmed the increased risk of negative health outcomes from swimming in waters containing these and other fecal indicator bacteria (FIB) (Pruss, 1998).…”

“…Recent studies of PMamended soils have reported survival of FIB and pathogens for weeks and months after application (Rogers et al, 2011;Cook et al, 2014), highlighting the potential for continued release of bacteria to subsurface waters.…”

Evaluating the long‐term persistence of Bacillus spores on common surfaces

“…The robustness of Gompertz models to predict microbial populations under different temperatures has made them versatile for modelling purposes. The broken‐line model (Carrera et al ., 2007; Rogers et al ., 2011) was used to account for abrupt changes in the non‐linearity in inactivation curve. Points of abrupt changes known as ‘break points’ were developed originally to provide a better understanding of the kinetic parameters at different points of departure in the inactivation curve.…”