Extended persistence of general and cattle-associated fecal indicators in marine and freshwater environment

Abstract

Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli, enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p: 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p: 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli, EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p: 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets.

Keywords: Cattle manure; Decay; Fecal indicators; Microbial source tracking markers.

Figure 1.

Cultured E. coli and enterococci measurements (mean log₁₀ CFU per 10mL) in mesocosm experiments in freshwater and marine water for cultured E. coli and enterococci measurements. Treatment A = Upper level, unfiltered watered; Treatment B = Lower level, unfiltered water; Treatment C = Upper level, filtered water; Treatment D = Lower level, filtered water. Error bars represent standard deviation. T₀ (0 h), T₁ (48 h), T₂ (96 h), and T₃ (144 h).

Figure 2.

Molecular fecal indicator bacteria measurements by qPCR (mean log₁₀ gene copies per 10mL) in mesocosm experiments in freshwater and marine water. Treatment A = Upper level, unfiltered watered; Treatment B = Lower level, unfiltered water; Treatment C = Upper level, filtered water; Treatment D = Lower level, filtered water. Error bars represent standard deviation. T₀ (0 h), T₁ (48 h), T₂ (96 h), and T₃ (144 h).

Figure 3.

Molecular MST marker measurements (mean log₁₀ gene copies per 10mL) in mesocosm experiments in freshwater and marine water. Treatment A = Upper level, unfiltered watered; Treatment B = Lower level, unfiltered water; Treatment C = Upper level, filtered Water; Treatment D = Lower level, filtered Water. Error bars represent standard deviation. T₀ (0 h), T₁ (48 h), T₂ (96 h), and T₃ (144 h).