Use of the ESP gene to detect human contamination with Enterococcus faecium of South Texas coastal waters

Microbial source tracking comprises a set of techniques to determine animal/human sources of fecal contamination. As new methods are developed, it is important to evaluate their effectiveness in a range of environments. The enterococcal surface protein gene (esp) of Enterococcus faecium, which has been used to indicate the presence of this human-associated opportunistic pathogen, was used in this study to detect human-source contamination in marine and freshwaters of south Texas. Several surface water segments in this area have been placed on the Texas 303 (d) list of impaired waters for bacteria contamination, as a result of elevated levels of enterococci or Escherichia coli. Detection of esp was compared with levels of fecal indicator bacteria in the water in order to determine whether contamination was of human origin and also to assess whether the contamination was detected at times of elevated indicator bacteria levels. Water samples were collected from marine and freshwater environments in the Texas Coastal Bend area, and fecal bacteria levels were determined using standard membrane filtration methods. For esp analysis, water was filtered onto membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI) and incubated at 41°C for 48 hours. Following enrichment in azide dextrose broth, DNA extraction and PCR analysis were performed. The esp gene was detected in both marine and freshwater samples by PCR. Inhibition frequently affected PCR analyses in initial samples from marine sites, so a protocol for inhibitory samples was used for the remainder of the study. The esp gene was only detected in marine waters when Enterococcus levels greatly exceeded water quality standards. The lowest enterococci concentration of an esp-positive freshwater sample was 83 CFU 100 ml-1. The esp gene showed potential for use in detecting human source contamination in Texas coastal waters.