Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for Differentiating Sources of Fecal Pollution

Since the passage of the Marine Mammal Protection Act in 1973, pinniped populations in coastal waters of the United States have increased exponentially. These high populations of large mammals could contribute to fecal contamination of recreational waters. Enterococci species counts are used to assess the degree of fecal contamination and elevated counts of this fecal indicator bacteria (FIB) force managers to close beaches; however, contribution of pinnipeds to high Enterococci counts is not known. This may reflect the high cost of methods of tracking the source of microbial contamination. Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) is a time and cost-effective way to identify bacteria through protein mass spectra analysis. MALDI-TOF can distinguish strains of bacteria of the same species but has not been evaluated as a tool for tracking Enterococci isolated from pinnipeds. In this study, Enterococci isolates were cultured from fresh seal scat samples taken from a pen housing Harbor seals. Enterococci were isolated by plating serial dilutions onto Rapid Enterococci ChromoSelect agar to generate a library of isolates from captive seals. Libraries of Enterococci were also generated from dog scat and a composite sample of human waste from a wastewater treatment plant, respectively. These isolates were identified with a MALDI-TOF system and cluster analysis was performed of all Enterococci isolates showed overall clusters between Enterococci isolated from a single source. Cluster analysis of isolates from harbor seal and dog scat reliably identified as E. faecalis with Bruker MALDI Biotyper system (> 2.3) was performed for source discrimination comparison. 22 E. faecalis isolates clustered into four separate MTUs, with two MTUs being harbor seal-specific. The distinct source-dependent MALDI-TOF MS clusters suggest that MALDI-TOF MS may be a valuable tool in microbial source tracking.