Selecting rep-PCR markers to source track fecal contamination in Laguna Lake, Philippines.

Kevin L Labrador,Christopher Rensing,Mae Ashley G Nacario,Gicelle T Malajacan,Windell L Rivera,Luiza H Galarion,Joseth Jermaine M Abello

doi:10.2166/wh.2019.042

Abstract

Fecal contamination is one of the factors causing deterioration of Laguna Lake. Although total coliform levels are constantly monitored, no protocol is in place to identify their origin. This can be addressed using the library-dependent microbial source tracking (MST) method, repetitive element sequence-based polymerase chain reaction (rep-PCR) fingerprinting. Serving as a prerequisite in developing the host-origin library, we assessed the discriminatory power of three fingerprinting primers, namely BOX-A1R, (GTG)5, and REP1R-1/2-1. Fingerprint profiles were obtained from 290 thermotolerant Escherichia coli isolated from sewage waters and fecal samples of cows, chickens, and pigs from regions surrounding the lake. Band patterns were converted into binary profiles and were classified using the discriminant analysis of principal components. Results show that: (1) REP1R-1/2-1 has a low genotyping success rate and information content; (2) increasing the library size led to more precise estimates of library accuracy; and (3) combining fingerprint profiles from BOX-A1R and (GTG)5 revealed the best discrimination (average rate of correct classification (ARCC) = 0.82 ± 0.06) in a two-way categorical split; while (4) no significant difference was found between the combined profiles (0.74 ± 0.15) and using solely BOX-A1R (0.76 ± 0.09) in a four-way split. Testing the library by identifying known isolates from a separate dataset has shown that a two-way classification performed better (ARCC = 0.66) than a four-way split (ARCC = 0.29). The library can be developed further by adding more representative isolates per host source. Nevertheless, our results have shown that combining profiles from BOX-A1R and (GTG)5 is recommended in developing the MST library for Laguna Lake.

Highlights

Laguna Lake, the largest lake in the Philippines, has experienced continued deterioration over the past years
Our objective was to assess the discriminatory power of three repetitive element sequencebased polymerase chain reaction (rep-PCR) fingerprinting primers in source tracking fecal contamination in Laguna Lake
We aimed to answer the following questions: (1) How did an increase in library size affect its performance? (2) Was a two-way split better than a four-way split? (3) Which among the primers or primer combinations were best suited in source tracking fecal contamination to Laguna Lake? (4) How accurate was the library in source tracking known isolates? To the best of our knowledge, this is the first initiative to perform microbial source tracking (MST) on the Philippines’ largest lake

Summary

Introduction

Laguna Lake, the largest lake in the Philippines, has experienced continued deterioration over the past years. Monitoring activities by the Laguna Lake Development Authority, the agency that is primarily responsible for the lake’s development, revealed fecal contamination in the lake and high total coliform counts in most of its tributaries. These pose serious public health problems since pathogens from infected sources can be introduced into the environment through feces, causing risks to public health and the economy (Ahmed et al ; Ballesté et al )

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