Microbial source tracking of fecal contamination in Laguna Lake, Philippines using the library-dependent method, rep-PCR.

Laguna Lake is an economically important resource in the Philippines, with reports of declining water quality due to fecal pollution. Currently, monitoring methods rely on counting fecal indicator bacteria, which does not supply information on potential sources of contamination. In this study, we predicted sources of Escherichia coli in lake stations and tributaries by establishing a fecal source library composed of rep-PCR DNA fingerprints of human, cattle, swine, poultry, and sewage samples (n = 1,408). We also evaluated three statistical methods for predicting fecal contamination sources in surface waters. Random forest (RF) outperformed k-nearest neighbors and discriminant analysis of principal components in terms of average rates of correct classification in two- (84.85%), three- (82.45%), and five-way (74.77%) categorical splits. Overall, RF exhibited the most balanced prediction, which is crucial for disproportionate libraries. Source tracking of environmental isolates (n = 332) revealed the dominance of sewage (47.59%) followed by human sources (29.22%), poultry (12.65%), swine (7.23%), and cattle (3.31%) using RF. This study demonstrates the promising utility of a library-dependent method in augmenting current monitoring systems for source attribution of fecal contamination in Laguna Lake. This is also the first known report of microbial source tracking using rep-PCR conducted in surface waters of the Laguna Lake watershed.

Gene-Centric Metagenome Analysis Reveals Gene Clusters for Carbon Monoxide Conversion and Validates Isolation of a Clostridial Acetogen for C2 Chemical Production.

Syngas fermentation is largely dependent on acetogens that occur in various anaerobic environmental samples including soil, sediment, and feces. Here the authors report the metagenomic isolation of acetogens for C2 chemical production from syngas. Screening acetogens for C2 chemical production typically involves detecting the presence of the Wood-Ljungdahl Pathway for carbon monoxide conversion. The authors collect samples from river-bed sediments potentially having conditions suitable for carbon monoxide-converting anaerobes, and enrich the samples under carbon monoxide selection pressure. Changes in the microbial community during the experimental procedure are investigated using both amplicon and shotgun metagenome sequencing. Combined next-generation sequencing techniques enabl in situ tracking of the major acetogenic bacterial group and lead to the discovery of a 16 kb of gene cluster for WLP. The authors isolat an acetogenic clostridial strain from the enrichment culture (strain H21-9). The functional activity of H21-9 is confirmed by its high level of production of C2 chemicals from carbon monoxide (77.4 mM acetate and 2.5 mM of ethanol). This approach of incorporating experimental enrichment with metagenomic analysis can facilitate the discovery of novel strains from environmental habitats by tracking target strains during the screening process, combined with validation of their functional activity.

Quantitative microbial risk assessment of Cryptosporidium in bivalve samples from Manila Bay, Philippines

In the Philippines, consumption of bivalves is very common due to its year-round availability and cheap price. However, many consume bivalves as lightly-cooked or raw. This might pose health hazards because bivalves are filter-feeders which act as vehicles for transmission of several pathogens such as the protozoan parasite Cryptosporidium, the causative agent of cryptosporidiosis. Cryptosporidiosis in humans is manifested by profuse diarrhea and abdominal pain. To determine the risk of acquiring cryptosporidiosis from consumption of bivalves, quantitative microbial risk assessment (QMRA) should be done. This study aimed to determine the risk associated with the consumption of bivalves which are contaminated with Cryptosporidium oocysts. The results indicate that consumption of at least 21 grams of cooked bivalves contaminated with at least 0.1% viable oocysts might pose a risk to consumers, especially to immunocompromised individuals. This estimated risk of infection exceeded the United States Environmental Protection Agency (US EPA) standards (1.0×10-4). Results call for drive of decision-makers to establish an educational or treatment program to reduce the incidence of gastrointestinal infections of the consumers. Improvement of sanitation techniques and hygienic practices will contribute to the decrease of occurrence of the disease.

Subtype analysis of Blastocystis sp. isolates from asymptomatic individuals in an urban community in the Philippines

Blastocystis sp. is a commonly reported enteric protistan parasite in faecal specimens with a worldwide distribution afflicting both humans and a wide range of animals. The aim of this study is to characterize the subtypes (STs) of Blastocystis sp. isolates from asymptomatic individuals in an urban community in Pateros, Metro Manila, Philippines. The 600-bp small subunit ribosomal RNA (SSU rRNA) barcoding region of Blastocystis sp. isolates was amplified and sequenced using the primers RD5 and BhRDr. Subtypes were identified by uploading the sequences onto the Basic Local Alignment and Search Tool (BLAST) websites, the Blastocystis Subtype (18S) and Sequence Typing (MLST) Database and by construction of a phylogenetic tree. Twenty-nine (29) out of 35 individuals were detected positive for Blastocystis sp. ST3 is the most common among the three STs detected (65.5%), followed by ST1 (31.0%) and ST4 (3.44%). This study showed that DNA barcoding can serve as a helpful tool to investigate the diversity of Blastocystis sp. in the Philippines.