Genotypic detection of ß-lactamase-producing Escherichia coli isolates obtained from Seven Crater Lakes of San Pablo, Laguna, Philippines.

The extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is becoming a global public health concern. More comprehensive surveillance of ß-lactam resistance in E. coli would improve monitoring strategies and control resistance transmission in contaminated environments. This study investigated the prevalence of ß-lactamase genes in E. coli isolated from the Seven Crater Lakes in San Pablo, Laguna, Philippines. Water samples from lakes were collected for the isolation of E. coli (n = 846) and molecular characterization by detecting the presence of the uidA gene. The isolates were then tested for the presence of ß-lactamase genes using PCR. Among the screened genes, blaAmpC was the most dominant (91%). Other ß-lactamase genes such as blaTEM, blaSHV, and blaCTXM were also detected with percentage occurrence of 34, 5, and 1%, respectively. Multiple genes within individual isolates were also observed, wherein blaTEM/AmpC was the most prevalent gene combination. Moreover, a significant negative correlation between blaAmpC with blaSHV and blaCTXM was depicted in this study. Overall, these findings demonstrate the presence of ß-lactamase genes in E. coli in the Seven Crater Lakes of San Pablo and can be used in developing effective strategies to control antibiotic resistance in environmental waters.

Genotypic virulence profiles and associations in Salmonella isolated from meat samples in wet markets and abattoirs of Metro Manila, Philippines.

BACKGROUND: Salmonella are pathogenic foodborne bacteria with complex pathogenicity from numerous virulence genes housed in Salmonella pathogenicity islands (SPIs), plasmids, and other gene cassettes. However, Salmonella virulence gene distributions and mechanisms remain unestablished. In the Philippines, studies mainly report Salmonella incidences and antimicrobial resistance, but little to none on virulence profiles, their associations to animal sources, collection sites and Salmonella serogroups. Hence, a total of 799 Salmonella isolates, previously obtained from pig, cow, and chicken meat samples in wet markets and abattoirs (wet markets: 124 chicken, 151 cow, and 352 pig meat isolates; abattoirs: 172 pig tonsil and jejunum isolates) in Metro Manila, Philippines, were revived and confirmed as Salmonella through invA gene polymerase chain reaction (PCR). Isolates were then screened for eight virulence genes, namely avrA, hilA, sseC, mgtC, spi4R, pipB, spvC and spvR, by optimized multiplex PCR and significant pair associations between virulence genes were determined through Fisher's exact test. Gene frequency patterns were also determined. Salmonella serogroups in addition to animal sources and location types were also used to predict virulence genes prevalence using binary logistic regression. RESULTS: High frequencies (64 to 98%) of SPI virulence genes were detected among 799 Salmonella isolates namely mgtC, pipB, avrA, hilA, spi4R and sseC, from most to least. However, only one isolate was positive for plasmid-borne virulence genes, spvC and spvR. Diversity in virulence genes across Salmonella serogroups for 587 Salmonella isolates (O:3 = 250, O:4 = 133, O:6,7 = 99, O:8 = 93, O:9 = 12) was also demonstrated through statistical predictions, particularly for avrA, hilA, sseC, and mgtC. mgtC, the most frequent virulence gene, was predicted by serogroup O:9, while sseC, the least frequent, was predicted by serogroup O:4 and chicken animal source. The highest virulence gene pattern involved SPIs 1-5 genes which suggests the wide distribution and high pathogenic potential of Salmonella. Statistical analyses showed five virulence gene pair associations, namely avrA and hilA, avrA and spi4R, hilA and spi4R, sseC and spi4R, and mgtC and pipB. The animal sources predicted the presence of virulence genes, sseC and pipB, whereas location type for hilA and spi4R, suggesting that these factors may contribute to the type and pathogenicity of Salmonella present. CONCLUSION: The high prevalence of virulence genes among Salmonella in the study suggests the high pathogenic potential of Salmonella from abattoirs and wet markets of Metro Manila, Philippines which poses food safety and public health concerns and threatens the Philippine food animal industry. Statistical associations between virulence genes and prediction analyses across Salmonella serogroups and external factors such as animal source and location type and presence of virulence genes suggest the diversity of Salmonella virulence and illustrate determining factors to Salmonella pathogenicity. This study recommends relevant agencies in the Philippines to improve standards in food animal industries and increase efforts in monitoring of foodborne pathogens.

DNA fingerprinting using BOX-A1R and (GTG)5 primers identify spatial variations of fecal contamination along Pasig River, Philippines.

Pasig River is one of the most economically important rivers in Metro Manila, Philippines. It traverses some of the region's major cities, and because of its strategic location, it is utilized as a means of transportation, as a source of water for domestic and industrial uses, and for recreational purposes. However, due to population growth, industrialization, and land use, the river's water quality is deteriorating. Wastes that pollute the river pose health risks to the people that benefit from it. To prevent the river's further degradation, it is essential to identify the origin of contamination. In this study, the sources of fecal contamination in Pasig River were identified using BOX-A1R and (GTG)5 primers in the DNA fingerprinting of Escherichia coli isolated from the river. Results showed the dominance of human contamination (percent composition = 65.55%), followed by agricultural sources (percent composition = 23.48%), and the lowest was from sewage (percent composition = 10.98%). The results of this research can help in evaluating public health risks and can be used as a scientific basis for policymaking and implementation for the rehabilitation and improvement of Pasig River.

Molecular surveillance of Cryptosporidium spp. for microbial source tracking of fecal contamination in Laguna Lake, Philippines.

Water quality deterioration in source waters poses increased health, environmental, and economic risks. Here, we genotyped Cryptosporidium spp. obtained from water samples of Laguna Lake, Philippines, and its tributaries for the purpose of source-tracking fecal contamination. A total of 104 surface water samples were collected over a 1-year period (March 2018 to April 2019). Detection of Cryptosporidium was carried out using genus-specific primers targeting a fragment of the small subunit (SSU) rRNA gene. The study revealed 8 (14%) tributary samples and 1 (2.77%) lake sample positive for contamination. The species were determined to be C. parvum (n = 4), C. muris (n = 2), C. hominis (n = 1), C. galli (n = 1), C. baileyi (n = 1), C. suis (n = 1), as well as rat genotype IV (n = 1). Two species were detected in duck (C. baileyi) and cattle (C. parvum) fecal samples. The data presented suggest that Cryptosporidium contamination is likely to come from sewage or human feces as well as various agricultural sources (i.e. cattle, swine, and poultry). This information reveals the importance of mitigating fecal pollution in the lake system and minimizing health risks due to exposure to zoonotic Cryptosporidium species.

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.

Library-independent source tracking of fecal contamination in selected stations and tributaries of Laguna Lake, Philippines.

Laguna Lake is the largest inland freshwater body in the Philippines. Although it is classified to be usable for agricultural and recreational purposes by the country's Department of Environment and Natural Resources (DENR), studies looking at lake ecology revealed severe fecal contamination which contributes to the deterioration of water quality. Determining the sources of fecal contamination is necessary for lake protection and management. This study utilized a library-independent method of microbial source tracking (LIM-MST) to identify sources of fecal contamination in selected Laguna Lake stations and tributaries. Genetic markers of the host-associated Escherichia coli, heat-labile toxin (LTIIA) and heat-stable II (STII), were used to identify cattle and swine fecal contaminations, respectively. Meanwhile, human mitochondrial DNA (mtDNA) was used to identify human fecal contamination. Results identified the presence of agricultural and human fecal contamination in Laguna Lake Stations 1 and 5, Mangangate River, and Alabang River. The selected sites are known to be surrounded by residential and industrial complexes, and most of their discharges find their way into the lake. The identification of the specific sources of fecal contamination will guide management practices that aim to regulate the discharges in order to improve the water quality of Laguna Lake.

Occurrence of Giardia duodenalis in selected stations and tributary rivers of Laguna Lake, Philippines.

Fecal pollution is a major contributor to the progressive degradation of Laguna Lake, the Philippines' largest inland lake used for aquaculture, recreation, and as a source of irrigation and domestic water. Consequently, intestinal parasites may be present in this body of water, posing risks to water safety and human health. This study was able to detect the protozoan parasite, Giardia duodenalis, in three Laguna Lake stations and seven tributary rivers in a 13-month monitoring period by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene of Giardia cysts concentrated from water samples. The pathogen was present in 37.7% of tributary samples (n = 69) and 16.7% of lake samples (n = 36). Notable frequencies of detection were observed in four tributary rivers -Bagumbayan, Taguig (66.7%); Santa Rosa, Laguna (55.6%); San Cristobal, Cabuyao, Laguna (44.4%); and Biñan, Laguna (42.9%). All test SSU rRNA gene sequences were identified as human-infective genotypes of G. duodenalis predominated by Assemblage A (94.1%). Furthermore, analysis of the glutamate dehydrogenase (gdh) gene revealed the possible presence of mixed genotypes in at least two samples. These results support the pressing need to include protozoan pathogen monitoring in Laguna Lake and its tributaries to prevent Giardia infection in humans and animals. This study also recommends microbial source tracking to identify fecal pollution sources and aid in regulation of waste discharges into the lake and its tributaries.

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

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.

Influences of carbohydrate, nitrogen, and phosphorus sources on the citric acid production by fungal endophyte Aspergillus fumigatus P3I6.

Fungi are ecologically ubiquitous organisms on earth and regarded as one of the prolific sources of natural products. Fungal endophytes may provide essential prerequisite molecules to plant biochemical pathways which allow the efficient synthesis of primary and secondary metabolites. This study characterized the influences of various combinations of process parameters namely, carbohydrate, nitrogen, and phosphorus sources on citric acid (CA) production by the isolated fungal endophyte Aspergillus fumigatus P3I6 from Citrus microcarpa. Aspergillus fumigatus P3I6 had higher CA concentration of 9.2 (± 0.9) g L-1 and 9.0 (± 5.0 × 10-15) g L-1 when supplemented with sucrose and white refined sugar, respectively, than A. niger NRRL 599. Response Surface Methodology (RSM) had shown that A. fumigatus P3I6 produced the highest CA (23.8 g L-1) in Combination 4 (18.0% sucrose, 0.3 g L-1 ammonium sulfate, and 5.0 g L-1 dipotassium phosphate (K2HPO4)). Analysis of variance showed that when K2HPO4 concentrations were increased, CA content in fermentation media was significantly elevated. Hence, K2HPO4 was the most critical variable in the quadratic model (p < 0.05); however, sucrose concentration still has its role in production. Aside from using A. niger in most fermentation processes, this discovered fungal strain can be potentially used in biotechnological applications.

Leptospira spp. and Toxoplasma gondii in stranded representatives of wild cetaceans in the Philippines.

BACKGROUND: The stranding events of cetaceans in the Philippines provide opportunities for gathering biological information and specimens, especially from the pelagic forms. As part of an effort to monitor the health of wild cetaceans, this study detected Leptospira spp. and Toxoplasma gondii, causative agents of the emerging zoonotic diseases leptospirosis and toxoplasmosis respectively, in their stranded representatives. From October 2016-August 2018, 40 cetaceans (representing 14 species) that stranded nationwide were sampled for brain, cardiac muscle, skeletal muscle, kidney, and blood tissues, urine, and sera. These were subjected to molecular, serological, culture, and histopathological analyses to detect the target pathogens. RESULTS: T. gondii was detected in 20 (71%) of the 28 cetaceans with biological samples subjected to either molecular detection through RE gene amplification or IgG antibodies detection through agglutination-based serological assay. On the other hand, Leptospira was detected in 18 (64%) of 28 cetaceans with biological samples subjected to bacterial culture, molecular detection through 16S rDNA amplification, or IgM antibodies detection through ELISA-based serological assay. CONCLUSIONS: There is the plausibility of toxoplasmosis and leptospirosis in cetacean populations found in the Philippines, however, acute or chronic phases of infections in sampled stranded individuals cannot be confirmed in the absence of supporting pathological observations and corroborating detection tests. Further studies should look for more evidences of pathogenicity, and explore the specific mechanisms by which pelagic cetacean species become infected by Leptospira spp. and T. gondii. As there is growing evidence on the role of cetaceans as sentinels of land-sea movement of emerging pathogens and the diseases they cause, any opportunity, such as their stranding events, should be maximized to investigate the health of their populations. Moreover, the role of leptospirosis or toxoplasmosis in these stranding events must be considered.

Molecular characterization and antimicrobial resistance of Salmonella enterica from swine slaughtered in two different types of Philippine abattoir.

Salmonella enterica is a well-known pathogen commonly acquired from the consumption of contaminated food. It has been estimated to affect millions of humans and cause hundreds of thousands of deaths per year globally. Pork, one of the most commonly consumed meats worldwide, has been identified as one of the main sources of human salmonellosis. In this study, we aimed to detect and characterize S. enterica from slaughtered swine and generate antimicrobial resistance profiles of select isolates. Tonsils and jejunum with mesenteric lymph nodes (MLN) were collected from a total of 240 swine from eight abattoirs (five accredited and three locally registered abattoirs) across Metro Manila. S. enterica were isolated using conventional culture methods and confirmed by PCR amplification of the invA gene. Isolates were further characterized based on somatic antigen by multiplex PCR. We report that there is no significant difference (P = 0.42) between the incidences of S. enterica in swine slaughtered in accredited (44.0%) and in locally registered abattoirs (46.7%). Most samples were contaminated with S. enterica under serogroup O:3,10. Antimicrobial susceptibility testing of 183 isolates using the VITEK® 2 system revealed high resistance to ampicillin (67.8%) and trimethoprim/sulfamethoxazole (80.3%). Multidrug-resistance was found in 124 (67.8%) isolates.

Detection of Class I and II integrons for the assessment of antibiotic and multidrug resistance among Escherichia coli isolates from agricultural irrigation waters in Bulacan, Philippines.

Contaminated irrigation water may greatly affect not only the quality of produce but also the people exposed to it. In this study, agricultural irrigation waters in Bulacan, Philippines were assessed and found to be contaminated with Escherichia coli (E. coli) ranging from 0.58 to 4.51 log10 CFU/mL. A total of 79 isolates of E. coli were confirmed through polymerase chain reaction (PCR) amplifying the uidA gene and were tested for phenotypic resistance using 10 antimicrobials through the Kirby-Bauer disc diffusion method. Forty-six isolates (58.22%) were noted to be multidrug resistant (MDR) with high resistance rate to cephalothin, tetracycline, streptomycin, ampicillin, trimethoprim, nalidixic acid, and chloramphenicol. Moreover, this study also examined the prevalence of Class I and II integrons accounting to 67.39% and 17.39%, respectively, of the MDR E. coli strains using multiplex PCR. The results imply that the agricultural water used in Bulacan is contaminated with the fecal material of man or other animals present in the area, and the presence of MDR bacteria, which pose a potential threat to individuals in these areas, is alarming. In addition, detection of integrons could be a good marker for the identification of MDR isolates. Lastly, this study could develop strategies for the proper management of farming sites leading to the detection of food-borne pathogens and prevention of infectious diseases.

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety.

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

Fecal contamination in irrigation water and microbial quality of vegetable primary production in urban farms of Metro Manila, Philippines.

Microbial contamination of fresh produce can present a severe risk to public health. By conducting a rigorous survey of irrigation waters, the impacts of fecal contamination on the quality of produce could be assessed. In this study, surface waters were observed to be contaminated with Escherichia coli, Salmonella spp., and somatic coliphages. Culture methods show that out of 373 irrigation water, soil, and vegetable samples collected for a 1-year period, 232 (62.20%) were found positive for E. coli, 213 (57.26%) for somatic coliphages, and 2 (0.53%) for Salmonella spp. Out of 190 water samples, 167 (87.9%) were found to have E.coli, 174 (91.6%) have somatic coliphages, and 1 (0.5%) with Salmonella spp. In soil samples, 36 of 91 (39.6%) have E. coli, 31 (34.0%) have somatic coliphages, and none with Salmonella spp. Lastly, out of 92 vegetable samples, 29 (31.5%), 8 (8.7%), and 1 (1.1%) were found to have E. coli, somatic coliphages, and Salmonella spp., respectively. Molecular analysis confirmed the presence of bacterial contaminants. Seasonal weather conditions were noted to have an effect on the presence and number of these fecal indicator organisms. The observed data suggest that contaminated irrigation water may greatly affect the quality of fresh produce from these agricultural operations.

Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

Microbiological quality of fresh produce from open air markets and supermarkets in the Philippines.

This study is the first in the Philippines to conduct a comprehensive assessment of the prevalence of bacterial pathogens and somatic phages in retailed fresh produce used in salad preparation, namely, bell pepper, cabbage, carrot, lettuce, and tomato, using culture and molecular methods. Out of 300 samples from open air and supermarkets, 16.7% tested positive for thermotolerant Escherichia coli, 24.7% for Salmonella spp., and 47% for somatic phages. Results show that counts range from 0.30 to 4.03 log10 CFU/g for E. coli, 0.66 to ≥ 2.34 log10 MPN/g for Salmonella spp., and 1.30 to ≥ 3.00 log 10 PFU/g for somatic phages. Statistical analyses show that there was no significant difference in the microbial counts between open air and supermarkets (α = 0.05). TaqMan and AccuPower Plus DualStar real-time polymerase chain reaction (RT-PCR) was used to confirm the presence of these organisms. The relatively high prevalence of microorganisms observed in produce surveyed signifies reduction in shelf-life and a potential hazard to food safety. This information may benefit farmers, consumers, merchants, and policy makers for foodborne disease detection and prevention.

Subtype-host patterns and genetic differentiation of Blastocystis sp. in the Philippines.

Blastocystis sp. is a gastrointestinal protozoan commonly encountered in humans and animals. Specificity to certain hosts may be associated with 38 known subtypes (STs) and 8 nonmammalian and avian STs (NMASTs). This can be determined by analyzing ST-host associations, ST-allele data, genetic variability analyses, and fixation index (FST) with sufficient data present. Thus, newly acquired and previously published data on Blastocystis sp. STs and NMASTs from the Philippines were compiled to determine the following: (1) ST-host associations, (2) ST-allele diversity per ST in certain hosts/sources, (3) intrasubtype diversity of certain STs found in different hosts using genetic variability analysis, and (4) comparison of similarities between specific ST populations to determine if these are the same circulating populations using FST. A total of 448 samples subtyped using both sequence-tagged site primers and the 600-bp barcoding region of the Blastocystis sp. SSU rRNA gene were analyzed in this study. Patterns of association for the Philippine samples were similar to those from neighboring Southeast Asian countries and around the world: ST1-ST4 were found in humans but ST3 was the most common, ST5 were found in pigs, and ST6 and ST7 were found in poultry. Blastocystis sp. from humans are mostly the same ST alleles (ST3 allele 34 and ST1 allele 4) while 3-5 ST alleles were found in the most common STs in pigs, macaques, and poultry. Also, ST1, ST3, ST5, and NMAST I are undergoing population expansion according to genetic variability analyses through possible addition of new alleles based on ST-allele diversity. Moreover, FST shows the same circulating population of ST1 in humans, pigs, and water indicating a possible waterborne route of cross-transmission. In contrast, ST3 found in humans possibly come from the same circulating population and is genetically distinct from those in nonhuman sources.

Source tracking of fecal contamination in Asian green mussels (Perna viridis) harvested in Manila Bay, Philippines by molecular detection and genotyping of Cryptosporidium spp.

Manila Bay, a multipurpose body of water located around Metro Manila, Philippines, is progressively deteriorating because of massive pollution. Reports have shown that the bay and its aquatic resources (i.e., seafood) are contaminated with fecal matter and enteric pathogens, posing a threat to public health and industry. This problem raises the need for a microbial source tracking methodology as a part of the rehabilitation efforts in the bay. Bivalve mollusks cultivated in water can serve as sentinel species to detect fecal pollution and can complement water monitoring. With the use of polymerase chain reaction and DNA sequence analysis, this study detected Cryptosporidium spp. in Asian green mussels (Perna viridis) cultivated and harvested in Manila Bay and sold in Bulungan Seafood Market, Parañaque, Philippines, from 2019 to 2021 with an overall occurrence of 8.77% (n = 57). The analysis of the 18S rDNA segment revealed three genotypes from Cryptosporidium-positive samples, namely, Cryptosporidium sp. rat genotype IV (60%), C. galli (20%), and C. meleagridis (20%). These findings suggest fecal pollution in bivalve cultivation sites coming from sewage, nonpoint, and agricultural sources. The presence of C. meleagridis, the third most common cause of human cryptosporidiosis, in mussels poses a threat to human health. Thus, there is a need to establish routine detection and source tracking of Cryptosporidium spp. in Manila Bay and to educate seafood consumers on food safety.

A brief history of metal recruitment in protozoan predation.

Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples.

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/µL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.