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.

Microeukaryotic plankton evolutionary constraints in a subtropical river explained by environment and bacteria along differing taxonomic resolutions.

Microeukaryotic plankton communities are keystone components for keeping aquatic primary productivity. Currently, variations in microeukaryotic plankton diversity have often been explained by local ecological factors but not by evolutionary constraints. We used amplicon sequencing of 100 water samples across five years to investigate the ecological preferences of the microeukaryotic plankton community in a subtropical riverine ecosystem. We found that microeukaryotic plankton diversity was less associated with bacterial abundance (16S rRNA gene copy number) than bacterial diversity. Further, environmental effects exhibited a larger influence on microeukaryotic plankton community composition than bacterial community composition, especially at fine taxonomic levels. The evolutionary constraints of microeukaryotic plankton community increased with decreasing taxonomic resolution (from 97% to 91% similarity levels), but not significant change from 85% to 70% similarity levels. However, compared with the bacterial community, the evolutionary constraints were shown to be more affected by environmental variables. This study illustrated possible controlling environmental and bacterial drivers of microeukaryotic diversity and community assembly in a subtropical river, thereby indirectly reflecting on the quality status of the water environment by providing new clues on the microeukaryotic community assembly.

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.

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.

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.

Host-Associated Bacteroides 16S rDNA-Based Markers for Source Tracking of Fecal Pollution in Laguna Lake, Philippines.

Sources of fecal contamination in Laguna Lake, Philippines, were identified using a library-independent microbial source tracking method targeting host-associated Bacteroides 16S rDNA-based markers. Water samples from nine lake stations were assessed for the presence of the fecal markers HF183 (human), BoBac (cattle), Pig-2-Bac (swine), and DuckBac (duck) from August 2019 to January 2020. HF183 (average concentration = 1.91 log10 copies/mL) was the most frequently detected, while Pig-2-Bac (average concentration = 2.47 log10 copies/mL) was the most abundant. The detected marker concentrations in different stations corresponded to the land use patterns around the lake. Generally, all marker concentrations were higher during the wet season (August-October), suggesting the effect of rainfall-associated factors on the movement and retention of markers from sources. There was a significant association (ρ = 0.45; p < 0.001) between phosphate and the concentration of HF183, suggesting domestic sewage-derived pollution. The markers had acceptable sensitivity and specificity, i.e., HF183 (S = 0.88; R = 0.99), Pig-2-Bac (S = 1.00; R = 1.00), and DuckBac (S = 0.94; R = 1.00), and therefore may be used for the continuous monitoring of fecal pollution in the lake and in designing interventions to improve the quality of the lake water.

Roles of phosphate-solubilizing bacteria in mediating soil legacy phosphorus availability.

Phosphorus (P), an essential macronutrient for all life on Earth, has been shown to be a vital limiting nutrient element for plant growth and yield. P deficiency is a common phenomenon in terrestrial ecosystems across the world. Chemical phosphate fertilizer has traditionally been employed to solve the problem of P deficiency in agricultural production, but its application has been limited by the non-renewability of raw materials and the adverse influence on the ecological health of the environment. Therefore, it is imperative to develop efficient, economical, environmentally friendly and highly stable alternative strategies to meet the plant P demand. Phosphate-solubilizing bacteria (PSB) are able to improve plant productivity by increasing P nutrition. Pathways to fully and effectively use PSB to mobilize unavailable forms of soil P for plants has become a hot research topic in the fields of plant nutrition and ecology. Here, the biogeochemical P cycling in soil systems are summarized, how to make full use of soil legacy P via PSB to alleviate the global P resource shortage are reviewed. We highlight the advances in multi-omics technologies that are helpful for exploring the dynamics of nutrient turnover and the genetic potential of PSB-centered microbial communities. Furthermore, the multiple roles of PSB inoculants in sustainable agricultural practices are analyzed. Finally, we project that new ideas and techniques will be continuously infused into fundamental and applied research to achieve a more integrated understanding of the interactive mechanisms of PSB and rhizosphere microbiota/plant to maximize the efficacy of PSB as P activators.

Phylogenetic relationship of nonmammalian and avian Blastocystis isolates and conventional subtypes.

Blastocystis is an intestinal protist commonly identified in human and animal feces. At present, there are 28 proposed subtypes (STs) identified based on the small subunit rRNA gene, 13 of which are found in both humans and animals. In general, nonmammalian and avian groups are infected by the nonmammalian and avian subtypes (NMASTs). However, NMASTs were also isolated from mammalian hosts, suggesting possible cross-contamination and transmission from nonmammalian and avian hosts to other animals and, potentially, humans. Thus, this study determined the possible relationship between NMAST sequences and conventional STs to provide new insights into Blastocystis classification, identification, and epidemiological significance. Phylogenetic trees were constructed using three statistical models, namely, Maximum Likelihood, Neighbor Joining, and Maximum Parsimony, based on the 30 NMAST sequences. The NMAST sequences formed groups clustered closely with other NMAST subtrees. Most sequences of nonmammalian and avian isolates formed distinct monophyletic clades based on their NMAST classification, with some clustering with mammalian and avian STs. These results indicate the close relationship between Blastocystis isolated from mammalian and avian hosts and nonmammalian and avian hosts. Supplementary Information: The online version contains supplementary material available at (10.1007/s12639-022-01554-7).

In Silico Approaches for the Identification of Aptamer Binding Interactions to Leptospira spp. Cell Surface Proteins.

Aptamers are nucleic acids that can bind with high affinity and specificity to a range of target molecules. However, their functionality relies on their secondary and tertiary structures such that the combination of nucleotides determines their three-dimensional conformation. In this study, the binding mechanisms of candidate aptamers and their interactions with selected target proteins found in the cell surface of Leptospira were predicted to select high-affinity aptamers. Four aptamers were evaluated through molecular modeling and docking using available software and web-based tools, following the workflow previously designed for in silico evaluation of DNA aptamers. The most predominant and highly conserved surface-exposed proteins among pathogenic Leptospira species were used as aptamer targets. The highest number of interactions was seen in aptamers AP5 and AP1. Hydrogen bonds, along with a few hydrophobic interactions, occur in most aptamer-protein complexes. Further analysis revealed serine, threonine, glutamine, and lysine as main protein residues. H-bond interactions occur mostly with polar amino acids, as reflected in the predicted interaction profiles of aptamer-protein complexes. In silico strategies allowed the identification of key residues crucial in aptamer-target interaction during aptamer screening. Such information can be used in aptamer modification for improved binding affinity and accuracy for diagnostics application.

Genomic analysis of Salmonella enterica from Metropolitan Manila abattoirs and markets reveals insights into circulating virulence and antimicrobial resistance genotypes.

The integration of next-generation sequencing into the identification and characterization of resistant and virulent strains as well as the routine surveillance of foodborne pathogens such as Salmonella enterica have not yet been accomplished in the Philippines. This study investigated the antimicrobial profiles, virulence, and susceptibility of the 105 S. enterica isolates from swine and chicken samples obtained from slaughterhouses and public wet markets in Metropolitan Manila using whole-genome sequence analysis. Four predominant serovars were identified in genotypic serotyping, namely, Infantis (26.7%), Anatum (19.1%), Rissen (18.1%), and London (13.3%). Phenotypic antimicrobial resistance (AMR) profiling revealed that 65% of the isolates were resistant to at least one antibiotic, 37% were multidrug resistant (MDR), and 57% were extended-spectrum ß-lactamase producers. Bioinformatic analysis revealed that isolates had resistance genes and plasmids belonging to the Col and Inc plasmid families that confer resistance against tetracycline (64%), sulfonamide (56%), and streptomycin (56%). Further analyses revealed the presence of 155 virulence genes, 42 of which were serovar-specific. The virulence genes primarily code for host immune system modulators, iron acquisition enzyme complexes, host cell invasion proteins, as well as proteins that allow intracellular and intramacrophage survival. This study showed that virulent MDR S. enterica and several phenotypic and genotypic AMR patterns were present in the food chain. It serves as a foundation to understand the current AMR status in the Philippines food chain and to prompt the creation of preventative measures and efficient treatments against foodborne pathogens.

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.

Development of a salivary IgA detection method for accurate diagnosis of amebiasis.

An amebiasis detection method was developed based on identifying anti-Entamoeba histolytica IgA in the saliva of infected individuals. The enzyme-linked immunosorbent assay (ELISA)-based detection method was tested along with microscopy and polymerase chain reaction (PCR) on saliva and stool samples from 110 asymptomatic individuals visiting the Manila Health Department - Public Health Laboratory of the City of Manila, Philippines. A receiver operating curve (ROC) was constructed to compare the ELISA results with PCR results. E. histolytica infection was detected in 18 of the 110 individuals. The developed method had an accuracy of 90%, sensitivity of 88.89%, specificity of 90.22%, positive predictive value of 64%, and negative predictive value of 97.65% if a 1:2 dilution of crude saliva sample in phosphate-buffered saline (PBS) was used for diagnosis when compared to PCR. The area under the curve (AUC) of the ROC was 0.9436 if a 1:2 dilution of a crude saliva sample was used. The developed assay presents an easy and accurate method of detecting amebiasis in infected individuals using saliva samples instead of stool or blood samples and has potential applications in both diagnosis and epidemiological studies.

Variations in active proteases of Blastocystis sp. obtained from water and animal isolates from the Philippines.

Blastocystis sp. is a commonly encountered gut protozoan with unclear pathogenicity. The presence of proteases in this organism may be related to its potential pathogenicity or other physiological differences. This study aimed to identify the various proteases that may be present in different subtypes (STs) of Blastocystis sp. using azocasein assay and gelatin zymography. In this study, cysteine, serine, aspartic protease, metalloproteases, and unknown proteases were identified in Blastocystis sp. cultures obtained from animal and water samples belonging to ST1-ST5 and ST7. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Cysteine protease was the most commonly encountered. Protease activity in general is not associated with ST or source (animal or water). However, the presence of cysteine protease alone was associated with animal samples, whereas the presence of more than two protease types was associated with water samples. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Protease activity and the types present may change over time. Blastocystis sp. shows high protease diversity, including possible novel types.

Recent Progress in Recombinant Influenza Vaccine Development Toward Heterosubtypic Immune Response.

Flu, a viral infection caused by the influenza virus, is still a global public health concern with potential to cause seasonal epidemics and pandemics. Vaccination is considered the most effective protective strategy against the infection. However, given the high plasticity of the virus and the suboptimal immunogenicity of existing influenza vaccines, scientists are moving toward the development of universal vaccines. An important property of universal vaccines is their ability to induce heterosubtypic immunity, i.e., a wide immune response coverage toward different influenza subtypes. With the increasing number of studies and mounting evidence on the safety and efficacy of recombinant influenza vaccines (RIVs), they have been proposed as promising platforms for the development of universal vaccines. This review highlights the current progress and advances in the development of RIVs in the context of heterosubtypic immunity induction toward universal vaccine production. In particular, this review discussed existing knowledge on influenza and vaccine development, current hemagglutinin-based RIVs in the market and in the pipeline, other potential vaccine targets for RIVs (neuraminidase, matrix 1 and 2, nucleoprotein, polymerase acidic, and basic 1 and 2 antigens), and deantigenization process. This review also provided discussion points and future perspectives in looking at RIVs as potential universal vaccine candidates for influenza.

Corrigendum: Recent Progress in Recombinant Influenza Vaccine Development Toward Heterosubtypic Immune Response.

[This corrects the article DOI: 10.3389/fimmu.2022.878943.].

Antimicrobial Susceptibility and Frequency of bla and qnr Genes in Salmonella enterica Isolated from Slaughtered Pigs.

Salmonella enterica is known as one of the most common foodborne pathogens worldwide. While salmonellosis is usually self-limiting, severe infections may require antimicrobial therapy. However, increasing resistance of Salmonella to antimicrobials, particularly fluoroquinolones and cephalosporins, is of utmost concern. The present study aimed to investigate the antimicrobial susceptibility of S. enterica isolated from pork, the major product in Philippine livestock production. Our results show that both the qnrS and the blaTEM antimicrobial resistance genes were present in 61.2% of the isolates. While qnrA (12.9%) and qnrB (39.3%) were found less frequently, co-carriage of blaTEM and one to three qnr subtypes was observed in 45.5% of the isolates. Co-carriage of blaTEM and blaCTX-M was also observed in 3.9% of the isolates. Antimicrobial susceptibility testing revealed that the majority of isolates were non-susceptible to ampicillin and trimethoprim/sulfamethoxazole, and 13.5% of the isolates were multidrug-resistant (MDR). MDR isolates belonged to either O:3,10, O:4, or an unidentified serogroup. High numbers of S. enterica carrying antimicrobial resistance genes (ARG), specifically the presence of isolates co-carrying resistance to both ß-lactams and fluoroquinolones, raise a concern on antimicrobial use in the Philippine hog industry and on possible transmission of ARG to other bacteria.

Bacteriological and histopathological findings in cetaceans that stranded in the Philippines from 2017 to 2018.

The relatively high frequency of marine mammal stranding events in the Philippines provide many research opportunities. A select set of stranders (n = 21) from 2017 to 2018 were sampled for bacteriology and histopathology. Pertinent tissues and bacteria were collected from individuals representing eight cetacean species (i.e. Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to histopathological examination and antibiotic resistance screening, respectively. The antibiotic resistance profiles of 24 bacteria (belonging to genera Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n = 24) and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n = 24). The resistance profiles of these bacteria show the extent of antimicrobial resistance in the marine environment, and may inform medical management decisions during rehabilitation of stranded cetaceans. Due to inadequate gross descriptions and limited data gathered by the responders during the stranding events, the significance of histopathological lesions in association with disease diagnosis in each cetacean stranding or mortality remained inconclusive; however, these histopathological findings may be indicative or contributory to the resulting debility and stress during their strandings. The findings of the study demonstrate the challenges faced by cetacean species in the wild, such as but not limited to, biological pollution through land-sea movement of effluents, fisheries interactions, and anthropogenic activities.

Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines.

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.