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Abstract Background Acinetobacter baumannii continued to be an important Gram-negative pathogen of concern in the clinical context. The resistance of this pathogen to carbapenems due to the production of carbapenemases is considered a global threat. Despite the efforts to track carbapenemase synthesis among A. baumannii in the Philippines, local data on its molecular features are very scarce. This study aims to characterize A. baumannii clinical isolates from a Philippine tertiary hospital through genotyping of the pathogen’s carbapenemase genes. Methods Antibiotic susceptibility profiling, phenotypic testing of carbapenemase production, and polymerase chain reaction assays to detect the different classes of carbapenemase genes (class A bla KPC, class B bla NDM, bla IMP, bla VIM, and class D bla OXA-23-like, bla OXA-24/40-like, bla OXA-48-like, bla OXA-51-like, ISAba1-bla OXA-51-like, bla OXA-58-like) were performed in all collected A. baumannii, both carbapenem resistant and susceptible (n = 52). Results Results showed that the majority of the carbapenem-resistant strains phenotypically produced carbapenemases (up to 84% in carbapenem inactivation methods) and possessed the ISAba1-bla OXA-51-like gene complex (80%). Meanwhile, both carbapenem-resistant and carbapenem-susceptible isolates possessed multi-class carbapenemase genes including bla NDM (1.9%), bla VIM (3.9%), bla OXA-24/40-like (5.8%), bla OXA-58-like (5.8%), bla KPC (11.5%), and bla OXA-23-like (94.2%), which coexist with each other in some strains (17.3%). In terms of the intrinsic bla OXA-51-like (oxaAb) genes, 23 unique alleles were reported (bla OXA-1058 to bla OXA-1080), the majority of which are closely related to bla OXA-66. Isolates possessing these alleles showed varying carbapenem resistance profiles. Conclusions In summary, this study highlighted the importance of molecular genotyping in the characterization of A. baumannii by revealing the carbapenemase profiles of the pathogen (which may not be captured accurately in phenotypic tests), in identifying potent carriers of transferrable carbapenemase genes (which may not be expressed straightforwardly in antimicrobial susceptibility testing), and in monitoring unique pathogen epidemiology in the local clinical setting.

The known intrinsic and polymorphic blaOXA-51-like genes of Acinetobacter baumannii were recently reported in other non-A. baumannii Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of blaOXA-51-like genes. Specifically, an alignment-based primer design, in silico primer screening, and in vitro assay confirmation were conducted. Both in silico and in vitro results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3′-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel blaOXA-51-like genes. Finally, it showed a superior limit of detection (as low as 101 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor blaOXA-51-like genes in the clinical setting and provided important insights into LAMP primer design and screening.

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.

The extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is becoming a global public health concern. More comprehensive surveillance of β-lactam resistance in E. coli would improve monitoring strategies and control resistance transmission in contaminated environments. This study investigated the prevalence of β-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 β-lactamase genes using PCR. Among the screened genes, blaAmpC was the most dominant (91%). Other β-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 β-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. HIGHLIGHTS blaAmpC was the predominant gene detected among the five screened β-lactamase genes.; A significant association was detected between blaAmpC with blaSHV and blaCTXM.; blaKPC was not detected in any E. coli isolates from the lakes.; Seasonal variations have an effect on the fecal coliform counts and occurrence of β-lactamase-resistant E. coli isolates.;

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 β-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.

Abstract 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.

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. HIGHLIGHTS DNA fingerprinting of E. coli, coupled with machine learning algorithms, can be used to discriminate fecal pollution sources in Laguna Lake, Philippines.; The majority of E. coli isolates can be attributed to sewage contamination, followed by human and agricultural sources.; Source-tracking methods can empower local agencies responsible for water quality management to minimize public health and economic risks.;

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. HIGHLIGHTS The Escherichia coli LTIIa and STII genes, as well as human NADH mtDNA, are useful biomarkers for detecting fecal contamination in Laguna Lake.; Fecal contamination from agricultural and human origins can be detected in some lake stations and rivers near Laguna Lake.; As indicated by the high detection of LTIIa gene, fecal contamination in the sampling sites was mostly of cattle origin.;

Lakes are one of the sinks of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs); however, information on ARB and ARGs in lakes in the Philippines is scarce. In this study, Escherichia coli was isolated from the largest freshwater lake in the Philippines, Laguna Lake, to detect antibiotic resistance and the presence of ARGs. Broth microdilution assay (BMA) and molecular identification of five environmentally prevalent ARGs (strA, blaCTX-M, blaSHV, blaTEM, and tetA) were performed. The majority (75.70%) of the isolates harbored at least one of the targeted antibiotic genes. Multiplex PCR detected about 49.07% of the isolates had genes for extended-spectrum β-lactamases (ESBL), which were mostly represented by blaTEM (47.66%). The genes strA and tetA were observed in this study with detection frequencies of 29.91 and 45.33%, respectively. About 95.69% of thermotolerant E. coli isolates were non-susceptible to six different antibiotics using BMA. Nearly 37% of the isolates were found to be multidrug-resistant (MDR) with most isolates resistant to ampicillin (81.72%). Furthermore, the occurrence of ESBL genes was significantly correlated with tetA genes (P = 0.013). To date, this study is the first to report on the presence of MDR and thermotolerant E. coli in Laguna Lake, Philippines. HIGHLIGHTS First report of the ecological presence of thermotolerant and multidrug-resistant Escherichia coli (37%) in Laguna Lake.; Most (75.70%) isolates harbored at least one of the targeted antibiotic resistance genes (strA, blaTEM, blaCTX-M, blaSHV, and tetA).; First report of the occurrence of aminoglycoside resistant gene (strA) and tetracycline efflux gene (tetA) in waterborne E. coli isolated in the 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. HIGHLIGHTS Zoonotic and pathogenic species of Cryptosporidium in a freshwater reservoir point to domestic and agricultural sources of fecal contamination.; Cryptosporidium can be a potential marker for microbial source tracking in the Laguna Lake watershed to augment current monitoring efforts.;

The current gold standard for Salmonella serotyping is costly, labor-intensive and time-consuming. However, proper identification is key to monitor Salmonella transmission and implementation of necessary control measures. The onset of advanced molecular techniques has lessened resource and labor requirements; however, it still remains complex, unestablished and plagued with insufficiencies. Hence, a simpler serotyping method with sufficient resolution is needed. In this study, the invA virulence gene, associated with Salmonella invasion into host cells and is considered as a marker for Salmonella detection, was amplified and sequenced among isolates from meat samples in Metro Manila, Philippines. This was followed by sequence alignments with reference sequences (Refseqs), oversaturation and model tests, phylogenetic tree analyses and signal detections. Unfortunately, alignment of a 229bp amplified and sequenced invA gene segment with Refseqs generated little to no base variations and consequently provided insufficient phylogenetic resolution for molecular serotyping (0 of the 17 serotypes tested). However, another segment of 1498bp, outside the amplified region, showed considerable base variation in alignment and consequently resolved a maximum of 13 out of 17 (76.47%) serotypes tested, all generated trees considered. These suggest the potential of the invA virulence gene as a single-gene marker for molecular serotyping of Salmonella through phylogenetic analyses.

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.

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.

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.

Abstract 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.

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.

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.

Due to the misuse and overuse of antibiotics, antibiotic residues accumulate in natural environments, leading to the development of antibiotic-resistant bacteria (ARBs). The presence of ARBs in bodies of water poses health hazards to the surrounding community. This study focused on Laguna Lake, the largest lake in the Philippines, which serves as a water source for agriculture and domestic purposes. We aimed to detect the presence of antibiotic-resistant Escherichia coli from the lake waters and potential reservoirs of resistance as well as determine the multiple antibiotic resistance (MAR) indices of the isolates. E. coli (n = 450) was isolated from fecal-associated samples (chicken, cow, pig, human, sewage) and water samples (sites in Laguna Lake and selected river tributaries). The isolates were subjected to an antibiotic resistance assay using VITEK 2®. Among the 16 antibiotics tested, the isolates exhibited varying resistance to 14, but complete susceptibility to amikacin and tigecycline was observed. Isolates were most frequently resistant to ampicillin (196/450, 43.6%). Among fecal-associated samples, chicken isolates exhibited the highest MAR index (0.174), whereas samples from Pila River exhibited the highest MAR index (0.152) among water samples. The results of this study demonstrate the presence of multidrug-resistant E. coli in samples collected around Laguna Lake and reveal fecal and sewage sources as potential reservoirs of ARBs in the water body. With this information, the public is urged to use antibiotics responsibly to help mitigate the spread of antibiotic resistance.

This study aimed to detect Salmonella from retail meat collected from nine wet markets in Metro Manila, and identify the subtypes of Salmonella isolates using molecular serotyping assays from previously developed primers. Of the 720 collected meat samples, 57.64% were found to be Salmonella-contaminated. The most predominant serogroup was Salmonella O:3, and Salmonella serogroups O:4, O:6,7, O:8, O:9, and undetermined serogroups were also found. Most frequently detected isolates in bovine meat were S. 3:e,h:1,6 (putative identity: S. Anatum) and S: 4:e,h:1,2 (putative identity: S. Saintpaul), in porcine meat was S. 3:e,h:1,6 (putative identity: S. Anatum), and S. 8:i:z6 (putative identity: S. Kentucky) was common in poultry products. This study also demonstrated retail meat samples were contaminated with multiple Salmonella serogroups and serovars. This is the first Philippine study that utilized PCR-based assays to characterize Salmonella isolates down to a serovar level and provides baseline information regarding Salmonella prevalence and serovar distribution in retail meat. Molecular serotyping performed in this study can be used as an alternative approach to traditional serotyping in surveillance of Salmonella in the Philippines since the latter is expensive, time-consuming, and requires skilled technicians.

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 β-lactams and fluoroquinolones, raise a concern on antimicrobial use in the Philippine hog industry and on possible transmission of ARG to other bacteria.

Background/Purpose: The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. Results: A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. Conclusion: This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate.

This data article contains transmission electron microscopy (TEM) images of psychrotolerant bacterium Shewanella olleyana sp. nov. Cells of S. olleyana were grown following an optimized culture conditions in liquid medium. Procedure for the preparation of cells suitable for TEM is described in detail.

Objective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16–33 mm inhibition diameter), minimum inhibitory concentration (19–156 μg/mL) and minimum bactericidal concentration (312 μg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing) in the disk diffusion (17–21 mm inhibition diameter), minimum inhibitory concentration (312–625 μg/mL) and minimum bactericidal concentration (312–625 μg/mL) assays. Conclusions: P. betle had the greatest potential value against both Gram-negative and Gram-positive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.

Highly-polluted surface waters are increasingly used for irrigation in different agricultural settings because they have high nutrient content and are readily available. However, studies showed that they are reservoirs for the emergence and dissemination of antibiotic-resistant bacteria in the environment. In this study, the resistance of 212 Escherichia coli isolates from irrigation water, soil, and vegetables in selected urban farms in Metro Manila, Philippines was evaluated. Results showed that antibiotic resistance was more prevalent in water (67.3%) compared to soil (56.4%) and vegetable (61.5%) isolates. Resistance to tetracycline was the highest among water (45.6%) and vegetable (42.3%) isolates while ampicillin resistance was the highest among soil isolates (33.3%). Multidrug-resistant (MDR) isolates were also observed and they were more prevalent in water (25.3%) compared to soil (2.8%) and vegetable (8.4%) isolates. Interestingly, there are patterns of antibiotic resistance that were common to isolates from different samples. Extended-spectrum beta-lactamase production (ESBL) was also investigated and genes were observed to be present in 13 isolates. This provides circumstantial evidence that highly-polluted surface waters harbor antibiotic-resistant and MDR E. coli that may be potentially transferred to primary production environments during their application for irrigation purposes.

Entamoeba histolytica is one of the most significant protozoan pathogens found in developing countries like the Philippines. This intestinal parasite causes the disease amebiasis, which has a yearly average mortality of about 100,000 people worldwide. Thus, it is essential to develop new diagnostic markers and possible treatment against this disease. The crude cell extract of E. histolytica, was used to induce polyclonal antibody response in mice. Balb/c mice were given immunizations of the prepared crude E. histolytica antigens for a period of twelve weeks. Indirect fluorescent antibody test showed the specificity of polyclonal IgG in recognizing the cytosolic components of E. histolytica trophozoites. Enzyme-linked immunosorbent assay was performed to determine the antibody titers in sera collected at various time intervals. Antibody titers for the mouse serum taken 10 and 20 days after the third booster immunization were known to be 16,384 and 4,096 respectively. SDS-PAGE profile of the crude E. histolytica antigens revealed three bands with molecular weights of 23, 41, and 47 kDa. Western immunoblot results indicated that the polyclonal IgG produced by mice targets the potentially novel 23 kDa antigen from an axenic E. histolytica culture.

The problem on the widespread occurrence of antibiotic resistant strains of bacteria calls for novel methods of control of bacterial activity. One of the new viable alternatives to antibiotics is the use of substances that inhibit quorum sensing (QS) – a bacterial communication system that has been known to regulate the expression of virulence genes during infection. In this study, epigallocatechin gallate (EGCG) from green tea, Camellia sinensis L. (Kuntze) was tested for its ability to inhibit QS in a test organism, Chromobacterium violaceum. This microorganism produces a violet-colored substance, violacein, through QS. This study aimed to detect inhibition of QS-regulated violacein production in C. violaceum by EGCG and to determine the dynamics of QS inhibition relative to the concentration of EGCG. The effect of increasing concentration of EGCG on both violacein production and cell density of treated and untreated C. violaceum was determined in a 96-well-microplate format and read at 570nm and 620nm for violacein production and growth, respectively. The results show that addition of EGCG increased the growth of the organism while there is concentration-dependent decrease in the QS-controlled production of violacein. This study thus establishes that EGCG is a potential QS inhibitor and can be further studied and developed for its use as an anti-pathogenic but non-toxic drug.

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 log10 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.

Trichomonads are obligate anaerobes generally found in the digestive and genitourinary tract of domestic animals. In this study, four trichomonad isolates were obtained from carabao, dog, and pig hosts using rectal swab. Genomic DNA was extracted using Chelex method and the 18S rRNA gene was successfully amplified through novel sets of primers and undergone DNA sequencing. Aligned isolate sequences together with retrieved 18S rRNA gene sequences of known trichomonads were utilized to generate phylogenetic trees using maximum likelihood and neighbor-joining analyses. Two isolates from carabao were identified as Simplicimonas similis while each isolate from dog and pig was identified as Pentatrichomonas hominis and Trichomitus batrachorum, respectively. This is the first report of S. similis in carabao and the identification of T. batrachorum in pig using 18S rRNA gene sequence analysis. The generated phylogenetic tree yielded three distinct groups mostly with relatively moderate to high bootstrap support and in agreement with the most recent classification. Pathogenic potential of the trichomonads in these hosts still needs further investigation.