Harnessing the Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular-matrix (ECM)-derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. Here, a chitosan/alginate (C/A) hydrogel is developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) are cultured with these constructs and proliferation and cytotoxicity assays confirm that these C/A hydrogels are bioactive. Sequential z-axis fluorescent imaging visualizes hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining shows that hDPCs cultured with the hydrogels display increased calcium-ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity is increased, as is expression of transforming growth factor-beta as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrates that both dentin and pulp ECM molecules act as chemoattractants for hDPCs. Data from this study demonstrate that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulates dental tissue repair processes in vitro.

Biodegradation and reduction of toxicity of Azo Trypan Blue dye by Amazonian strains of gasteroid fungi (Basidiomycota).

Amazonian strains of Cyathus spp. and Geastrum spp. were studied for the ability to discolor the trypan blue azo dye and reduce its toxicity. Discoloration of trypan blue dye (0.05%) was evaluated in solid and aqueous medium over different periods. The reduction of dye toxicity after treatment was assessed by seed germination and the development of lettuce seedlings (Lactuca sativa L.) and toxicity test in Artemia salina (L.) larvae. All evaluated strains showed the potential to reduce the color intensity of trypan blue dye. Cyathus strains reached 96% discoloration, and C. albinus and C. limbatus also reduced dye toxicity. Geastrum strains showed a high efficiency degree in color reduction, reaching 98% discoloration, however, the by-products generated during the process presented toxicity and require further investigation. For the first time, Amazonian strains of gasteroid fungi degrading trypan blue are reported, some even reducing its toxicity. Thus, making them promising sources of enzymes of interest to bioremediation scenarios involving synthetic dyes.

In Vitro Models Used in the Formation of Root Caries Lesions-A Review of the Literature.

The management of root caries remains a challenge for clinicians due to its unique anatomical location and structure. There is increasing interest in utilising artificial root caries lesions to develop new strategies for remineralisation. An ideal protocol has not yet been agreed upon. The aim of this review is to provide a structured overview of previously reported in vitro root caries models. The literature was screened and mined for information mainly on substrate selection, model systems utilised, and variables used in the models. Human roots (60%) were the most frequently used substrates, followed by bovine roots (40%). Chemical models (69%) were the most frequently utilised model systems, followed by microbiological models (27%), to form root caries lesions. Acetate buffer solution (80%), pH 5.0 or above (40%), and a demineralisation time of five days (25%) were the common variables used in the chemical systems, while mono-species biofilm was most frequently used (73%) in microbiological models and Streptococcus mutans was the most common bacterial strain utilised in these models (80%). This review highlights the variability amongst the experimental approaches, discusses the advantages and limitations of these approaches, and emphasises that standardisation of experimental conditions along with sustained research will benefit root caries research.

Effects of glycation end-products on the dental pulp in patients with type 2 diabetes.

AIM: This ex vivo study aimed to compare protein expression of advanced glycation end-products (AGE) and receptor (RAGE), and the levels of selected genes associated with inflammation and collagen within dental pulp tissue from patients with type 2 (T2D) diabetes and non-T2D. METHODOLOGY: Noncarious extracted permanent molar teeth from patients with well-controlled T2D (n = 19) and non-T2D (controls) (n = 19) were collected and compared. The coronal pulp was examined using immunohistochemistry (IHC) (n = 10 per group) for anti-AGE and anti-RAGE. Quantitative PCR (n = 9 per group) was used to analyse the gene expression levels of NFKB, S100A12 and COLIA1. Data analyses were performed between the groups using GraphPad Prism using Pearson correlation, Shapiro-Wilk and Mann-Whitney U-tests, and multiple regression using SPSS. RESULTS: AGEs were distributed diffusely throughout the pulp extracellular matrix associated with collagen fibres and were present on several cell types. RAGE was expressed at the pulp-dentine interface and was observed on odontoblasts, immune cells, endothelial cells and fibroblasts. Semi-quantitative analysis of IHC samples showed significantly increased expression of AGE (p < .0001) and RAGE (p = .02) in T2D samples compared with controls. The expression of NFKB (p < .0001), S100A12 (p < .0001) and COLIA1 (p = .01) genes were significantly higher in the T2D pulp, and multivariate logistic regression analysis showed that these findings were not affected by age. CONCLUSION: T2D may exert a similar glycation response in the dental pulp to other body sites. This could occur through activation of NF-κB pathways with a concomitant increase in genes associated with inflammation and collagen.

Characteristics and outcomes of cases with methylphenidate abuse, dependence or withdrawal: an analysis of spontaneous reports in EudraVigilance.

Methylphenidate (MPH) is a central nervous stimulant, which is mainly used in attention deficit hyperactivity disorder (ADHD) and narcolepsy. In recent years, rising MPH prescription volumes have drawn attention to possible misuse. We analyzed data on suspected MPH abuse, dependence or withdrawal reported to the EudraVigilance database (1996-2019), comparing case characteristics (e.g. age, indications and outcome). In 1531 cases from 35 countries (median: 29 years, 57.6% male), 42.3% had been diagnosed with ADHD and 8.0% with narcolepsy. Narcolepsy cases were older than ADHD cases (mean age: 47 vs. 22 years), and used co-medications more frequently (79.4 vs. 47.8%). Intravenous MPH administration was most common among individuals not diagnosed with ADHD or narcolepsy. A history of abuse, dependence or withdrawal of any substance was more often documented in fatal than in nonfatal cases (49.0 vs. 22.5%), whereas differences regarding the route of administration were less pronounced. Minors, who presumably received MPH for ADHD treatment, presented less frequently with serious outcomes than older cases or those without an approved indication. Prescribers should exercise caution in adult MPH users and should make a thorough co-medication assessment. Finally, more research on substance abuse in narcolepsy patients is required, which should include comorbidities and co-medication.

Predation risk effects on larval development and adult life of Aedes aegypti mosquito.

Biological control is one of the methods available for control of Aedes aegypti populations. We used experimental microcosms to evaluate the effects of actual predation and predation risk by dragonfly larvae (Odonata) on larval development, adult longevity, and adult size of Ae. aegypti. We used six treatments: control, removal, variable density cues (Cues VD), fixed density cues (Cues FD), variable density predator (Predator VD), and fixed density predator (Predator FD) (n = 5 each). Predator treatments received one dragonfly larva. Cue treatments were composed of crushed Ae. aegypti larvae released into the microcosm. For the FD treatments, we maintained a larval density of 200 individuals. The average mortality of Ae. aegypti larvae in the Predator VD treatment was used as the standard mortality for the other treatments. Mosquitoes from the Predator VD and Cues VD treatments developed faster, and adults were larger and had greater longevity compared to all other treatments, likely due to the higher food availability from larval density reduction. High larval density negatively affected larval developmental time, adult size, and longevity. Males were less sensitive to density-dependent effects. Results from this study suggest that the presence of predators may lead to the emergence of adult mosquitoes with greater fitness, causing an overall positive effect on Ae. aegypti population growth rates.

Methanolic Extracts of Chiococca alba in Aedes aegypti Biorational Management: Larvicidal and Repellent Potential, and Selectivity against Non-Target Organisms.

The use of formulations containing botanical products for controlling insects that vector human and animal diseases has increased in recent years. Plant extracts seem to offer fewer risks to the environment and to human health without reducing the application strategy's efficacy when compared to synthetic and conventional insecticides and repellents. Here, we evaluated the potential of extracts obtained from caninana, Chiococca alba (L.) Hitchc. (Rubiaceae), plants as a tool to be integrated into the management of Aedes aegypti, one of the principal vectors for the transmission of arborviruses in humans. We assessed the larvicidal and repellence performance against adult mosquitoes and evaluated the potential undesired effects of the extracts on non-target organisms. We assessed the susceptibility and predatory abilities of the nymphs of Belostoma anurum, a naturally occurring mosquito larva predator, and evaluated the C. alba extract's cytotoxic effects in mammalian cell lines. Our chromatographic analysis revealed 18 compounds, including rutin, naringin, myricetin, morin, and quercetin. The methanolic extracts of C. alba showed larvicidal (LC50 = 82 (72-94) mg/mL) activity without killing or affecting the abilities of B. anurum to prey upon mosquito larvae. Our in silico predictions revealed the molecular interactions between rutin and the AeagOBP1 receptor to be one possible mechanism for the repellent potential recorded for formulations containing C. alba extracts. Low cytotoxicity against mammalian cell lines reinforces the selectivity of C. alba extracts. Collectively, our findings highlight the potential of C. alba and one of its constituents (rutin) as alternative tools to be integrated into the management of A. aegypti mosquitoes.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

OBJECTIVES: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). METHODS: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. CONCLUSION: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.

Carbon conundrums: Do United States' current carbon market baselines represent an undesirable ecological threshold?

Relative frequency distribution of observed annual mortality expressed in aboveground (AG) carbon (C) (Mg CO2 e ha-1 year-1 ) summarized across supersections by forest type [Hardwood (HW) vs. Softwood (SW)] and site class (Low vs. High) based on approximately 130,000 remeasured USDA Forest Service Forest Inventory and Analysis plots across the US. Top panel summarizes conditions in plots that do and do not meet the California Air Resources Board standards based on total basal area, whereas bottom panel summarizes conditions in plots falling inside and outside of optimum relative density levels. The latter represents a biophysically-informed approach accounting for changes in tree (and carbon) packing over forest development.

Technological, sensory, nutritional and bioactive potential of pan breads produced with refined and whole grain buckwheat flours.

The nutritional quality and bioactive potential of breads made with partial replacement of refined wheat flour (RWF) with 30% or 45% refined buckwheat flour (RBF) or whole buckwheat flour (WGBF) was assessed through mineral bioaccessibility, starch digestibility, dietary fiber content and bioactive potential by determining rutin and quercetin levels during processing. Moreover, technological quality and sensory acceptance were also evaluated. Breads made with 30% or 45% WGBF showed higher mineral and fiber contents compared to the control, while the formulations with RBF showed higher bioaccessibility. No changes were observed in the rutin levels of the dough before and after fermentation, but after baking, rutin and quercetin levels increased. The highest starch hydrolysis was found in the formulation containing 45% RBF. The formulations made with 30% RBF or 30% WGBF were well accepted by consumers. Our study shows interesting results, as few studies report the effect of processing on bioactive compounds.

Type 2 diabetes and the clinically normal pulp: An in vitro study.

AIM: The aim of this study was to investigate the effect of type 2 diabetes (T2D) on clinically normal dental pulp tissue by using special stains and immunohistochemistry (IHC) to determine the morphology of the coronal pulp and distribution of immune markers in non-T2D and T2D groups. METHODOLOGY: Ethics approval for this in vitro pilot study was obtained from the University of Otago Human Ethics Committee (16/069). Twenty extracted permanent molar teeth diagnosed as having clinically normal pulp status were collected. Ten teeth were from participants with well-controlled T2D and ten from participants without diabetes (non-T2D). Each tooth was sectioned transversely at the cemento-enamel junction before the crowns were decalcified and embedded in paraffin. Sections were stained with haematoxylin and eosin, Massons trichrome, and van Gieson stains for histological and morphological evaluation. IHC using anti-CD4, anti-CD68 and anti-CD83 and anti-IL1ß, anti-IL6, anti-IL17, anti-TNF-α, anti-TLR2, anti-TLR4 and anti-FOXP3 identified proteins of interest. Qualitative and semi-quantitative analyses evaluated the morphology of the dental pulp and protein expression. Data analyses were performed with GraphPad Prism, using Student's t-test and multiple regression using SPSS at p < .05. RESULTS: Special stains demonstrated morphological differences in the T2D dental pulp compared with non-T2D. Qualitative analysis indicated that the pulp in the T2D samples was consistently less cellular, less vascular, showed evidence of thickened blood vessel walls, increased pulp calcification and collagen deposition. Semi-quantitative analysis of IHC samples showed the T2D pulp had significantly increased expression of macrophage and dendritic cell markers CD68 (p < .001) and CD83 (p = .04), and there was significantly greater expression of inflammatory cytokines IL1ß (p = .01), IL6 (p < .0001), IL17 (p < .0001) and TNF-α (p = .01). T2D samples showed a significant increase in markers of innate inflammation, TLR2 (p < .001) and TLR4 (p < .001) and decreased expression of regulatory T-cell marker, FOXP3 (p = .01). Multiple regression showed that age-corrected differences were statistically significant. CONCLUSION: Preliminary findings suggest that T2D may exert a similar response in the pulp to complications in other body sites. Hyperglycaemia is associated with changes in the morphology of the clinically normal dental pulp with altered immune cell and cytokine expression.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Immunohistochemical and immunofluorescence expression profile of lymphatic endothelial cell markers in oral cancer.

Lymphangiogenesis makes an important contribution to the tumour microenvironment (TME), but little is known about this in oral squamous cell carcinoma (OSCC). Archival formalin-fixed paraffin-embedded specimens (28 OSCC, 10 inflamed and 6 normal oral mucosa controls) were processed using immunohistochemistry (IHC) with antibodies against lymphatic markers D2-40 (podoplanin), LYVE-1, VEGFR3 and Prox1. After the endothelial cells had been highlighted by the various markers for lymphatic endothelium, the positive stained cells and vessels were identified and counted in a systematic manner to determine microvessel density. Double-labelling immunofluorescence (DLIF) was used to investigate the specificity of D2-40 and LYVE-1 to lymphatic endothelial cells (LECs) as opposed to blood ECs. There was higher D2-40 and Prox1 lymphatic vessel density (P = .001) in the OSCC group when compared with both control groups. Some malignant keratinocytes expressed lymphatic markers, as did a much smaller number of epithelial cells in the control groups. DLIF showed that no vessels co-expressed D2-40/CD34 or LYVE/CD34. Some D2/40+ LVs were LYVE- . D2-40 was the most specific LEC marker in OSCC tissues. These results establish that the OSCC TME contains significantly more lymphatic vessels expressing D2-40 and Prox1 than the control groups, which may play a role in facilitating lymphatic invasion and metastases.

Genomic surveillance of Pseudomonas aeruginosa in the Philippines, 2013-2014.

Pseudomonas aeruginosa is an opportunistic pathogen that often causes nosocomial infections resistant to treatment. Rates of antimicrobial resistance (AMR) are increasing, as are rates of multidrug-resistant (MDR) and possible extensively drug-resistant (XDR) infections. Our objective was to characterize the molecular epidemiology and AMR mechanisms of this pathogen. We sequenced the whole genome for each of 176 P. aeruginosa isolates collected in the Philippines in 2013-2014; derived the multilocus sequence type (MLST), presence of AMR determinants and relatedness between isolates; and determined concordance between phenotypic and genotypic resistance. Carbapenem resistance was associated with loss of function of the OprD porin and acquisition of the metallo-ß-lactamase (MBL) gene bla VIM. Concordance between phenotypic and genotypic resistance was 93.27% overall for six antibiotics in three classes, but varied among aminoglycosides. The population of P. aeruginosa was diverse, with clonal expansions of XDR genomes belonging to MLSTs ST235, ST244, ST309 and ST773. We found evidence of persistence or reintroduction of the predominant clone ST235 in one hospital, and of transfer between hospitals. Most of the ST235 genomes formed a distinct lineage from global genomes, thus raising the possibility that they may be unique to the Philippines. In addition, long-read sequencing of one representative XDR ST235 isolate identified an integron carrying multiple resistance genes (including bla VIM-2), with differences in gene composition and synteny from the P. aeruginosa class 1 integrons described previously. The survey bridges the gap in genomic data from the Western Pacific Region and will be useful for ongoing surveillance; it also highlights the importance of curtailing the spread of ST235 within the Philippines.

Genomic surveillance of Neisseria gonorrhoeae in the Philippines, 2013-2014.

Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health and is of particular concern in the Western Pacific Region, where the incidence of gonorrhoea is high. The Antimicrobial Resistance Surveillance Program (ARSP) has been capturing information on resistant gonorrhoea since 1996, but genomic epidemiology studies on this pathogen are lacking in the Philippines. We sequenced the whole genomes of 21 N. gonorrhoeae isolates collected in 2013-2014 by ARSP. The multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance and relatedness among the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined. Ten of 21 isolates were resistant to penicillin, ciprofloxacin and tetracycline, due mainly to the presence of the blaTEM gene, the S91F mutation in the gyrA gene and the tetM gene, respectively. None of the isolates was resistant to ceftriaxone or cefixime. The concordance between phenotypic and genotypic resistance was 92.38% overall for five antibiotics in four classes. Despite the small number of isolates studied, they were genetically diverse, as shown by the sequence types, the N. gonorrhoeae multiantigen sequence typing types and the tree. Comparison with global genomes placed the Philippine genomes within global lineage A and led to the identification of an international transmission route. This first genomic survey of N. gonorrhoeae isolates collected by ARSP will be used to contextualize prospective surveillance. It highlights the importance of genomic surveillance in the Western Pacific and other endemic regions for understanding the spread of drug-resistant gonorrhoea worldwide.

Fermentation and the microbial community of Japanese koji and miso: A review.

Miso is a well-known traditional Japanese fermented food, with a characteristic savory flavor and aroma, known predominately as the seasoning in miso soup. Miso production involves a two-stage fermentation, where first a mold, such as Aspergillus oryzae, is inoculated onto a substrate to make koji. A subsequent fermentation, this time by bacteria and yeast, occurs when the koji is added to a salt and soybean mash, with the miso left to ferment for up to 2 years. The microbial community of miso is considered essential to the development of the unique taste, texture, and nutritional profile of miso. Despite the importance of microorganisms in the production of miso, very little research has been undertaken to characterize and describe the microbial process. In this review, we provide an overview of the two-stage fermentation process, describe what is currently known about the microbial communities involved and consider any potential health benefits associated with the consumption of miso, along with food safety concerns. As the popularity of miso continues to expand globally and is produced under new environmental conditions, understanding the microbiological processes involved will assist to ensure that global production of miso is safe as well as delicious.

Genomic surveillance of Acinetobacter baumannii in the Philippines, 2013-2014.

OBJECTIVE: Acinetobacter baumannii is an opportunistic nosocomial pathogen that has increasingly become resistant to carbapenems worldwide. In the Philippines, rates of carbapenem resistance and multidrug resistance are above 50%. We undertook a genomic study of carbapenem-resistant A. baumannii in the Philippines to characterize the population diversity and antimicrobial resistance mechanisms. METHODS: We sequenced the whole genomes of 117 A. baumannii isolates recovered by 16 hospitals in the Philippines between 2013 and 2014. From the genome sequences, we determined the multilocus sequence type, presence of acquired determinants of antimicrobial resistance and relatedness between isolates. We also compared the phenotypic and genotypic resistance results. RESULTS: Carbapenem resistance was mainly explained by acquisition of the class-D ß-lactamase gene blaOXA-23. The concordance between phenotypic and genotypic resistance to imipenem was 98.15%, and it was 94.97% overall for the seven antibiotics analysed. Twenty-two different sequence types were identified, including 7 novel types. The population was dominated by the high-risk international clone 2 (i.e. clonal complex 92), in particular by ST195 and ST208 and their single locus variants. Using whole-genome sequencing, we identified local clusters representing potentially undetected nosocomial outbreaks, as well as multihospital clusters that indicated interhospital dissemination. Comparison with global genomes suggested that the establishment of carbapenem-resistant international clone 2 in the Philippines is likely the result of clonal expansion and geographical dissemination, and at least partly explained by inadequate hospital infection control and prevention. DISCUSSION: This is the first extensive genomic study of carbapenem-resistant A. baumannii in the Philippines, and it underscores the importance of hospital infection control and prevention measures to contain high-risk clones.

Genomic surveillance of Pseudomonas aeruginosa in the Philippines, 2013-2014

@#Pseudomonas aeruginosa is an opportunistic pathogen often causing nosocomial infections that are resilient to treatment due to an extensive repertoire of intrinsic and acquired resistance mechanisms. In recent years, increasing resistance rates to antibiotics such as carbapenems and extended-spectrum cephalosporins have been reported, as well as multi-drug resistant and possible extremely drug-resistant rates of approximately 21% and 15%, respectively. However, the molecular epidemiology and AMR mechanisms of this pathogen remains largely uncharacterized. We sequenced the whole genomes of 176 P. aeruginosaisolates collected in 2013-2014 by the Antimicrobial Resistance Surveillance Program. The multi-locus sequence type, presence of antimicrobial resistance (AMR) determinants, and relatedness between the isolates were derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined. Carbapenem resistance was associated namely with loss-of function of the OprD porin, and acquisition of the metallo-?-lactamase VIM. The concordance between phenotypic and genotypic resistance was 93.27% overall for 6 antibiotics in 3 classes, but varied widely between aminoglycosides. The population of P. aeruginosain the Philippines was diverse, with clonal expansions of XDR genomes belonging to multi-locus sequence types ST235, ST244, ST309, and ST773. We found evidence of persistence or reintroduction of the predominant clone ST235 in one hospital, as well as transfer between hospitals. Most of the ST235 genomes formed a distinct Philippine lineage when contextualized with international genomes, thus raising the possibility that this is a lineage unique to the Philippines. This was further supported by long-read sequencing of one representative XDR isolate, which revealed the presence of an integron carrying multiple resistance genes, including blaVIM-2, with differences in gene composition and synteny to other P. aeruginosaclass 1 integrons described before. We produced the first comprehensive genomic survey of P. aeruginosain the Philippines, which bridges the gap in genomic data from the Western Pacific region and will constitute the genetic background to contextualize ongoing prospective surveillance. Our results also highlight the importance of infection control interventions aimed to curtail the spread of international epidemic clone ST235 within the country.

Genomic surveillance of Neisseria gonorrhoeae in the Philippines, 2013–2014

@#Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health and is of particular concern in the Western Pacific Region, where the incidence of gonorrhoea is high. The Antimicrobial Resistance Surveillance Program (ARSP) has been capturing information on resistant gonorrhoea since 1996, but genomic epidemiology studies on this pathogen are lacking in the Philippines. We sequenced the whole genomes of 21 N. gonorrhoeae isolates collected in 2013–2014 by ARSP. The multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance and relatedness among the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined. Ten of 21 isolates were resistant to penicillin, ciprofloxacin and tetracycline, due mainly to the presence of the blaTEM gene, the S91F mutation in the gyrA gene and the tetM gene, respectively. None of the isolates was resistant to ceftriaxone or cefixime. The concordance between phenotypic and genotypic resistance was 92.38% overall for five antibiotics in four classes. Despite the small number of isolates studied, they were genetically diverse, as shown by the sequence types, the N. gonorrhoeae multiantigen sequence typing types and the tree. Comparison with global genomes placed the Philippine genomes within global lineage A and led to the identification of an international transmission route. This first genomic survey of N. gonorrhoeae isolates collected by ARSP will be used to contextualize prospective surveillance. It highlights the importance of genomic surveillance in the Western Pacific and other endemic regions for understanding the spread of drug-resistant gonorrhoea worldwide.