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BackgroundKlebsiella pneumoniae is a critically important pathogen in the Philippines. Isolates are commonly resistant to at least two classes of antibiotics, yet mechanisms and spread of its resistance are not well studied.MethodsA retrospective sequencing survey was performed on carbapenem-, extended spectrum beta-lactam- and cephalosporin-resistant Klebsiella pneumoniae isolated at 20 antimicrobial resistance (AMR) surveillance sentinel sites from 2015-2017. We characterized 259 isolates using biochemical methods, antimicrobial susceptibility testing, and whole genome sequencing (WGS). Known AMR mechanisms were identified. Potential outbreaks were investigated by detecting clusters from epidemiologic, phenotypic and genome-derived data.ResultsPrevalent AMR mechanisms detected include blaCTX-M-15 (76.8%) and blaNDM-1 (37.5%). An epidemic IncFII(Yp) plasmid carrying blaNDM-1 was also detected in 46 isolates from 6 sentinel sites and 14 different sequence types (ST). This plasmid was also identified as the main vehicle of carbapenem resistance in 2 previously unrecognized local outbreaks of ST348 and ST283 at 2 different sentinel sites. A third local outbreak of ST397 was also identified but without the IncFII(Yp) plasmid. Isolates in each outbreak site showed identical STs, K- and O-loci, and similar resistance profiles and AMR genes. All outbreak isolates were collected from blood of children aged ConclusionWGS provided an in-depth understanding of the epidemiology of AMR in the Philippines, which was not possible with only phenotypic and epidemiologic data. The identification of three previously unrecognized Klebsiella outbreaks highlights the utility of WGS in outbreak detection, as well as its importance in public health and in implementing infection control programs.summaryWhole genome sequencing identified three distinct previously unrecognized local outbreaks in a retrospective study in the Philippines, along with an epidemic plasmid carrying antimicrobial resistance genes, highlighting its importance in antimicrobial resistance surveillance, outbreak detection and infection control.

Performing whole genome sequencing (WGS) for the surveillance of antimicrobial resistance offers the ability to determine not only the antimicrobials to which rates of resistance are increasing, but also the evolutionary mechanisms and transmission routes responsible for the increase at local, national, and global scales. To derive WGS-based outputs, a series of processes are required, beginning with sample and metadata collection, followed by nucleic acid extraction, library preparation, sequencing, and analysis. Throughout this pathway there are many data-related operations required (informatics) combined with more biologically focused procedures (bioinformatics). For a laboratory aiming to implement pathogen genomics, the informatics and bioinformatics activities can be a barrier to starting on the journey; for a laboratory that has already started, these activities may become overwhelming. Here we describe these data bottlenecks and how they have been addressed in laboratories in India, Colombia, Nigeria, and the Philippines, as part of the National Institute for Health Research Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance. The approaches taken include the use of reproducible data parsing pipelines and genome sequence analysis workflows, using technologies such as Data-flo, the Nextflow workflow manager, and containerization of software dependencies. By overcoming barriers to WGS implementation in countries where genome sampling for some species may be underrepresented, a body of evidence can be built to determine the concordance of antimicrobial sensitivity testing and genome-derived resistance, and novel high-risk clones and unknown mechanisms of resistance can be discovered.

Increasing antimicrobial resistance (AMR) in Salmonella has been observed in the Philippines. This study aims to utilize whole genome sequencing (WGS) to characterize the population and AMR mechanisms of Salmonella captured by the Philippine Antimicrobial Resistance Surveillance Program (ARSP) and contrast to traditional laboratory methods.We sequenced the whole genomes of 148 Salmonella Typhi (S. Typhi) and 65 non-typhoidal Salmonella (NTS) collected in the Philippines in 2013–2014. From the genome sequences, we determined the serotype, multilocus sequence type, presence of determinants of antimicrobial resistance and relatedness between isolates. We also compared the genotypic predictions of serotype and AMR to the phenotypic data.AMR rates in S. Typhi were low, with sparse acquisition of mutations associated with reduced susceptibility to fluoroquinolones or extended-spectrum beta-lactamases (ESBL) genes. In contrast, three quarters of NTS isolates were insusceptible to at least one antimicrobial, with more than half carrying mutations and/or genes linked to resistance to fluoroquinolones. ESBL genes were detected in five genomes that also carried other AMR determinants. The population of S. Typhi was dominated by the likely endemic genotype 3.0, which also caused of a putative local outbreak susceptible to antibiotics. The main NTS clades were global epidemic S. Enteritidis ST11 and the monophasic variant of S. Typhimurium (I 4,[5],12:i:-) ST34, which had frequently been serotyped as S. Typhimurium in the laboratory.This was the first time that Salmonella isolated from the Philippines were characterized by WGS and we provide evidence of its utility for ongoing surveillance at the ARSP.

This work was supported by a Newton Fund award from the Medical Research Council (United Kingdom) MR/N019296/1 and the Philippine Council for Health Research and Development. Additional support provided by National Institute for Health Research (UK) Global Health Research Unit on genomic Surveillance of AMR (16/136/111) and by research grant U01CA207167 from the U.S. National Institutes of Health. Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. In the Philippines, MRSA rates have remained above 50% since 2010, but resistance to other antibiotics, including vancomycin, is low. The MRSA burden can be partially attributed to pathogen-specific characteristics of the circulating clones, but little was known about the S. aureus clones circulating in the Philippines. We sequenced the whole genomes of 116 S. aureus isolates collected in 2013-2014 within the Antimicrobial Resistance Surveillance Program. The multilocus sequence type, spa type, SCCmec type, presence of antimicrobial resistance (AMR) determinants and virulence genes and relatedness between the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined. The MRSA population in the Philippines comprised a limited number of genetic clones, including several international epidemic clones, such as CC30-spa-t019-SCCmec-IV-PVL+, CC5-SCCmec-typeIV and ST239-spa-t030-SCCmec-typeIII. The CC30 genomes were related to the South-West Pacific clone but formed a distinct, diverse lineage, with evidence of global dissemination. We showed independent acquisition of resistance to sulfamethoxazole/trimethoprim in various locations and genetic clones but mostly in paediatric patients with invasive infections. The concordance between phenotypic and genotypic resistance was 99.68% overall for eight antibiotics in seven classes. We have made the first comprehensive genomic survey of S. aureus in the Philippines, which bridges the gap in genomic data from the Western Pacific Region and will constitute the genetic background for contextualizing prospective surveillance. Publisher PDF

We sequenced the whole genomes of 117Carbapenem resistance was mainly explained by acquisition of the class-D β-lactamase gene blaThis is the first extensive genomic study of carbapenem-resistant

Acinetobacter baumannii is an opportunistic nosocomial pathogen that has increasingly become resistant to carbapenems worldwide. In the Philippines, carbapenem resistance and multi-drug resistance (MDR) rates are above 50%. We undertook a genomic study of carbapenem resistant A. baumannii in the Philippines to characterize the population diversity and antimicrobial resistance (AMR) mechanisms.We sequenced the whole genomes of 117 A. baumannii isolates recovered by 16 hospitals in the Philippines between 2013 and 2014. We determined the multi-locus sequence type (MLST), presence of acquired AMR determinants and relatedness between isolates from the genome sequences. We also compared the phenotypic and genotypic resistance results.Carbapenem resistance was mainly explained by the acquisition of class-D beta-lactamase gene blaOXA-23. The concordance between phenotypic and genotypic resistance to imipenem was 98.15% and 94.97% overall for the seven antibiotics analysed. Twenty-two different sequence types (ST) were identified, including 7 novel STs. The population was dominated by high-risk international clone 2 (i.e., clonal complex 92), in particular by ST195 and ST208 and their single locus variants. With WGS we identified local clusters representing potential undetected nosocomial outbreaks, as well as multi-hospital clusters indicating inter-hospital transmission. Comparison with global genomes suggested that the establishment of carbapenem-resistant IC2 clones 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.This study is the first extensive genomic study of carbapenem-resistant A. baumannii in the Philippines and underscores the importance of hospital infection control and prevention to contain high-risk clones.

This work was supported by a Newton Fund award from the Medical Research Council (United Kingdom) MR/N019296/1 and the Philippine Council for Health Research and Development. Additional support was provided by the National Institute for Health Research (United Kingdom), the Global Health Research Unit on genomic surveillance of AMR (16/136/111) and by research grant U01CA207167 from the US National Institutes of Health. 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. Publisher PDF

National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor resistance trends and disseminate these data to AMR stakeholders. Whole-genome sequencing (WGS) can support surveillance by pinpointing resistance mechanisms and uncovering transmission patterns. However, genomic surveillance is rare in low- and middle-income countries. Here, we implement WGS within the established Antimicrobial Resistance Surveillance Program of the Philippines via a binational collaboration. In parallel, we characterize bacterial populations of key bug-drug combinations via a retrospective sequencing survey. By linking the resistance phenotypes to genomic data, we reveal the interplay of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying the expansion of specific resistance phenotypes that coincide with the growing carbapenem resistance rates observed since 2010. Our results enhance our understanding of the drivers of carbapenem resistance in the Philippines, while also serving as the genetic background to contextualize ongoing local prospective surveillance., Whole-genome sequencing (WGS) can support drug resistance surveillance, but is rare in low- and middle-income countries. Here, the authors integrate WGS within the national antimicrobial resistance surveillance program in the Philippines and conduct a retrospective sequencing survey to characterize bacterial populations and dissect resistance phenotypes.

Objective. Scarcity of data on the serotype composition and antibiotic resistance of invasive pneumococci from developing countries has been noted. We describe in this study the serogroup distribution and antimicrobial resistance patterns of Streptococcus pneumoniae in the Philippines from 2004-2011. Methodology. S. pneumoniae isolated from patients with invasive pneumococcal disease were referred to the Antimicrobial Resistance Surveillance Reference Laboratory from 2004 to 2011. Typing of isolates was done through slide agglutination and antimicrobial susceptibility was determined following CLSI methods. Results. The penicillin-resistant meningitis isolates were of serotypes 1, 5 and 14 which are covered by PCV10 and 13. The erythromycin resistant isolates were serotype 9 while cotrimoxazole resistant isolates were serotypes 1, 5, 6, 12 and 14. Forty-one percent of the cotrimoxazole resistant isolates are covered by PCV7, and 88% are covered by both PCV10 and PCV13. Levofloxacin resistant isolates were of serotypes 5 and 23 with PCV7 coverage of 50% and PCV10 and PCV13 coverages of 100%. Conclusion. S. pneumoniae serotypes causing IPD in the country is largely similar to the dominant IPD serotypes worldwide. The serotype distribution in the Philippines remained stable from 2004 to 2011 and antimicrobial resistance among the isolates remained low. The serotypes of antibiotic resistant S. pneumoniae in this study were not similar with known serotype resistance profiles in other Asian countries. With the inclusion of PCV in the national free national immunization program of the country beginning 2013, continued surveillance of prevailing pneumococcal serotypes should be done to monitor any shift in the prevalence of PCV associated serotypes to guide disease control measures including control of emergence of resistant pneumococcal isolates.

Methicillin Resistant Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. In the Philippines, MRSA rates have remained above 50% since 2010, but resistance to other antibiotics, including vancomycin, is low. The MRSA burden can be partially attributed to pathogen-specific characteristics of the circulating clones, but little was known about the S. aureus circulating clones in the Philippines.We sequenced the whole genomes of 116 S. aureus isolates collected in 2013-2014 by the Antimicrobial Resistance Surveillance Program. The multi-locus sequence type, spa type, SCC-mec type, presence of antimicrobial resistance (AMR) determinants and virulence genes, and relatedness between the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.The MRSA population in the Philippines was composed of a limited number of genetic clones, including several international epidemic clones, such as CC30-spa-t019-SCCmec-IV-PVL+, CC5-SCCmec-typeIV, and ST239-spa-t030-SCCmec-typeIII. The CC30 genomes were related to the South West Pacific clone, but formed a distinct and diverse lineage, with evidence of global dissemination. We showed the independent acquisition of resistance to sulfamethoxazole/trimethoprim across different locations and genetic clones, but mostly in pediatric patients with invasive infections. The concordance between phenotypic and genotypic resistance was 99.68% overall for 8 antibiotics in 7 classes.We produced the first comprehensive genomic survey of S. aureus in 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.

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. aeruginosa isolates 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-β-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. aeruginosa in 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. aeruginosa class 1 integrons described before.We produced the first comprehensive genomic survey of P. aeruginosa in 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.

Drug-resistant bacterial infections constitute a growing threat to public health globally 1. National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor the emergence and spread of resistance and are central to the dissemination of these data to AMR stakeholders 2. Whole-genome sequencing (WGS) can support these efforts by pinpointing resistance mechanisms and uncovering transmission patterns 3, 4. However, genomic surveillance is rare in low- and middle-income countries (LMICs), which are predicted to be the most affected by AMR 5. We implemented WGS within the established Antimicrobial Resistance Surveillance Program (ARSP) of the Philippines via ongoing technology transfer, capacity building in and binational collaboration. In parallel, we conducted an initial large-scale retrospective sequencing survey to characterize bacterial populations and dissect resistance phenotypes of key bug-drug combinations, which is the focus of this article. Starting in 2010, the ARSP phenotypic data indicated increasing carbapenem resistance rates for Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli. We first identified that this coincided with a marked expansion of specific resistance phenotypes. By then linking the resistance phenotypes to genomic data, we revealed the diversity of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying this expansion. We discovered a previously unreported plasmid-driven hospital outbreak of carbapenem-resistant K. pneumoniae, uncovered the interplay of carbapenem resistance genes and plasmids in the geographic circulation of epidemic K. pneumoniae ST147, and found that carbapenem-resistant E. coli ST410 consisted of diverse lineages of global circulation that carried both international and local plasmids, resulting in a combination of carbapenemase genes variants previously unreported for this organism. Thus, the WGS data provided an enhanced understanding of the interplay between strains, genes and vehicles driving the dissemination of carbapenem resistance in the Philippines. In addition, our retrospective survey served both as the genetic background to contextualize local prospective surveillance, and as a comprehensive dataset for training in bioinformatics and genomic epidemiology. Continued prospective sequencing, capacity building and collaboration will strengthen genomic surveillance of AMR in the Philippines and the translation of genomic data into public-health action. We generated a blueprint for the integration of WGS and genomic epidemiology into an established national system of laboratory-based surveillance of AMR through international collaboration that can be adapted and utilized within other locations to tackle the global challenge of AMR.