Epidemiological trends in serotypes distribution and antimicrobial resistance in Salmonella from humans in Taiwan, 2004-2022.

Objectives: Salmonella, a zoonotic pathogen, significantly impacts global human health. Understanding its serotype distribution and antimicrobial resistance is crucial for effective control measures and medical interventions. Methods: We collected Salmonella isolates and demographic data from Taiwanese hospitals between 2004 and 2022, analyzing their serotypes and antimicrobial susceptibility. Results: Among 40,595 isolates, salmonellosis predominated in children aged 0-4 (61.2%) years and among males (55.2%). Males also showed higher rates of extraintestinal infections (18.1% vs 16.0%, P <0.001), particularly, in the ≥65 years age group (52.4%). The top five serovars were S. Enteritidis (32.8%), S. Typhimurium (21.7%), S. Newport (6.2%), S. Stanley (4.7%), and S. Anatum (4.0%). Notably, S. Enteritidis prevalence increased from 23.9% (2004-2005) to 43.6% (2021-2022). Antimicrobial resistance was high, with a 51.6% multidrug resistance (MDR) rate. Disturbingly, MDR rates exceeded 90% in serovars Albany, Schwarzengrund, Choleraesuis, and Goldcoast. Resistance to key therapeutic agents, azithromycin, cefotaxime, and ciprofloxacin, exhibited concerning upward trends, and the surge in cefotaxime and ciprofloxacin resistance was closely linked to the emergence and spread of MDR S. Anatum and S. Goldcoast clones. Conclusions: Prioritizing control measures against S. Enteritidis and closely monitoring the prevalence and spread of MDR clones are imperative to mitigate Salmonella infections in Taiwan.

Epidemiological trends and antimicrobial resistance in Salmonella enterica serovar Typhimurium clones in Taiwan between 2004 and 2019.

OBJECTIVES: We investigated the temporal trends of Salmonella enterica serovar Typhimurium (S. Typhimurium) clones in Taiwan from 2004 to 2019, focusing on antimicrobial resistance (AMR), resistance genetic determinants, and plasmid types. METHODS: Salmonella isolates were characterized using pulsed-field gel electrophoresis (PFGE), whole-genome sequencing, and antimicrobial susceptibility testing. Clones were defined using PFGE clustering and the hierarchical cgMLST clustering (HierCC) assignments. RESULTS: Seven major S. Typhimurium clones, HC100_2, 13, 41, 305, 310, 501, and 46261, accounted for 97.6% (8079/8275) of human isolates in Taiwan. Each clone displayed a unique AMR profile, resistance genetic determinants, and plasmid types. Four highly resistant clones (HC100_2, 41, 305, and 310) exhibited multiple resistance in 86.5% to 96.1% of isolates. HC100_305 and HC100_2 were pandemic multidrug-resistant clones, characterized by resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) and ASSuT, respectively. The prevalence of the ACSSuT clone decreased from 68.7% of S. Typhimurium isolates in 2004 to 1.7% in 2019, while the ASSuT clone emerged in 2007 and became the largest clone after 2010. Several plasmids, including IncHI2-IncHI2A, IncC, IncFIB(K), and IncI1-1(α), carried multiple resistance genes or were associated with the carriage of mph(A), blaCMY-2, and blaDHA-1. CONCLUSIONS: Between 2004 and 2019, Taiwan experienced the emergence, prevalence, and subsequent decline of several highly resistant S. Typhimurium clones. The clones defined using the HierCC approach have global comparability. The increasing resistance to third-generation cephalosporins, cephamycins, ciprofloxacin, and azithromycin in recent years poses a significant medical concern.

Persistence and dynamic structures of diverse cephalosporinase genes in nontyphoidal Salmonella in cross-sectional surveillance in Taiwan.

OBJECTIVES: Nontyphoidal Salmonella (NTS) is a major foodborne pathogen causing from acute gastroenteritis to bacteraemia, particularly in paediatric and elderly patients. Antimicrobial resistance of NTS, especially resistance to extended-spectrum cephalosporins, has emerged over the past decades. METHODS: Thirteen NTS isolates resistant to ceftriaxone or cefotaxime were collected from a teaching hospital in Taipei, and another three from a tertiary hospital, in New Taipei City, Taiwan, from September 2018 to December 2019. Ten other archived isolates from 2000 to 2017 were also obtained. Complete genomes of the 26 isolates were obtained. Serovars, sequence types, resistomes, genetic relatedness, and sequence comparison of plasmids were analyzed. RESULTS: Serogroups B, C2 and E were significantly associated with ampicillin resistance. Over 90% of these 26 isolates are susceptible to carbapenems and colistin. Genomic epidemiology of these isolates shows that blaCMY-2-harbouring isolates in different serovars were prevalent over two decades, presumably resulting from highly mobile IncI1 plasmid harbouring blaCMY-2. One type of the IncI1 plasmids contained a mobile element, IS26, which might be involved in the acquisition of antimicrobial resistance genes. Two emerging serovars, S. Goldcoast ST358 harbouring blaCTX-M-55 on IncHI2 plasmids and S. Anatum ST64 harbouring blaDHA-1 on IncA/C2 plasmids persisted in Taiwan, possibly through the clonal spread. Integration of complete or partial plasmid sequences into host chromosomes or multiplications of the antimicrobial resistance genes also appears to be mediated by IS26, in the two emerging clones. CONCLUSION: The dynamic movement of cephalosporinase genes mediated by IS26 in NTS is of great concern.

High-level levofloxacin-resistant emm12 group A Streptococcus, 2012-2018: A multicenter study in Taiwan.

High-level levofloxacin-resistant group A Streptococcus emerged in Taiwan in 2012. Among the 24 isolates identified, 23 belonged to emm12/ST36, most harbored the same GyrA and ParC mutations and were highly clonal. wgMLST showed them to be closely related to the Hong Kong scarlet fever outbreak strains. Continuous surveillance is warranted.

Effects of colonization-associated gene yqiC on global transcriptome, cellular respiration, and oxidative stress in Salmonella Typhimurium.

BACKGROUND: yqiC is required for colonizing the Salmonella enterica serovar Typhimurium (S. Typhimurium) in human cells; however, how yqiC regulates nontyphoidal Salmonella (NTS) genes to influence bacteria-host interactions remains unclear. METHODS: The global transcriptomes of S. Typhimurium yqiC-deleted mutant (ΔyqiC) and its wild-type strain SL1344 after 2 h of in vitro infection with Caco-2 cells were obtained through RNA sequencing to conduct comparisons and identify major yqiC-regulated genes, particularly those involved in Salmonella pathogenicity islands (SPIs), ubiquinone and menaquinone biosynthesis, electron transportation chains (ETCs), and carbohydrate/energy metabolism. A Seahorse XFp Analyzer and assays of NADH/NAD+ and H2O2 were used to compare oxygen consumption and extracellular acidification, glycolysis parameters, adenosine triphosphate (ATP) generation, NADH/NAD+ ratios, and H2O2 production between ΔyqiC and SL1344. RESULTS: After S. Typhimurium interacts with Caco-2 cells, yqiC represses gene upregulation in aspartate carbamoyl transferase, type 1 fimbriae, and iron-sulfur assembly, and it is required for expressing ilvB operon, flagellin, tdcABCD, and dmsAB. Furthermore, yqiC is required for expressing mainly SPI-1 genes and specific SPI-4, SPI-5, and SPI-6 genes; however, it diversely regulates SPI-2 and SPI-3 gene expression. yqiC significantly contributes to menD expression in menaquinone biosynthesis. A Kyoto Encyclopedia of Genes and Genomes analysis revealed the extensive association of yqiC with carbohydrate and energy metabolism. yqiC contributes to ATP generation, and the analyzer results demonstrate that yqiC is required for maintaining cellular respiration and metabolic potential under energy stress and for achieving glycolysis, glycolytic capacity, and glycolytic reserve. yqiC is also required for expressing ndh, cydA, nuoE, and sdhB but suppresses cyoC upregulation in the ETC of aerobically and anaerobically grown S. Typhimurium; priming with Caco-2 cells caused a reversed regulation of yiqC toward upregulation in these ETC complex genes. Furthermore, yqiC is required for maintaining NADH/NAD+ redox status and H2O2 production. CONCLUSIONS: Specific unreported genes that were considerably regulated by the colonization-associated gene yqiC in NTS were identified, and the key role and tentative mechanisms of yqiC in the extensive modulation of virulence factors, SPIs, ubiquinone and menaquinone biosynthesis, ETCs, glycolysis, and oxidative stress were discovered.

Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6')-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25-2 µg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 µg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25-2 µg/mL), thus providing insights into mechanisms underlying CIP resistance.

Evolution and Population Structures of Prevalent Methicillin-Resistant Staphylococcus aureus in Taiwan.

Global methicillin-resistant Staphylococcus aureus (MRSA) strains were dominated by few genetic lineages, suggesting their inherent advantage of competitive fitness. The information of genome evolution and population structures of prevalent MRSA strains can help gain a better understanding of the success of the pandemic clones. Whole-genome sequencing was performed in 340 MRSA isolates belonging to three prevalent lineages, including ST59 (129 isolates), ST239/241 (140 isolates), and ST5 (71 isolates), collected from 1996 to 2016 in Taiwan. The time-scaled phylogeny and evolutionary pathways were estimated by Bayesian analysis using Markov chain Monte Carlo. The toxome, resistome, and plasmids were characterized by screening the raw reads with a public database. ST59, ST239/241, and ST5 MRSA were estimated to emerge in 1974, 1979, and 1995, respectively, in Taiwan. ST59 evolved through two major pathways, generating two subclones in 1980 and 1984. Both ST59 subclones remained prevalent in the healthcare and community environments in late 2010s. ST239/241 diverged into three subclones, respectively, in 1989, 1993, and 1995. The 1995-emerging ST239 subclone predominated after 2000 by replacing two previous early subclones. ST5 could be subdivided into two clades within 3 years of introduction, but no substantial difference of genomic profiles was identified in the strains of distinct clades. Each of the three pandemic MRSA lineages harbored its own specific toxome, resistome, and plasmids. The frequently identified genetic diversities between the subclones of the same lineage were genes mediating immune evasion, leukocidins, enterotoxins, and resistance to aminoglycosides. In conclusion, MRSA ST59 and ST239/241 emerged in the 1970s and evolved drastically during 1980 and 1995, resulting in three successful subclones prevailing in Taiwan. ST5 was introduced late in 1995 without a significant genetic drift during 20 years of evolution.

Temporal Trend of ST131 Clone among Urinary Escherichia coli Isolates in the Community: A Taiwan National Surveillance from 2002 to 2016.

Sequence type (ST) 131 is a multidrug-resistant pandemic lineage of E. coli responsible for extraintestinal infections. Few surveillance data of ST131 included all antimicrobial-susceptible and -resistant isolates or focused on community-acquired urinary tract infection (UTI). From a population-based surveillance pool of 2997 outpatient urine E. coli isolates, 542 were selected for detection of ST131 based on ciprofloxacin and/or cefotaxime resistance. Pulsed-field gel electrophoresis (PFGE) was performed on all ST131 isolates to further determine their relatedness. The estimated overall ST131 prevalence in this community UTI cohort increased from 11.2% (in 2002-2004), 12.2% (in 2006-2008), 13.6% (in 2010-2012), to 17.4% in 2014-2016 (p < 0.01). In the ciprofloxacin-resistant/cefotaxime-resistant group, ST131 increased from 33.3% in 2002-2004 to 72.1% in 2014-2016 (p < 0.01). In the ciprofloxacin-resistant/cefotaxime-susceptible group, ST131 was found in 24.3% overall without significant increase in its prevalence over time. PFGE showed emergence of a cluster of ciprofloxacin-resistant/cefotaxime-resistant ST131 carrying Gr. 1 CTX-M ESBL in 2014-2016, especially 2016. Multivariate analysis revealed that age (≥65 y.o) and ciprofloxacin resistance were independent factors associated with ST131. This longitudinal surveillance showed that ciprofloxacin-resistant/cefotaxime-susceptible ST131 has been circulating in the community since 2002 but ciprofloxacin-resistant/cefotaxime-resistant ST131 increased rapidly in the later years.

In vitro activity of imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam and other novel antibiotics against imipenem-non-susceptible Gram-negative bacilli from Taiwan.

OBJECTIVES: To investigate the susceptibility of imipenem-non-susceptible Escherichia coli (INS-EC), Klebsiella pneumoniae (INS-KP), Acinetobacter baumannii (INS-AB) and Pseudomonas aeruginosa (INS-PA) to novel antibiotics. METHODS: MICs were determined using the broth microdilution method. Carbapenemase and ESBL phenotypic testing and PCR for genes encoding ESBLs, AmpCs and carbapenemases were performed. RESULTS: Zidebactam, avibactam and relebactam increased the respective susceptibility rates to cefepime, ceftazidime and imipenem of 17 INS-EC by 58.8%, 58.8% and 70.6%, of 163 INS-KP by 77.9%, 88.3% and 76.1% and of 81 INS-PA by 45.7%, 38.3% and 85.2%, respectively. Vaborbactam increased the meropenem susceptibility of INS-EC by 41.2% and of INS-KP by 54%. Combinations of ß-lactams and novel ß-lactamase inhibitors or ß-lactam enhancers (BLI-BLE) were inactive against 136 INS-AB. In 58 INS-EC and INS-KP with exclusively blaKPC-like genes, zidebactam, avibactam, relebactam and vaborbactam increased the susceptibility of the partner ß-lactams by 100%, 96.6%, 84.5% and 75.9%, respectively. In the presence of avibactam, ceftazidime was active in an additional 85% of 20 INS-EC and INS-KP with exclusively blaOXA-48-like genes while with zidebactam, cefepime was active in an additional 75%. INS-EC and INS-KP with MBL genes were susceptible only to cefepime/zidebactam. The ß-lactam/BLI-BLE combinations were active against INS-EC and INS-KP without detectable carbapenemases. For INS-EC, INS-KP and INS-AB, tigecycline was more active than omadacycline and eravacycline but eravacycline had a lower MIC distribution. Lascufloxacin and delafloxacin were active in <35% of these INS isolates. CONCLUSIONS: ß-Lactam/BLI-BLE combinations were active in a higher proportion of INS-EC, INS-KP and INS-PA. The susceptibility of novel fluoroquinolones and tetracyclines was not superior to that of old ones.

Detection, spread and phylogeny of meticillin-resistant Staphylococcus aureus sequence type 45 in Taiwan.

Meticillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 was reported in the literature to have been first identified in 2006 in Taiwan. The present study was carried out to explore and trace the emergence, transmission and evolutional dynamics of MRSA ST45 in Taiwan. We identified MRSA ST45 isolates retrospectively from two collections of MRSA isolates, namely TSAR (Taiwan Surveillance of Antimicrobial Resistance) surveys and the CGMH (Chang Gung Memorial Hospital)-based laboratory collection. Representative ST45 isolates were selected for whole-genome sequencing (WGS) analysis. A total of 9554 MRSA isolates was included in this study. Among the 3766 MRSA isolates biennially collected from TSAR surveys between 1998 and 2014, ST45 accounted for 133 (3.53 %) MRSA isolates, was first identified in 2004, and the prevalence rate peaked in 2010 (up to 10.77 %). Among the 5788 MRSA isolates collected between 1995 and 2017 by the CGMH-based laboratory, 257 isolates (4.44 %) were characterized as ST45, with most identified from nursing homes since 2012. Of the 75 isolates randomly selected for WGS, two clades were identified. The major clade, clade II, comprised 63 isolates and was phylogenetically relatively close to those isolates identified from Singapore. All but one of the isolates in clade I, the minor clade, were identified from non-Taiwanese people, mostly from newly recruited foreign workers in 2017, and were phylogenetically relatively close to one isolate from the USA (CA-347). Conclusively, the emergence of MRSA ST45 strain in Taiwan can be traced back to 2004 and the strain is connected to South-East Asian countries. Since its emergence, transmission and spread of MRSA ST45 has occurred in Taiwan.

Increasing New Delhi metallo-ß-lactamase-positive Escherichia coli among carbapenem non-susceptible Enterobacteriaceae in Taiwan during 2016 to 2018.

New Delhi metallo-ß-lactamase (NDM) had been reported to be the predominant carbapenemase among Escherichia coli in Taiwan. However, studies focusing on the clonal background and epidemiology of plasmids carrying NDM genes were limited. Between 2016 and 2018, all clinical E. coli and Klebsiella pneumoniae isolates that were non-susceptible to ertapenem, meropenem, and imipenem were tested for carbapenemase-encoding genes (CEGs) and antimicrobial susceptibilities. Molecular typing was performed on all carbapenemase-producing isolates. Whole genome sequencing (WGS) was performed on all NDM-positive E. coli isolates. Twenty-three (29.5%) of 78 carbapenem non-susceptible E. coli and 108 (35.3%) of 306 carbapenem non-susceptible K. pneumoniae isolates carried CEGs. The most prevalent CEGs in carbapenemase-producing E. coli (CPEc) were blaNDM (39.1%) and blaIMP-8 (30.4%), while that in carbapenemase-producing K. pneumoniae was Klebsiella pneumoniae carbapenemase (KPC) (72.2%). Fifteen sequence types were identified among 23 CPEc, and 55.6% of NDM-positive E. coli isolates belonged to ST410. WGS showed ST410 isolates were highly clonal and similar to those from other countries. All NDM-5-positive E. coli isolates carried identical IncX3 plasmid harboring blaNDM-5 but no other antimicrobial resistance (AMR) genes. In each of the four NDM-1-positive E. coli isolates, the blaNDM-1 was present in a ∼ 300 kb IncHI2/IncHI2A plasmid which carried an array of AMR genes. NDMs are the most prevalent carbapenemase among CPEc in Taiwan. Awareness should be raised as the prevalence of NDM-positive E. coli might increase rapidly with IncX3 plasmid and globally distributed strain ST410 being the potential vectors for wide dissemination.

Total synthesis of landomycins Q and R and related core structures for exploration of the cytotoxicity and antibacterial properties.

Herein, we report the total synthesis of landomycins Q and R as well as the aglycone core, namely anhydrolandomycinone and a related core analogue. The synthesis features an acetate-assisted arylation method for construction of the hindered B-ring in the core component and a one-pot aromatization-deiodination-denbenzylation procedure to streamline the global functional and protecting group manuipulation. Subsequent cytotoxicity and antibacterial studies revealed that the landomycin R is a potential antibacterial agent against methicillin-resistant Staphylococcus aureus.

Susceptibility of Elizabethkingia spp. to commonly tested and novel antibiotics and concordance between broth microdilution and automated testing methods.

OBJECTIVES: We aimed to determine susceptibilities of Elizabethkingia spp. to 25 commonly tested and 8 novel antibiotics, and to compare the performance of different susceptibility testing methods. METHODS: Clinical isolates of Elizabethkingia spp., Chryseobacterium spp. and Flavobacterium spp. collected during 2002-18 (n = 210) in a nationwide surveillance programme in Taiwan were speciated by 16S rRNA sequencing. MICs were determined by broth microdilution. The broth microdilution results of 18 common antibiotics were compared with those obtained by the VITEK 2 automated system. RESULTS: Among the Elizabethkingia spp. identified (n = 108), Elizabethkingia anophelis was the most prevalent (n = 90), followed by Elizabethkingia meningoseptica (n = 7) and Elizabethkingia miricola cluster [E. miricola (n = 6), Elizabethkingia bruuniana (n = 3) and Elizabethkingia ursingii (n = 2)]. Most isolates were recovered from respiratory or blood specimens from hospitalized, elderly patients. PFGE showed two major and several minor E. anophelis clones. All isolates were resistant to nearly all the tested ß-lactams. Doxycycline, minocycline and trimethoprim/sulfamethoxazole inhibited >90% of Elizabethkingia spp. Rifampin inhibited E. meningoseptica (100%) and E. anophelis (81.1%). Fluoroquinolones and tigecycline were active against E. meningoseptica and E. miricola cluster isolates. Novel antibiotics, including imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, delafloxacin, eravacycline and omadacycline were ineffective but lascufloxacin inhibited half of Elizabethkingia spp. The very major discrepancy rates of VITEK 2 were >1.5% for ciprofloxacin, moxifloxacin and vancomycin. Major discrepancy rates were >3% for amikacin, tigecycline, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. CONCLUSIONS: MDR, absence of standard interpretation criteria and poor intermethod concordance necessitate working guidelines to facilitate future research of emerging Elizabethkingia spp.

Serotype Distribution and Antimicrobial Susceptibility of Streptococcus pneumoniae in Pre- and Post- PCV7/13 Eras, Taiwan, 2002-2018.

In Taiwan, the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in 2006 and a PCV13 national childhood catchup program was implemented in 2013. To delineate the trend of serotype distribution and antimicrobial susceptibility following vaccination programs, we investigated a total of 1845 Streptococcus pneumoniae isolates collected biennially between 2002 and 2018 over a 3-month period from 25 hospitals. The number of isolates collected over the years decreased significantly in all age groups, from a total of 320 isolates in 2002 (pre-PCV), to 196 in 2010 (post-PCV7/pre-PCV13), to 89 in 2018 (post-PCV13). Overall, PCV7/PCV13 serotypes comprised 66.9%/76.3%, 53.1%/78.1%, and 15.7%/31.5% of isolates in 2002, 2010, and 2018, respectively. The leading serotypes in the pre-PCV era were 23F, 19F, 6B, and 14, while serotype 19A predominated in the post-PCV7/pre-PCV13 era, but non-vaccine serotypes (NVT) 15A (18.0%) and 23A (15.7%) surpassed 19A (10.1%) to become the top two leading serotypes in 2018. All the major serotypes, including the emergent serotypes 15A and 23A, were multidrug-resistant with high rates of non-susceptibility to ß-lactam (except serotype 3) and several non-ß-lactam agents. PFGE and MLST revealed that while meropenem-susceptible serotype 15A-ST3058 isolates and a serotype 23A-ST338 clone existed in earlier years, rise and spread of meropenem-non-susceptible serotype 15A-ST63 and serotype 23A-ST166 clones occurred in recent years. We conclude that successive implementation of PCVs has led to a marked decrease in pneumococcal isolate burden, but the replacement by meropenem-non-susceptible NVT 15A and 23A highlights the need for continued local surveillance to track pneumococcal evolution in each region to help vaccine polyvalency decisions.

nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples.

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant Staphylococcus aureus isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.