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Objectives: Campylobacter is a significant zoonotic pathogen primarily transmitted through poultry. Our study aimed to assess antimicrobial resistance and genetic relationships among Campylobacter isolates from retail chicken meat and humans in Taiwan. Methods: Campylobacter isolates were analysed using whole-genome sequencing to investigate their antimicrobial resistance, genetic determinants of resistance, and genotypes. Results: Campylobacter coli and Campylobacter jejuni accounted for 44.9% and 55.1% of chicken meat isolates, and 11.4% and 88.6% of human isolates, respectively. C. coli displayed significantly higher resistance levels. Furthermore, isolates from chicken meat exhibited higher levels of resistance to most tested antimicrobials compared to isolates from humans. Multidrug resistance was observed in 96.3% of C. coli and 43.3% of C. jejuni isolates from chicken meat and 80.6% of C. coli and 15.8% of C. jejuni isolates from humans. Macrolide resistance was observed in 85.5% of C. coli isolates, primarily attributed to the erm(B) rather than the A2075G mutation in 23S rRNA. Among the 511 genomes, we identified 133 conventional MLST sequence types, indicating significant diversity among Campylobacter strains. Notably, hierarchical Core-genome multilocus sequence typing clustering, including HC0, HC5, and HC10, revealed a significant proportion of closely related isolates from chicken meat and humans. Conclusions: Our research highlights significant associations in antimicrobial resistance and genetic relatedness between Campylobacter isolates from chicken meat and humans in Taiwan. The genetic analysis data suggest that campylobacteriosis outbreaks may occur more frequently in Taiwan than previously assumed. Our study emphasizes the need for strategies to control multidrug-resistant strains and enhance outbreak prevention.

While the incidence of shigellosis has decreased in developed nations due to improved living conditions and healthcare systems, it remains prevalent in economically developing regions. In recent years, a resurgence of shigellosis has been observed in the United States, Europe, and Taiwan, primarily among men having sex with men and people living with human immunodeficiency virus, along with a rise in antimicrobial resistance. This study aims to review the historical epidemiological trends and drug resistance in shigellosis, with a focus on Taiwan. A comprehensive search was conducted using various databases and sources, including non-English literature in Japanese and Chinese. In developed countries, Shigella sonnei and Shigella flexneri are the most common species, while Shigella dysenteriae infections are sporadic. In Taiwan, the classification and prevalence of Shigella species have evolved over time, with S. flexneri and S. sonnei being the predominant strains. Fluoroquinolone resistance and azithromycin non-susceptibility are the ongoing threat. In conclusion, shigellosis remains a significant global health concern, with recent increases in certain populations and antimicrobial resistance. Further research is necessary to understand the clinical significance and risk factors associated with asymptomatic carriers and to assess the impact of behavioral modifications and interventions in high-risk populations.

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes blaKPC-2, blaIMP-8, and predominantly blaOXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a blaOXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harboring a blaIMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel blaKPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

ABSTRACT Bacterial genotyping through whole-genome sequencing plays a crucial role in disease surveillance and outbreak investigations in public health laboratories. This study assessed the effectiveness of Oxford Nanopore Technologies (ONT) sequencing in the genotyping of Listeria monocytogenes and Salmonella enterica serovar Enteritidis. Our results indicated that ONT sequences, generated with the R10.4.1 flow cell and basecalled using the Dorado 0.5.0 Super Accurate 4.3 model, exhibited comparable accuracy to Illumina sequences, effectively discriminating among bacterial strains from outbreaks. These findings suggest that ONT sequencing has the potential to be a promising tool for rapid whole-genome sequencing of bacterial pathogens in public health laboratories for epidemiological investigations.IMPORTANCEThis study unveils that Oxford Nanopore Technologies sequencing, by itself, holds the potential to serve as a whole-genome sequencing-based genotyping tool in public health laboratories, enabling routine subtyping of bacterial isolates for disease surveillance and outbreak investigations

ABSTRACT: 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.

Abstract The accuracy of Oxford Nanopore Technology (ONT) sequencing has significantly improved thanks to new flowcells, sequencing kits, and basecalling algorithms. However, novel modification types untrained in the basecalling models can seriously reduce the quality. Here we reports a set of ONT-sequenced genomes with unexpected low quality due to novel modification types. Demodification by whole-genome amplification significantly improved the quality but lost the epigenome. We also developed a reference-based method, Modpolish, for correcting modification-mediated errors while retaining the epigenome when a sufficient number of closely-related genomes is publicly available (default: top 20 genomes with at least 95% identity). Modpolish not only significantly improved the quality of in-house sequenced genomes but also public datasets sequenced by R9.4 and R10.4 (simplex). Our results suggested that novel modifications are prone to ONT systematic errors. Nevertheless, these errors are correctable by nucleotide demodification or Modpolish without prior knowledge of modifications.

A CTX-M-65‒producing Salmonella enterica serovar Infantis clone, probably originating in Latin America and initially reported in the United States, has emerged in Taiwan. Chicken meat is the most likely primary carrier. Four of the 9 drug resistance genes have integrated into the chromosome: blaCTX-M-65, tet(A), sul1, and aadA1.

ABSTRACT We report that carbapenem resistance developed in an extensively drug-resistant Salmonella enterica serovar Agona strain during antimicrobial therapy and in an AmpC β-lactamase-producing S. Infantis strain. Whole-genome sequence analysis indicated that carbapenem resistance in the S. Agona strain was associated with the insertion of a 160-kb IS15DI composite transposon in ompC_378, which encodes a porin consisting of 378 amino acids, along with a 397-bp deletion in the ompD. The transposon harbored 12 resistance genes including bla CTX-M-55 and an efflux pump regulatory gene, ramAp. Carbapenem resistance in the bla CMY-2-carrying S. Infantis strain was linked to the insertion of a 22,905-bp segment located 10 bp upstream of the ompC_378 coding sequence, as well as a 254-bp deletion in ompD. Our findings indicate that the loss of both ompC_378 and ompD is necessary for carbapenem resistance in extended-spectrum β-lactamase and AmpC β-lactamase-producing Salmonella strains. IMPORTANCE Carbapenem resistance arising from the loss of porins is commonly observed in extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase-producing strains of certain Enterobacteriaceae genera, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. However, this resistance mechanism is rarely reported in the Salmonella genus. To address this knowledge gap, our study offers genetic evidence demonstrating that the loss of two specific porins (OmpC_378 and OmpD) is crucial for the development of carbapenem resistance in Salmonella ESBL and AmpC β-lactamase-producing strains. Furthermore, our findings reveal that most Salmonella serovars carry seven porin parathologs, with OmpC_378 and OmpD being the key porins involved in the development of carbapenem resistance in Salmonella strains.

Carbapenem-resistant ST11_KL64 Klebsiella pneumoniae emerged as a significant public health concern in Taiwan, peaking between 2013 and 2015, with the majority of isolates exhibiting OXA-48 as the sole carbapenemase. In this study, we employed whole-genome sequencing to investigate the molecular underpinnings of ST11_KL64 isolates collected from 2013 to 2021. Phylogenomic analysis revealed a notable genetic divergence between the ST11_KL64 strains in Taiwan and those in China, suggesting an independent evolutionary trajectory. Our findings indicated that the ST11_KL64_Taiwan lineage originated from the ST11_KL64 lineage in Brazil, with recombination events leading to the integration of ICEKp11 and a 27-kb fragment at the tRNAASN sites, shaping its unique genomic landscape. To further elucidate this unique sublineage, we examined the plasmid contents. In contrast to ST11_KL64_Brazil strains, which predominantly carried blaKPC-2, ST11_KL64_Taiwan strains exhibited the acquisition of an epidemic blaOXA-48-carrying IncL plasmid. Additionally, ST11_KL64_Taiwan strains consistently harbored a multi-drug resistance IncC plasmid, along with a collection of gene clusters that conferred resistance to heavy metals and the phage shock protein system via various Inc-type plasmids. Although few, there were still rare ST11_KL64_Taiwan strains that have evolved into hypervirulent CRKP through the horizontal acquisition of pLVPK variants. Comprehensive characterization of the high-risk ST11_KL64 lineage in Taiwan not only sheds light on its epidemic success but also provides essential data for ongoing surveillance efforts aimed at tracking the spread and evolution of ST11_KL64 across different geographical regions. Understanding the molecular underpinnings of CRKP evolution is crucial for developing effective strategies to combat its emergence and dissemination.

Shigellosis appears to increase in certain at-risk populations in developed countries. Based on the nationwide surveillance, the annual incidence of shigellosis in Taiwan (1999-2019) was 0.38-5.77 cases per 100,000 people. Indigenous shigellosis has mostly affected men who have sex with men (MSM) and people living with HIV (PLWH) since 2015. In this retrospective study, compared with those diagnosed before 2015, indigenous cases diagnosed during 2015–2019 mostly occurred in male adults (96.0% vs 47.1%, P

Background: Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. Methods: Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. Results: NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. Conclusion: NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.

ABSTRACT: Objectives: A nosocomial salmonellosis outbreak caused by Salmonella enterica serovar Goldcoast occurred in a respiratory care ward (RCW) of a hospital in central Taiwan between December 24, 2020, and January 21, 2021. Ten isolates recovered from 10 RCW residents were resistant to extended-spectrum cephalosporins. The resistance mechanism needs to be investigated. Methods: Whole-genome sequencing and antimicrobial susceptibility testing were conducted to determine the genetic resistance determinants and the phenotypic resistance in the isolates. Results: Each of the 10 outbreak isolates harbored an IncHI2 plasmid that carried 15 antimicrobial resistance genes aac(3)-IId, aadA22, aph(3’)-Ia, aph(6)-Id, arr-2, blaCTX-M-55, blaLAP-2, blaTEM-1, dfrA14, floR, lnu(F), qnrS13, sul2, sul3, tet(A), an efflux pump regulatory gene ramAp and an IncL plasmid carried a blaOXA-48. The outbreak strains were expected to be resistant to numerous antimicrobials, including aminoglycosides, b-lactams /inhibitors, tetracycline, rifamycin, lincosamide, sulfonamides, trimethoprim, phenicols, fluoroquinolones, and carbapenems. Two outbreak isolates displayed higher minimum inhibitory concentrations than the other eight isolates to cefmetazole and carbapenems, which was linked to a deficiency of a major facilitator superfamily transporter in the two isolates. Conclusion: The carbapenem-resistant outbreak strains could have been derived from extensively drug-resistant S. enterica Goldcoast strains, which have been a major pathogen in Taiwan since 2018, through the acquisition of a blaOXA-48-carrying plasmid. Special efforts are needed in Taiwan to monitor the spread of extremely resistant strains.

ABSTRACT Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan from 2017 to 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 selected isolates using PCR and confirmed in 81 selected isolates using whole-genome sequencing. Multidrug resistance was found in 47.3% of total isolates and 96.2% of Salmonella enterica serovar Anatum and 81.7% of S. enterica serovar Typhimurium isolates. Resistance to the conventional first-line drugs (ampicillin, chloramphenicol, and cotrimoxazole), cefotaxime and ceftazidime, and ciprofloxacin was found in 32.5 to 49.0%, 20.3 to 20.4%, and 3.2% of isolates, respectively. A total of 76 (3.1%) isolates were resistant to azithromycin, which was associated with mph(A), erm(42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR. mph(A) was found in 53% of the 76 azithromycin-resistant isolates from 11 serovars and located in an IS26-mph(A)-mrx(A)-mphR(A)-IS6100 unit in various incompatibility plasmids and the chromosomes. erm(42) in S. enterica serovar Albany was carried by an integrative and conjugative element, ICE_erm42, and in S. enterica serovar Enteritidis and S. Typhimurium was located in IS26 composite transposons in the chromosomes. erm(B) was carried by IncI1-I(α) plasmids in S. Enteritidis and S. Typhimurium. ramAp was a plasmid-borne ramA, a regulatory activator of efflux pump(s), found in only S. enterica serovar Goldcoast. Since the azithromycin resistance determinants are primarily carried on mobile genetic elements, they could easily be disseminated among human bacterial pathogens. The ramAp-carrying S. Goldcoast isolates displayed azithromycin MICs of 16 to 32 mg/L. Thus, the epidemiological cutoff value of ≤16 mg/L of azithromycin proposed for wild-type NTS should be reconsidered. IMPORTANCE Antimicrobial resistance in NTS isolates is a major public health concern in Taiwan, and the mechanisms of azithromycin resistance are rarely investigated. Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains. Our study reports the epidemiological trend of resistance in NTS in Taiwan and the genetic determinants involved in azithromycin resistance. We point out that nearly half of NTS isolates from 2017 to 2018 are MDR, and 20% are resistant to third-generation cephalosporins. The azithromycin resistance rate (3.1%) for the NTS isolates from Taiwan is much higher than those for the NTS isolates from the United States and Europe. Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are frequently carried on mobile genetic elements. Thus, the azithromycin resistance determinants could be expected to be disseminated among diverse bacterial pathogens.

Background/purpose: Salmonella Panama was considered an invasive non-typhoidal Salmonella (iNTS) serovar. Comprehensive clinical, microbiological, and genomic studies on S. Panama are scarce. We aimed to characterize the clinical and microbiological characteristics of S. Panama infection. Virulence mechanism of S. Panama and other iNTS serovars were also examined. Methods: Based on data from the longitudinal surveillance system for Salmonella deployed in Taiwan since 2004, a case–control study was undertaken to evaluate clinical characteristics of S. Panama infection during an outbreak in 2015–2016. Cellular experiments were conducted to compare pathogenicity of S. Panama and other iNTS with S. Typhimurium. Results: Most patients (41/44, 93.2%) infected by S. Panama were

Background: Cholera, a rapidly dehydrating diarrheal disease caused by toxigenic Vibrio cholerae, is a leading cause of morbidity and mortality in some regions of the world. Core genome multilocus sequence typing (cgMLST) is a promising approach in generating genetic fingerprints from whole-genome sequencing (WGS) data for strain comparison among laboratories. Methods: We constructed a V. cholerae core gene allele database using an in-house developed computational pipeline, a database with cgMLST profiles converted from genomic sequences from the National Center for Biotechnology Information, and built a REST-based web accessible via the Internet. Results: We built a web service platform—cgMLST@Taiwan and installed a V. cholerae allele database, a cgMLST profile database, and computational tools for generating V. cholerae cgMLST profiles (based on 3,017 core genes), performing rapid global strain tracking, and clustering analysis of cgMLST profiles. This web-based platform provides services to researchers, public health microbiologists, and physicians who use WGS data for the investigation of cholera outbreaks and tracking of V. cholerae strain transmission across countries and geographic regions. The cgMLST@Taiwan is accessible at http://rdvd.cdc.gov.tw/cgMLST.

ABSTRACT Listeria monocytogenes is a life-threatening foodborne pathogen. Here, we report the genomic characterization of a nationwide dataset of 411 clinical and 82 food isolates collected in Taiwan between 2014 and 2019. The observed incidence of listeriosis increased from 0.83 to 7 cases per million population upon implementation of mandatory notification in 2018. Pregnancy-associated cases accounted for 2.8% of human listeriosis and all-cause 7-day mortality was of 11.9% in nonmaternal-neonatal listeriosis. L. monocytogenes was isolated from 90% of raw pork and 34% of chicken products collected in supermarkets. Sublineages SL87, SL5, and SL378 accounted for the majority (65%) of clinical cases. SL87 and SL378 were also predominant (57%) in food products. Five cgMLST clusters accounted for 57% clinical cases, suggesting unnoticed outbreaks spanning up to 6 years. Mandatory notification allowed identifying the magnitude of listeriosis in Taiwan. Continuous real-time genomic surveillance will allow reducing contaminating sources and disease burden. IMPORTANCE Understanding the phylogenetic relationship between clinical and food isolates is important to identify the transmission routes of foodborne diseases. Here, we performed a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes. We show a 9-fold increase in listeriosis reporting upon implementation of mandatory notification. We found that sublineages SL87 and SL378 predominated among both clinical (50%) and food (57%) isolates, and identified five cgMLST clusters accounting for 57% of clinical cases, suggestive of potential protracted sources of contamination over up to 6 years in Taiwan. These findings highlight that mandatory declaration is critical in identifying the burden of listeriosis, and the importance of genome sequencing for a detailed characterization of the pathogenic L. monocytogenes genotypes circulating in Asia.

ABSTRACT CG23-I lineage constitutes the majority of hypervirulent Klebsiella pneumoniae. A diabetic patient suffered six episodes of infections caused by CG23-I K. pneumoniae. A total of nine isolates were collected in 2020. We performed whole-genome sequencing to elucidate the within-patient evolution of CG23-I K. pneumoniae. The maximum pairwise difference among the nine longitudinally collected isolates was five single nucleotide polymorphisms. One of the mutations was at the Asp87 position of GyrA. Four indels were identified, including an initiator tRNAfMet duplication, a tRNAArg deletion, a 7-bp insertion, and a 22-bp deletion. All 9 isolates had the genomic features of CG23-I K. pneumoniae, a chromosome-borne ICEKp10, and a large virulence plasmid. The carriage of a complete set of genes for the biosynthesis of colibactin by ICEKp10 gave the nine isolates an ability to cause DNA damage to RAW264.7 cells. Compared with the initial isolate, the last isolate with an additional copy of initiator tRNAfMet grew faster in a nutrient-limiting condition and exhibited enhanced virulence in BALB/c mice. Collectively, we characterized the within-patient microevolution of CG23-I K. pneumoniae through an in-depth comparison of genome sequences. Using the in vitro experiments and mouse models, we also demonstrated that these genomic alterations endowed the isolates with advantages to pass through in vivo selection. IMPORTANCE CG23-I is a significant lineage of hypervirulent Klebsiella pneumoniae. This study characterizes the within-patient microevolution of CG23-I K. pneumoniae. Selective pressures from continuous use of antibiotics favored point mutations contributing to bacterial resistance to antibiotics. The duplication of an initiator tRNAfMet gene helped CG23-I K. pneumoniae proliferate to reach a maximal population size during infections. For longer persistence inside a human host, the large virulence plasmid evolved with more flexible control of replication through duplication of the iteron-1 region. With the genomic alterations, the last isolate had a growth advantage over the initial isolate and exhibited enhanced virulence in BALB/c mice. This study gives us a deeper understanding of the genome evolution during the within-patient pathoadaptation of CG23-I K. pneumoniae.

The first imported case of XDR typhoid fever in Taiwan contracted with a bacterial strain, which was most closely related to the blaCTX-M-15-carrying strains linked to Pakistan. Meropenem, in combination with an antimicrobial with intracellular activity against Salmonella, should be used for the treatment of XDR typhoid fever.

ABSTRACT We present the demographic features of invasive meningococcal disease (IMD) in Taiwan between 1993 and 2020 and the genetic characteristics of Neisseria meningitidis isolates recovered from 2003 to 2020. IMD was rare in Taiwan between 1993 and 2020, with an annual incidence ranging from 0.009 to 0.204 per 100,000 people. The case fatality rate (CFR) declined from 18.1% for patients in 1993 to 2002 to 9.8% in 2003 to 2020. Infants less than 12 months were most susceptible to the disease. N. meningitidis serogroup B (NmB) was most predominant, responsible for 81.2% (134/165) of the IMD cases in 2003 to 2020. The majority of the isolates recovered from 2003 to 2020 belonged to 4 worldwide-spread hyperinvasive clonal complexes (cc), cc4821 (30.3%), cc32 (19.4%), cc41/44 (12.7%), cc23 (7.3%), and also a newly assigned clonal complex, cc3439 (10.3%). Core genome multilocus sequence typing (cgMLST) profile comparisons revealed that the cc4821 isolates with a T-to-I substitution at position 91 in gyrA were closely related to those originating from China. Of the 165 isolates, 20.0% and 53.3% were predicted to be covered by the Bexsero and Trumenba vaccines, respectively, whereas, 77.0% and 46.7% remained indeterminate. In conclusion, N. meningitidis isolates recovered in Taiwan between 2003 and 2020 were mostly highly diverse. Most IMD cases appeared sporadically and were caused by localized strains, although some patients were infected by recently introduced strains. cgMLST is a powerful tool for the rapid comparison of genetic relatedness among a large number of isolates. cgMLST profiling, based on 1,241 core genes, and strain tracking can be performed on the website of cgMLST@Taiwan (http://rdvd.cdc.gov.tw/cgMLST/). IMPORTANCE N. meningitidis can cause life-threatening invasive meningococcal disease (IMD), including meningitis and sepsis, resulting in a high CFR and long-term sequelae in survivors. Here, we report the demographic features of IMD in Taiwan over a 28-year period (1993 to 2020) and the genetic characteristics of N. meningitidis isolates recovered from patients with IMD over an 18-year period (2003 to 2020). We conducted a whole-genome sequence analysis to characterize the genetic features of the isolates and developed a cgMLST scheme for epidemiological investigation and strain tracking. The findings can be beneficial in understanding the epidemiology of IMD in Taiwan, the genetic characteristics of the bacterial strains, and the distribution of vaccine antigens for vaccine development and implementation.

With the reduction in the cost of next-generation sequencing, whole-genome sequencing (WGS)–based methods such as core-genome multilocus sequence type (cgMLST) have been widely used. However, gene-based methods are required to assemble raw reads to contigs, thus possibly introducing errors into assemblies. Because the robustness of cgMLST depends on the quality of assemblies, the results of WGS should be assessed (from sequencing to assembly). In this study, we investigated the robustness of different read lengths, read depths, and assemblers in recovering genes from reference genomes. Different combinations of read lengths and read depths were simulated from the complete genomes of three common food-borne pathogens: Escherichia coli, Listeria monocytogenes, and Salmonella enterica. We found that the quality of assemblies was mainly affected by read depth, irrespective of the assembler used. In addition, we suggest several cutoff values for future cgMLST experiments. Furthermore, we recommend the combinations of read lengths, read depths, and assemblers that can result in a higher cost/performance ratio for cgMLST.

We investigated the epidemiology of cholera in Taiwan during 2002–2018. Vibrio cholerae sequence type (ST) 75 clone emerged in 2009 and has since become more prevalent than the ST69 clone from a previous pandemic. Closely related ST75 strains have emerged in 4 countries and may now be widespread in Asia.

In 2011, a Salmonella enterica serovar Anatum clone emerged in Taiwan. During 2016–2017, infections increased dramatically, strongly associated with emergence and spread of multidrug-resistant strains with a plasmid carrying 11 resistance genes, including blaDHA-1. Because these resistant strains infect humans and food animals, control measures are urgently needed.

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Sequence type (ST) 11 is one of the major lineages of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although the gastrointestinal (GI) carriage of CRKP predisposes individuals to subsequent infections, little is known for its impact on gut homeostasis. In this study, we investigated the association between ST11 CRKP colonization and colorectal cancer (CRC). Two ST11 CRKP, KPC160111 (KL47) and KPC160132 (KL64), were selected as the representative strains. We used azoxymethane (AOM) and dextran sodium sulfate (DSS) to initiate a colitis-associated CRC model. Both strains established prolonged colonization in the GI tract of the AOM-DSS-treated BALB/c mice and aggravated gut dysbiosis. Under this AOM-DSS-induced setting, ST11 K. pneumoniae colonization significantly promoted the growth and progression of colorectal adenomas to high-grade dysplasia. Numerous crypts were formed inside the enlarged adenomas, in which CD163+ tumor-associated macrophages accumulated. Similarly, ST11 K. pneumoniae also increased the population size of the CD163+ macrophages with the M2 phenotype in the peritoneal cavity of LPS-primed BALB/c mice. When applied to RAW264.7 cells, ST11 K. pneumoniae polarized the macrophages toward an M2 phenotype through the inhibition of IKK-NFκB and the activation of STAT6-KLF4-IL-10. Through the M2-skewing ability, ST11 K. pneumoniae promoted the accumulation of CD163+ macrophages in the adenomatous crypts to create an immunosuppressive niche, which not only accommodated the extended stay for its own sake but also deteriorated colorectal tumorigenesis.

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Background/Purpose: Group G Streptococcus (GGS) infections in human have increased. Treatment relied on antibiotic therapy, including erythromycin. However, information regarding the dominant strains and erythromycin susceptibility in GGS bacteremia is limited. Methods: A total of 134 GGS were isolated from patients with bacteremia in a university hospital of southern Taiwan during 1993â2010. The erythromycin susceptibility was determined by disc diffusion and agar dilution assays. The bacterial species was determined by MALDI-TOF. The presence of erythromycin-resistant genes and emm types were determined by polymerase chain reaction and sequence. The clonal spreading was analyzed by pulsed-field gel electrophoresis with SmaI or SgrAI digestion. Results: The annual erythromycin non-susceptible rate varied, with an average of 40.3%. All erythromycin non-susceptible strains belonged to the Streptococcus dysgalactiae. No erythromycin non-susceptible strains belong to the anginosus group. The most prevalent erythromycin-resistant gene was mefA (57.4%), followed by ermB (37%), and ermA (3.7%). The N terminal hyper variable region of emm was sequenced to determine the emm type, and only S. dysgalactiae had the emm gene. The most prevalent emm types were emmSTG840.0 (17.2%), emmSTG485.0 (10.4%), and emmSTC839.0 (9.0%). 73% and 47% of the strains with only mefA and ermB belonged to emmSTG840.0 and emmSTC839.0 types, respectively. Pulsed-field gel electrophoresis showed that different clones of emmSTG840.0 and emmSTC839.0 strains were spread in this region during the 18 years of surveillance. Conclusion: Our data indicate that there were dominant emm types with erythromycin non-susceptibility in S. dysgalactiae isolated from bacteremia in Taiwan, and thus constant surveillance is warranted. Keywords: emm, Epidemiology, Erythromycin, Group G Streptococcus

The aim of this study is to characterize antibiotic-nonsusceptible Salmonella isolates in Taiwan. Methods: A total of 76 Salmonella isolates showing lower susceptibility to cephalosporins or quinolones were identified from 1416 clinical isolates from 1999 to 2008. Minimal inhibitory concentrations for selected antimicrobial agents were tested by the agar dilution method. Antibiotic resistance-related genes were determined by polymerase chain reaction (PCR) combined with sequencing. Southern blotting, conjugation tests, and transformation tests were used to characterize plasmid-mediated quinolone resistance (PMQR) determinants. Results: The observed nonsusceptible phenotypes of 76 isolates were against cefoxitin (57.9%), cefotaxime (43.4%), ceftazidime (40.8%), ceftriaxone (42.1%), cefepime (5.3%), ciprofloxacin (80.3%), and levofloxacin (81.6%). Among 44 cephalosporin-resistant isolates, TEM-1, CMY-2, CMY-14, CTX-M-3-like and CTX-M-15-like determinants were present in 31 (70.5%), 32 (72.7%), 1 (2.3%), 1 (2.3%), and 1 (2.3%) of isolates, respectively. PCR screening for PMQR genes of 62 quinolone-nonsusceptible isolates revealed the presence of qnrS, qnrD, aac(6′)-Ib-cr, and oqxAB in 3 (4.8%), 2 (3.2%), 1 (1.6%), and 10 (16.1%) isolates, respectively. Among 36 isolates showing high resistance to quinolones, S83F/D87N and S83F/D87G amino acid substitutions of GyrA were found in 29 (80.6%) and 6 (16.7%) isolates, respectively. Moreover, among quinolone highly resistant isolates, eight (22.2%) of isolates showed over-expression of the PAβN-sensitive efflux pump. Transformants and transconjugants harboring qnrD- or oqxAB-plasmids showed decreased susceptibility to quinolones. Conclusion: GyrA mutations are the major mechanisms associated with quinolone-resistant Salmonella isolates in Taiwan. Overproduction of efflux pump genes and the presence of qnr and oqxAB play additional roles in reduced susceptibility to quinolones.

We built a pan-genome allele database with 395 genomes of Salmonella enterica serovar Enteritidis and developed computer tools for analysis of whole genome sequencing (WGS) data of bacterial isolates for disease cluster identification. A web server (http://wgmlst.imst.nsysu.edu.tw) was set up with the database and the tools, allowing users to upload WGS data to generate whole genome multilocus sequence typing (wgMLST) profiles and to perform cluster analysis of wgMLST profiles. The usefulness of the database in disease cluster identification was demonstrated by analyzing a panel of genomes from 55 epidemiologically well-defined S. Enteritidis isolates provided by the Minnesota Department of Health. The wgMLST-based cluster analysis revealed distinct clades that were concordant with the epidemiologically defined outbreaks. Thus, using a common pan-genome allele database, wgMLST can be a promising WGS-based subtyping approach for disease surveillance and outbreak investigation across laboratories.

An increase in group D Salmonella isolates with high antimicrobial resistant rates is being seen in Taiwan. This study aimed to determine the multidrug-resistant (MDR, more than three antibiotics) phenotype, genotype, and the correlation between the presence of class 1 integrons and its invasiveness of Salmonella panama and Salmonella enteritidis isolated from children. Methods: Twenty S. panama and 59 S. enteritidis isolates were examined for minimal inhibitory concentrations of ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline by agar dilution method. The presence of blaPSE-1, floR, aadA2, sul1, and tet(G) resistance genes, class 1 integrons, and Salmonella genomic island 1 (SGI1) was identified by polymerase chain reaction. The adhesion and invasion assays of S. panama to Caco-2 cells were determined using the pour plate method. Results: All S. panama and 15 (25.4%) of the S. enteritidis isolates displayed MDR phenotype. Furthermore, MDR genotype was present in 70.0% of S. panama and 6.8% of S. enteritidis. Class 1 integrons were present in 40.0% of S. panama and 11.9% of S. enteritidis. None contained SGI1 or SGI1 variants. Strains carrying class 1 integrons were more frequently isolated from bacteria with MDR (73.3% vs. 37.5%; odds ratio, 4.6; 95% confidence interval, 1.3-16.0; p=0.01) and isolated from blood and cerebrospinal fluid (46.7% vs. 21.9%; odds ratio, 3.1; 95% confidence interval, 1.0-10.1; p=0.05) than noncarriers. S. panama carrying class 1 integrons were more invasive to Caco-2 cells than those without (p=0.01). Conclusion: S. panama and S. enteritidis with class 1 integrons are significantly related to the presence of MDR phenotype. Moreover, S. panama with class 1 integrons may present more invasiveness than those without.

We report an outbreak of azithromycin-nonsusceptible Shigella flexneri 3a infection in Taiwan associated with men who have sex with men. The bacterial strains belonged to the sublineage A of a recently reported outbreak lineage associated with men who have sex with men, characterized by reduced azithromycin susceptibility and circulation in shigellosis low-risk regions.

Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas) and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson's index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.

We report the prevalence and characteristics of Salmonella strains resistant to ciprofloxacin and extended-spectrum cephalosporins in Taiwan from January to May 2004. All isolates resistant to extended-spectrum cephalosporins carried blaCMY-2, and all ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates were genetically related.

To investigate global epidemiology of Shigella sonnei, we performed multilocus variable number tandem repeat analysis of 1,672 isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three major clonal groups were identified; 2 were globally spread. Type 18 and its derivatives have circulated worldwide in recent decades.

We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96%) were multidrug resistant (MDR) and 48 (44%) had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8-11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.