The usefulness of nanopore sequencing in whole-genome sequencing-based genotyping of Listeria monocytogenes and Salmonella enterica serovar Enteritidis.

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. IMPORTANCE: This 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.

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.

Chromosome-Borne CTX-M-65 Extended-Spectrum ß-Lactamase-Producing Salmonella enterica Serovar Infantis, Taiwan.

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.

Molecular typing and epidemiology of Clostridium difficile in respiratory care wards of central Taiwan.

BACKGROUND/PURPOSE: In industrialized countries, Clostridium difficile is the major cause of nosocomial diarrhea. This study involved a broad overview of baseline epidemiology for C. difficile in Taiwan. MATERIALS AND METHODS: Point prevalence was estimated from a prospective survey conducted in the respiratory care wards of six hospitals in central Taiwan. Polymerase chain reaction (PCR) ribotyping and multiple-locus variable-number tandem-repeat analysis (MLVA) were performed on all toxigenic C. difficile isolates, including asymptomatic and symptomatic strains. RESULTS: A total of 149 patients were screened for C. difficile; the point prevalence for C. difficile infection (CDI) and C. difficile colonization was 4% and 19%, respectively. CDI cases were significantly related to end-stage renal disease, and C. difficile colonization cases were significantly associated with previous admission to an acute-care facility. No hypervirulent PCR ribotype 027 strain was found. MLVA detected two clusters of CDI-related and three clusters of asymptomatic C. difficile strains circulating in wards. CONCLUSION: Our results demonstrate a high prevalence of toxigenic C. difficile colonization in hospitals. Infection control personnel should pay attention to the increasing numbers of CDI cases, and molecular typing for C. difficile should be performed when necessary.

Salmonella enterica serovar Typhi variants in long-term carriers.

Long-term typhoid carriers can simultaneously excrete Salmonella enterica serovar Typhi variants with considerable genetic differences, a situation that complicates the interpretation of the subtyping data used in outbreak investigations and disease surveillance.

Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan.

BACKGROUND: The number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE) method. National weekly report data were collected for investigating epidemiological trends. RESULTS: A total of 23 emm types were identified in 1,218 S. pyogenes isolates. The five most prevalent emm types were emm12 (50.4%), emm4 (23.2%), emm1 (16.4%), emm6 (3.8%) and emm22 (3.0%). PFGE analysis with SmaI suggested that, with a few exceptions, strains with a common emm type belonged to the same clone. There were two large emm12 clones, one with DNA resistant to cleavage by SmaI. Each prevalent emm clone had major PFGE strain(s) and many minor strains. Most of the minor strains emerged in the population and disappeared soon after. Even some major strains remained prevalent for only 2-3 years before declining. The large fluctuation of scarlet fever cases between 2000 and 2006 was associated with the shuffling of six prevalent emm clones. In 2003, the dramatic drop in scarlet fever cases in central Taiwan and throughout the whole country was associated with the occurrence of a severe acute respiratory syndrome (SARS) outbreak that occurred between late-February and mid-June in Taiwan. CONCLUSION: The occurrences of scarlet fever in central Taiwan in 2000-2006 were primarily caused by five emm types, which accounted for 96.8% of the isolates collected. Most of the S. pyogenes strains (as defined by PFGE genotypes) emerged and lasted for only a few years. The fluctuation in the number of scarlet fever cases during the seven years can be primarily attributed to the shuffling of six prevalent emm clones and to the SARS outbreak in 2003.

Epidemiology and evolution of genotype and antimicrobial resistance of an imported Shigella sonnei clone circulating in central Taiwan.

Shigella sonnei replaced Shigella flexneri to become the predominant species for shigellosis in 2001 to 2003 in central Taiwan. A total of 425 S. sonnei isolates collected from 1996 to 2004 were available for characterization by pulsed-field gel electrophoresis (PFGE), inter-IS1 spacer typing (IST), and antimicrobial susceptibility testing. The results showed that at least 21 IST clones had emerged for the S. sonnei infections in 1996 to 2004. Most IST clones lasted for a short time; some circulated for 2 to 3 years. An IST1 clone, detected for the first time in 2000, was the most prevalent and responsible for the shigellosis epidemic in 2001 to 2003. Over 3 years of sustained transmission, the IST1 clone evolved into many strains with different PFGE genotypes and antibiograms. The ancestor, with a J16N09.0019 PFGE genotype, remained to be the predominant circulating strain in the period studied; however, new strains with certain PFGE genotypes and antibiograms could become major circulating strains for subsequent infections.

Usefulness of inter-IS1 spacer polymorphisms for subtyping of Shigella sonnei isolates.

Shigella sonnei contains numerous IS1 elements. The existence of polymorphisms in the length of the inter-IS1 spacer is a basis for the development of a PCR-based method for the subtyping of S. sonnei strains. The usefulness of inter-IS1 spacer typing (IST) was evaluated and compared with that of pulsed-field gel electrophoresis (PFGE) by characterization of S. sonnei isolates from epidemiologically nonrelated cases and outbreaks and of isolates that were indistinguishable by PFGE and that were collected from independent infection events. IST was less discriminatory than PFGE, with discriminatory indices of 0.96 and 0.63, respectively, but was able to compensate for the drawbacks of PFGE. PFGE exhibited a high level of discriminatory power for S. sonnei isolates; however, PFGE was also, at times, too discriminatory, which was a disadvantage in constructing the clonal relationships among strains circulating over a period of months or years. Furthermore, IST provided greater subtyping information for isolates indistinguishable by PFGE. The present study indicates that IST is more useful than PFGE for investigating the genetic relationships among S. sonnei strains circulating over a longer time span and also for discriminating certain strains which are indistinguishable by PFGE.