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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.

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

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 ICE

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

The accuracy of Oxford Nanopore Technology (ONT) sequencing has significantly improved thanks to new flowcells, sequencing kits, and basecalling algorithms. However, novel modifications untrained in the basecalling models can seriously reduce the quality. This paper reports a set of ONT-sequenced genomes with unexpected low quality (∼Q30) due to extensive new modifications. Demodification by whole-genome amplification (WGA) significantly improved the quality of all genomes (∼Q50-60) while losing the epigenome. We developed a computational method, Modpolish, for correcting modification-mediated errors without WGA. Modpolish produced high-quality genomes and uncovered the underlying modification motifs without loss of epigenome. Our results suggested that novel modifications are prone to ONT errors, which are correctable by WGA or Modpolish without additional short-read sequencing.

Campylobacter coli and Campylobacter Jejuni are highly resistant to most therapeutic antimicrobials in Taiwan; rapid diagnostics of resistance in bacterial isolates is crucial for the treatment of campylobacteriosis. We characterized 219 (40 C. coli and 179 C. jejuni) isolates recovered from humans from 2016 to 2019 using whole-genome sequencing to investigate the genetic diversity among isolates and the genetic resistance determinants associated with antimicrobial resistance. Susceptibility testing with 8 antimicrobials was conducted to assess the concordance between phenotypic resistance and genetic determinants. The conventional and core genome multilocus sequence typing analysis revealed diverse clonality among the isolates. Mutations in gyrA (T86I, D90N), rpsL (K43R, K88R), and 23S rRNA (A2075G) were found in 91.8%, 3.2%, and 6.4% of the isolates, respectively. The horizontally transferable resistance genes ant(6)-I, aad9, aph(3′)-IIIa, aph(2″), bla(OXA), catA/fexA, cfr(C), erm(B), lnu, sat4, and tet were identified in 24.2%, 21.5%, 33.3%, 11.9%, 96.3%, 10.0%, 0.9%, 6.8%, 3.2%, 13.2%, and 96.3%, respectively. High-level resistance to 8 antimicrobials in isolates was 100% predictable by the known resistance determinants, whereas low-level resistance to azithromycin, clindamycin, nalidixic acid, ciprofloxacin, and florfenicol in isolates was associated with sequence variations in CmeA and CmeB of the CmeABC efflux pump. Resistance-enhancing CmeB variants were identified in 62.1% (136/219) of isolates. In conclusion, an extremely high proportion of C. coli (100%) and C. jejuni (88.3%) were multidrug-resistant, and a high proportion (62.5%) of C. coli isolates were resistant to azithromycin, erythromycin, and clindamycin, which would complicate the treatment of invasive campylobacteriosis in this country.

In investigating the epidemiological trends of Salmonella enterica serovar Goldcoast, we previously identified several closely related strains with different MICs to azithromycin and quinolones. Genome sequencing and comparison of two very similar multidrug-resistant (MDR) strains, R18.0877 and R18.1656, has led to the identification of an extra plasmid-borne ramA gene, ramAp, on the large IncHI2 plasmid carried by R18.0877. The ramAp gene is located in a 953-bp region on the plasmid, which is identical to that of the Klebsiella quasipneumoniae chromosomal ramA loci. A truncated ISEcp1 located at the adjacent upstream area of the putative regulatory region of ramAp may likely contribute to its mobilization and expression. Introducing the ramAp gene and the truncated ISEcp1 into Escherichia coli has resulted in elevated expression of efflux pump genes and elevated MICs to chloramphenicol, azithromycin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, tetracycline, and tigecycline. The ramAp is an extra efflux pump activator gene that potentially could be transmitted with the IncHI2 plasmid among bacteria. It is plausible that, with high interspecific conservation, the plasmid-encoded regulator reduces drug susceptibility by activating existing efflux pump systems of the host and thus can be regarded as a new type of auxiliary antimicrobial resistance determinant. Sequences of similar plasmids were found worldwide. Its impact on the emergence of antimicrobial resistance among bacterial pathogens is worrisome.

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.

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 identified an erm42 -carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 in Taiwan. ICE_erm42-carrying strains displayed high-level resistance to azithromycin, and the element could move into the phylogenetically distant species Vibrio cholerae via conjugation.

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.

Objectives The global spread of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has become a massive threat to human health. We investigated the clonal relatedness of CR-Kp strains in central Taiwan. Methods CR-Kp strains were prospectively collected from inpatients referred to Chung Shan Medical University Hospital (CSMUH) during September 2011 to December 2015. The presence of carbapenemase genes, including blaKPC-2, blaVIM-1, blaNDM-1, and blaOXA-48, was analysed with polymerase chain reaction (PCR) and sequence determination. Clonal relatedness was determined by pulse-field gel electrophoresis and multilocus sequence typing. Capsule synthesis loci were typed based on the variation of the wzi gene. Results A total of 174 CR-Kp strains were collected. KPC-2 and OXA-48 were present in 63 (36.2%) and 22 (12.6%) CR-Kp strains, respectively. Two strains isolated in 2014 coproduced KPC-2 and OXA-48. Nearly all (98%) carbapenemase-producing K. pneumoniae strains belonged to the ST11 clone and could be further grouped into distinct sub-lineages. Intriguingly, the first sub-lineage, designated ST11-Clade I, contained all KPC-2 strains; OXA-48 strains were mostly included in the second sub-lineage, ST11-Clade II. Furthermore, a variation on the capsule synthesis loci was detected between these two sub-lineages: KL-47 was assigned to ST11-Clade I, whereas KL-64 or KL-9 were the main types for the ST11-Clade II strains. Conclusions Clonal expansion of ST11 was responsible for the dissemination of carbapenemase-producing K. pneumoniae. Although KPC-2 still predominates, OXA-48 has emerged rapidly. Co-existence of KPC-2 and OXA-48 in two ST11-Clade I K. pneumoniae highlights the urgency to unravel mechanisms that contribute to this highly transmissible lineage.

We identified an erm42-carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 in Taiwan. ICE_erm42-carrying strains displayed high-level resistance to azithromycin, and the element could move into the phylogenetically distant species Vibrio cholerae via conjugation.

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.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.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.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.

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 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 .

Volume 63, no. 6, e02282-18, 2019, [https://doi.org/10.1128/AAC.02282-18][1]. In the originally published article, one institutional affiliation was missing for the first author. The corrected affiliation line is shown above. [1]: /lookup/doi/10.1128/AAC.02282-18

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.

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.

Salmonella enterica serovar Goldcoast infection was rare in Taiwan; it was not detected in routine surveillance from 2004 to 2013. This serovar was first identified in 2014, but the frequency of infection remained low until 2017. From 2014 to 2016, all but one isolate was pan-susceptible. S. Goldcoast infections abruptly increased in 2018, and all isolates were multidrug-resistant (MDR). All MDR isolates harbored an IncHI2 plasmid, and the majority carried 14 antimicrobial resistance genes, aac(3)-IId, aadA22, aph(3′)-Ia, aph(6)-Id, bla(TEM-1B), bla(CTX-M-55), lnu(F), floR, qnrS13, arr-2, sul2, sul3, tet(A), and dfrA14. S. Goldcoast strains recovered in Taiwan and 96 of 99 strains from Germany, the Netherlands, the United Kingdom, and the United States belonged to sequence type 358 (ST358). Whole-genome single-nucleotide polymorphism and core genome multilocus sequence type analyses revealed that all strains of the ST358 clone shared a high degree of genetic relatedness. The present study highlighted that a dramatic increase in S. Goldcoast infections followed the emergence of MDR strains and indicated that a genetically closely related S. Goldcoast ST358 clone may have widespread significance internationally.

Salmonella is one of the most common pathogens of waterborne and foodborne disease-causing pathogens. In this study, we collected 172 surface water samples from Puzih River and Kaoping River between the years 2010 and 2011. Salmonella was detected in 31.7% (32/101) and 42.2% (30/71) of the samples from the two rivers, respectively. From these positive samples, 44 Salmonella isolates were obtained from these positive samples and were characterized using serotyping and pulsed-field gel electrophoresis (PFGE) genotyping. The isolates were found with 17 serovars and 32 PFGE patterns. Salmonella enterica Newport, Bareilly, Kedougou, Albany and subspecies IIIb 50:k:z were the five most common serovars in aquatic environmental Salmonella isolates. In addition, of the total clinical samples from Chiayi and Kaohsiung, 33.7% (60/178) Newport serovars were isolated. After conducting categorical analysis, we found that the serovar Newport was not uniformly distributed cross the cities. The serovar Newport was over-represented (p

Salmonella spp. is a foodborne pathogen that causes zoonotic disease worldwide. The aim of this study was to investigate the prevalence of antimicrobial resistance of Salmonella isolated from turkey farms in Taiwan. During the past 2 yr, 243 strains of Salmonella were isolated from 2,040 samples (11.9%) from turkey farms, including 32.5% (52/160) from the intestines of 12-day-old turkey poults, 14.2% (119/840) from feces collected from the turkey growing periods, and 6.9% (72/1,040) from finishing periods. S. Albany (35.0%, 85/243), S. Schwarzengrund (23.0%, 56/243), and S. Hadar (19.3%, 47/243) were the most common serovars on turkey farms. For these strains, a high frequency of resistance was observed against florfenicol (97.5%), oxytetracycline (89.3%), doxycycline (78.6%), colistin (77.8%), ampicillin (75.7%), amoxicillin (75.3%), trimethoprim-sulfamethoxazole (73.7%), chloramphenicol (69.1%), and nalidixic acid (67.9%). floR (63.8%), tet (A) (60.5%), blaPSE (57.6%), blaTEM (42.0%), blaCTX-M (34.2%), cmlA (34.2%), and tet (D) (29.2%) were the most common resistance genes found in this study. The int1 gene was identified in 72.4% (176/243) of Salmonella isolates in which the conserved region 3’ of class 1 integrons also was amplified, whereas none had the int2 gene. This study demonstrates that imported and fattening turkeys could be a reservoir for Salmonella isolates resistant to multiple antimicrobials. These results also reinforce the need to develop strategies and implement specific control procedures to reduce the development of antimicrobial resistance.

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

Objectives Epidemic spread of OXA-48-producing Klebsiella pneumoniae, mainly mediated by the transmission of a blaOXA-48-carrying plasmid, has threatened global health during the last decade. Since its introduction to Taiwan in 2013, OXA-48 has become the second most common carbapenemase. We described the transmission and evolution of an OXA-producing K. pneumoniae clone in a single hospital. Methods Twenty-two OXA-48 K. pneumoniae were isolated between October 2013 and December 2015. Comparative genomic analysis was performed based on the WGS data generated with Illumina and MinION techniques. Results Seventeen of the 22 OXA-48 K. pneumoniae that belonged to ST11, with the same capsular genotype, KL64, and differed from each other by seven or fewer SNPs, were considered outbreak strains. Eight of the 17 outbreak strains harboured a 65 499 bp blaOXA-48-carrying IncL plasmid (called pOXA48). pOXA48 was absent from the remaining nine strains. Instead, a 24.9 kb blaOXA-48-carrying plasmid fragment was integrated into a prophage region of their chromosomes. Transmission routes of the ST11_KL64 K. pneumoniae sublineages, which carried either pOXA48 or chromosomally integrated blaOXA-48, were reconstructed. Conclusions Clonal expansion of ST11_KL64 sublineages contributed to the nosocomial outbreak of OXA-48 K. pneumoniae. The chromosome-borne blaOXA-48 lineage emerged during a 2 year period in a single hospital. Dissemination of OXA-48, which is vertically transmitted in K. pneumoniae even in the absence of selective pressure from antimicrobials, deserves public health attention.

Salmonella Typhimurium and S. Stanley are the most prevalent serogroup B serovars to infect humans in Taiwan. The aim was to determine possible factors to influence the prevalence between S. Typhimurium and S. Stanley. Genotypes were determined by pulsed field gel electrophoresis (PFGE) analysis and the intracellular survival, phagocytosis, reactive oxygen species (ROS) production of human monocyte THP-1 cell and tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and IL-1βexpression in peripheral blood CD14+ cells after infection were analyzed. 182 S. Stanley was clonal disseminated with main pulsotypes 2 from 2004 to 2007. Overall S. Typhimurium evolved more genotypes, while S. Stanley conserved in genotypes. Human blood CD14+ monocytes expressed TNF-α, IL-6 and IL-1β differently among serovars and bacterial conditions (live vs. killed). Live S. Stanley and S. Typhimurium suppressed the TNF-α and IL-6 expression compared to killed bacteria. However, live S. Typhimurium stimulated more IL-1β expression than the killed bacteria, but S. Stanley expressed similar IL-1β levels in both conditions. Furthermore, S. Stanley and S. Typhimurium differed in intracellular survival in the THP-1 cells, an early decrease for S. Stanley, not for S. Typhimurium. Additionally, higher reactive oxygen species (ROS) production in THP-1 cells was found agsinst S. Stanley infection, not found in S. Typhimurium. However, some isolates of S. Stanley could recover from early loss to become more in the monocytes than S. Typhimurium. Difference in phagocytized number, intracellular survival, ROS production and IL-1β expression may contribute to prevalence different between two serovars.

We identified 20 to 22 resistance genes, carried in four incompatibility groups of plasmids, in each of five genetically closely related Salmonella enterica serovar Typhimurium strains recovered from humans, pigs, and chickens. The genes conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, fluoroquinolones, extended-spectrum cephalosporins and cefoxitin, and azithromycin. This study demonstrates the transmission of multidrug-resistant Salmonella strains among humans and food animals and may be the first identification of mphA in azithromycin-resistant Salmonella strains in Taiwan.

The global spread of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has become a massive threat to human health. We investigated the clonal relatedness of CR-Kp strains in central Taiwan.CR-Kp strains were prospectively collected from inpatients referred to Chung Shan Medical University Hospital (CSMUH) during September 2011 to December 2015. The presence of carbapenemase genes, including blaA total of 174 CR-Kp strains were collected. KPC-2 and OXA-48 were present in 63 (36.2%) and 22 (12.6%) CR-Kp strains, respectively. Two strains isolated in 2014 coproduced KPC-2 and OXA-48. Nearly all (98%) carbapenemase-producing K. pneumoniae strains belonged to the ST11 clone and could be further grouped into distinct sub-lineages. Intriguingly, the first sub-lineage, designated ST11-Clade I, contained all KPC-2 strains; OXA-48 strains were mostly included in the second sub-lineage, ST11-Clade II. Furthermore, a variation on the capsule synthesis loci was detected between these two sub-lineages: KL-47 was assigned to ST11-Clade I, whereas KL-64 or KL-9 were the main types for the ST11-Clade II strains.Clonal expansion of ST11 was responsible for the dissemination of carbapenemase-producing K. pneumoniae. Although KPC-2 still predominates, OXA-48 has emerged rapidly. Co-existence of KPC-2 and OXA-48 in two ST11-Clade I K. pneumoniae highlights the urgency to unravel mechanisms that contribute to this highly transmissible lineage.

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.

We characterized Salmonella enterica serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam to investigate their genetic relatedness and antimicrobial resistance. The isolates from Bangladesh and Vietnam were genetically closely related but were distant from those from Indonesia and Taiwan. All but a few isolates from Indonesia and Taiwan were susceptible to all antimicrobials tested. The majority of isolates from Bangladesh and Vietnam were multidrug resistant (MDR) and belonged to the widespread haplotype H58 clone. IncHI1 plasmids were detected in all MDR S . Typhi isolates from Vietnam but in only 15% of MDR isolates from Bangladesh. Resistance genes in the majority of MDR S . Typhi isolates from Bangladesh should reside in the chromosome. Among the isolates from Bangladesh, 82% and 40% were resistant to various concentrations of nalidixic acid and ciprofloxacin, respectively. Several resistance mechanisms, including alterations in gyrase A, the presence of QnrS, and enhanced efflux pumps, were involved in the reduced susceptibility and resistance to fluoroquinolones. Intensive surveillance is necessary to monitor the spread of chromosome-mediated MDR and fluoroquinolone-resistant S . Typhi emerging in Bangladesh.

From 2007 to 2009, we collected a total of 83 bacteraemic isolates of Escherichia coli with reduced susceptibility or resistance to third-generation cephalosporins (TGCs). Isolates were genotyped by PFGE and multilocus sequence typing (MLST). The PFGE patterns revealed two highly correlated clusters (cluster E: nine isolates; cluster G: 22 isolates) associated with this prolonged clonal spreading. Compared with cluster E isolates, cluster G isolates were significantly more likely to harbour aac(6')-Ib-cr (PPE. coli cases caused by cluster E and G isolates were significantly associated with having stayed in our hospital’s respiratory care ward (Paac(6')-Ib-cr and bla CTX-M-14 positivity, and were more likely to belong to ST68/S68-like (all PE. coli ST68 associated with a stay in a long-term care facility. Using epidemiological investigations and PFGE and MLST analyses, we have identified long-term clonal spreading caused by E. coli ST68, with extra antimicrobial-resistance genes possibly acquired during the prolonged spreading period.

We created a database consisting of a large number of Salmonella pulsed-field gel electrophoresis (PFGE) profiles covering a wide range of different serovars. This database was used for the prediction of the serovars based on the PFGE profiles for isolates from Taiwan and Denmark. The PFGE profiles proved very useful in the determination of a serovar although serovar prediction was more efficient for local isolates than those from a distant geographic area. To use a highly stringent band matching tolerance in the BioNumerics software is also important for the grouping of serovars.

ObjectivesOutbreaks of shigellosis among men who have sex with men (MSM) have been reported since the late 1990s. HIV infection is an important risk factor. Since 2014, the global shigellosis epidemic has intensified. Whether chemsex (the use of crystal methamphetamine, γ-hydroxybutyrate or mephedrone to enhance sex) is a new risk factor has not been previously examined.MethodsWe conducted a population-based, case–control study in Taiwan. Acute shigellosis cases diagnosed during the 2015 outbreak among MSM living with HIV were compared with those without shigellosis. CD4+ counts, plasma viral load (pVL), gonorrhoea, syphilis and amoebiasis records were obtained from the Notifiable Disease Surveillance System database. We invited cases/controls to provide information on illicit drug use and sexual behaviours, using a structured questionnaire.ResultsSeventy-five shigellosis cases were compared with 225 controls. High pVL (>100 000 copies/mL; adjusted OR (aOR): 4.9, 95% CI 1.4 to 16.9), gonorrhoea (aOR: 29.4, 95% CI 2.3 to 340.2) and syphilis (aOR: 4.3, 95% CI 1.6 to 11.6) were independent risk factors of shigellosis. Twenty shigellosis cases and 59 controls completed the questionnaire. Oral-to-anal sex (aOR: 15.5, 95% CI 3.6 to 66.7), chemsex (aOR: 5.6, 95% CI 1.4 to 22.7) and poppers use (aOR: 10.9, 95% CI 1.9 to 64.2) within 12 months were independent behavioural risk factors of shigellosis.ConclusionsChemsex is a new risk factor for shigellosis among MSM living with HIV, as identified in the 2015–2016 outbreak. Additional risk factors include poppers use, sexual risk behaviours and high pVL. Further studies on chemsex among MSM, which is a rising public health concern, are urgently required.

We detected the colistin resistance gene mcr-1 in four Salmonella serovars isolated from humans and animals with diarrhea. The resistance gene was carried on different plasmids. One mcr-1-carrying conjugative plasmid, a variant of pHNSHP45, was disseminated among Salmonella isolates recovered from humans, pigs, and chickens.

Food sold over the internet is an emerging business that also presents a concern with regard to food safety. A nationwide foodborne disease outbreak associated with sandwiches purchased from an online shop in July 2010 is reported. Consumers were telephone interviewed with a structured questionnaire and specimens were collected for etiological examination. A total of 886 consumers were successfully contacted and completed the questionnaires; 36.6% had become ill, with a median incubation period of 18 h (range, 6-66 h). The major symptoms included diarrhea (89.2%), abdominal pain (69.8%), fever (47.5%), headache (32.7%), and vomiting (17.3%). Microbiological laboratories isolated Salmonella enterica serovar Enteritidis, Salmonella Virchow, Staphylococcus aureus, Bacillus cereus, and enterotoxigenic Escherichia coli from the contaminated sandwiches, Salmonella Enteritidis and Salmonella Virchow from the patients, and Salmonella Enteritidis and Staphylococcus aureus from food handlers. Pulsed-field gel electrophoresis genotyping suggested a common origin of Salmonella bacteria recovered from the patients, food, and a food handler. Among the pathogens detected, the symptoms and incubation period indicated that Salmonella, likely of egg origin, was the probable causative agent of the outbreak. This outbreak illustrates the importance of meticulous hygiene practices during food preparation and temperature control during food shipment and the food safety challenges posed by online food-shopping services.

a b s t r a c t Multilocus variable-number tandem repeat analysis (MLVA) is a promising subtyping tool to comple- ment pulsed-field gel electrophoresis for discriminating closely related strains of some monomorphic organisms, including Shigella sonnei, which is one of the major foodborne pathogens. However, MLVA results are usually difficult to compare directly between laboratories, impeding the application of MLVA as a subtyping tool for disease surveillance and investigation of common outbreaks across regions or countries. It has long been a big challenge in seeking an approach that can be implemented to obtain comparable MLVA results across laboratories. By implementing a panel of calibration strains in each par- ticipating laboratory for data normalization, the MLVA results of 20 test strains were comparable even though some analytical conditions were different among the laboratories. This approach is simple, pro- tocol independent, and easy to implement in every laboratory, and a small calibration set is sufficient to generate mathematical equations for accurate copy number conversion.

A salmonella genomic island, designated SGI11, was found in 18 of 26 multidrug-resistant Salmonella enterica serovar Typhi isolates from Bangladesh. SGI11 was an IS 1 composite transposon and carried 7 resistance genes that conferred resistance to 5 first-line antimicrobials. Eleven of the 18 SGI11-carrying S . Typhi isolates had developed resistance to high levels of ciprofloxacin.

Clostridium difficile PCR ribotype 027 is a hypervirulent strain that has caused significant nosocomial diarrhea in many countries but has not yet been reported or isolated in Taiwan previously. Here, we present the characteristics of a case of C. difficile PCR ribotype 027 identified in Taiwan. Taiwan is located in a key transportation center of Asia. This report is important for alerting hospitals and public health departments in Asia about the emergence of this hypervirulent strain so that close monitoring may be enacted to prevent potential outbreaks.

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10⁰ CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

Background/Purpose 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 bla PSE-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.

Salmonella Schwarzengrund is one of the frequent serovars isolated from chicken meat in Taiwan. This organism is also one of the invasive Salmonella serovars which may cause human salmonellosis and animal infections. In this study, a total of 466 strains of S. Schwarzengrund including 232 retail chicken meat isolates and 234 human isolates in Taiwan were analyzed for their antibiotic resistance and pulsed field gel electrophoresis (PFGE) patterns. For XbaI-digested DNA, a total of 110 PFGE patterns were obtained. When patterns from both origins were analyzed, of these patterns, 21 were shared by isolates from chicken meat samples and humans. In these 21 patterns, 153 (32.8%) isolates from both origins shared the top five patterns. Since ACSSXTT R-type strains are the major concern worldwide and they accounted for 74.5% of total strains used in this study, such R-type strains in the top five XbaI-digested patterns were then further analyzed with AvrII digestion followed by PFGE and PCR assay targeted to 10 Salmonella virulence genes, i.e., avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC. When PFGE patterns and virulence gene profiles were combined for the analysis of ACSSXTT R-type strains of S. Schwarzengrund, 29 strains from both origins showed the same pattern combinations. Such results suggested the possible transmission of S. Schwarzengrund from chicken meat to humans.

We evaluated 11 variable number tandem repeat (VNTR) markers for the epidemiological investigation of Salmonella enterica serovar Typhi (S. Typhi) infection and compared the results to those obtained by PFGE. PFGE, using one or two restriction enzymes (XbaI and BlnI), was insufficient to differentiate between some isolates that were epidemiologically unlinked. Multilocus variable-number tandem repeat analysis (MLVA)-8, based on analysis of the eight most variable VNTRs, displayed a high level of discrimination when distinguishing between epidemiologically unlinked isolates that could not be discerned by PFGE with two enzymes. An MLVA-8 typing scheme could be implemented as a routine subtyping tool for the epidemiological investigation of S. Typhi infections. Because seven of the 11 VNTRs are highly variable, the VNTR markers may only be useful in determining genetic relationships among very closely related isolates in short-term epidemiological studies and not for discerning S. Typhi clones.

International audience; Salmonella genomic island I (variant SGII-J3) has been previously identified in multi-drug resistant (MDR) Salmonella enterica serovar Virchow isolated from humans in 1994. In this study, antimicrobial resistance, genotypes and genetic relationship were investigated in 96 S. Virchow isolates collected from humans in 2004-2006. XbaI-PFGE analysis separated 96 isolates into two main related-clusters, I and II, which consisted of four major pulsotypes differing in prevalence by year. The majority of isolates were MDR to chloramphenicol, sulfonamide, trimethoprim and tetracyclines associated with antimicrobial resistance genes dfrA1, floR2, sull and tet(G) of variant SGII-J3. Among nine variants, we determined two novel variants SGII-J4 and -J5, which have undergone different homologous recombinational events resulting in partial deletions of the MDR region. The first one contained an empty integron structure and the second presented a deletion extending from the IS6100 element to the adjacent SGII backbone. SGII-J3 is largely encountered in clonally related MDR S. Virchow isolates collected from humans which spread vertically. The genomic island SGII appears to be largely responsible for the diversity of MDR phenotypes among S. Virchow isolates in Taiwan.

The aim of this study was to investigate the high level of pulmonaryMycobacterium abscessusinfections and implement a surveillance programme among 43 ventilator-dependent patients, 15 with pulmonaryM. abscessusinfections, in a hospital long-term respiratory care ward (RCW) in central Taiwan.M. abscessusisolates were obtained from 35 patients in the RCW of hospital A, 6 patients in the RCWs of another three hospitals (B, C and D), and from 4 water sources in two of the hospitals (A and B). Strains were characterized by methods includinghsp65 PCR–RFLP and PFGE. The patients were followed-up by chest X-ray for 1 year. All clinical isolates were type I and II, and belonged to ten distinct clusters of PFGE patterns. Five clinical strains in two hospitals belonged to a single cluster, whilst four clinical strains in the other two hospitals belonged to a single unique cluster. The strains from hospital A fell into nine clusters and were distinct from the strains isolated from the water supply. Patients infected with type I strains showed a significantly more rapid progression of disease. The number of different strains involved suggested either that there had been a polyclonal outbreak or that a high level of endemic infections was present in the RCW of hospital A. This and the lack of homology between the clinical and environmental isolates from hospital A raised the possibility that pulmonaryM. abscessusinfections may have been spread by the movement of patients between RCWs, a routine practice in Taiwan's integrated delivery system.

Pulsed-field gel electrophoresis (PFGE) analysis revealed that the genomes of some pathogenic Escherichia coli O157:H7 strains, including EDL933, were resistant to NotI digestion. An amino acid sequence comparison suggested that the z2389 gene carried on prophage CP-933R in strain EDL933 is likely to encode a C 5 -cytosine methyltransferase. The z2389 -equivalent gene was found in the NotI-resistant strains tested, but it was not detected in the NotI-susceptible strains. PFGE analysis of the wild-type EDL933 strain and of a z2389 null mutant revealed that z2389 was associated with full genome protection against NotI digestion and partial protection against EagI digestion. In vitro methylation experiments with purified recombinant protein demonstrated that Z2389 is capable of methylating NotI and EagI sites. Sequencing of bisulfite-treated DNA indicated that the methylation occurred at the first cytosine residue of the NotI recognition sequence, whereas EagI sites remained unmethylated or were methylated at the first cytosine residue. Thus, z2389 encodes a DNA cytosine methyltransferase that confers full protection to NotI sites.