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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 bla KPC-2 , bla VIM-1 , bla NDM-1 , and bla OXA-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., (Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vibrio parahaemolyticus is the most prevalent foodborne pathogen in Taiwan and it is frequently recovered from seafood. In this study, V. parahaemolyticus that was isolated in recent years from aquacultural environments and clinical specimens were comparatively analyzed by NotI-restricted pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction, targeting common toxin genes (tdh, trh, ureC), MTase gene, toxR regulator, markers for pandemic strains (ORF8, group-specific toxRS) and representative genes of type three secretion systems T3SS1 (vcrD1, VP1680, vopD) and T3SS2α (vcrD2, vopD2, vopB2, vopP, vopC, vopT). Among the 48 clinical isolates and 93 environmental isolates that were analyzed by PFGE, a total of 26 and 76 pulsetypes were identified and grouped into six and nine clusters, respectively, at 80% similarity. The pandemic O3:K6 clones and other clinical and environmental isolates were further characterized according to the distribution of these examined target genes. The MTase gene and the vopB2, vopP, vopC and vopT genes of T3SS2α were present at a significantly higher frequency (>90%) in the pandemic clones than in other clinical isolates. The MTase gene and some other virulence-associated genes were also present in a few of the environmental isolates, and these results suggest the horizontal transfer of these genes in the clinical and environmental isolates of this species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium is a major zoonotic pathogen with a high prevalence of antimicrobial resistance. This pathogen can disseminate across borders and spread far distances via the food trade and international travel. In this study, we compared the genotypes and antimicrobial resistance of 378 S. Typhimurium isolates collected in Taiwan and Denmark between 2009 and 2010. Genotyping revealed that many S. Typhimurium strains were concurrently circulating in Taiwan, Denmark and other countries in 2009 and 2010. When compared to the isolates collected from Denmark, the isolates from Taiwan displayed a significantly higher level of resistance to 11 of the 12 tested antimicrobials. Seven genetic clusters (A-G) were designated for the isolates. A high percentage of the isolates in genetic clusters C, F and G were multidrug-resistant. Of the isolates in cluster C, 79.2% were ASSuT-resistant, characterized by resistance to ampicillin, streptomycin, sulfamethoxazole, and tetracycline. In cluster F, 84.1% of the isolates were ACSSuT-resistant (resistant to ASSuT and chloramphenicol). Cluster G was unique to Taiwan and characterized in most isolates by the absence of three VNTRs (ST20, ST30 and STTR6) as well as a variety of multidrug resistance profiles. This cluster exhibited very high to extremely high levels of resistance to several first-line drugs, and among the seven clusters, it displayed the highest levels of resistance to cefotaxime and ceftazidime, ciprofloxacin and gentamicin. The high prevalence of antimicrobial resistance in S. Typhimurium from Taiwan highlights the necessity to strictly regulate the use of antimicrobials in the agriculture and human health care sectors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We identified 16 variable number tandem repeat (VNTR) loci for Salmonella enterica serovar Typhimurium. These VNTRs were evaluated with panels of 183 diverse isolates, 203 closely related isolates and 54 isolates from seven outbreaks. The evaluations revealed that five of the 16 VNTRs had diversity values greater than 0.5, and three (STTR5, STTR6 and STTR10) were hypervariable. The results obtained from the outbreak isolates suggested that the 16 VNTRs were considerably stable in isolates recovered during a normal outbreak time course. Multilocus VNTR analysis (MLVA) based on four most variable VNTRs (MLVA4), exhibited a better resolving power over pulsed-field gel electrophoresis (PFGE) in discriminating among isolates, in particular among the closely-related isolates. An MLVA5, which is based on five VNTRs and has been widely used in many European laboratories, displayed a level of discrimination close to MLVA4. The phylogenetic tree established using the MLVA16 profiles presented four distinct clusters, which were associated with four different phage types. Therefore, MLVA based on four or five highly variable VNTRs is sufficiently powerful to supplement or replace PFGE for outbreak investigation and surveillance of S. Typhimurium infections, and MLVA data based on 16 VNTRs can be useful in establishing clonal structures among isolates., (Copyright 2010 Elsevier B.V. All rights reserved.)

We reported four foodborne disease outbreaks in Taiwan caused by sucrose-nonfermenting and by beta-galactosidase-deficient variants of non-O1, non-O139 Vibrio cholerae. The sucrose-nonfermenting vibrios collected from three outbreaks were biochemically identified to be V. mimicus and the beta-galactosidase-deficient vibrios from an outbreak to be V. alginolyticus. However, molecular methods including DNA-DNA hybridization, fatty acid profile analysis, and sequence analysis of 16S rRNA, oriC, pyrH, recA, and rpoA indicated that these vibrios should be V. cholerae. These V. cholerae variants carried two hemolysin genes, hlyA and hlx, but contained neither cholera toxin gene, ctx, V. mimicus hemolysin gene, vmh, nor thermo-directed hemolysin, tdh. The sucrose-nonfermenting variants of V. cholerae shared a high level of genetic relatedness; they could derive from a common clone. In our record from 1995 to date, this was the first time that V. cholerae variants were discovered as etiologic agents for foodborne disease outbreaks in Taiwan.

Staphylococcal enterotoxins (SEs) are superantigenic toxins. They are five major classical types, i.e., SEA, SEB, SEC, SED, SEE, and new SEs or SE-like superantigens, such as SEG to SEU. Only the staphylococcal superantigens (SAgs) that induce emesis following oral administration in a monkey model are designated as SEs while other related toxins are called SE-like (SEl) superantigens. To survey the enterotoxin genotypes for S. aureus strains isolated from food-poisoning cases in Taiwan, we developed PCR primers specific for SEN, SEO, SEP, SEQ, SER, and SEU genes. The complete SE sequences and their expression potential for strains positive to sen, seo, sep, seq, ser, and seu specific primers were also determined. These strains were used as reference strains. With the PCR primers specific for all SEs or SAgs, including toxic shock syndrome toxin I (TSST-1), we assayed the genotypes of 147 S. aureus strains isolated from patients associated with staphylococcal food-poisoning outbreaks occurred during 2001-2003. For these 147 strains, 135 (91.8%) were found positive for one or more SE or SAg genes. For classical enterotoxin and TSST-1 types, the major one was tsst-1 (59.1%) following by sea (29.2%), seb (19.7%), sec (6.8%), and sed (2.0%). For new SE and SAg types, the major one was sei (29.9%) and sep (27.9%) followed by, sek (16.3%), seo (14.3%), seu (14.2%), sem (11.6%), sen (10.9%), seq (10.9%), seh (8.2%), sel (6.8%), and ser (5.4%) etc. This report reveals the whole SE and SAg genotypes for S. aureus strains isolated from staphylococcal food-poisoning cases in Taiwan.

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other maritime Asian countries where seafood is frequently consumed. A total of 535 strains of V. parahaemolyticus were recovered mostly (97%) from clinical samples obtained in Taiwan or in 14 other countries. These strains were typed by pulsed-field gel electrophoresis following SfiI digestion and a typing scheme was generated. The 115 different patterns identified were grouped into 13 types with dissimilarity values less than 15, plus 16 miscellaneous patterns not grouped into any of the types. Types I, A, D and J contained the most patterns, with the numbers of patterns being 17, 13, 12, and 11, respectively. However, types I, B, D, A, H and C contained the most strains, with the numbers of strains being 204, 73, 71, 54, 29 and 25, respectively. Type I consisted exclusively of the pandemic O3:K6 strains and genetically closely related strains. This PFGE typing scheme for V. parahaemolyticus could be used for the characterization of pathogenic isolates.

Data regarding the incidence of the newly found enterotoxigenic Staphylococcus aureus strains in food poisoning cases and in food samples were to date not available in Taiwan. In this study, PCR primers specific for the detection of SEG, H and I genes, i.e., seg, seh and sei, were used for the assay of 55 human isolates of S. aureus negative to the classical enterotoxins (SEA-->SEE) detection. These isolates were from the fecal specimens of the patients suffering from food poisoning outbreaks. Only eight strains were found to have the seg, seh and sei. The presence of other bacterial pathogens, such as Vibrio parahaemolyticus, Bacillus cereus and Salmonella spp. and perhaps, strains producing other new staphylococcal enterotoxins, in the fecal specimens of these patients, may account for these food poisoning cases. For 139 strains from food samples, such as frozen Chinese foods, Chinese sausages and lunch meals, sea strains accounted for the major portion and it seemed to be the most common SE type to coexist with seg, seh and sei. Only two strains had sec and none of them had seg, seh or sei. For strains without the classical SE genes, only 13 strains had seg, seh and/or sei. The above results imply that seg, seh and sei S. aureus strains play only a minor role in food-borne outbreaks in Taiwan.