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.

Antimicrobial Resistance in Campylobacter coli and Campylobacter jejuni from Human Campylobacteriosis in Taiwan, 2016 to 2019.

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″), blaOXA, 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.

Emergence of Multidrug-Resistant Salmonella enterica Serovar Goldcoast Strains in Taiwan and International Spread of the ST358 Clone.

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, blaTEM-1B, blaCTX-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.

Usefulness of pulsed-field gel electrophoresis profiles for the determination of Salmonella serovars.

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.

Antimicrobial resistance in Salmonella enterica Serovar Typhi isolates from Bangladesh, Indonesia, Taiwan, and Vietnam.

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.

An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan.

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.

A simple approach to obtain comparable Shigella sonnei MLVA results across laboratories.

Multilocus variable-number tandem repeat analysis (MLVA) is a promising subtyping tool to complement 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 participating 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, protocol independent, and easy to implement in every laboratory, and a small calibration set is sufficient to generate mathematical equations for accurate copy number conversion.

Human isolates of Salmonella enterica serovar Typhimurium from Taiwan displayed significantly higher levels of antimicrobial resistance than those from Denmark.

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.

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.

Use of multilocus variable-number tandem repeat analysis in molecular subtyping of Salmonella enterica serovar Typhi isolates.

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.

Global Distribution of Shigella sonnei Clones.

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.

Comparison of multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in molecular subtyping of Salmonella enterica serovars Paratyphi A.

Salmonella enterica serotype Paratyphi A is a highly clonal organism; pulsed-field gel electrophoresis (PFGE) is insufficient in discriminating isolates. A multilocus variable-number tandem repeat analysis (MLVA) was developed, and its usefulness in discriminating isolates was compared. PFGE analysis with XbaI and BlnI discriminated 55 isolates into 14 types, with a discriminatory index (DI) of 0.741 (confidence interval [CI], 0.635-0.847). MLVA divided the isolates into 23 types, with a DI of 0.937 (CI, 0.909-0.964), which was significantly higher than that for PFGE. Clustering analysis of PFGE and MLVA patterns indicated that S. Paratyphi A isolates recovered from 2000 to 2010 in South and Southeast Asia were highly clonal. Although MLVA is not sufficiently powerful in discriminating epidemiologically unrelated isolates, it can complement PFGE for epidemiologic investigation of S. Paratyphi A infections.

Development and evaluation of multilocus variable number tandem repeat analysis for fine typing and phylogenetic analysis of Salmonella enterica serovar Typhimurium.

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.

Significant association of Ku80 single nucleotide polymorphisms with bladder cancer susceptibility in Taiwan.

UNLABELLED: The aim of the present study was to evaluate the association between the polymorphisms of the Ku80 DNA repair gene, which plays an important role in maintaining genome stability, and the risk of bladder cancer in Central Taiwan. MATERIALS AND METHODS: In this hospital-based case-control study, the association of Ku80 G-1401T rs828907, Ku80 C-319T rs11685387 and Ku80 intron 19 rs9288518 polymorphisms with bladder cancer risk in a central Taiwanese population was investigated. In total, 288 patients with bladder cancer and 288 age- and gender-matched healthy controls recruited from the China Medical Hospital in central Taiwan were genotyped. RESULTS: A significantly different distribution was found in the frequency of the Ku80 G-1401T polymorphism genotypes, but not the Ku80 C-319T or intron 19 polymorphism genotypes, between the bladder cancer and control groups. The T allele of Ku80 G-1401T conferred a significant (p=0.0055) increased risk of bladder cancer. Gene-environment interactions with smoking, but not with alcohol consumption, were significant for the Ku80 G-1401T polymorphism. The Ku80 G-1401T GT and TT genotypes, in association with smoking, conferred an increased risk of 2.053-fold (95% confidence interval=1.232-3.419) for bladder cancer. CONCLUSION: The first evidence that the T allele of the Ku80 G-1401T may be associated with the development of bladder cancer and may be a novel useful marker for primary prevention and anticancer intervention is provided.

Lung cancer susceptibility and genetic polymorphisms of Exo1 gene in Taiwan.

AIM: To evaluate the association between the polymorphisms of the Exo1 gene and the risk of lung cancer in central Taiwan. PATIENTS AND METHODS: In this hospital-based study, the association of Exol A-1419G (rs3754093), C-908G (rs10802996), A238G (rs1776177), C498T (rs1635517), K589E (rs1047840), G670E (rs1776148), C723R (rs1635498), L757P (rs9350) and C3114T (rs851797) polymorphisms with lung cancer risk in a central Taiwanese population was investigated. In total, 358 patients with lung cancer and 358 age- and gender-matched healthy controls recruited from the China Medical Hospital in central Taiwan were genotyped. RESULTS: A significantly different distribution was found in the frequency of the Exo1 K589E genotype, but not the other genotypes, between the lung cancer and control groups. The A allele Exo1 K589E conferred a significantly (p = 0.0097) increased risk of lung cancer. As for the rest of the polymorphisms, there was no difference in distribution between the lung cancer and control groups. Gene environment interactions with smoking were significant for Exo1 K589E polymorphism. The Exo1 K589E AG and AA genotype in association with smoking conferred an increased risk of 1.7208 (95% confidence interval = 1.2188-2.4295) for lung cancer. CONCLUSION: Our results provide the first evidence that the A allele of Exo1 K589E may be associated with the development of lung cancer and may be a novel useful marker for primary prevention and anticancer intervention.

Association between DNA double strand break gene Ku80 polymorphisms and oral cancer susceptibility.

The DNA double strand break repair gene Ku80 is thought to play a major role in the caretaking of the overall genome stability. It is very possible that defective in double strand break repair capacity can lead to human carcinogenesis. Thus, the polymorphic variants of Ku80 were firstly investigated regarding their association with oral cancer susceptibility. In this hospital-based case-control study, the association of Ku80 promoter G-1401T (rs828907), promoter C-319T (rs11685387), and intron19 (rs9288518) polymorphisms with oral cancer risk in a Taiwanese population was investigated. 600 patients with oral cancer and 600 age- and gender-matched healthy controls recruited were genotyped and analyzed by PCR-RFLP method. There were significant differences between oral cancer and control groups in the distributions of their genotypes (P=0.0038) and allelic frequencies (P=0.0044) in the Ku80 promoter G-1401T polymorphism. In the other two polymorphisms, there was no difference between both groups in the distribution of either genotype or allelic frequency. There is a synergistic gene-environmental interaction between Ku80 and areca chewing. Compared with G/G genotype in Ku80 promoter G-1401T, the G/T plus T/T significantly enhanced the risk only in the areca chewers (odds ratio=1.603; 95% confidence interval=1.053-2.011), not in the non-areca chewers. In conclusion, the Ku80 promoter G-1401T is correlated with oral cancer susceptibility and this polymorphism may be a useful marker for oral cancer prevention and early detection.

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri.

BACKGROUND: Shigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri. RESULTS: Thirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b. CONCLUSIONS: The 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable.

The role of XRCC4 in carcinogenesis and anticancer drug discovery.

In the past decades, the incidence of cancer keeps its rapid increasing step all over the world and cancer is always an important threat to public health. It is believed that cancer is resulted from a series of genetic alterations leading to progressive disorder of the normal mechanisms controlling cell proliferation, differentiation, death, and/or genomic stability. The response of the cell to genetic injury and its ability to maintain genomic stability by means of a variety of DNA repair mechanisms are therefore essential in preventing tumor initiation and progression. From the same viewpoint, the relative role of DNA repair as a biomarker for prognosis, predicator of drug and therapy responses, or indeed as target for novel gene therapy is recently patented and very promising. In this review, we have summarized the studies investigating the association between XRCC4, one of the NHEJ genes, and the susceptibility to multiple cancers, and discussed its role in carcinogenesis and application in anticancer drug discovery.

Association of XRCC4 codon 247 polymorphism with oral cancer susceptibility in Taiwan.

BACKGROUND: The DNA repair gene XRCC4, an important caretaker of overall genome stability, is thought to play a major role in the development of human carcinogenesis. However, the association of the polymorphic variants of XRCC4 with oral cancer susceptibility has never been reported. MATERIALS AND METHODS: In this hospital-based case-control study, the association of XRCC4 codon 247 (rs3734091), G-1394T (rs6869366), intron 7 (rs28360317) and intron 7 (rs1805377) polymorphisms with oral cancer risk in a Central Taiwanese population was investigated. In total, 318 patients with oral cancer and 318 age- and gender-matched healthy controls recruited from the China Medical Hospital in Central Taiwan were genotyped. RESULTS: A significantly different distribution was found in the frequency of the XRCC4 codon 247 genotype, but not the XRCC4 G-1394T or intron 7 genotypes, between the oral cancer and control groups. A/C heterozygosity at XRCC4 codon 247 conferred a significant (2.04-fold) increased risk of oral cancer. As for XRCC4 G-1394T and intron 7 polymorphisms, there was no difference in distribution between the oral cancer and control groups. Gene-environment interactions with smoking, but not with betel quid chewing or alcohol consumption, were significant for XRCC4 codon 247 polymorphism. The XRCC4 codon 247 A/C genotype in association with smoking conferred an increased risk of 3.44 (95% confidence interval = 1.24-9.60) for oral cancer. CONCLUSION: Our results provide the first evidence that the heterozygous A allele of the XRCC4 codon 247 may be associated with the development of oral cancer and may be a novel useful marker for primary prevention and anticancer intervention.