Distinct evolution of ST11 KL64 Klebsiella pneumoniae in Taiwan.

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

Shigellosis in Taiwan: An old enteric pathogen with changing epidemiology and increasing antimicrobial resistance.

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

Correcting modification-mediated errors in nanopore sequencing by nucleotide demodification and reference-based correction.

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

Carbapenem resistance in extensively drug-resistant Salmonella enterica serovar Agona and AmpC ß-lactamase-producing S. Infantis.

IMPORTANCE: Carbapenem resistance arising from the loss of porins is commonly observed in extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase-producing strains of certain Enterobacteriaceae genera, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. However, this resistance mechanism is rarely reported in the Salmonella genus. To address this knowledge gap, our study offers genetic evidence demonstrating that the loss of two specific porins (OmpC_378 and OmpD) is crucial for the development of carbapenem resistance in Salmonella ESBL and AmpC ß-lactamase-producing strains. Furthermore, our findings reveal that most Salmonella serovars carry seven porin parathologs, with OmpC_378 and OmpD being the key porins involved in the development of carbapenem resistance in Salmonella strains.

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 and Mechanisms of Azithromycin Resistance in Nontyphoidal Salmonella Isolates in Taiwan, 2017 to 2018.

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

Integrative and Conjugative Element-Mediated Azithromycin Resistance in Multidrug-Resistant Salmonella enterica serovar Albany.

We identified an erm42-carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from 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 Vibrio cholerae via conjugation.

Genomic Surveillance of Listeria monocytogenes in Taiwan, 2014 to 2019.

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

Microevolution of CG23-I Hypervirulent Klebsiella pneumoniae during Recurrent Infections in a Single Patient.

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

Demographic Features of Invasive Meningococcal Disease in Taiwan, 1993 to 2020, and Genetic Characteristics of Neisseria meningitidis Isolates, 2003 to 2020.

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

A nosocomial salmonellosis outbreak caused by blaOXA-48-carrying, extensively drug-resistant Salmonella enterica serovar Goldcoast in a hospital respiratory care ward in Taiwan.

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

Changing epidemiology of shigellosis in Taiwan, 2010-2019: an emerging threat to HIV-infected patients and men who have sex with men.

Shigellosis appears to increase in certain at-risk populations in developed countries. Based on the nationwide surveillance, the annual incidence of shigellosis in Taiwan (1999-2019) was 0.38-5.77 cases per 100,000 people. Indigenous shigellosis has mostly affected men who have sex with men (MSM) and people living with HIV (PLWH) since 2015. In this retrospective study, compared with those diagnosed before 2015, indigenous cases diagnosed during 2015-2019 mostly occurred in male adults (96.0% vs 47.1%, P < 0.001), with a longer hospital stay (median 5.0 vs 3.5 days, P = 0.029) and different coinfections. The predominant strains in 2015 and 2016 were ciprofloxacin-resistant Shigella sonnei and azithromycin non-susceptible Shigella flexneri (S. flexneri) 3a, which had been replaced by ciprofloxacin-resistant S. flexneri 2a since 2018. Notably, six indigenous cases were caused by cefotaxime-resistant S. flexneri. Inappropriate use of empiric antibiotic treatment was common. In conclusion, there is an ongoing spread of ciprofloxacin-resistant shigellosis among PLWH and MSM and cefotaxime-resistant S. flexneri is an emerging threat in Taiwan.

RamAp Is an Efflux Pump Regulator Carried by an IncHI2 Plasmid.

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.

Microbiological and genomic investigations of invasive Salmonella enterica serovar Panama from a large outbreak in Taiwan.

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 <5 years old (median, 1.3 years). The case-control study showed that 28 out of the 41 (68.3%) manifested as bacteremia, compared to S. Typhimurium (11.1%). Patients infected by S. Panama had longer durations of fever (P = 0.005) and hospitalization (P < 0.001). Genomic analyses split the isolates into three clades: two clones caused the outbreak, whereas another one accounted for the sporadic infections before 2015. Cellular experiments revealed that S. Panama and other iNTS serovars showed higher monolayer penetration and intracellular survival within macrophages, compared to S. Typhimurium. CONCLUSION: This study confirmed that S. Panama is a clinically invasive serovar. Different iNTS serovars express common virulence phenotypes, but they may acquire invasiveness through distinct expression or combinations of virulence genes.

cgMLST@Taiwan: A web service platform for Vibrio cholerae cgMLST profiling and global strain tracking.

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.

Association between Escherichia coli with NotI-restriction resistance and urinary tract infections.

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

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.

Two ST11 Klebsiella pneumoniae strains exacerbate colorectal tumorigenesis in a colitis-associated mouse model.

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.

Assessment of metrics in next-generation sequencing experiments for use in core-genome multilocus sequence type.

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.