Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

IMPORTANCE: By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

In vitro and in vivo efficacy of minocycline-based therapy for Elizabethkingia anophelis and the impact of reduced minocycline susceptibility.

OBJECTIVES: Elizabethkingia anophelis is inherently resistant to multiple antibiotics, except minocycline. This study aimed to determine the in vitro and in vivo efficacy of minocycline monotherapy and combination therapy against susceptible strains and the impact of reduced minocycline susceptibility. METHODS: Three clinical isolates and one laboratory-induced mutant with reduced minocycline susceptibility were included. Time-kill and checkerboard assays were used to assess in vitro efficacy and synergy, respectively. Galleria mellonella infection and mouse pneumonia models were used to assess in vivo efficacy, and a mouse thigh infection model was used to determine the bacterial load. RESULTS: Minocycline monotherapy exerted a modest inhibitory effect on three clinical minocycline-susceptible E. anophelis isolates in vitro, but delayed G. mellonella death and improved infected mouse survival; it also significantly reduced the in vivo bacterial load. Minocycline had decreased efficacy on G. mellonella and mice infected by the mutant with reduced minocycline susceptibility. Genome comparison revealed several spontaneous mutations associated with reduced minocycline susceptibility. Among eight antibiotics tested in combination with minocycline, rifampin consistently showed in vitro synergy. The addition of rifampin (1 mg/L) reduced the mutant prevention concentration of minocycline from 2-4 mg/L to < 0.5 mg/L. However, compared with monotherapy, the combination of rifampin and minocycline did not further reduce the bacterial load or improve the survival of G. mellonella or mice. CONCLUSION: Minocycline monotherapy was in vivo effective against susceptible E. anophelis. Reduced minocycline susceptibility due to spontaneous mutation decreased its therapeutic efficacy. In combination with rifampin, it prevented the in vitro emergence of reduced susceptibility but did not provide additional in vivo survival benefit.

Distribution and Genomic Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli Isolated from a Single Family and Home Environment: A 2-Year Longitudinal Study.

Third-generation cephalosporin-resistant Escherichia coli (CREC), particularly strains producing extended-spectrum ß-lactamases (ESBLs), are a global concern. Our study aims to longitudinally assemble the genomic characteristics of CREC isolates from fecal samples from an index patient with recurrent CREC-related urinary tract infections and his family and swabs from his home environment 12 times between 2019 and 2021 to investigate the distribution of antibiotic resistance genes. CREC identified using the VITEK 2 were subjected to nanopore whole-genome sequencing (WGS). The WGS of 27 CREC isolates discovered in 137 specimens (1 urine, 123 feces, and 13 environmental) revealed the predominance of ST101 and ST131. Among these sequence types, blaCTX-M (44.4%, n = 12) was the predominant ESBL gene family, with blaCTX-M-14 (n = 6) being the most common. The remaining 15 (55.6%) isolates harbored blaCMY-2 genes and were clonally diverse. All E. coli isolated from the index patient's initial urine and fecal samples belonged to O25b:H4-B2-ST131 and carried blaCTX-M-14. The results of sequence analysis indicate plasmid-mediated household transmission of blaCMY-2 or blaCTX-M-55. A strong genomic similarity was discovered between fecal ESBL-producing E. coli and uropathogenic strains. Furthermore, blaCMY-2 genes were widely distributed among the CREC isolated from family members and their home environment.

Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination.

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.

Increasing New Delhi metallo-ß-lactamase-positive Escherichia coli among carbapenem non-susceptible Enterobacteriaceae in Taiwan during 2016 to 2018.

New Delhi metallo-ß-lactamase (NDM) had been reported to be the predominant carbapenemase among Escherichia coli in Taiwan. However, studies focusing on the clonal background and epidemiology of plasmids carrying NDM genes were limited. Between 2016 and 2018, all clinical E. coli and Klebsiella pneumoniae isolates that were non-susceptible to ertapenem, meropenem, and imipenem were tested for carbapenemase-encoding genes (CEGs) and antimicrobial susceptibilities. Molecular typing was performed on all carbapenemase-producing isolates. Whole genome sequencing (WGS) was performed on all NDM-positive E. coli isolates. Twenty-three (29.5%) of 78 carbapenem non-susceptible E. coli and 108 (35.3%) of 306 carbapenem non-susceptible K. pneumoniae isolates carried CEGs. The most prevalent CEGs in carbapenemase-producing E. coli (CPEc) were blaNDM (39.1%) and blaIMP-8 (30.4%), while that in carbapenemase-producing K. pneumoniae was Klebsiella pneumoniae carbapenemase (KPC) (72.2%). Fifteen sequence types were identified among 23 CPEc, and 55.6% of NDM-positive E. coli isolates belonged to ST410. WGS showed ST410 isolates were highly clonal and similar to those from other countries. All NDM-5-positive E. coli isolates carried identical IncX3 plasmid harboring blaNDM-5 but no other antimicrobial resistance (AMR) genes. In each of the four NDM-1-positive E. coli isolates, the blaNDM-1 was present in a ∼ 300 kb IncHI2/IncHI2A plasmid which carried an array of AMR genes. NDMs are the most prevalent carbapenemase among CPEc in Taiwan. Awareness should be raised as the prevalence of NDM-positive E. coli might increase rapidly with IncX3 plasmid and globally distributed strain ST410 being the potential vectors for wide dissemination.

Clinical and molecular characterization of Acinetobacter seifertii in Taiwan.

OBJECTIVES: Acinetobacter seifertii, a new member of the Acinetobacter baumannii group, has emerged as a cause of severe infections in humans. We investigated the clinical and molecular characteristics of A. seifertii. PATIENTS AND METHODS: This retrospective study enrolled 80 adults with A. seifertii bloodstream infection (BSI) at four medical centres over an 8 year period. Species identification was confirmed by MALDI-TOF MS, rpoB sequencing and WGS. Molecular typing was performed by MLST. Clinical information, antimicrobial susceptibility and the mechanisms of carbapenem and colistin resistance were analysed. Transmissibility of the carbapenem-resistance determinants was examined by conjugation experiments. RESULTS: The main source of A. seifertii BSI was the respiratory tract (46.3%). The 28 day and in-hospital mortality rates of A. seifertii BSI were 18.8% and 30.0%, respectively. High APACHE II scores and immunosuppressant therapy were independent risk factors for 28 day mortality. The most common MLST type was ST553 (58.8%). Most A. seifertii isolates were susceptible to levofloxacin (86.2%), and only 37.5% were susceptible to colistin. Carbapenem resistance was observed in 16.3% of isolates, mostly caused by the plasmid-borne ISAba1-blaOXA-51-like genetic structure. A. seifertii could transfer various carbapenem-resistance determinants to A. baumannii, Acinetobacter nosocomialis and other A. seifertii isolates. Variations of pmrCAB and lpxCAD genes were not associated with colistin resistance of A. seifertii. CONCLUSIONS: Levofloxacin and carbapenems, but not colistin, have the potential to be the drug of choice for A. seifertii infections. A. seifertii can transfer carbapenem-resistance determinants to other species of the A. baumannii group and warrants close monitoring.

Susceptibility of Elizabethkingia spp. to commonly tested and novel antibiotics and concordance between broth microdilution and automated testing methods.

OBJECTIVES: We aimed to determine susceptibilities of Elizabethkingia spp. to 25 commonly tested and 8 novel antibiotics, and to compare the performance of different susceptibility testing methods. METHODS: Clinical isolates of Elizabethkingia spp., Chryseobacterium spp. and Flavobacterium spp. collected during 2002-18 (n = 210) in a nationwide surveillance programme in Taiwan were speciated by 16S rRNA sequencing. MICs were determined by broth microdilution. The broth microdilution results of 18 common antibiotics were compared with those obtained by the VITEK 2 automated system. RESULTS: Among the Elizabethkingia spp. identified (n = 108), Elizabethkingia anophelis was the most prevalent (n = 90), followed by Elizabethkingia meningoseptica (n = 7) and Elizabethkingia miricola cluster [E. miricola (n = 6), Elizabethkingia bruuniana (n = 3) and Elizabethkingia ursingii (n = 2)]. Most isolates were recovered from respiratory or blood specimens from hospitalized, elderly patients. PFGE showed two major and several minor E. anophelis clones. All isolates were resistant to nearly all the tested ß-lactams. Doxycycline, minocycline and trimethoprim/sulfamethoxazole inhibited >90% of Elizabethkingia spp. Rifampin inhibited E. meningoseptica (100%) and E. anophelis (81.1%). Fluoroquinolones and tigecycline were active against E. meningoseptica and E. miricola cluster isolates. Novel antibiotics, including imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, delafloxacin, eravacycline and omadacycline were ineffective but lascufloxacin inhibited half of Elizabethkingia spp. The very major discrepancy rates of VITEK 2 were >1.5% for ciprofloxacin, moxifloxacin and vancomycin. Major discrepancy rates were >3% for amikacin, tigecycline, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. CONCLUSIONS: MDR, absence of standard interpretation criteria and poor intermethod concordance necessitate working guidelines to facilitate future research of emerging Elizabethkingia spp.

nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples.

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant Staphylococcus aureus isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

The Oxford Nanopore MinION is an affordable and portable DNA sequencer that can produce very long reads (tens of kilobase pairs), which enable de novo bacterial genome assembly. Although many algorithms and tools have been developed for base calling, read mapping, de novo assembly, and polishing, an automated pipeline is not available for one-stop analysis for circular bacterial genome reconstruction. In this paper, we present the pipeline CCBGpipe for completing circular bacterial genomes. Raw current signals are demultiplexed and base called to generate sequencing data. Sequencing reads are de novo assembled several times by using a sampling strategy to produce circular contigs that have a sequence in common between their start and end. The circular contigs are polished by using raw signals and sequencing reads; then, duplicated sequences are removed to form a linear representation of circular sequences. The circularized contigs are finally rearranged to start at the start position of dnaA/repA or a replication origin based on the GC skew. CCBGpipe implemented in Python is available at https://github.com/jade-nhri/CCBGpipe. Using sequencing data produced from a single MinION run, we obtained 48 circular sequences, comprising 12 chromosomes and 36 plasmids of 12 bacteria, including Acinetobacter nosocomialis, Acinetobacter pittii, and Staphylococcus aureus. With adequate quantities of sequencing reads (80×), CCBGpipe can provide a complete and automated assembly of circular bacterial genomes.

Molecular Epidemiology of Emerging Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter pittii in Taiwan, 2010 to 2014.

This study investigated the molecular epidemiology of carbapenem-resistant Acinetobacter nosocomialis and Acinetobacter pittii (ANAP). Clinical isolates of Acinetobacter spp. collected by the biennial nationwide Taiwan Surveillance of Antimicrobial Resistance program from 2010 to 2014 were subjected to species identification, antimicrobial susceptibility testing, and PCR for detection of carbapenemase genes. Whole-genome sequencing or PCR mapping was performed to study the genetic surroundings of the carbapenemase genes. Among 1,041 Acinetobacter isolates, the proportion of ANAP increased from 11% in 2010 to 22% in 2014. The rate of carbapenem resistance in these isolates increased from 7.5% (3/40) to 22% (14/64), with a concomitant increase in their resistance to other antibiotics. The blaOXA-72 and blaOXA-58 genes were highly prevalent in carbapenem-resistant ANAP. Various genetic structures were found upstream of blaOXA-58 in different plasmids. Among the plasmids found to contain blaOXA-72 flanked by XerC/XerD, pAB-NCGM253-like was identified in 8 of 10 isolates. Conjugations of plasmids carrying blaOXA-72 or blaOXA-58 to A. baumannii were successful. In addition, three isolates with chromosome-located blaOXA-23 embedded in AbGRI1-type structure with disruption of genes other than comM were detected. Two highly similar plasmids carrying class I integron containing blaIMP-1 and aminoglycoside resistance genes were also found. The universal presence of blaOXA-272/213-like on A. pittii chromosomes and their lack of contribution to carbapenem resistance indicate its potential to be a marker for species identification. The increase of ANAP, along with their diverse mechanisms of carbapenem resistance, may herald their further spread and warrants close monitoring.

Effect of a Point Mutation in mprF on Susceptibility to Daptomycin, Vancomycin, and Oxacillin in an MRSA Clinical Strain.

We previously reported the sequential recovery of daptomycin-nonsusceptible MRSA clinical isolates with an L431F substitution in the MprF protein. The aim of the present study is to determine the effect of this mutation by replacing the mprF gene on the chromosome of a daptomycin-susceptible progenitor strain, CGK5, to obtain CGK5mut having the L431F MprF mutation. Compared to CGK5, the daptomycin and vancomycin MICs of CGK5mut increased from 0.5 to 3 µg/ml and from 1.5 to 3 µg/ml, respectively; however, its oxacillin MIC decreased from 128 to 1 µg/ml in medium without added 2% NaCl. The expression levels of vraSR and several other cell-wall synthesis-related genes were significantly increased in CGK5mut, and the mutant also had significantly reduced negative cell membrane charge, thicker cell wall, and longer doubling time. These features were abolished in the reverse mutant carrying F431L MprF, confirming the pleiotropic effects of the L431F MprF mutation. We believe that this is the first work that shows a single MprF missense mutation can lead to not only changes in the cell membrane but also increased expression of vraSR and subsequently increased resistance to daptomycin and vancomycin while simultaneously conferring increased susceptibility to oxacillin in an isogenic MRSA strain.

Role of the mecA gene in oxacillin resistance in a Staphylococcus aureus clinical strain with a pvl-positive ST59 genetic background.

The most prevalent community-associated methicillin-resistant Staphylococcus aureus (C-MRSA) strains in Taiwan, sequence type 59 (ST59) clones, carry staphylococcal cassette chromosome mec (SCCmec) type V and, to a lesser extent, type IV. These strains show wide variation in sensitivity to oxacillin, but the reasons for this variation are unknown. Here we compared the sequences of the mecA genes from clinical strains of different SCCmec types and found that they contain different mecA promoter mutations. Analysis of mecA promoter activity by reporter gene fusions showed that single base substitutions in the promoter have a strong influence on mecA transcription. The different mecA variants, including promoter sequences, were expressed in the methicillin-sensitive Staphylococcus aureus (MSSA) strain C195 (ST59 background). PBP 2a production among the parental strains and strains with promoter mutant mecA genes showed a close correlation with mecA transcription levels. Furthermore, the quantity of PBP 2a also closely correlated with the level of oxacillin resistance in the C195 background. Our data suggest that mecA promoter mutations play an important role in determining the level of oxacillin resistance. The mecA promoter mutation G-25A (25 bases upstream of the mecA translation start site) was found to be associated with a high oxacillin MIC (256 µg/ml), G-7T conferred a moderate oxacillin MIC (32 to 64 µg/ml), strains with C-33T showed a low oxacillin MIC (4 to 8 µg/ml), and A-38G reversed the effect of the C-33T mutation, restoring the oxacillin resistance level in the A-38G C-33T double mutant. These observations may explain why C-MRSA strains in Taiwan carrying SCCmec type IV or V have such enormous variations in oxacillin MICs.

Complete Genome Sequence of Staphylococcus aureus Z172, a Vancomycin-Intermediate and Daptomycin-Nonsusceptible Methicillin-Resistant Strain Isolated in Taiwan.

We report the complete genome sequence of Z172, a representative strain of sequence type 239-staphylococcal cassette chromosome mec type III (ST239-SCCmec type III) hospital-associated methicillin-resistant Staphylococcus aureus in Taiwan. Strain Z172 also exhibits a vancomycin-intermediate and daptomycin-nonsusceptible phenotype.