Antimicrobial efficacy and amino acid substitutions associated with susceptibility to the tellurium compound AS101 against Haemophilus influenzae and Haemophilus parainfluenzae.

The tellurite toxicity in Haemophilus influenzae and H. parainfluenzae remains unclear. To understand the potential of tellurite as a therapeutic option for these bacteria, we investigated the antimicrobial efficacy of AS101, a tellurium compound, against H. influenzae and H. parainfluenzae and the molecular basis of their differences in AS101 susceptibility. Through broth microdilution, we examined the minimum inhibitory concentration (MIC) of AS101 in 51 H. influenzae and 28 H. parainfluenzae isolates. Whole-genome sequencing was performed on the H. influenzae isolates to identify genetic variations associated with AS101 susceptibility. The MICs of AS101 were ≦ 4, 16-32, and ≧ 64 µg/mL in 9 (17.6%), 12 (23.5%), and 30 (58.8%) H. influenzae isolates, respectively, whereas ≦ 0.5 µg/mL in all H. parainfluenzae isolates, including multidrug-resistant isolates. Time-killing kinetic assay and scanning electron microscopy revealed the in vitro bactericidal activity of AS101 against H. parainfluenzae. Forty variations in nine tellurite resistance-related genes were associated with AS101 susceptibility. Logistic regression, receiver operator characteristic curve analysis, Venn diagram, and protein sequence alignment indicated that Val195Ile substitution in TerC, Ser93Gly in Gor (glutathione reductase), Pro44Ala/Ala50Pro in NapB (nitrate reductase), Val307Leu in TehA (tellurite resistance protein), Cys105Arg in CysK (cysteine synthase), and Thr364Ser in Csd (Cysteine desulfurase) were strongly associated with reduced AS101 susceptibility, whereas Ser155Pro in TehA with increased AS101 susceptibility. In conclusions, the antimicrobial efficacy of AS101 is high against H. parainfluenzae but low against H. influenzae. Genetic variations and corresponding protein changes relevant to AS101 non-susceptibility in H. influenzae were identified.

Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Clinical Impact of the Revised 2019 CLSI Levofloxacin Breakpoints in Patients with Enterobacterales Bacteremia.

The Clinical and Laboratory Standards Institute (CLSI) revised the fluoroquinolone MIC breakpoints for Enterobacterales in 2019, based on pharmacokinetic/pharmacodynamic analyses. However, clinical evidence supporting these breakpoint revisions is limited. A retrospective study was conducted at 3 hospitals in Taiwan between January 2017 and March 2019. Patients treated with levofloxacin for bacteremia caused by members of the Enterobacterales with high MICs (1 or 2 µg/ml; levofloxacin susceptible by pre-2019 CLSI breakpoints) were compared with those with low-MIC bacteremia (≤0.5 µg/ml; levofloxacin susceptible by 2019 CLSI breakpoints) to assess therapeutic effectiveness by multivariable logistic regression. The primary outcome was 30-day mortality, and the secondary outcome was the emergence of levofloxacin-resistant isolates within 90 days after levofloxacin initiation. A total of 308 patients were eligible for the study. Kaplan-Meier analysis showed that patients infected with high-MIC isolates (n = 63) had a significantly lower survival rate than those infected with low-MIC isolates (n = 245) (P = 0.001). Multivariable logistic regression revealed that high levofloxacin MIC was a predictor of 30-day mortality (odds ratio [OR], 6.05; 95% confidence interval [CI], 1.51 to 24.18; P = 0.011). We consistently found similar results in a propensity score-matched cohort (OR, 5.38; 95% CI, 1.06 to 27.39; P = 0.043). The emergence of levofloxacin-resistant isolates was more common in the high-MIC group than the low-MIC group (25.0% versus 7.5%; P = 0.065). An estimated area under the concentration-time curve/MIC ratio of ≥87 was significantly associated with better survival (P = 0.002). In conclusion, patients infected with isolates with levofloxacin MICs within the pre-2019 CLSI susceptible range of 1 or 2 µg/ml exhibited higher mortality than those infected with isolates with MICs of ≤0.5 µg/ml.

Emergence of aac(6')-Ie-aph(2'')-Ia-positive enterococci with non-high-level gentamicin resistance mediated by IS1216V: adaptation to decreased aminoglycoside usage in Taiwan.

OBJECTIVES: To explore the mechanisms mediating the different levels of gentamicin resistance in enterococci. METHODS: Susceptibility testing with gentamicin and PCR of resistance determinants were performed in 149 enterococcal isolates. Genetic relatedness was characterized by MLST and PFGE analysis. Sequences of the aac(6')-Ie-aph(2'')-Ia gene and its surrounding environment were determined by Illumina sequencing. Stability assays of gentamicin resistance were carried out to evaluate the probability of loss of the high-level gentamicin resistance (HLGR) phenotype. RESULTS: A total of 17 (11.4%) aac(6')-Ie-aph(2'')-Ia-positive enterococcal isolates (2 Enterococcus faecalis and 15 Enterococcus faecium) with non-HLGR phenotype were found. MLST analysis revealed that the 2 E. faecalis belonged to ST116 and ST618, while all the 15 E. faecium belonged to clonal complex 17. Sequence analysis demonstrated that IS1216V was inserted into the 5'-end of aac(6')-Ie-aph(2'')-Ia, leading to loss of HLGR phenotype. Three IS1216V insertion types were found, and type II and III were frequently found in E. faecium. Interestingly, a total of 38 aac(6')-Ie-aph(2'')-Ia-positive E. faecium with HLGR phenotype also had type II or type III IS1216V insertion. Sequencing of the aac(6')-Ie-aph(2'')-Ia-positive HLGR E. faecium E37 revealed that an intact aac(6')-Ie-aph(2'')-Ia was located adjacent to IS1216V-disrupted aac(6')-Ie-aph(2'')-Ia. In a non-antibiotic environment, E37 tended to lose HLGR phenotype with a probability of 1.57 × 10-4, which was largely attributed to homologous recombination between the intact and disrupted aac(6')-Ie-aph(2'')-Ia. CONCLUSIONS: This is first study to elucidate that the E. faecium is capable of changing its HLGR phenotype, which may contribute to adaptation to hospital environments with decreased usage of gentamicin.

Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection.

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004-2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.

Novel 11-norbetaenone isolated from an entomopathogenic fungus Lecanicillium antillanum.

A novel nor-betaenone compound, 11-norbetaenone (1), was isolated from the culture broth of an entomopathogenic fungus Lecanicillium antillanum. The structure was determined on the basis of 1D and 2D NMR spectroscopic data. The absolute stereochemistry of 1 was further confirmed by X-ray single crystallography analysis. It is the first secondary metabolite reported from the species Lecanicillium antillanum. And it is also the first time that a betaenone-type compound was isolated from the genus Lecanicillium. Furthermore, 11-norbetaenone (1) displayed significant anti-angiogenic effect by suppressing tube formation.

5-Episinuleptolide Decreases the Expression of the Extracellular Matrix in Early Biofilm Formation of Multi-Drug Resistant Acinetobacter baumannii.

Nosocomial infections and increasing multi-drug resistance caused by Acinetobacter baumannii have been recognized as emerging problems worldwide. Moreover, A. baumannii is able to colonize various abiotic materials and medical devices, making it difficult to eradicate and leading to ventilator-associated pneumonia, and bacteremia. Development of novel molecules that inhibit bacterial biofilm formation may be an alternative prophylactic option for the treatment of biofilm-associated A. baumannii infections. Marine environments, which are unlike their terrestrial counterparts, harbor an abundant biodiversity of marine organisms that produce novel bioactive natural products with pharmaceutical potential. In this study, we identified 5-episinuleptolide, which was isolated from Sinularia leptoclados, as an inhibitor of biofilm formation in ATCC 19606 and three multi-drug resistant A. baumannii strains. In addition, the anti-biofilm activities of 5-episinuleptolide were observed for Gram-negative bacteria but not for Gram-positive bacteria, indicating that the inhibition mechanism of 5-episinuleptolide is effective against only Gram-negative bacteria. The mechanism of biofilm inhibition was demonstrated to correlate to decreased gene expression from the pgaABCD locus, which encodes the extracellular polysaccharide poly-ß-(1,6)-N-acetylglucosamine (PNAG). Scanning electron microscopy (SEM) indicated that extracellular matrix of the biofilm was dramatically decreased by treatment with 5-episinuleptolide. Our study showed potentially synergistic activity of combination therapy with 5-episinuleptolide and levofloxacin against biofilm formation and biofilm cells. These data indicate that inhibition of biofilm formation via 5-episinuleptolide may represent another prophylactic option for solving the persistent problem of biofilm-associated A. baumannii infections.

Topical simvastatin promotes healing of Staphylococcus aureus-contaminated cutaneous wounds.

Cutaneous wounds are prompt to be contaminated by bacteria, but the clinical benefits of applying antibiotics and antiseptics in wound management have not been proven. Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors commonly used to lower cholesterol levels. Studies indicated that statins, especially simvastatin, promote wound healing in experimental models. As Staphylococcus aureus is one of the most important microorganism responsible for wound infections, the aims of this study were to characterise the anti-staphylococcal activity of simvastatin and to evaluate the application of simvastatin as a topical therapy for S. aureus-contaminated wounds. In the present study, simvastatin was bacteriostatic against S. aureus at sub-inhibitory concentrations up to 8 hours after exposure. Further increased concentrations of simvastatin above the minimal inhibitory concentration (MIC) did not enhance the growth inhibitory effect. By contrast, the ability of simvastatin to inhibit S. aureus biofilm formation was concentration dependent. Topical application of simvastatin at its MIC against S. aureus accelerated the healing and bacterial clearance of S. aureus-contaminated wounds in an excisional mice wound model. This effective concentration is well below the safe concentration for topical use. Collectively, topical application of simvastatin has the potential as a novel modality for managing wound infections and promoting wound healing.

First Report of bla(IMP-8) in Raoultella planticola.

Two carbapenem-resistant Raoultella planticola clinical isolates were isolated from patients with pneumonia and Port-A catheter-related bacteremia, respectively, in Taiwan. These isolates remained susceptible to fluoroquinolone, aminoglycoside, and colistin. Though the two isolates had the same antibiogram, plasmidic carbapenemase blaIMP-8, class 1 integron cassette (dfrA12-orfF-aadA2), and qnrB2, they had different pulsed-field gel electrophoresis patterns, plasmid sizes, and outer membrane protein loss profiles. To our knowledge, this is the first report of blaIMP-8 found in R. planticola. Interestingly, blaIMP-8 is the most common carbapenemase found in Klebsiella pneumoniae in Taiwan. In the literature, carbapenemase genes in R. planticola in each country were also found in carbapenem-resistant Enterobacteriaceae in the same country.

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.

Gemella parahaemolysans sp. nov. and Gemella taiwanensis sp. nov., isolated from human clinical specimens.

Four Gram-staining-positive, catalase-negative, coccoid isolates, designated NTUH_1465(T), NTUH_2196, NTUH_4957 and NTUH_5572(T), were isolated from human specimens. The four isolates displayed more than 99.6% 16S rRNA gene sequence similarity with Gemella haemolysans ATCC 10379(T), and 96.7 to 98.6% similarity with Gemella sanguinis ATCC 700632(T), Gemella morbillorum ATCC 27824(T) or Gemella cuniculi CCUG 42726(T). However, phylogenetic analysis of concatenated sequences of three housekeeping genes, groEL, rpoB and recA, suggested that the four isolates were distinct from G. haemolysans ATCC 10379(T) and other species. Isolates NTUH_2196, NTUH_4957 and NTUH_5572(T) clustered together and formed a stable monophyletic clade. DNA-DNA hybridization values among strains NTUH_1465(T) and NTUH_5572(T) and their phylogenetically related neighbours were all lower than 49%. The four isolates could be distinguished from G. haemolysans and other species by phenotypic characteristics. Based on the phylogenetic and phenotypic results, two novel species Gemella parahaemolysans sp. nov. (type strain NTUH_1465(T) = BCRC 80365(T) = JCM 18067(T)) and Gemella taiwanensis sp. nov. (type strain NTUH_5572(T) = BCRC 80366(T) = JCM 18066(T)) are proposed.

New structure of phage-related islands carrying fusB and a virulence gene in fusidic acid-resistant Staphylococcus epidermidis.

Nucleotide sequencing of the fusB-flanking regions in two fusidic acid-resistant Staphylococcus epidermidis isolates with the type IV aj1-leader peptide (LP)-fusB structure (lacking aj1) revealed that their fusB gene was located on novel phage-related islands inserted downstream of smpB and are here referred to as SeRIfusB-3692 and SePIfusB-857. The novel SePIfusB-857 structure was followed by SeCI857, forming a composite pathogenicity island which contained a putative virulence gene, vapE. The linkage of fusB and vapE may contribute to bacterial adaption.

In Vitro Evaluation of Tellurium-Based AS101 Compound against Neisseria gonorrhoeae Infectivity.

Neisseria gonorrhoeae (GC) is a obligate human pathogen responsible for gonorrhea, one of the most common sexually transmitted infections. The yearly increased multidrug resistance in GC has led to treatment failure clinically, suggesting an urgent need for novel therapy to combat this global health issue. AS101 [ammonium trichloro(dioxoethylene-O,O'-)tellurate], a tellurium-based compound previously used as an immunomodulatory agent, was found to have antimicrobial effects against Klebsiella pneumoniae via a high-throughput drug screening and showed antibacterial activity against Acinetobacter spp. This study aimed to evaluate the in vitro anti-gonococcal activity of AS101, including its antimicrobial activity, biofilm and infectivity inhibition, and potential underlying mechanisms. The agar-dilution-based MIC was used. The inhibition of GC microcolony formation and continual growth by AS101 was assessed by microscopy. The effect of AS101 on GC infectivity was evaluated by infecting endocervical ME180 and colorectal T84 epithelial cell lines. The mode of action was evaluated by a time-killing curve, transmission electron microscopy (TEM), and the level of reactive oxygen species (ROS). The MICs of MS11 and WHO GC isolates were both found to be 0.05 µg/mL. The biofilm formation, continual growth, and infectivity of two epithelial cell lines were significantly decreased with AS101 treatment. The time-kill curve, similar to that of azithromycin, suggested that AS101 is a bacteriostatic antimicrobial. However, TEM and ROS levels implied a mode of action different from that of azithromycin. Our findings highlighted the robust anti-gonococcal activities of AS101, which potentiates its use as a future antimicrobial for GC. IMPORTANCE Neisseria gonorrhoeae is an obligate human pathogen responsible for gonorrhea, one of the most common sexually transmitted infections. The yearly increased multidrug resistance in GC has led to treatment failure clinically, suggesting an urgent need for novel therapy to combat the global health issue. This study aimed to evaluate the in vitro anti-gonococcal activity of a previous immunomodulatory agent, AS101, and its underlying mechanisms. Here, we report that AS101 possesses remarkable anti-gonococcal activity. These findings supported further studies on in vivo experiments and formulations for the clinical application of AS101 as an anti-gonococcal agent.

Potentials of organic tellurium-containing compound AS101 to overcome carbapenemase-producing Escherichia coli.

BACKGROUND: The issue of carbapenem-resistant Escherichia coli was aggravated yearly. The previous studies reported the varied but critical epidemiology of carbapenem-resistant E. coli among which the carbapenemase-producing strains were regarded as one of the most notorious issues. AS101, an organic tellurium-containing compound undergoing clinical trials, was revealed with antibacterial activities. However, little is known about the antibacterial effect of AS101 against carbapenemase-producing E. coli (CPEC). MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of AS101 against the 15 isolates was examined using a broth microdilution method. The scanning electron microscopy, pharmaceutical manipulations, reactive oxygen species level, and DNA fragmentation assay were carried out to investigate the antibacterial mechanism. The sepsis mouse model was employed to assess the in vivo treatment effect. RESULTS: The blaNDM (33.3%) was revealed as the dominant carbapenemase gene among the 15 CPEC isolates, followed by the blaKPC gene (26.7%). The MICs of AS101 against the 15 isolates ranged from 0.5 to 32 µg/ml, and 99.9% of bacterial eradication was observed at 8 h, 4 h, and 2 h for 1×, 2×, and 4 × MIC, respectively. The mechanistic investigations suggest that AS101 would enter the bacterial cell, and induce ROS generation, leading to DNA fragmentation. The in vivo study exhibited that AS101 possessed a steady treatment effect in a sepsis mouse model, with an up to 83.3% of survival rate. CONCLUSION: The in vitro activities, mechanisms, and in vivo study of AS101 against CPEC were unveiled. Our finding provided further evidence for the antibiotic development of AS101.

In Vitro Analysis of Matched Isolates from Localized and Disseminated Gonococcal Infections Suggests That Opa Expression Impacts Clinical Outcome.

Gonorrhea is the second most common sexually transmitted infection, which is primarily localized but can be disseminated systemically. The mechanisms by which a localized infection becomes a disseminated infection are unknown. We used five pairs of Neisseria gonorrhoeae isolates from the cervix/urethra (localized) and the blood (disseminated) of patients with disseminated gonococcal infection to examine the mechanisms that confine gonococci to the genital tract or enable them to disseminate to the blood. Multilocus sequence analysis found that the local and disseminated isolates from the same patients were isogenic. When culturing in vitro, disseminated isolates aggregated significantly less and transmigrated across a polarized epithelial monolayer more efficiently than localized isolates. While localized cervical isolates transmigrated across epithelial monolayers inefficiently, those transmigrated bacteria self-aggregated less and transmigrated more than cervical isolates but comparably to disseminating isolates. The local cervical isolates recruited the host receptors of gonococcal Opa proteins carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on epithelial cells. However, the transmigrated cervical isolate and the disseminated blood isolates recruit CEACAMs significantly less often. Our results collectively suggest that switching off the expression of CEACAM-binding Opa(s), which reduces self-aggregation, promotes gonococcal dissemination.

Activities of imipenem-relebactam combination against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

BACKGROUND: Imipenem-relebactam is a new ß-lactam and ß-lactamase inhibitor combination to treat carbapenem-resistant gram-negative bacteria infections. However, difference in carbapenem resistant mechanisms existed with geographic variations. OBJECTIVE: To evaluate the susceptibility of imipenem-relebactam to 660 carbapenem-nonsusceptible Enterobacteriaceae isolates in Taiwan and to identify the in vivo efficacy with a Caenorhabditis elegans model. METHODS: 188 carbapenem-nonsusceptible Escherichia coli isolates and 472 carbapenem-nonsusceptible Klebsiella pneumoniae isolates were collected from a national surveillance study in Taiwan. The antimicrobial susceptibility profiles and carbapenemase distributions were determined. An agar dilution method was performed to evaluate the in vitro activities of imipenem monotherapy and imipenem-relebactam combination. Contributions of metallo-carbapenemase to imipenem-relebactam susceptibility was investigated via EDTA treatment. A C. elegans model was used to evaluate the in vivo efficacy of imipenem-relebactam combination. RESULTS: 87.8% and 82.2% susceptibility to imipenem-relebactam was observed for 188 carbapenem-nonsusceptible E. coli and 472 carbapenem-nonsusceptible K. pneumoniae, respectively. However, poor activities of imipenem-relebactam was observed against 23 metallo-carbapenemase producers tested in this study. In the in vivo C. elegans model, imipenem-relebactam significantly rescued nematodes from the infection of a blaKPC-producing K. pneumoniae isolate. CONCLUSION: Our study supports that imipenem-relebactam is a potential therapy against carbapenem-nonsusceptible Enterobacteriaceae, and to our knowledge, this is the first report of evaluation for imipenem-relebactam efficacy against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

The first case of monkeypox virus infection detected in Taiwan: awareness and preparation.

OBJECTIVES: Monkeypox has recently been detected outside African countries. This study aimed to report and analyze the first case of monkeypox virus infection in Taiwan. METHODS: The global epidemiological information was collected from the World Health Organization (WHO) and US Centers for Disease Control and Prevention (CDC). The data from the first confirmed Taiwanese monkeypox case was obtained from Taiwan Centers for Disease Control. Monkeypox diagnosis and prevention strategies were obtained from WHO guidelines on monkeypox. Phylogenetic tree analysis and sequence alignment and comparison were used to identify the phylogeny and single nucleotide polymorphism (SNP) characterization. RESULTS: Epidemiological data indicated that since 2013, monkeypox has caused outbreaks outside African countries through contact with infected animals and international travels. Recently, two confirmed monkeypox cases were reported in Singapore and South Korea. On June 24, 2022, Taiwan CDC reported the first confirmed case of monkeypox virus infection in a 20-year-old man who returned from Germany, from January to June 2022. This is the third confirmed case of an imported monkeypox infection in Asia. Phylogenetic analysis demonstrated that this imported monkeypox virus belonged to the West African clade and is clustered with the 2022 European outbreak monkeypox isolates. Full-length sequence analysis indicates that this virus contains 51 SNPs, and has five variant SNPs compared with the recent outbreak strains. CONCLUSION: This study suggests that active surveillance, enhancing border control, and the development of vaccines and antiviral drugs are urgently required to prevent and control the burden of monkeypox disease.

Identification of fusB-mediated fusidic acid resistance islands in Staphylococcus epidermidis isolates.

To understand the high prevalence of fusB genes in fusidic acid-resistant Staphylococcus epidermidis, analysis of resistance elements in 34 isolates was performed. First, sequence analysis of the aj1-LP-fusB region indicated that at least three types were present. Type I contained full-length aj1, type II contained a partial aj1 truncated from nucleotide position 93 to 421, and type III contained a more truncated aj1 that retained only the last 37 bp. Isolates with type I or type II aj1 displayed slightly higher levels of resistance to fusidic acid (MICs, 8 to 32 µg/ml) than did those with type III aj1 (MICs, 4 to 16 µg/ml). Subsequent sequencing of the flanking regions of fusB from four selected isolates carrying different types of aj1-LP-fusB regions revealed that the fusB genes were all located on phage-related resistance islands (RIs), referred to as SeRI(fusB)(-2793), SeRI(fusB)(-704), SeRI(fusB)(-5907), and SeRI(fusB)(-7778), respectively. Among them, three islands (SeRI(fusB)(-2793), SeRI(fusB)(-704), and SeRI(fusB)(-5907)) were located downstream of groEL (corresponding to the 44-min position based on Staphylococcus aureus whole genomic sequences), and one (SeRI(fusB)(-7778)) was located downstream of rpsR (corresponding to the 8-min position). All of the RIs were inserted into integrase-recognized att sites. Among 34 isolates, the insertion sites of fusB RIs were mostly (28/34, 82%) located downstream of groEL and two were located downstream of rpsR, but four remained unidentified. The pulsotype distribution indicated that fusB-containing S. epidermidis isolates were heterogeneous. In conclusion, the fusB resistance determinant in S. epidermidis was highly associated with phage-related RIs. This is the first report of fusB RI in S. epidermidis.

In Vitro and In Vivo Activity of AS101 against Carbapenem-Resistant Acinetobacter baumannii.

The increasing trend of carbapenem-resistant Acinetobacter baumannii (CRAB) worldwide has become a concern, limiting therapeutic alternatives and increasing morbidity and mortality rates. The immunomodulation agent ammonium trichloro (dioxoethylene-O,O'-) tellurate (AS101) was repurposed as an antimicrobial agent against CRAB. Between 2016 and 2018, 27 CRAB clinical isolates were collected in Taiwan. The in vitro antibacterial activities of AS101 were evaluated using broth microdilution, time-kill assay, reactive oxygen species (ROS) detection and electron microscopy. In vivo effectiveness was assessed using a sepsis mouse infection model. The MIC range of AS101 for 27 CRAB isolates was from 0.5 to 32 µg/mL, which is below its 50% cytotoxicity (approximately 150 µg/mL). Bactericidal activity was confirmed using a time-kill assay. The antibacterial mechanism of AS101 was the accumulation of the ROS and the disruption of the cell membrane, which, in turn, results in cell death. The carbapenemase-producing A. baumannii mouse sepsis model showed that AS101 was a better therapeutic effect than colistin. The mice survival rate after 120 h was 33% (4/12) in the colistin-treated group and 58% (7/12) in the high-dose AS101 (3.33 mg/kg/day) group. Furthermore, high-dose AS101 significantly decreased bacterial population in the liver, kidney and spleen (all p < 0.001). These findings support the concept that AS101 is an ideal candidate for further testing in future studies.

Synergistic Combination of AS101 and Azidothymidine against Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae.

Owing to the over usage of carbapenems, carbapenem resistance has become a vital threat worldwide, and, thus, the World Health Organization announced the carbapenem-resistant Enterobacteriaceae (CRE) as the critical priority for antibiotic development in 2017. In the current situation, combination therapy would be one solution against CRE. Azidothymidine (AZT), a thymidine analog, has demonstrated its synergistically antibacterial activities with other antibiotics. The unexpected antimicrobial activity of the immunomodulator ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) has been reported against carbapenem-resistant Klebsiella pneumoniae (CRKP). Here, we sought to investigate the synergistic activity between AS101 and AZT against 12 CRKP clinical isolates. According to the gene detection results, the blaOXA-1 (7/12, 58.3%), blaDHA (7/12, 58.3%), and blaKPC (7/12, 58.3%) genes were the most prevalent ESBL, AmpC, and carbapenemase genes, respectively. The checkerboard analysis demonstrated the remarkable synergism between AS101 and AZT, with the observable decrease in the MIC value for two agents and the fractional inhibitory concentration (FIC) index ≤0.5 in all strains. Hence, the combination of AS101 and azidothymidine could be a potential treatment option against CRKP for drug development.