Tracing the footprints of MPXV in Asia: phylogenetic insights and lineage dynamics.

OBJECTIVES: The 2022 global outbreak of monkeypox virus (MPXV), previously confined to Central and West Africa, necessitates an enhanced understanding of its spread. Comprehensive genomic surveillance to understand the virus's evolution and spread is needed, particularly in Asia. METHODS: Genomic data from 169 MPXV genome sequences in Asia were analysed. Through advanced genomic sequencing of clinical samples, we analysed the distribution and mutations of MPXV lineages in Asia. RESULTS: Phylogenetic analysis revealed a distinct clustering of C.1 strains rise in Northeast Asia in 2023, while genomic examination identified specific consensus mutations like R84K, R665C and L16F in C.1 strains. The mutations, coupled with an increased rate of apolipoprotein B mRNA-editing catalytic polypeptide-like 3 motif G-to-A mutations in C.1 (OR 24.87±8.81), indicate a potential adaptation mechanism. CONCLUSIONS: Our findings underscore the need for ongoing surveillance and provide vital insights into MPXV's evolving dynamics, aiding in public health strategy formulation against this emerging infectious threat.

SARS-CoV-2 primed platelets-derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive. METHODS: We explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting. RESULTS: COVID-19 patient-derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet-derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1ß/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement. CONCLUSIONS: We identified SARS-CoV-2 triggered platelets-derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

In Vitro and In Vivo Antimicrobial Activities of Vancomycin and Rifampin against Elizabethkingia anophelis.

Elizabethkingia anophelis has emerged as a critical human pathogen, and a number of isolated reports have described the successful treatment of Elizabethkingia infections with vancomycin, a drug that is typically used to target Gram-positive bacteria. This study employed in vitro broth microdilution checkerboard and time-kill assays, as well as in vivo zebrafish animal models to evaluate the individual and combination antimicrobial effects of vancomycin and rifampin against E. anophelis. The minimum inhibitory concentration ranges of vancomycin and rifampin against 167 isolates of E. anophelis were 16-256 mg/L and 0.06-128 mg/L, respectively. The checkerboard assay results revealed a synergistic effect between vancomycin and rifampin in 16.8% (28/167) of the isolates. Time-kill assays were implemented for 66 isolates, and the two-drug combination had a synergistic interaction in 57 (86.4%) isolates. In vivo zebrafish studies revealed that treatment with vancomycin monotherapy, rifampin monotherapy, or vancomycin-rifampin combination therapy yielded a higher survival rate than the control group treatment with 0.9% saline. The results of this study support the use of vancomycin to treat E. anophelis infections.

Mutant Prevention Concentrations of Ciprofloxacin and Levofloxacin and Target Gene Mutations of Fluoroquinolones in Elizabethkingia anophelis.

Fluoroquinolones are potentially effective against Elizabethkingia anophelis. We investigated the MIC, mutant prevention concentration (MPC), and target gene mutations of fluoroquinolones in E. anophelis. Eighty-five E. anophelis isolates were collected from five hospitals in Taiwan. The MIC and MPC of ciprofloxacin and levofloxacin were examined for all E. anophelis except 17 isolates, in which ciprofloxacin MPC could not be determined due to drug precipitation caused by overly high drug concentration. Mutations in the quinolone resistance-determining regions of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) in the clinical isolates and fluoroquinolone-selected mutants were examined. Overall, 23.5% and 71.8% of the isolates tested were susceptible to ciprofloxacin and levofloxacin, respectively. The MPC50 of ciprofloxacin was 128 mg/L, and the MPC50 of levofloxacin was 51.2 mg/L. The MPC50/MIC50 ratio for ciprofloxacin was 64, whereas that for levofloxacin was 25.6. The coefficient of determination between the MPC and MIC for ciprofloxacin and levofloxacin was 0.72 and 0.56, respectively, in the linear regression analysis. Preexisting mutations in GyrA (S83I, S83R, and D87Y) were identified in 18 clinical isolates, all of which were resistant to both ciprofloxacin and levofloxacin. Additional amino acid substitutions in GyrA were identified in all ciprofloxacin- and levofloxacin-selected mutants. Furthermore, GyrB alterations (D431N or D431H) were found in nine levofloxacin-treated isolates. Given that maintaining the serum concentrations of fluoroquinolones above MPCs is impossible under presently recommended doses, the selection of mutant E. anophelis strains seems inevitable.

Blood Reflux-Induced Epigenetic Factors HDACs and DNMTs Are Associated with the Development of Human Chronic Venous Disease.

Blood reflux and metabolic regulation play important roles in chronic venous disease (CVD) development. Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) serve as repressors that inhibit metabolic signaling, which is induced by proatherogenic flow to promote aortic endothelial cell (EC) dysfunction and atherosclerosis. The aim of this study was to elucidate the relationship between blood reflux and epigenetic factors HDACs and DNMTs in CVD. Human varicose veins with different levels of blood reflux versus normal veins with normal venous flow were examined. The results show that HDAC-1, -2, -3, -5, and -7 are overexpressed in the endothelium of varicose veins with blood reflux. Blood reflux-induced HDACs are enhanced in the varicose veins with a longer duration time of blood reflux. In contrast, these HDACs are rarely expressed in the endothelium of the normal vein with normal venous flow. Similar results are obtained for DNMT1 and DNMT3a. Our findings suggest that the epigenetic factors, HDACs and DNMTs, are induced in venous ECs in response to blood reflux but are inhibited in response to normal venous flow. Blood reflux-induced HDACs and DNMTs could inhibit metabolic regulation and promote venous EC dysfunction, which is highly correlated with CVD pathogenesis.

A Fatal Case of Chlorfenapyr Poisoning and the Therapeutic Implications of Serum Chlorfenapyr and Tralopyril Levels.

Chlorfenapyr is a new contact and stomach insecticide derived from natural pyrroles secreted by Streptomyces spp. It is a pro-insecticide and acts after metabolic transformation to its active metabolite tralopyril. Tralopyril is an uncoupler of oxidative phosphorylation in the mitochondria of the target insects and of experiment animals, leading to the disruption of adenosine triphosphate synthesis and death. Several fatal human poisonings had been reported and no blood chlorfenapyr or tralopyril measurements were available. The treatment remains supportive. A 32-year-old healthy man ingested 200 mL of 10% chlorfenapyr as a suicide attempt. Unfortunately, he succumbed at 157 h post-ingestion, shortly after having fever and seizures. His serum level of chlorfenapyr at 4 h post-exposure was 77.4 ng/mL, and was undetectable at 113 and 156 h, respectively. The serum levels of tralopyril were 723.6, 14,179, and 9654.2 ng/mL at 4, 113, and 156 h post-ingestion, respectively. The delay in the rise of serum tralopyril levels was noticeable, which seems to correlate with the patient's signs and symptoms. The information may have therapeutic implications in the management of this deadly poisoning.

High prevalence of HIV-1 transmitted drug resistance and factors associated with time to virological failure and viral suppression in Taiwan.

BACKGROUND: Integrase strand transfer inhibitor (InSTI)-based regimens have become the major first-line treatment for HIV-1-infected patients in Taiwan. Transmitted drug resistance (TDR) and several clinical characteristics are associated with time to virological failure or viral suppression; however, these have not been investigated in Taiwan. OBJECTIVES: To determine the impact of several factors on treatment outcomes in HIV-1-infected patients in Taiwan. METHODS: The cohort included 164 HIV-1 treatment-naive patients in Taiwan from 2018 to 2020. Blood specimens were collected to determine the genotypic drug resistance using the Stanford University HIV drug resistance database. Cox proportional hazards models were used to identify factors associated with time to virological failure or viral suppression. RESULTS: The prevalence of TDR in Taiwan was 27.4% and an increasing trend was seen from 2018 to 2020. TDR mutations related to NNRTIs were the most prevalent (21%) while TDR to InSTIs remained at a relatively low level (1.3%). A baseline HIV-1 viral load of ≥100 000 copies/mL was associated with a shorter time to virological failure [multivariate hazard ratio (mHR) 7.84; P = 0.018] and longer time to viral suppression (mHR 0.46; P < 0.001). Time to viral suppression was shorter in patients receiving InSTI-based regimens (mHR 2.18; P = 0.006). Different InSTI-based regimens as initial treatment did not affect the treatment outcomes. CONCLUSIONS: This study found an increasing trend of HIV-1 TDR prevalence from 2018 to 2020 in Taiwan. Baseline HIV-1 viral load and receiving InSTI-based regimens are important factors associated with time to virological failure or viral suppression.

National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART).

A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

Comparative Genomics Reveals Pathogenicity-Related Loci in Shewanella algae.

Shewanella algae is an emerging marine zoonotic pathogen and accounts for considerable mortality and morbidity in compromised hosts. However, there is scarce literature related to the understanding of the genetic background of virulence determinants in S. algae. In this study, we aim to determine the occurrence of common virulence genes in S. algae using whole-genome sequence and comparative genomic analysis. Comparative genomics reveals putative-virulence genes related to bile resistance, chemotaxis, hemolysis, and motility. We detected the existence of hlyA, hlyD, and hlyIII involved in hemolysis. We also found chemotaxis gene cluster cheYZA operon and cheW gene. The results provide insights into the genetic basis underlying pathogenicity in S. algae.

Identification of TMAO-producer phenotype and host-diet-gut dysbiosis by carnitine challenge test in human and germ-free mice.

OBJECTIVE: The gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance. DESIGN: A pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation. RESULTS: The OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT. CONCLUSION: The OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment. TRIAL REGISTRATION NUMBER: NCT02838732; Results.

Variations in Gut Microbiota of Siberian Flying Squirrels Correspond to Seasonal Phenological Changes in Their Hokkaido Subarctic Forest Ecosystem.

Gut microbial communities of animals are influenced by diet and seasonal weather changes. Since foraging strategies of wild animals are affected by phenological changes, gut microbial communities would differ among seasons. However, interactions of plant-animal-microbiota with seasonal changes have not been well characterized. Here, we surveyed gut microbial diversity of Siberian flying squirrels (Pteromys volans orii) from a natural forest in Hokkaido during spring and summer of 2013 and 2014. Additionally, we compared microbial diversity to temperature changes and normalized difference vegetation index (NDVI). Changes in both seasonal temperature and phenology were significantly associated with alterations in gut microbiota. There were two clusters of OTUs, below and above 20 °C that were significantly correlated with low and high temperatures, respectively. Low-temperature cluster OTUs belonged to various phyla, whereas the high-temperature cluster was only constituted by Firmicutes. In conclusion, gut microbiota of Siberian flying squirrels varied with environmental changes on an ecological scale.

Genome Sequence of Colistin-Resistant Bacteremic Shewanella algae Carrying the Beta-Lactamase Gene bla OXA-55.

Shewanella algae is an emerging pathogen widely distributed in aquatic environment. Bacteremia is a major manifestation of S. algae infections, and there are increasing reports of antibiotic-resistant strains. However, little is known about the genomic characteristics of human bacteremic S. algae. Here, we report the results of the whole-genome sequencing of colistin-resistant S. algae TYL, a blood isolate. Chromosome-encoded pmrC associated with colistin resistance and bla OXA-55 gene intrinsic to S. algae was identified. Continuous surveillance for the emergence of S. algae is needed.

Comparative genomics reveals diverse capsular polysaccharide synthesis gene clusters in emerging Raoultella planticola.

Raoultella planticola is an emerging zoonotic pathogen that is associated with rare but life-threatening cases of bacteremia, biliary tract infections, and urinary tract infections. Moreover, increasing antimicrobial resistance in the organism poses a potential threat to public health. In spite of its importance as a human pathogen, the genome of R. planticola remains largely unexplored and little is known about its virulence factors. Although lipopolysaccharides has been detected in R. planticola and implicated in the virulence in earlier studies, the genetic background is unknown. Here, we report the complete genome and comparative analysis of the multidrug-resistant clinical isolate R. planticola GODA. The complete genome sequence of R. planticola GODA was sequenced using single-molecule real-time DNA sequencing. Comparative genomic analysis reveals distinct capsular polysaccharide synthesis gene clusters in R. planticola GODA. In addition, we found bla TEM-57 and multiple transporters related to multidrug resistance. The availability of genomic data in open databases of this emerging zoonotic pathogen, in tandem with our comparative study, provides better understanding of R. planticola and the basis for future work.

Characteristics and Phylogeny of Shewanella haliotis Isolated from Cultivated Shellfish in Taiwan.

Shewanella haliotis is an emerging human pathogen. Many infectious cases were linked to shellfish ingestion or aquatic exposure. Therefore, it is important to study the phylogeny and distribution of S. haliotis in shellfish aquaculture. We investigated the distribution of S. haliotis in cultivated shellfish farming in Taiwan in which S. haliotis was found in the shellfish from all sampling sites. S. haliotis was identified in cultivated shellfish by 16S rRNA gene sequencing, such as abalone (Haliotis diversicolor), clam (Meretrix lusoria), and oyster (Crassostrea gigas). This study highlighted the contamination of S. haliotis in cultivated shellfish and importance of further study regarding the biodiversity and pathogenesis of S. haliotis.

The Pathogenicity of Shewanella algae and Ability to Tolerate a Wide Range of Temperatures and Salinities.

Shewanella algae is a rod-shaped Gram-negative marine bacterium frequently found in nonhuman sources such as aquatic ecosystems and has been shown to be the pathogenic agent in various clinical cases due to the ingestion of raw seafood. The results of this study showed that S. algae was present in approximately one in four samples, including water and shellfish samples. Positive reactions (API systems) in S. algae strains were seen for gelatinase (gelatin); however, negative reactions were found for indole production (tryptophan). S. algae is adapted to a wide range of temperatures (4°C, 25°C, 37°C, and 42°C) and salinity. Temperature is a key parameter in the pathogenicity of S. algae as it appears to induce hemolysis at 25°C and 37°C. S. algae exhibits pathogenic characteristics at widely varying temperatures, which suggests that it may have the ability to adapt to climate change.

Volatile Oils of Nepeta tenuifolia (Jing Jie) as an Alternative Medicine against Multidrug-Resistant Pathogenic Microbes.

Essential oils from the dried spikes of Nepeta tenuifolia (Benth) are obtained by steam distillation. Pulegone was identified as the main component in the spikes of N. tenuifolia through analysis, with greater than 85% purity obtained in this study. The essential oils are extremely active against all Gram-positive and some Gram-negative reference bacteria, particularly Salmonella enterica, Citrobacter freundii, and Escherichia coli. The minimum inhibitory concentration was found to be between 0.08 and 0.78% (against S. enterica), 0.39 and 0.78% (against C. freundii), and 0.097 and 0.39% (against E. coli), whereas the minimum bactericidal concentration varied in range from 0.097% to 1.04%. In general, the essential oils show a strong inhibitory action against all tested reference strains and clinical isolates. However, the antibacterial activity of EOs against both Pseudomonas aeruginosa reference strains and clinical isolates was relatively lower than other Gram-negative pathogens. The essential oils of N. tenuifolia also displayed bactericidal activities (MBC/MIC < 4) in this study. These findings reflect the bactericidal activity of the essential oils against a wide range of multidrug-resistant clinical pathogens in an in vitro study. In addition, we propose the fragmentation pathways of pulegone and its derivatives by LC-ESI-MS/MS in this study.

Using genotyping to delineate tuberculosis transmission in long-term care facilities: single facility 4-year experience.

BACKGROUND: Residents in long-term care facilities (LTCFs) are vulnerable to tuberculosis (TB) transmission; however, to delineate possible routes of TB transmission in LTCFs is difficult. This study aimed to address the use of regular genotyping surveillance to delineate TB transmission in LTCFs. METHODS: All of Mycobacterium tuberculosis isolates in the reported 620-bed LTCF between July 2011 and August 2015 were genotyped, and we retrospectively compared epidemiological data and genotyping results. RESULTS: A total of 42 subjects were diagnosed with culture-positive pulmonary TB infection during the 4-year period. Their median age was 76.5 years, and 64.3% (27/42) of them were male. Genotyping identified 5 clustered TB infections involving 76.2% (32/42) of all TB subjects. In a multivariate logistic regression model adjusted for age, sex, chronic obstructive pulmonary disease, and body mass index, subjects with clustered TB infection were less likely to be Activities of Daily Living (ADL)-dependence (adjOR 0.073, 95% CI 0.007-0.758) when compared with subjects having individual TB infections. Prolonged surveillance is essential given that the median interval to diagnose secondary subjects was 673 days. Finally, only 63.0% (17/27) of the 27 secondary TB subjects in this study had contact history with index subject in the same ward. CONCLUSIONS: In conclusion, possible routes of TB transmission in a complex TB outbreak at LTCFs might be delineated by routine genotyping surveillance and regular health check-up.

Colistin Resistance of Pseudomonas aeruginosa Isolated from Snakes in Taiwan.

This study included fifty-eight isolates of P. aeruginosa from the oral cavity of snakes that were recruited from clinical cases, captive and wild snakes. The minimum inhibitory concentrations (MICs) for the determination of susceptibility were identified by the broth microdilution method. Polymerase chain reaction (PCR) was employed to detect ß-lactamases genes. With regard to antipseudomonal antibiotics, the lowest nonsusceptible rates were in aztreonam (15%), piperacillin/tazobactam (12%), and amikacin (9%). The nonsusceptible rates were high in gentamicin (33%) and colistin (55%). Meanwhile, blaTEM presented in 100% of isolates where blaAmpC, blaOXA-1, and blaOXA-10 came at 94.8%, 89.7%, and 27.6%, respectively. Emergence of multidrug resistant (MDR) strains and colistin-resistant strains highlights the potential breach of public health as P. aeruginosa could be transmitted through either direct contact or indirect dissemination through the environment. This study reports that the highly resistant P. aeruginosa from snakes' oral cavity were discovered for the very first time in Taiwan.