Stability of mpox virus on different commonly contacted surfaces.

Mpox is still spreading globally and is mostly reported to be transmitted by skin and mucosal contact. However, transmission through contact with fomites, contaminated objects, or surfaces has been reported in general population. Evaluation of the stability of mpox virus (MPXV) on different surfaces is important to minimize mpox transmission. In the study, the stability of MPXV on different kinds of commonly contacted surfaces was determined. MPXV was observed to have a surface-dependent stability pattern. Viable virus was detected on both glass and stainless steel for up to 5 days, and on plastic surfaces for up to 3 days. In contrast, no viable MPXV was detected on wooden board and cardboard, which are porous and water-absorbent surfaces, after 1 and 2 days of incubation, respectively. In addition, MPXV nucleic acids were more stable and showed better correlation with viral titers on stainless steel, plastic, and glass. The results indicate that fomite transmission of MPXV is plausible. Moreover, the stability of MPXV was highly surface-dependent and more stable on smooth surfaces, which could provide more information for minimizing the transmission of mpox and emphasize the significance of environmental disinfection in mpox prevention and control.

Public health implications of Yersinia enterocolitica investigation: an ecological modeling and molecular epidemiology study.

BACKGROUND: Yersinia enterocolitica has been sporadically recovered from animals, foods, and human clinical samples in various regions of Ningxia, China. However, the ecological and molecular characteristics of Y. enterocolitica, as well as public health concerns about infection in the Ningxia Hui Autonomous Region, remain unclear. This study aims to analyze the ecological and molecular epidemiological characteristics of Y. enterocolitis in order to inform the public health intervention strategies for the contains of related diseases. METHODS: A total of 270 samples were collected for isolation [animals (n = 208), food (n = 49), and patients (n = 13)], then suspect colonies were isolated and identified by the API20E biochemical identification system, serological tests, biotyping tests, and 16S rRNA-PCR. Then, we used an ecological epidemiological approach combined with machine learning algorithms (general linear model, random forest model, and eXtreme Gradient Boosting) to explore the associations between ecological factors and the pathogenicity of Y. enterocolitis. Furthermore, average nucleotide identity (ANI) estimation, single nucleotide polymorphism (SNP), and core gene multilocus sequence typing (cgMLST) were applied to characterize the molecular profile of isolates based on whole genome sequencing. The statistical test used single-factor analysis, Chi-square tests, t-tests/ANOVA-tests, Wilcoxon rank-sum tests, and Kruskal-Wallis tests. RESULTS: A total of 270 isolates of Yersinia were identified from poultry and livestock (n = 191), food (n = 49), diarrhoea patients (n = 13), rats (n = 15), and hamsters (n = 2). The detection rates of samples from different hosts were statistically different (χ2 = 22.636, P < 0.001). According to the relatedness clustering results, 270 isolates were divided into 12 species, and Y. enterocolitica (n = 187) is a predominated species. Pathogenic isolates made up 52.4% (98/187), while non-pathogenic isolates made up 47.6% (89/187). Temperature and precipitation were strongly associated with the pathogenicity of the isolates (P < 0.001). The random forest (RF) prediction model showed the best performance. The prediction result shows a high risk of pathogenicity Y. enterocolitica was located in the northern, northwestern, and southern of the Ningxia Hui Autonomous Region. The Y. enterocolitica isolates were classified into 54 sequence types (STs) and 125 cgMLST types (CTs), with 4/O:3 being the dominant bioserotype in Ningxia. The dominant STs and dominant CTs of pathogenic isolates in Ningxia were ST429 and HC100_2571, respectively. CONCLUSIONS: The data indicated geographical variations in the distribution of STs and CTs of Y. enterocolitica isolates in Ningxia. Our work offered the first evidence that the pathogenicity of isolates was directly related to fluctuations in temperature and precipitation of the environment. CgMLST typing strategies showed that the isolates were transmitted to the population via pigs and food. Therefore, strengthening health surveillance on pig farms in high-risk areas and focusing on testing food of pig origin are optional strategies to prevent disease outbreaks.

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles.

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100-120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.

Super Dominant Pathobiontic Bacteria in the Nasopharyngeal Microbiota Cause Secondary Bacterial Infection in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease responsible for many infections worldwide. Differences in respiratory microbiota may correlate with disease severity. Samples were collected from 20 severe and 51 mild COVID-19 patients. High-throughput sequencing of the 16S rRNA gene was used to analyze the bacterial community composition of the upper and lower respiratory tracts. The indices of diversity were analyzed. When one genus accounted for >50% of reads from a sample, it was defined as a super dominant pathobiontic bacterial genus (SDPG). In the upper respiratory tract, uniformity indices were significantly higher in the mild group than in the severe group (P < 0.001). In the lower respiratory tract, uniformity indices, richness indices, and the abundance-based coverage estimator were significantly higher in the mild group than in the severe group (P < 0.001). In patients with severe COVID-19, SDPGs were detected in 40.7% of upper and 63.2% of lower respiratory tract samples. In patients with mild COVID-19, only 10.8% of upper and 8.5% of lower respiratory tract samples yielded SDPGs. SDPGs were present in both upper and lower tracts in seven patients (35.0%), among which six (30.0%) patients possessed the same SDPG in the upper and lower tracts. However, no patients with mild infections had an SDPG in both tracts. Staphylococcus, Corynebacterium, and Acinetobacter were the main SDPGs. The number of SDPGs identified differed significantly between patients with mild and severe COVID-19 (P < 0.001). SDPGs in nasopharyngeal microbiota cause secondary bacterial infection in COVID-19 patients and aggravate pneumonia. IMPORTANCE The nasopharyngeal microbiota is composed of a variety of not only the true commensal bacterial species but also the two-face pathobionts, which are one a harmless commensal bacterial species and the other a highly invasive and deadly pathogen. In a previous study, we found that the diversity of nasopharyngeal microbiota was lost in severe influenza patients. We named the genus that accounted for over 50% of microbiota abundance as super dominant pathobiontic genus, which could invade to cause severe pneumonia, leading to high fatality. Similar phenomena were found here for SARS-CoV-2 infection. The diversity of nasopharyngeal microbiota was lost in severe COVID-19 infection patients. SDPGs in nasopharyngeal microbiota were frequently detected in severe COVID-19 patients. Therefore, the SDPGs in nasopharynx microbiota might invade into low respiratory and be responsible for secondary bacterial pneumonia in patients with SARS-CoV-2 infection.

Inhibitor screening using microarray identifies the high capacity of neutralizing antibodies to Spike variants in SARS-CoV-2 infection and vaccination.

Rationale: Mutations of SARS-CoV-2, which is responsible for coronavirus disease 2019 (COVID-19), could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. Methods: The SARS-CoV-2 spike variant protein microarrays were prepared by printing 72 S variants onto a chemically-modified glass slides. The neutralization potential of purified anti-S antibodies and serum from convalescent COVID-19 patients and vaccinees to S variants were assessed with the mSAIS assay. Results: We identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n = 104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and five prevalent S variants (D614G, B.1.1.7, B.1.351, P.1, B.1.617.2), thus demonstrating that high antibody diversity is associated with high NAb titers. Conclusions: Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica.

AIMS: To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. METHODS AND RESULTS: A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20 min), PCR pre-amplification (60 min), CRISPR-based detection (10 min), can be completed within 90 min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1 pg DNA per reaction in pure cultures and 105  CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. CONCLUSIONS: The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents. SIGNIFICANCE AND IMPACT OF THE STUDY: In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method.

A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology.

The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.

Whole genome sequencing reveals the genomic diversity, taxonomic classification, and evolutionary relationships of the genus Nocardia.

Nocardia is a complex and diverse genus of aerobic actinomycetes that cause complex clinical presentations, which are difficult to diagnose due to being misunderstood. To date, the genetic diversity, evolution, and taxonomic structure of the genus Nocardia are still unclear. In this study, we investigated the pan-genome of 86 Nocardia type strains to clarify their genetic diversity. Our study revealed an open pan-genome for Nocardia containing 265,836 gene families, with about 99.7% of the pan-genome being variable. Horizontal gene transfer appears to have been an important evolutionary driver of genetic diversity shaping the Nocardia genome and may have caused historical taxonomic confusion from other taxa (primarily Rhodococcus, Skermania, Aldersonia, and Mycobacterium). Based on single-copy gene families, we established a high-accuracy phylogenomic approach for Nocardia using 229 genome sequences. Furthermore, we found 28 potentially new species and reclassified 16 strains. Finally, by comparing the topology between a phylogenomic tree and 384 phylogenetic trees (from 384 single-copy genes from the core genome), we identified a novel locus for inferring the phylogeny of this genus. The dapb1 gene, which encodes dipeptidyl aminopeptidase BI, was far superior to commonly used markers for Nocardia and yielded a topology almost identical to that of genome-based phylogeny. In conclusion, the present study provides insights into the genetic diversity, contributes a robust framework for the taxonomic classification, and elucidates the evolutionary relationships of Nocardia. This framework should facilitate the development of rapid tests for the species identification of highly variable species and has given new insight into the behavior of this genus.

Case Report: Identification of SARS-CoV-2 in Cerebrospinal Fluid by Ultrahigh-Depth Sequencing in a Patient With Coronavirus Disease 2019 and Neurological Dysfunction.

We reported that the complete genome sequence of SARS-Coronavirus-2 (SARS-CoV-2) was obtained from a cerebrospinal fluid (CSF) sample by ultrahigh-depth sequencing. Fourteen days after onset, seizures, maxillofacial convulsions, intractable hiccups and a significant increase in intracranial pressure developed in an adult coronavirus disease 2019 patient. The complete genome sequence of SARS-CoV-2 obtained from the cerebrospinal fluid indicates that SARS-CoV-2 can invade the central nervous system. In future, along with nervous system assessment, the pathogen genome detection and other indicators are needed for studying possible nervous system infection of SARS-CoV-2.

Direct detection of Corynebacterium striatum, Corynebacterium propinquum, and Corynebacterium simulans in sputum samples by high-resolution melt curve analysis.

BACKGROUND: Pulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus. METHODS: Three reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay's performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay. RESULTS: The melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5-99.9%) sensitivity and 100% (95% CI, 82.8-100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79-0.99]) was noted. CONCLUSIONS: The HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

Mce1C and Mce1D facilitate N. farcinica invasion of host cells and suppress immune responses by inhibiting innate signaling pathways.

The mammalian cell entry (Mce) family of proteins consists of invasin-like membrane-associated proteins. The roles of Mce1C and Mce1D proteins in host-pathogen interactions have not been investigated. In this study, we demonstrate that Mce1C and Mce1D protein is localized in the cell wall fraction of N. farcinica. Both N. farcinica Mce1C and Mce1D proteins are expressed at the level of protein and mRNA and elicit antibody responses during infection. Mce1C and Mce1D facilitate the internalization of Escherichia coli expressing Mce1C protein or latex beads coated with Mce1D protein by HeLa cells, respectively. We further demonstrate that Mce1C and Mce1D can suppress the secretion of the proinflammatory factors TNF-α and IL-6 in macrophages infected with Mycobacterium smegmatis expressing Mce1C or Mce1D and promote the survival of M. smegmatis expressing Mce1C or Mce1D in macrophages. In addition, Mce1C and Mce1D supress the activation of the NF-κB and MAPK signaling pathways by blocking the phosphorylation of AKT, P65, ERK1/2, JNK, or P38 in macrophages. These findings suggest that Mce1C and Mce1D proteins facilitate N. farcinica invasion of HeLa cells and suppress host innate immune responses by manipulating NF-κB and MAPK signaling pathways, which may provide a target for N. farcinica treatment.

Efficient differentiation of Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia beijingensis by high-resolution melting analysis using a novel locus.

Accurate identification of Nocardia species remains a challenge due to the complexities of taxonomy and insufficient discriminatory power of traditional techniques. We report the development of a molecular technique that utilizes real-time PCR-based high-resolution melting (HRM) analysis for differentiation of the most common Nocardia species. Based on a novel fusA-tuf intergenic region sequence, Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia beijingensis were clearly distinguished from one another by HRM analysis. The limit of detection of the HRM assay for purified Nocardia spp. DNA was at least 10 fg. No false positives were observed for specificity testing of 20 non-target clinical samples. In comparison to established matrix-assisted laser desorption/ionization-time of flight MS, the HRM assay improved the identification of N. beijingensis. Additionally, all the products of PCR were verified by direct sequencing. In conclusion, the developed molecular assay allows simultaneous detection and differentiation of N. farcinica, N. cyriacigeorgica and N. beijingensis with high sensitivity and specificity.

The Heparin-Binding Hemagglutinin of Nocardia cyriacigeorgica GUH-2 Stimulates Inflammatory Cytokine Secretion Through Activation of Nuclear Factor κB and Mitogen-Activated Protein Kinase Pathways via TLR4.

Heparin-binding hemagglutinin (HBHA) from mycobacteria is involved in the dissemination of infection and the activation of the host immune response. However, the interaction of Nocardia cyriacigeorgica HBHA with the host cells remains unknown. In the present study, we describe N. cyriacigeorgica HBHA interactions with epithelial cells and organ colonization. We then investigate the mechanisms by which HBHA induces the production of inflammatory cytokines in macrophages. Immunofluorescent microscopy showed that HBHA adhered to A549 cells and HeLa cells and that the C-terminal fragment, which contains a Pro-Ala-Lys-rich domain, was responsible for adhesion. The deletion of the hbha gene in N. cyriacigeorgica mutant strains impaired adhesion to A549 cells and HeLa cells. In addition, the HBHA protein activated the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways and promoted the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 in macrophages. HBHA-mediated TNF-α production was dependent on the activation of the c-Jun N-terminal kinase (JNK) signal pathways, and the IL-6 and IL-10 production was dependent on the activation of extracellular regulated kinase (ERK) 1/2, MAPK p38 (p38), JNK, and nuclear NF-κB signaling pathways. Additionally, the HBHA-mediated activation of innate immunity was dependent on Toll-like receptor 4 (TLR4). Taken together, these results indicate that N. cyriacigeorgica HBHA not only adheres to epithelial cells and may be involved in organ colonization, but also plays a critical role in the modulation of innate immunity through the MAPK and NF-κB signaling pathways via TLR4.

Microbiological profile of distinct virulence of Nocardia cyriacigeorgica strains in vivo and in vitro.

There are significant differences between different Nocardia species regarding geographical distribution, biochemical features, phenotypic characterization, and drug sensitivity. In this study, we explored the differences in virulence and pathogenic mechanisms of two Nocardia cyriacigeorgica strains. We examined the difference in virulence between N. cyriacigeorgica ATCC14759 and N. cyriacigeorgica GUH-2 by measuring cytotoxicity, animal survival after infection, the ability of host cell invasion, and viability in host cells. Western blotting was used to compare the differences in activation of MAPKs, including p38, ERK, and JNK, the NF-κB signaling pathway, and the PI3K/Akt signaling pathway in A549 and RAW264.7 cells. We measured the difference in stimulatory effects on production of the cytokines IL-6, IL-10, and TNF-α by ELISA. We found that N. cyriacigeorgica ATCC14759 causes higher cytotoxicity in cultured cells and higher lethality in mice, and exhibits superior invasion ability and viability in host cells compared with N. cyriacigeorgica GUH-2. Moreover, these two strains show marked differences in activation of the expression of cytokines and signaling pathways. N. cyriacigeorgica ATCC14759 is more virulent than N. cyriacigeorgica GUH-2. Furthermore, there is a significant difference in pathogenesis between the two strains. Our results provide a theoretical basis for the prevention and treatment of Nocardia infection.

Nfa34810 Facilitates Nocardia farcinica Invasion of Host Cells and Stimulates Tumor Necrosis Factor Alpha Secretion through Activation of the NF-κB and Mitogen-Activated Protein Kinase Pathways via Toll-Like Receptor 4.

The mechanism underlying the pathogenesis of Nocardia is not fully known. The Nfa34810 protein of Nocardia farcinica has been predicted to be a virulence factor. However, relatively little is known regarding the interaction of Nfa34810 with host cells, specifically invasion and innate immune activation. In this study, we aimed to determine the role of recombinant Nfa34810 during infection. We demonstrated that Nfa34810 is an immunodominant protein located in the cell wall. Nfa34810 protein was able to facilitate the uptake and internalization of latex beads coated with Nfa34810 protein into HeLa cells. Furthermore, the deletion of the nfa34810 gene in N. farcinica attenuated the ability of the bacteria to infect both HeLa and A549 cells. Moreover, stimulation with Nfa34810 triggered macrophages to produce tumor necrosis factor alpha (TNF-α), and it also activated mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways by inducing the phosphorylation of ERK1/2, p38, JNK, p65, and AKT in macrophages. Specific inhibitors of ERK1/2, JNK, and NF-κB significantly reduced the expression of TNF-α, which demonstrated that Nfa34810-mediated TNF-α production was dependent upon the activation of these kinases. We further found that neutralizing antibodies against Toll-like receptor 4 (TLR4) significantly inhibited TNF-α secretion. Taken together, our results indicated that Nfa34810 is a virulence factor of N. farcinica and plays an important role during infection. Nfa34810-induced production of TNF-α in macrophages also involves ERK, JNK, and NF-κB via the TLR4 pathway.

Prevention and Control of Pathogens Based on Big-Data Mining and Visualization Analysis.

Morbidity and mortality caused by infectious diseases rank first among all human illnesses. Many pathogenic mechanisms remain unclear, while misuse of antibiotics has led to the emergence of drug-resistant strains. Infectious diseases spread rapidly and pathogens mutate quickly, posing new threats to human health. However, with the increasing use of high-throughput screening of pathogen genomes, research based on big data mining and visualization analysis has gradually become a hot topic for studies of infectious disease prevention and control. In this paper, the framework was performed on four infectious pathogens (Fusobacterium, Streptococcus, Neisseria, and Streptococcus salivarius) through five functions: 1) genome annotation, 2) phylogeny analysis based on core genome, 3) analysis of structure differences between genomes, 4) prediction of virulence genes/factors with their pathogenic mechanisms, and 5) prediction of resistance genes/factors with their signaling pathways. The experiments were carried out from three angles: phylogeny (macro perspective), structure differences of genomes (micro perspective), and virulence and drug-resistance characteristics (prediction perspective). Therefore, the framework can not only provide evidence to support the rapid identification of new or unknown pathogens and thus plays a role in the prevention and control of infectious diseases, but also help to recommend the most appropriate strains for clinical and scientific research. This paper presented a new genome information visualization analysis process framework based on big data mining technology with the accommodation of the depth and breadth of pathogens in molecular level research.

An immunoproteomic approach to identify antigenic proteins in Nocardia farcinica IFM 10152.

Nocardia farcinica is the etiological agent of nocardiosis, leading to serious pulmonary or systemic infections. To uncover virulence factors and early diagnostic markers, secreted proteins of N. farcinica IFM 10152 were analyzed using an immunoproteome-based approach. A total of 5 proteins were identified by matrix-assisted laser desorption (MALDI-TOF-MS). Bioinformatic analyses showed that the identified proteins were involved in defense against the host innate immune system and required for pathogenesis. All proteins were expressed in E. coli and antigenicity was analyzed with Western blot. To our knowledge, these proteins with antigenicity were identified for the first time in N. farcinica and they may help elucidate the pathogenesis underlying Nocardia and provide potential future diagnostic markers.

Whole-Genome Sequencing Reveals a Prolonged and Persistent Intrahospital Transmission of Corynebacterium striatum, an Emerging Multidrug-Resistant Pathogen.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals. In this study, a total of 192 C. striatum isolates obtained from 14 September 2017 to 29 March 2018 in a hospital in Beijing, China, were analyzed by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was conducted on 91 isolates. Nearly all isolates (96.3%, 183/190) were MDR. The highest resistance rate was observed for ciprofloxacin (99.0%, 190/192), followed by cefotaxime (90.6%, 174/192) and erythromycin (89.1%, 171/192). PFGE separated the 192 isolates into 79 pulsotypes, and differences in core genome single-nucleotide polymorphisms (SNPs) partitioned the 91 isolates sequenced into four clades. Isolates of the same pulsotype were identical or nearly identical at the genome level, with some exceptions. Two dominant subclones, clade 3a, and clade 4a, were responsible for the hospital-wide dissemination. Genomic analysis further revealed nine resistance genes mobilized by eight unique cassettes. PFGE and whole-genome sequencing revealed that the C. striatum isolates studied were the result mainly of predominant clones spreading in the hospital. C. striatum isolates in the hospital progressively acquired resistance to antimicrobial agents, demonstrating that isolates of C. striatum may adapt rapidly through the acquisition and accumulation of resistance genes and thus evolve into dominant and persistent clones. These insights will be useful for the prevention of C. striatum infection in hospitals.

A novel isothermal amplification-based method for detection of Corynebacterium striatum.

Corynebacterium striatum is an emerging multidrug-resistant pathogen causing increasing numbers of infections and nosocomial outbreaks worldwide. Thus, a simple, rapid and accurate method for C. striatum is urgently required for improving diagnosis efficiency. In this study, a C. striatum-multiple cross displacement amplification (MCDA) with visual detection reagent (VR) assay (C. striatum-MCDA-VR), which was a novel isothermal amplification-based method, was established to detect the species-specific ftr1 gene of C. striatum. Amplification was performed at a constant temperature (68 °C) for only 40 min, and the reaction results could be easily elucidated by observation of reaction mixture color when employing the VR. The limit of detection of this method was 10 fg of pure C. striatum DNA. No cross-reaction was observed with non-C. striatum strains. In testing of clinical sputum samples, the C. striatum-MCDA-VR assay showed excellent sensitivity and specificity when compared with sputum smear tests and PCR. The C. striatum-MCDA-VR assay is a simple, rapid and cost-effective approach for identifying C. striatum in microbiological laboratories, especially in resource-limited settings.

Biosafety and biosecurity.

With the profound changes in the international security situation, the progression of globalization, and the continuous advancement of biotechnology, the risks and challenges posed by major infectious diseases and bioterrorism to the international community are also increasing. Biosafety, therefore, presents new opportunities for international cooperation and global governance. The world has become more integrated and now shares a common destiny in terms of biosafety. In the face of the current risks and challenges, the international community must work together to avert threats, advance mutual interests, and safeguard global biosecurity. In the context of the current situation regarding biosafety and biosecurity, we conducted the present analysis, and present here some appropriate countermeasures.