Distribution of common and rare drug resistance patterns in Mycobacterium tuberculosis clinical isolates revealed by GenoType MTBDRplus and MTBDRsl assay.

Background: Tuberculosis (TB) remains a significant global health emergency caused by Mycobacterium tuberculosis (Mtb). The epidemiology, transmission, genotypes, mutational patterns, and clinical consequences of TB have been extensively studied worldwide, however, there is a lack of information regarding the epidemiology and mutational patterns of Mtb in Pakistan, specifically concerning the prevalence of multi-drug resistant TB (MDR-TB). Methods: This study aimed to investigate the incidence of Mtb and associated mutational patterns using the line probe assay (LPA). Previous studies have reported a high frequency of mutations in the rpoB, inhA, and katG genes, which are associated with resistance to rifampicin (RIF) and isoniazid (INH). Therefore, the current study utilized LPA to detect mutations in the rpoB, katG, and inhA genes to identify multi-drug resistant Mtb. Results: LPA analysis of a large pool of Mtb isolates, including samples from 241 sputum-positive patients, revealed that 34.85% of isolates were identified as MDR-TB, consistent with reports from various regions worldwide. The most prevalent mutations observed were rpoB S531L and inhA promoter C15T, which were associated with resistance to RIF and INH, respectively. Conclusions: This study highlights the effectiveness of GenoType MTBDRplus and MTBDRsl assays as valuable tools for TB management. These assays enable rapid detection of resistance to RIF, INH, and fluoroquinolones (FQs) in Mtb clinical isolates, surpassing the limitations of solid and liquid media-based methods. The findings contribute to our understanding of MDR-TB epidemiology and provide insights into the genetic profiles of Mtb in Pakistan, which are essential for effective TB control strategies.

Amino acid 17 in QRDR of Gyrase A plays a key role in fluoroquinolones susceptibility in mycobacteria.

IMPORTANCE: Fluoroquinolones (FQs) play a key role in the treatment regimens against tuberculosis and non-tuberculous mycobacterial infections. However, there are significant differences in the sensitivities of different mycobacteria to FQs. In this study, we proved that this is associated with the polymorphism at amino acid 17 of quinolone resistance-determining region of Gyrase A by gene editing. This is the first study using CRISPR-associated recombination for gene editing in Mycobacterium abscessus to underscore the contribution of the amino acid substitutions in GyrA to FQ susceptibilities in mycobacteria.

Type I Interferon Pathway-Related Hub Genes as a Potential Therapeutic Target for SARS-CoV-2 Omicron Variant-Induced Symptoms.

BACKGROUND: The global pandemic of COVID-19 is caused by the rapidly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The clinical presentation of SARS-CoV-2 Omicron variant infection varies from asymptomatic to severe disease with diverse symptoms. However, the underlying mechanisms responsible for these symptoms remain incompletely understood. METHODS: Transcriptome datasets from peripheral blood mononuclear cells (PBMCs) of COVID-19 patients infected with the Omicron variant and healthy volunteers were obtained from public databases. A comprehensive bioinformatics analysis was performed to identify hub genes associated with the Omicron variant. Hub genes were validated using quantitative RT-qPCR and clinical data. DSigDB database predicted potential therapeutic agents. RESULTS: Seven hub genes (IFI44, IFI44L, MX1, OAS3, USP18, IFI27, and ISG15) were potential biomarkers for Omicron infection's symptomatic diagnosis and treatment. Type I interferon-related hub genes regulated Omicron-induced symptoms, which is supported by independent datasets and RT-qPCR validation. Immune cell analysis showed elevated monocytes and reduced lymphocytes in COVID-19 patients, which is consistent with retrospective clinical data. Additionally, ten potential therapeutic agents were screened for COVID-19 treatment, targeting the hub genes. CONCLUSIONS: This study provides insights into the mechanisms underlying type I interferon-related pathways in the development and recovery of COVID-19 symptoms during Omicron infection. Seven hub genes were identified as promising biological biomarkers for diagnosing and treating Omicron infection. The identified biomarkers and potential therapeutic agent offer valuable implications for Omicron's clinical manifestations and treatment strategies.

Characterization of Genetic Variants Associated with Rifampicin Resistance Level in Mycobacterium tuberculosis Clinical Isolates Collected in Guangzhou Chest Hospital, China.

Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF. Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay. Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to >64 µg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426-452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284-1289 CCAGCT), (1295-1303 AATTCATGG), and insertion at (1300-1302 TTC) were detected within RRDR of three RIFR strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIFR strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively. Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIFR clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

CD19+CD1dhiCD5hi B Cells Can Downregulate Malaria ITV Protection by IL-10 Secretion.

Infection treatment vaccine (ITV) can lead to sterile protection against malaria infection in mice and humans. However, parasite breakthrough is frequently observed post-challenge. The mechanism of rapid decline in protection after the last immunization is unclear. Herein, C57BL/6 mice were immunized with 103, 105, or 107 ITV thice at 14-day intervals. Mice were challenged with 103 parasites at 1, 3, and 6 months after last immunization and the protection was checked using blood smear. The phenotypes of B cells were analyzed by flow cytometry. The levels of serum cytokines were quantified using cytometric bead array. The 103 ITV vaccination group exhibited 100% protection at 1 month after last immunization, and the 105 group showed sterile protection at 3 months after last immunization. However, the 107 group showed only partial protection. Further, the protection declined to 16.7% at 6 months after last immunization in 105 and 107 groups, whereas it maintained for more than 60% in 103 group. The number of memory B cells (MBC) decreased along with the decline in protection. However, programmed cell death protein 1 (PD-1) expressed on MBCs did not show significant variation among the three groups. Interestingly, CD19+CD1dhiCD5hi B cells, defined as B10 cells, exhibited negative regulation with respect to protection. The numbers of CD19+CD1dhiCD5hi B cells in the 103 group at 1 months and in the 105 group at 3 months post-immunization were the lowest compared to those in the other groups. Moreover, the serum levels of interleukin 10 (IL-10) in these two groups were also significantly lower than those in other groups. We conclude that higher immunization dose may not lead to better protection with the malaria vaccine as CD19+CD1dhiCD5hi B cells can downregulate ITV protection against malaria via IL-10 secretion. These results could facilitate the design of an effective long-lasting malaria vaccine with the aim of maintaining MBC function.