Medical Microbiology and Immunology最新文献

Advanced platforms, such as whole genome sequencing (WGS), should be employed to enhance and refine microbial identification compared to phenotypic methods, including miniaturized biochemical tests and MALDI-TOF. The application of WGS has led to the reclassification of clinical bacterial pathogens previously misidentified by phenotypic techniques. In this study, eight clinical isolates initially identified as Pseudomonas fragi by VITEK MS were subjected to WGS and bioinformatics analysis. The results revealed one strain as Pseudomonas lundensis, while average nucleotide identity and phylogenetic reconstruction suggested that the remaining seven strains represent novel species within the Pseudomonas fluorescens superclade. The strains harbored four antimicrobial resistance genes conferring resistance to β-lactams, fluoroquinolones, phenicols, and tetracyclines, yet in vitro assays indicated susceptibility to carbapenems and intermediate susceptibility to colistin. Additionally, the strains possessed virulence factor genes associated with alginate biosynthesis, flagellar formation, pilus assembly, and iron uptake, with Type III Secretion System (T3SS)-related genes detected only in P. lundensis. Notably, the isolated Pseudomonas spp., exhibited multiple haplotypes, a closely related pan-genome, and similar phenotypic characteristics. These findings underscore the necessity of integrating multiple approaches, including molecular methods such as 16S rRNA gene amplification, sanger sequencing, and WGS, alongside traditional phenotypic techniques, to improve the accuracy of microbial identification in clinical settings.

The objective of this study was to characterize the metabolic profile of tuberculous meningitis (TBM) in the cerebrospinal fluid (CSF) of a South African pediatric cohort using two-dimensional gas chromatography linked to time-of-flight mass spectrometry (GCxGC-TOFMS). A metabolite extraction and derivatization protocol was performed using CSF samples from 21 cases of bacteriologically confirmed TBM and 24 controls without meningitis, and analyzed by GCxGC-TOFMS, followed by univariate and multivariate statistical comparisons to identify differentiating compounds. A total of 48 metabolites (involved in glycolysis and amino acid and fatty acid metabolism) were identified that differentiated the two groups (effect size d-value > 0.5 and partial least squares discriminant analysis VIP value > 1.0, with 16 metabolites highlighted as significant (p-value < 0.05). Eight novel metabolites (linked to imines, alkenes, and volatile organic compounds), never before identified in a TBM study, characterize TBM in this cohort. This study confirms existing metabolomics research aimed at characterizing the metabolic profile of TBM. Some affected metabolic pathways include glycolysis, pentose phosphate pathway, amino acids, and fatty acid metabolism. New information is provided on the effect TBM has on neurometabolism, how TBM can reduce the neuroprotective function of some metabolites in the brain, resulting in the neurological symptoms typically associated with this disease, and contribute to our understanding of the pathogenesis of TBM; ultimately, contributing to the development of a metabolic model of TBM.

Continuous exposure to Plasmodium falciparum (Pf) has been associated with alterations in B cells. We investigated the effect of controlled human malaria infection (CHMI) on B cell phenotypes in individuals with different Pf immunity status: malaria-naïve, immunized with PfSPZ-CVac and semi-immune (lifelong-exposed) volunteers. Compared to naïve, semi-immune but not vaccinated individuals, had increased baseline frequencies of immature B cells (CD19⁺CD10⁺), active naive (IgD⁺CD27^-CD21^-) B cells, active atypical (IgD^-CD27^-CD21^-) memory B cells (MBCs), active classical (IgD^-CD27⁺CD21^-) MBCs and CD1c⁺-B cells but lower frequencies of some IgG⁺-B cells. The frequencies of CD1c⁺ active atypical MBCs correlated positively with anti-Pf antibodies and negatively with circulating eotaxin levels, while the opposite was observed for IgG⁺ resting atypical MBCs. During early blood-stage infection (day 11 after CHMI), there was an expansion of resting classical (IgD^-CD27⁺CD21⁺) MBCs in all three groups. Vaccination, compared to placebo, altered the effect of CHMI on B cells, showing a positive association with resting classical MBCs (β = 0.190, 95% CI 0.011-0.368) and active naïve-PD1⁺ (β = 0.637, 95% CI 0.058 to 1.217) frequencies, and a negative one with CD1c⁺ resting atypical MBCs (β = - 0.328, 95% CI - 0.621 to - 0.032). In addition, the sickle cell trait in semi-immune subjects altered the effect of CHMI on several B cells. In conclusion, lifelong but not vaccine exposure to malaria was associated with increased frequencies of multiple B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells, respectively. A single infection (CHMI) induces changes in B cell frequencies and is modulated by sickle cell trait and malaria-immunity status.Clinical Trials Registration NCT01624961, NCT02115516, and NCT02237586.

As regulators of innate and adaptive immunity, microRNAs (miRNAs) could aid in the discrimination between tuberculosis disease (TB) and (latent) TB infection (TBI). We analysed 754 circulating miRNAs in participants diagnosed with TB and TBI using TaqMan™ Advanced miRNA Human A and B cards. MiRNAs were normalized exogenously and endogenously via geometric means of selected reference miRNAs. Expression analysis was used to identify miRNAs that were significantly differentially expressed between individuals with TB and those with TBI. We utilised recursive feature elimination with a Random Forest model to identify the miRNAs most effective at discriminating TB from TBI and subsequently validated the miRNA in another group. 95 persons diagnosed with TB or TBI was divided into a discovery group (n = 36) and a validation group (n = 59). In the discovery group, we identified 495 distinct miRNAs in 36 persons with TB or TBI and by recursive feature elimination identified hsa-miR-148a-3p, hsa-miR-204-5p and hsa-miR-584-5p and created a three-miRNA-diagnostic model. In the validation group, the three-miRNA-diagnostic model had poorer performance. Expression analysis revealed 13 significantly differentially expressed miRNAs, including hsa-miR-148a-3p and hsa-miR-204-5p. Subsequent analysis in a validation group consisting of 59 persons revealed that six of the 14 miRNAs, including hsa-miR-148a-3p, exhibited the same pattern, albeit without statistical significance. Three circulating miRNAs showed potential for differentiating TB from TBI in the discovery cohort, but these differences were less pronounced in the validation cohort.

Tuberculosis remains a severe global health threat, exacerbated by the rising prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis. Despite the urgent need for effective interventions, the development of anti-tuberculosis drugs has been slow, and the emergence of pan-drug-resistant strains underscores the critical need for innovative therapeutic strategies. This study introduces Bacillus sonorensis PMC204, a novel probiotic strain with potent anti-tuberculosis properties identified through extensive screening. PMC204 significantly reduced M. tuberculosis H37Rv and XDR strains within Raw 264.7 macrophage cells. Moreover, membrane vesicles (MVs) derived from this strain exhibited superior inhibitory effects against both standard and XDR strains of M. tuberculosis. Proteomic analysis of the isolated MVs revealed a high abundance of flagellin proteins, which are hypothesized to play a pivotal role in the observed anti-tuberculosis effects. These findings also suggest a close link between the therapeutic efficacy of PMC204 and autophagy activation. Safety assessments further demonstrated the feasibility of PMC204 as a potential anti-tuberculosis therapeutic. The anti-tuberculosis activity of bacterial MVs represents an innovative approach in microbiome therapeutics, positioning PMC204 as a next-generation probiotic distinct from conventional strains. This study contributes to advancing the field of microbiome-based therapeutics and presents promising avenues for managing drug-resistant tuberculosis.

This comprehensive study provides insight into the antibacterial action of a recently published 2-chloro-N-(oxazol-2-yl)isonicotinamide (AB15), intending to assess its potential as a candidate adjuvant molecule to support existing antibacterial drugs. Within the determination of the antibacterial effect, a promising activity against a member of the ESKAPE group with reduced treatment options, biofilm producer, Acinetobacter baumannii, was recognized (MIC of AB15 ranged from 15.63 to 62.5 µM). In addition, AB15 exhibited bactericidal activity and non/low-toxicity in vitro (IC₅₀ > 1000 µM using HK-2 cells) and in vivo (LD₅₀ > 500 mg/kg of body weight of the Galleria mellonella larvae, for both intra-hemocoel and per oral administration routes). Checkerboard assay revealed additive and synergistic interactions of AB15 and last-resort antibiotic drug, colistin (CST). Moreover, attention was also given to a frequently overlooked antibiofilm activity - the ability to suppress bacterial dissemination from microbial biofilms, and parameter MBDC (minimum biofilm dissemination concentration) was introduced. The study of the antibiofilm activity of AB15 and CST, both acting individually, or in AB15 + CST combination, revealed that AB15 has significant potential to suppress bacterial dissemination from biofilm formed by a clinical isolate Acinetobacter baumannii and that it contributes to this effect when combined with CST. Finally, AB15 + CST combination demonstrated significantly greater biocompatibility towards human erythrocytes than CST acting individually at an equivalent antibiofilm-effective concentration. The role of AB15 as a promising adjuvant molecule to CST is also supported by its distinct mechanism of action, which reduces the risk of antimicrobial resistance emergence. To conclude, AB15 exhibits several essential attributes that support its designation as a promising antibiotic adjuvant.

Galectins exert a wide range of effects on immune cells in acute and chronic pathologies, although their effects are less described in chronic infections such as tuberculosis-TB. We assessed galectin-1 (Gal-1) and galectin-3 (Gal-3) concentrations and immune mediators in plasma from pulmonary TB-PTB cases (n=38), healthy controls-Hco (n=24), and patients with pleural TB-PLTB (n=11) in which pleural fluid-PLF was also evaluated. Galectin transcripts expression, together with glycosyltransferases, that positively (MGAT5, GCNT1) or negatively (ST6GAL1) control galectins activity, were assessed in mononuclear cells (MC). We also evaluated Gal-1 production, along with other immune-mediators, in Mtb-stimulated MCs. Both patient groups presented elevated circulating levels of pro- and anti-inflammatory mediators and reduced cell proliferation, but a marked T-cell response at the pleural compartment. PTB patients had increased Gal-1 in levels in plasma and higher Gal-1 mRNA levels in MCs (p<0.01, vs. Hco). Both TB groups showed high plasma Gal-3 concentrations and increased expression in MCs (p<0.01 vs. HCo). PLF showed the lowest levels of both galectins, as did their expressions on MCs from pleural effusion. Only PBMCs from PTB exhibited increased expression of GCNT1 (p<0.04) together with diminished ST6GAL1 suggesting enhanced availability of galectin ligands. Mtb-stimulated MCs from both patient groups showed increased Gal-1 production compared to HCo. Moreover, unstimulated cultures from PTB presented a major basal production of Gal-1. Thus, a balance of circulating levels of galectins, pro- and anti-inflammatory mediators, and the differential expression of these lectins as well as glycosylation-related enzymes in MCs, may condition cell function particularly in PTB cases.

Infections caused by multidrug-resistant Acinetobacter baumannii are an emerging global health threat. Although phages have shown promising results in treating bacterial infections, the mechanisms of the combined effect of phages and innate immunity on clearing A. baumannii remain unclear. Here, we report a synergistic effect of the complement system and phages on clearing multidrug-resistant A. baumannii. We show that A. baumannii rapidly adapts and becomes resistant to phage or serum complement by modifying the expression of capsule and lipooligosaccharides, which can be regulated through reversible transposon mutagenesis in the K locus. Compared to the encapsulated phenotype, the non-encapsulated, phage-resistant A. baumannii showed a higher level of membrane attack complex deposition and were susceptible to killing by complement. In contrast, the encapsulated phenotype escaped the complement system by shedding the membrane attack complex to the environment. Thus, while the complement system targets the non-encapsulated phenotype, the phage infects and eliminates the encapsulated subpopulation. These results suggest means of combatting antibiotic-resistant A. baumannii by a simultaneous treatment with phages and complement, a combination which can be supplemented further with antibacterial antibodies.

Influenza virus and Streptococcus pneumoniae are major respiratory pathogens responsible for significant global mortality. While influenza causes seasonal flu, pneumococcus is associated with pneumonia, meningitis, sepsis, and otitis, often worsening influenza cases through secondary infections. Aiming to develop a bivalent vaccine against these two pathogens, we used reverse genetics to construct a recombinant influenza virus that carries the gene of the pneumococcal PspA protein (Flu-PspA). This study assessed the safety and efficacy of a heterologous prime-boost vaccine protocol consisting of Flu-PspA prime and followed by a boost with recombinant PspA plus alum (Flu-PspA/PspA4 + Alum), administered intramuscularly in C57BL/6 mice. Following immunization, anti-PspA and anti-influenza antibody titers in serum and bronchoalveolar lavage fluid (BALF) were quantified by ELISA. The breadth of the immune response was evaluated by measuring complement deposition across multiple pneumococcal strains. Vaccine protection and efficacy were evaluated 21 days after final immunization by challenging mice with a lethal dose of 7 × LD50 of S. pneumoniae (strain ATCC6303) or 100 × LD50 of influenza A/PR8/34 virus. Mid-term pneumococcal protection was assessed similarly 90 days post-boost. Three days post-pneumococcal challenge, bacterial loads in BALF and blood were quantified. Additionally, bacterial colonization and secondary infection dynamics were evaluated and quantified after nasal colonization challenge (strain EF3030), infection with influenza H3N2 virus and secondary infection with pneumococcus and after nasal colonization with strain EF3030 and H3N2 virus infection. The results demonstrated that the vaccination regimen elicited a robust humoral immune response, conferring broad protection against diverse pneumococcal strains. Following lethal challenge with either pathogen, the vaccine provided 100% survival, by significantly reducing bacterial burden in blood and lungs. Notably, even 90 days post-boost, mice retained partial protection with minimal weight loss. Furthermore, the protocol reduced bacteremia following secondary infections and mitigated weight loss in H3N2-infected colonized mice. These findings demonstrate that the vaccine confers robust protection with broad-spectrum efficacy when administered intramuscularly, highlighting its potential as a viable preventive strategy against both influenza and pneumococcal infections, including co-infection scenarios.