Frontiers in Microbiology最新文献

Klebsiella pneumoniae is an opportunistic pathogen causing severe hospital-acquired infections, and it rapidly acquires multidrug resistance. Its robust biofilm formation further complicates treatment and drives interest in phage therapy. A phage UHKP was isolated from hospital sewage using an MDR K. pneumoniae strain (KP-03). UHKP formed clear plaques and having phage titer 2.3 × 10⁹ PFU/mL. Host-range testing on 19 clinical isolates showed a narrow spectrum. Only four MDR strains (KP-03, KP-05, KP-08, KP-11) and one K-17 serotype were lysed, with no activity on other strains or species. One-step growth analysis yielded a 30 min latent period and 85 PFU burst size. In planktonic culture, UHKP at MOI 1 stopped bacterial growth by 4 h and cleared cultures by 8 h, whereas at MOI 0.1 killing was delayed and incomplete. In static biofilm assays, UHKP eradicated 98% of 24-h and 96% of 48-h biofilm biomass by 24 h (MOI 1). Clearance of 72 - 96 h biofilms was limited (≤ 87% by 24 h). UHKP possesses an icosahedral head of 56 ± 3 nm and a short, non-contractile tail measuring around 15 ± 2 nm. Genome sequencing revealed a 62,542 bp dsDNA genome (56.6% GC) encoding 77 ORFs, and phylogenetic analysis placed UHKP in the genus Lastavirus. UHKP carries no lysogeny, toxin or antibiotic-resistance genes.

This study aimed to evaluate the antimicrobial and antibiofilm potential of 15 LAB strains using five types of LAB-derived preparations: cell-free supernatants (CFS), sonicated-inactivated cells (IC), their combination (ICS), and their neutralized variants (CFS N, ICS N) to identify the most effective strain-extract combinations for potential application as natural biocontrol agents in dairy systems. Antimicrobial activity was assessed through agar diffusion assays, growth and biofilm inhibition, and determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations. The extracts were further characterized by pH and organic acid profiles using high-performance liquid chromatography (HPLC), and their mechanisms of action were investigated through cellular leakage assays, time-kill kinetics, and scanning electron microscopy (SEM). Several LAB-derived extracts exhibited strong antagonistic and antibiofilm activity against both spoilage and pathogenic bacteria. Non-neutralized CFS and ICS showed pronounced bactericidal activity, confirming the central role of organic acids in microbial inhibition. Z-score ranking and leakage assays identified Lactiplantibacillus plantarum Q4C3, Lactococcus lactis subsp. lactis biovar diacetylactis SBR4, Weissella cibaria W21, and Weissella viridescens W23 as the most effective strains. Time-kill assays demonstrated rapid microbial reductions (>3 log CFU/mL) within 4 h by CFS, whereas ICS required longer exposure. SEM analysis revealed severe membrane disruption in CFS-treated cells and the presence of LAB-derived debris surrounding ICS-exposed cells. These findings demonstrate that acidic LAB-derived extracts, particularly CFS, efficiently disrupt microbial cells and support their use as safe and effective natural biocontrol agents for improving the microbial safety and quality of dairy products.

Background: Listeria monocytogenes (LM), a zoonotic intracellular pathogen, causes fatal neurological infections in ruminants (e.g., goats) and humans. However, the mechanisms by which LM breaches the blood-brain barrier (BBB) and regulates neuronal programmed cell death (apoptosis/autophagy) remain unclear in caprine models-knowledge that is critical for livestock disease control. This study aimed to investigate the spatiotemporal invasion pathway of LM in goat central nervous system, its brain region-specific effects on apoptosis and autophagy, and the role of the Pink1/Parkin pathway in mitochondrial autophagy during LM infection.

Methods: Goats were intravenously infected with LM to establish an intracranial infection model. Bacterial loads in brain tissues were quantified, and multiple techniques (immunofluorescence, TUNEL, immunohistochemistry, Western blot, qRT-PCR) were used to detect BBB integrity, apoptotic/autophagic markers, and related pathway proteins (E-cadherin/c-Met, Bcl-2/Bax, LC3B, Pink1/Parkin).

Results: LM showed tropism for brainstem regions (midbrain, pons, medulla oblongata) with focal colonization in neurons and glial cells. BBB tight junction proteins (ZO-1, Claudin-1, Occludin) exhibited region-specific dysregulation; notably, an upregulation of Claudin-1 and Occludin was observed in the medulla, suggesting a localized compensatory response. LM infection was associated with the activation of the E-cadherin/c-Met pathway, potentially facilitating transendothelial and neuronal invasion. Apoptosis (Bcl-2/Bax imbalance) and autophagy (LC3B, Pink1/Parkin) were heterogeneously regulated across brain regions, with significant quantitative changes observed in the cerebrum, cerebellum, midbrain, and medulla.

Conclusion: LM invades goat brain tissues coinciding with BBB disruption, exhibits brainstem tropism, and modulates apoptosis and autophagy through region-specific pathways, providing novel insights into LM-induced neurological pathogenesis in ruminants.

Background: Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (MTB) is a major cause of treatment failure in multidrug-resistant tuberculosis (MDR-TB). This resistance primarily results from mutations within the quinolone resistance-determining region (QRDR) of the gyrA gene encoding DNA gyrase. Conventional phenotypic drug susceptibility testing (DST) is labor-intensive and time-consuming, making it unsuitable for rapid clinical decision-making. Therefore, developing a rapid, sensitive, and point-of-care testing (POCT) assay is of great importance.

Methods: A cartridge-based POCT dual one-step recombinase-aided PCR (POCT-DO-RAP) assay was established for rapid detection of FQ resistance-associated mutations in MTB. Locked nucleic acid (LNA) probes were designed to enhance single-nucleotide discrimination for gyrA A90V and D94G mutations. Magnetic bead-based extraction enabled fully automated nucleic acid purification, while recombinase-aided amplification (RAA) and quantitative PCR (qPCR) were sequentially performed within a real-time PCR-based POCT device. The analytical performance of the POCT-DO-RAP assay was evaluated using recombinant plasmids (1-105 copies/μL), H37Rv-simulated sputum samples and 128 clinical isolates. The POCT-DO-RAP assay was further validated using 88 clinical samples and the results were compared with the conventional qPCR and the nested PCR followed by Sanger sequencing.

Results: The optimized POCT-DO-RAP assay achieved limits of detection of 1 copy/reaction for the wild-type (WT) tube and 10 CFU/mL for the mutant-type (MT) tube, representing a 10-fold increase in sensitivity compared with conventional qPCR. The assay reliably detected mutant alleles even when they represented only 1% of mixed templates. Among 128 clinical isolates, the assay accurately differentiated 50 wild-type and 78 resistant strains, showing complete concordance with Sanger sequencing and no cross-reactivity. In clinical validation,9 samples negative by qPCR were confirmed as positive by both DO-RAP assay and nested PCR followed by Sanger sequencing.

Conclusion: The POCT-DO-RAP assay developed in this study achieves a fully integrated "sample-in, result-out" workflow on a single device, offering ultra-high sensitivity, precise mutation discrimination, and excellent clinical concordance. This approach provides a promising molecular diagnostic tool for rapid detection of drug-resistant tuberculosis, particularly suitable for primary healthcare and resource-limited settings.

Introduction: Our previous research demonstrated that a high-fat diet (HFD) induced jejunal inflammation and hepatic steatosis, suggesting that small bowel microbiota contribute to these pathologies. This study investigated age- and sex-specific alterations in jejunal microbiota following a high-fructose, high-fat diet (HFHFD) in F344 rats.

Methods: Six-week-old and two-year-old rats of both sexes were fed an HFHFD for 8 weeks, after which jejunal contents were collected for metagenomic analysis. Taxonomic profiling and linear discriminant analysis were performed, and Spearman's rank correlation analysis was used to evaluate associations with jejunal inflammation and hepatic steatosis. Beta-diversity analysis was conducted to assess group separation. In vitro, HIEC-6 human intestinal epithelial cells were used to test the protective effect of Lactobacillus intestinalis under palmitic acid-induced lipotoxic stress.

Results: HFHFD reduced the Firmicutes/Bacteroidetes ratio in young females and in aged rats of both sexes. Notably, Lactobacillus intestinalis-which supports barrier function-decreased in young males and aged females. In contrast, Akkermansia muciniphila increased across all HFHFD groups, particularly in young females and aged rats. Bacteroides vulgatus increased in aged HFHFD-fed rats of both sexes, while Bacteroides caccae was elevated in females across both age groups. Furthermore, the Lactobacillus reuteri group decreased only in young HFHFD rats. L. intestinalis and L. reuteri groups negatively correlated with jejunal inflammation and hepatic steatosis, whereas B. caccae and A. muciniphila showed positive correlations with both pathogenic phenotypes. Beta-diversity revealed a pronounced diet- and sex-dependent separation in young rats, which was attenuated in aged groups. In HIEC-6 cells, L. intestinalis significantly restored viability under palmitic acid-induced lipotoxic stress, though its conditioned medium did not.

Discussion: Collectively, HFHFD induces age- and sex-dependent dysbiosis in the jejunum, and L. intestinalis may serve as a potential probiotic for metabolic dysfunction-associated steatotic liver disease.

Cotton monoculture is widespread in the oasis cotton-growing region of Xinjiang. Long-term continuous cropping has led to declines in soil fertility and imbalances in microbial communities, constraining sustainable, green production. Crop rotation is an effective agronomic practice to mitigate the deleterious effects of continuous cropping; however, the selection of rotation crops and the regulatory mechanisms by which rotation reshapes the soil micro-ecology require systematic clarification. Using continuous cotton (CK) cropping as the control, we combined high-throughput amplicon sequencing with soil physicochemical analyses to evaluate the effects of four previous-crop schemes-cotton → peanut (CPC), cotton → soybean (CSC), cotton → rapeseed (CRC), and cotton → maize (CMC)-on soil properties and the microbial community structure. Relative to CK, the CPC, CSC, and CRC treatments led to significantly reductions in yield and gross output value ranging from 38.72 to 62.23% and 34.54 to 55.35%, respectively. Although the net profit under CPC treatment decreased by 36.27% relative to CK, the benefit-cost ratio showed no significant difference. CPC significantly increased soil organic matter, available phosphorus, NH4⁺-N, and NO3^--N, while decreasing the pH and electrical conductivity, demonstrating the best overall improvement in soil fertility. In the fungal community, under CPC, Basidiomycota and Mortierellomycota significantly increased by 17.15 and 52.37%, respectively, whereas Basidiomycota significantly increased under CSC and CRC (by 17.15 and 20.58%). Functional guild analysis indicated that all four rotation schemes significantly reduced the relative abundance of plant pathogen fungi, with the greatest decrease under CPC (36.80%), with statistically significant differences. In the bacterial community, CPC significantly increased Actinobacteriota, Gemmatimonadota, and Firmicutes by 16.20, 15.75, and 29.73%, respectively, while CRC rose substantially Bacteroidota by 28.58%. Bacterial metabolism constituted the major predicted functional category (79.27-79.68%), no significant differences between treatments. Redundancy analysis identified soil moisture and the N/P ratio as key drivers of the variation in the fungal community, while bacterial communities were regulated by N/P, pH, and organic matter. Overall, rotation alleviated continuous-cropping constraints by optimizing soil properties and the soil microbial structure. The results provide an empirical basis for improving soil microbiomes and designing sustainable planting strategies in oasis cotton systems.

Background: Integrated genomic-phenotypic frameworks provide a more comprehensive approach to probiotic characterization, resolving longstanding ambiguities in strain safety assessment. Crucially, extracellular metabolites-not cell-bound components-emerge as dominant mediators of bacterial antioxidant activity, thereby refining the mechanistic understanding of microbial reactive oxygen species mitigation.

Methods: This study evaluated the probiotic potential and safety of Lacticaseibacillus rhamnosus SAL2 using whole-genome sequencing and functional validation. Hybrid Illumina/PacBio sequencing enabled complete de novo assembly, annotation, and pathogenicity assessment of this independently isolated strain. We benchmarked L. rhamnosus SAL2 against the reference strain L. rhamnosus GG through multidimensional assessment of safety and probiotic characterization. Antioxidant capacity was further evaluated through H₂O₂ tolerance assays and free radical scavenging tests.

Results: The genome of L. rhamnosus SAL2 comprises a single circular chromosome of 2,989,570 bp with no plasmids. Annotation using the KEGG, GO, COG, CAZy, and TCDB databases revealed that the majority of genes are involved in carbohydrate metabolism, cellular nitrogen compound metabolic process, carbohydrate transport and metabolism, glycoside hydrolases, and primary active transporters, respectively. Virulence factors were primarily limited to immune evasion mechanisms. L. rhamnosus SAL2 lacked hemolytic activity and was susceptible to multiple antibiotic classes. The strain exhibited high viability under simulated human gastrointestinal conditions while displaying strong mucosal adhesion potential. Notably, both intact cells and cell-free fermentation supernatants of L. rhamnosus SAL2 exhibited significant antioxidant activity. When comprehensively benchmarked against the classic probiotic reference strain L. rhamnosus GG, L. rhamnosus SAL2 matched its core safety profile and probiotic properties while demonstrating quantitatively superior antioxidant activity.

Conclusion: Lacticaseibacillus rhamnosus SAL2 showed safety and probiotic characteristics comparable to the reference strain, with enhanced antioxidant performance. These findings highlight its potential as a probiotic candidate for developing microecological preparations or functional foods, particularly due to its exceptional gastrointestinal tolerance, adhesive capacity, and free radical scavenging efficacy.