MicrobiologyOpen最新文献

This study investigated the usefulness of the IR Biotyper, which types bacterial strains using Fourier transform infrared (FT-IR) spectroscopy, against extended-spectrum β-lactamase (ESBL)- and metallo-β-lactamase (MBL)-producing Enterobacterales. Sixty-six clinical isolates (20 ESBL-producing Klebsiella pneumoniae, 15 IMP-producing K. pneumoniae, and 31 IMP-producing Enterobacter cloacae complex isolates) were analyzed using the IR Biotyper, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and whole-genome single-nucleotide polymorphism (wgSNP) analyses and the results were compared. Of the 20 ESBL-producing K. pneumoniae strains analyzed, the IR Biotyper detected three clusters. Of these clusters, two were determined as respective clusters by PFGE and wgSNP analyses, and the strains included in each cluster showed the same STs. The IR Biotyper detected three clusters in the analysis of 15 IMP-producing K. pneumoniae strains. Of these clusters, strains in the two clusters showed high concordance with PFGE, MLST, and wgSNP analyses. The IR Biotyper identified six clusters among the IMP-producing E. cloacae complex isolates. These results were fully concordant with those of PFGE, MLST, and wgSNP analyses in the two clusters. The range of adjusted Rand index was 0.734-0.967 between the IR Biotyper and PFGE and 0.658-0.857 between the IR Biotyper and MLST or wgSNP analyses. This study demonstrated the performance of IR Biotyper for the detection of clonal similarities in ESBL- and IMP-producing Enterobacterales and it might be useful for outbreak investigation.

Feline calicivirus (FCV) is a primary cause of upper respiratory tract infections and oral ulcerative disease in cats and exhibits substantial genetic diversity that complicates prevention and control. In this study, we isolated the FCV-BJ616 strain, established a reverse-genetics system, and investigated its pathogenic mechanisms, thereby providing a foundation for antibody-based therapies and broad-spectrum vaccine development. The virus was purified by three rounds of plaque cloning, and its morphology was examined by electron microscopy. VP1 expression was confirmed by immunofluorescence and Western blotting. Using integrated systems-biology and reverse-genetics approaches, an infectious clone of rFCV-BJ616 was successfully assembled and rescued, exhibiting genetic stability comparable to that of the parental strain. In vivo infection experiments showed that rFCV-BJ616 retained wild-type virulence, causing persistent high fever, weight loss, and multiorgan pathology in infected cats. Proteomic analysis indicated that infection with FCV-BJ616 or rFCV-BJ616 markedly activated cytokine-mediated inflammatory signaling pathways. Both FCV-BJ616 and rFCV-BJ616 significantly upregulated the expression of IL-8, S100A8/A9, and TLR3, which are associated with acute inflammation and tissue damage. Furthermore, elevated IFN-β levels concomitant with STAT1 downregulation suggested a transient attenuation of antiviral signaling during early immune activation. These findings were corroborated by ELISA-based validation of serum cytokine profiles. Collectively, this study provides new insights into the molecular pathogenesis and evolution of FCV-BJ616 and establishes a robust reverse-genetics platform for precise genome manipulation and future vaccine development.

Pseudomonas aeruginosa poses a significant therapeutic challenge in pediatric patients with cystic fibrosis (CF) due to increasing multidrug resistance (MDR) and carbapenem resistance, underscoring the need for surveillance to guide treatment strategies. In this study, sputum and throat swab samples were collected from inpatient and outpatient CF children with pulmonary infection at the Children's Medical Center in Tehran, Iran. Isolates were identified using standard culture and biochemical methods, followed by antimicrobial susceptibility testing. Carbapenemase production was assessed phenotypically and by molecular detection of resistance genes, and genetic diversity was also evaluated using Random Amplified Polymorphic DNA (RAPD)-polymerase chain reaction (PCR). A total of 117 P. aeruginosa isolates were recovered (prevalence 17.41%), of which 94.9% were nonsusceptible to at least one antimicrobial agent. Carbapenem-resistant P. aeruginosa (CRPA) and MDR isolates accounted for 24.8% and 23.1% of isolates, respectively. Carbapenemase gene coexistence was significantly associated with MDR (ρ = 0.227, p = 0.014) and CRPA (ρ = 0.314, p = 0.001). Metallo-β-lactamase production was detected in 13.7% of isolates, while blaVIM was the most frequently identified carbapenemase gene (59%). RAPD-PCR demonstrated marked genetic heterogeneity, grouping isolates into 24 distinct clusters. Overall, the substantial burden of MDR and CRPA identified at this tertiary pediatric center highlights an urgent need for stricter antimicrobial stewardship, enhanced infection control measures, and ongoing surveillance to mitigate resistance spread and preserve therapeutic effectiveness in this vulnerable population. These findings warrant multicenter investigation to determine whether similar patterns exist across other Iranian pediatric CF facilities.

Gut microbe cultivation is essential for studying host-microbiota interactions. Traditional cultivation methods often fail to recover microbial species at low abundance (< 0.1%). To overcome this limitation, we employed the bent-capillary-centrifugal-driven (BCCD) method to encapsulate and cultivate fecal microbes in microdroplets. Fecal bacterial cells were distributed into ~50 nL microdroplets via the BCCD generator, and the microdroplets were dispersed in the oil phase and further incubated under controlled conditions. The BCCD method significantly increased the frequency of microbes at low abundance. Compared to the plate-based method, BCCD-based cultivation produced distinct microbial community structures and exhibited significantly lower temporal variation during cultivation (p < 0.05). Lineage-specific effect size (LEfSe) analysis revealed that BCCD-based cultivation enriched 29 low-abundant bacterial genera, whereas the plate-based method enriched 26. Using this method, we isolated 1,049 bacterial strains representing 123 species and 58 genera, including 8 novel species. Among the isolated and cultivated genera, 62.1% (36/58) were microbes of low abundance in the original fecal sample, and 41.4% (12/29) of the BCCD-specific enriched genera were successfully obtained. Notably, comparison with four major gut microbial culture studies revealed 45 species were exclusively recovered in this work. Taken together, the results demonstrated that our BCCD-based cultivation method effectively enriched and facilitated the isolation and cultivation of microbes at low abundance and novel gut bacterial species.

Haloarcula rubripromontorii BS2, an extremely halophilic archaeon was obtained from the solar salt pans of Goa, India. It grew luxuriantly on EHM medium with 25% NaCl with a bright orange pigmentation. This study aimed to extract and characterize the carotenoids from Haloarcula rubripromontorii BS2 and evaluate their antioxidant properties, biocompatibility and provide insight into their potential therapeutic applications. Preparative high-performance liquid chromatography (HPLC) was employed to fractionate and separate the haloarchaeal carotenoids. Further detailed characterization using liquid chromatography-mass spectrometry (LC-MS), Raman spectroscopy and nuclear magnetic resonance (NMR) spectroscopy for each fraction confirmed the presence of C50 carotenoids primarily bacterioruberin, monoanhydrobacterioruberin, and their different isomeric forms. Our results indicate that these carotenoids are more stable in oil compared to solvents. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid (ABTS) assays resulted in an IC₅₀ of 4.31 ± 0.07 µgmL^-1 and 2.04 ± 0.02 µgmL^-1 respectively, indicating their potential as excellent antioxidants. Haloarchaeal carotenoids were found to be biocompatible with human keratinocyte skin cells (HaCaT). C50 carotenoids from Har. rubripromontorii BS2 represent promising, eco-friendly alternatives to synthetic antioxidants for use in high-value cosmetic and dermatological applications.

Stenotrophomonas maltophilia is a nosocomial and opportunistic microorganism with increasing antibiotic resistance rates. This study aimed to assess its biofilm production capacity, antibiotic resistance distribution, and the prevalence of biofilm- and resistance-related genes in clinical isolates. In this multiinstitutional study, 230 isolates were collected from hospitals across Iran between 2022 and 2024. Resistance trends were evaluated using disc diffusion and minimal inhibitory concentration E test methods, per Clinical and Laboratory Standards Institute guidelines. Crystal violet staining assessed biofilm production, while polymerase chain reaction (PCR) sequencing identified biofilm- and resistance-related genes. Real-time PCR was used to evaluate the relative expression of the smeD, smeE, and smeT genes, calibrated against TMP/SMX-sensitive control strains. Susceptibility rates to trimethoprim/sulfamethoxazole (TMP/SMX), levofloxacin, and minocycline were 97.39%, 93.47%, and 93.04%, respectively. TMP/SMX-resistant strains showed 19.8- and 16-fold higher expression of smeD and smeE, compared with sensitive isolates. The spgM gene was detected in all isolates, and 93.04% (n = 214) were biofilm producers, with most showing moderate-biofilm formation (n = 89, 38.70%). Additionally, the rpfF gene was closely associated with strong-biofilm formation (p ≤ 0.05). The L2, L1, smqnr, sul2, and sul1 resistance genes were identified in 214 (93.04%), 181 (78.69%), 135 (58.7%), 136 (59.1%), and 127 (55.2%) isolates, respectively. Our findings demonstrate that most isolates remain sensitive to TMP/SMX, while resistance to alternative antibiotics is rising. Moreover, biofilm production appears significantly associated with the rpfF gene.

To identify potential therapeutic strategies for relapsed or refractory lymphoma (R/RL) by examining differences in gut microbiota composition and metabolic profiles between patients with R/RL and those with primary, treatment-naïve lymphoma (PL), using fecal microbiota analysis and metabolomics. A total of 21 patients with lymphoma were enrolled at the Department of Lymphoma and Oncology, Shanxi Bethune Hospital, between November 2023 and December 2024. The cohort included 14 patients with R/RL and 7 with PL, who served as the control group. Pretreatment fecal samples and clinical data were collected from all participants. Gut microbiota profiling was conducted using 16S rDNA sequencing, including alpha diversity, beta diversity, species composition, and differential abundance. Untargeted metabolomics was employed to identify and analyze differentially expressed metabolites between the groups. Patients with R/RL exhibited increased relative abundances of Actinobacteriota and Alphaproteobacteria and decreased levels of Erysipelotrichales, Morganellaceae, Faecalibacterium, Clostridium, Klebsiella, and Ruminococcus. Seven metabolites were significantly upregulated in the R/RL group (p < 0.05): 3-amino-4-methylpentanoic acid (p = 0.028), 2-hydroxybutyric acid (p = 0.020), UDP-N-acetylglucosamine (UDP-N-AG) (p = 0.011), pantothenic acid (p = 0.037), isoleucine (p = 0.028), glycine (p = 0.044), and alanine (p = 0.025). Literature review and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated enhanced central carbon metabolism and amino acid metabolism in cancer. Alterations in gut microbiota and metabolic activity may contribute to the pathophysiology of R/RL. Therapeutic modulation of the gut microbiota, including the use of fecal microbiota transplantation, may improve the intestinal immune microenvironment in this patient population. The present work is hypothesis-generating and requires large-scale validation.

The ability of gut microbes to degrade host- and diet-derived glycans is central to microbiome ecology and host interactions, yet predicting these functions in silico remains challenging. Hyaluronan (HA), a glycosaminoglycan (GAG) abundant in host tissues and dietary supplements, is depolymerized by specialized polysaccharide utilization loci (PULs) in Bacteroides. Here, we combined comparative protein analysis, functional assays, and quantitative proteomics to evaluate the reliability of sequence-based predictions of HA utilization. Clustering of more than 3900 PL8 and GH88 protein sequences from 54 Bacteroides species did not distinguish known HA degraders from nondegraders, underscoring the limited predictive power of these enzymes alone. Experimental validation in Bacteroides acidifaciens DSM 111135 and Bacteroides thetaiotaomicron DSM 2079 confirmed HA degradation, as HA-derived fragments were identified by liquid chromatography-mass spectrometry. Proteomic profiling revealed coordinated induction of both canonical GAG-specific PULs-encoded proteins and noncanonical accessory proteins (BT4410/BT4411) in response to HA in both species. Incorporating such noncanonical components into comparative frameworks may improve prediction of glycan utilization potential and help link microbial genomic content to ecological function in the gut.

Gallid alphaherpesvirus 1 (GaAHV-1), also referred to as infectious laryngotracheitis virus (ILTV), primarily targets the upper respiratory tract of chickens. This infection leads to significant economic setbacks worldwide in the poultry sector, driven by reductions in egg output, weight gain, and increased mortality rates. Even with the broad implementation of vaccination programs, ILTV outbreaks remain a challenge, as vaccine strains can revert to a virulent form under field conditions. This underscores the need to explore targeted therapeutic options, including a deeper understanding of GaAHV-1's nuclear trafficking mechanisms, critical for viral replication. The herpesvirus large tegument protein UL36 contains N-terminal nuclear localization signals (NLSs) that are essential for capsid routing to the nuclear pore complex (NPC). However, the mechanisms by which UL36 of GaAHV-1 mediates nuclear import remain poorly understood. In this study, we identified the NLS of GaAHV-1 UL36 and elucidated their binding mechanism with human nuclear import proteins. Using high-resolution crystal structures and quantitative assays, we mapped the specific residues and regions within UL36's N-terminal domain that facilitate binding to importin (IMP) α. Moreover, we revealed variations in binding affinities among different importin isoforms. Our biochemical and structural analyses demonstrate that the predicted N-terminal NLS of GaAHV-1 UL36 is critical for IMPα binding. These findings provide detailed molecular insights into the interaction between the GaAHV-1 large tegument protein and IMPs, paving the way for the development of targeted antiviral therapies.

Brucella-induced neuroinflammation represents a key mechanism in the development of neurobrucellosis. The objective of this investigation was to clarify the molecular pathways through which the BvrR contributes to neuroinflammation and cognitive dysfunction. Human microglial clone 3 (HMC3) cells were transfected with pcDNA3.1-BvrR-His to examine the effects of BvrR from Brucella abortus on endoplasmic reticulum (ER) function and the activation of activating transcription factor 2 (ATF2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65. The role of phosphorylated inositol-requiring enzyme 1 (p-IRE1) in mediating BvrR-induced activation of ATF2 and NF-κB p65 was assessed by applying the IRE1 activator IXA4 and the IRE1 inhibitor GSK2850163, followed by evaluation with western blotting and RT-qPCR. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) concentrations in cell culture supernatants were quantified using ELISA. For in vivo analysis, HBAAV2/9-IBA-1-BvrR-6*HIS-ZsGreen was stereotactically delivered into the right hippocampus of mice. Expression of BvrR in HMC3 cells induced phosphorylation of IRE1 and expansion of the ER. This activation enhanced ATF2 and NF-κB p65 phosphorylation, facilitated their nuclear translocation, and significantly increased IL-6 and TNF-α expression at both the protein and mRNA levels. Inhibition of IRE1 with GSK2850163 suppressed these responses, whereas IRE1 activation with IXA4 reproduced the effects of BvrR. Findings indicate that BvrR from B. abortus activates IRE1, which subsequently stimulates ATF2 and NF-κB p65, leading to increased expression of IL-6 and TNF-α and the induction of inflammatory responses in HMC3 cells.