MicrobiologyOpen最新文献

Multi-Locus Sequence Typing (MLST) is a key method for allocation of Sequence Types (STs) for bacterial isolates. Traditionally, this is performed by the Sanger sequencing method, which can be highly time-consuming and laborious. In this study, we present NanoMLST, a high-throughput MLST workflow using multiplex PCR, Oxford Nanopore Technologies Next-Generation Sequencing, and the Krocus program for typing ESKAPE + E pathogens (Enterococcus faecium [E. faecium], Staphylococcus aureus, Klebsiella pneumoniae [K. pneumoniae], Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). Bacterial isolates were obtained from the Hospital Universitario La Paz's Microbiology Department and the Centro Nacional de Microbiología. Primers that can be multiplexed in a single PCR reaction were designed for the seven housekeeping genes for each species. DNA was extracted from single colonies by heating at 95°C for 10 min, mechanical lysis at 4.20 m/s for 2 min, and then by the MagCore extraction system. Multiplex PCRs were then performed with the respective primer mixes for each species, and libraries were prepared for sequencing by ONT Flongle cells. The Krocus program was then used to determine the STs from the raw FastQ reads. STs for 221 isolates were obtained through this workflow with an average time of 12 h per 24 isolates. In line with local data, the K. pneumoniae and E. faecium isolates were relatively oligoclonal, while the rest were polyclonal. STs from representative isolates showed 100% concordance between Sanger sequencing and the proposed workflow. NanoMLST offers a fast, cheaper, and less labor-intensive alternative for large-scale MLST applications targeting clinically important pathogens.

Fungal plant biomass conversion (FPBC) is of great importance to the global carbon cycle and has been increasingly applied for the production of biofuel and biochemicals from lignocellulose. However, the comprehensive understanding of relevant molecular mechanisms in different fungi remains challenging. Here, we comparatively analyzed the transcriptome, proteome and metabolome profile of four ascomycetes and one basidiomycete fungi during their growth on two common agricultural feedstocks (soybean hulls and corn stover). We revealed strong time-, substrate- and species-specific responses at multi-omics levels for the tested fungi, highlighting species-specific carbon utilization approaches and evolutionary adaptation to environmental niches. Notably, a remarkable expressional diversity of lignocellulose degrading enzymes, sugar transporter and metabolic genes, as well as industrially relevant metabolites were identified across different fungi and cultivation conditions. The findings improves our understanding of complex molecular networks underlying FPBC and fungal ecological roles, offering novel insights that can guide future genetic engineering of fungi for valorization of agriculture waste into value-added bioproducts.

Candida albicans is a fungal commensal of humans that often causes mucosal infections in otherwise healthy individuals and also serious infections in immunocompromised patients. The capacity of this fungus to colonize and cause disease relies on its ability to grow within the host, adapting to various nutrient restrictions and physicochemical conditions. The presence of alternative carbon sources, such as the lactate produced by the local microbiota, influences C. albicans antifungal drug resistance and immune evasion. In this study, we used genome-wide transcriptomic analysis to investigate the effect of lactate exposure upon metabolic rewiring. We provide evidence that C. albicans cells respond to growth in the presence of lactate at pH 5 by regulating genes encoding micronutrient transporters, notably iron transporters. More specifically, lactate triggers the downregulation of genes on the reductive iron uptake pathway, inferring a diminished requirement for high-affinity iron uptake. This is supported by the observation that lactate promotes the intracellular accumulation of iron by C. albicans cells. Lactate even enhances the growth of iron-transport defective C. albicans cells under iron-limited conditions. Lactate is known to activate protein kinase A (PKA) signaling. However, lactate-induced iron assimilation is PKA-independent. This study provides new insights into the role of lactate in iron homeostasis—two important factors that promote C. albicans virulence in the mammalian host, where nutritional immunity is a key antimicrobial strategy.

Bloodstream infection (BSI) is a severe and often fatal condition, and a major cause of mortality in patients with hematological malignancies due to underlying conditions and anticancer therapy-induced immunodeficiency. Rapid identification of the causative pathogens is essential as BSI results in worsened prognosis, extended hospitalization, delays or dose reductions in therapy, and may progress to sepsis and septic shock if untreated. Shotgun metagenomics is a culture-independent technique capable of detecting a wide range of fungal, viral, and bacterial organisms along with their antimicrobial resistance genes. Several studies showed that shotgun metagenomics enables the diagnosis of BSI, specifically in cases where conventional methods/culture-dependent techniques fail to identify the causative pathogens. However, evaluation of the accuracy of the applied bioinformatics pipelines remains incomplete. This study aimed to compare and optimize four commonly used bioinformatics pipelines (BLAST, Kraken, Metaphlan, RTG Core) for shotgun metagenomics by assessing their accuracy in identifying pathogens in blood samples from patients with hematological malignancies and suspected BSI, with blood culture serving as the reference standard. Our work shows that the selection of bioinformatics pipelines for diagnosing BSI strongly affects the precision of the findings, and an optimized BLAST pipeline was superior to the alternatives, as it was the only method that accurately identified the causative pathogens.

Patients in intensive care units, especially those immunocompromised, are prone to opportunistic infections, such as respiratory and urinary tract infections. Extended antibiotic use disrupts the production of microbiome-derived metabolites, including those involved in colonization resistance to Pseudomonas aeruginosa, which is known for its multidrug resistance. Hence, prior antibiotic treatment has been shown to increase susceptibility to P. aeruginosa infection, but the role of microbiota-derived metabolic cues in this context is still elusive. This study investigates how tryptophan metabolites from the indigenous microbiota affect P. aeruginosa virulence. In vitro tests on motility, biofilm production, and pigment quantification (pyocyanin and pyoverdine) were performed on P. aeruginosa strains (PAO1, PA103, PA14) and clinical isolates. Additionally, gene expression related to virulence was analyzed, and the effects of tryptophan metabolites on experimental lung infection in mice were evaluated. Indole, indoleacetic acid (IAA), and indoleacrylic acid (IA) reduced motility and pigment production. IAA and indole promoted biofilm formation, with indole having a stronger effect. Clinical isolates showed significant phenotypic diversity, and IAA was more effective at inhibiting virulence traits than indole or IA. Mice infected with bacteria grown in the presence of IAA had lower lethality and fewer polymorphonuclear leukocyte influx compared to the control group. This suggests that tryptophan metabolites, especially IAA, can modulate P. aeruginosa virulence and may help control infection progression.

Detecting pathogens in environmental samples using molecular-based technologies can be challenging, particularly in low biomass environments or where pathogens represent a low percentage of the community. Multiple displacement amplification (MDA) is a whole genome amplification (WGA) method that has been developed for low biomass samples. However, there is a lack of information on how MDA could improve PCR and sequence-based detection and genomic characterization of pathogens in challenging environmental samples. In this study, MDA was evaluated on low template samples of the Salmonella LT2 isolate, a foodborne and waterborne environmental pathogen. MDA was also evaluated on a variety of low template mixed-microbial mock, environmental communities containing a range of Salmonella genome percentages to simulate different levels of Salmonella in the environment. Using MDA starting inputs of 1.8 × 10⁴–1.8 × 10¹ Salmonella LT2 genome copies, > 99% of the Salmonella genome was recovered following MDA at > 16X depth of coverage from as few as 500,000 merged, 250 bp paired-end reads. For the mock microbial communities, moderately high levels of genome abundance distortion were evident following MDA across all communities when compared to the expected compositions, which could not be attributed to either genome size or GC content alone. Overall, MDA may provide a useful method for increasing Salmonella detection sensitivity in low target environmental samples where downstream selective targeted applications such as real-time PCR or targeted amplicon sequencing are used, but MDA may not be appropriate for identification and detection of Salmonella when using untargeted, metagenomic sequencing.

The increasing prevalence of antibiotic resistance among clinically significant pathogens necessitates the discovery of novel antimicrobial agents. This study investigated 29 Bacillus and Paenibacillus isolates from the soil for antimicrobial activity against multidrug-resistant clinical pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacterales (CRE). In both agar- and broth-based antimicrobial assays, Paenibacillus profundus strains 7.5 and M4.5 exhibited potent broad-spectrum activity, including significant inhibition of many CREs. Species identification was performed through 16S rRNA sequencing, and genome mining of three producer strains using antiSMASH revealed biosynthetic gene clusters associated with a variety of nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While many of these clusters were not associated with known antimicrobial compounds, several of them displayed high similarity to known compounds such as polymyxin B, paenilan, colistin, and paenibacterin. These findings reinforce numerous previous studies highlighting the potential of soil-derived Bacillus and Paenibacillus species as valuable sources of novel antimicrobials to address the global antibiotic resistance crisis.

Fungal colonization is a known carcinogenic accomplice in lung and colon cancer but has not been implicated in breast cancer. Here, we attempt to explore the mechanism behind fungal colonization and carcinogenesis by Malassezia globosa in breast cancer. To begin with, we found an increased abundance of the fungus in tumor tissues of breast cancer patients and the fungal inhibitor Amphotericin-B impeded tumor growth in patient-derived breast cancer xenograft models. On the other hand, Malassezia globosa enhanced the proliferative, migratory, and invasive abilities of breast cancer cells, and facilitated tumor growth in vivo. The positive effect of Malassezia globosa on tumor growth occurred via M2 macrophage polarization resulting in the activation of the pro-inflammatory MBL-C3a-C3aR signaling cascade which was reversed with the knockout of MBL expression. The proliferative, migratory, and invasive capacities of breast cancer cells were enhanced by culture medium from Malassezia globosa-treated THP-1 cells, which were rescued by a C3aR antagonist. In conclusion, Malassezia globosa activates MBL-C3a-C3aR signaling to trigger M2 macrophage polarization, promoting breast cancer progression and this study unravels a novel paradigm for breast cancer treatment.

Pseudomonas aeruginosa is a model organism for biofilm formation research, as it forms biofilms under diverse environmental conditions. At the same time, numerous studies have reported impaired-biofilm formation in clinical isolates; however, the genetic basis of these impairments remains unexplored. In this study, we assessed the ability of P. aeruginosa clinical isolates from a laboratory collection to form biofilms. Among these isolates, three demonstrated biofilm formation impairment. A comparative genomic analysis revealed genetic determinants associated with biofilm formation impairment, including mutations in the pelA and fleQ genes, and psl operon deletion. Interestingly, the identified loss-of-function mutations in regulatory genes involved in biofilm formation did not appear to affect the ability to form biofilms.

Multi-Resistant Bacteria (MRB) is a threatening biomedical problem, whose solution is of paramount importance. Due to the antibiotics resistance there is an emerging need for novel treatment strategies and protocolls. As bacteria tolerance in modern chemotherapeytic agents expands, the introduction of alternative methods is fundamental. The use of High voltage Electric Pulses, through a process known as Irreversible Electroporation (IRE), is an effective alternative bacterial control method. This paper describes a new prototype high voltage nanosecond pulser and validates its effectiveness in the in-vitro growth inhibition of a clinical resistant Staphylococcus aureus strain. Radiofrequency (RF) pulses of 100 ns and 450 ns pulse width and 1 Hz and 1 kHz repetition rate respectively were tested for therapy time in the range of 20–200 s. Increasing the electric field strength up to 11.5 kV/cm and the duration of therapy time up to 200 s results in 3.5 log scale reduction in bacterial cells. Nanosecond electric pulsed fields from our prototype device inhibite S. aureus growth in in-vitro test. It is sugested to test our prototype device in ex-vivo studies and propose a therapeutic protocol for infected skin wounds.