BMC Microbiology

Pseudomonas aeruginosa (P. aeruginosa) is the primary pathogen responsible for morbidity and mortality in patients with cystic fibrosis (CF). Its genomic plasticity and constant selective pressure from antimicrobial treatments have favored the emergence of multidrug-resistant clones. This study conducted a comparative genomic analysis of 41 P. aeruginosa isolated from pediatric patients with CF in Mexico from 2015 to 2024, with the aim of characterizing their evolutionary dynamics, resistome, and virulome. Whole-genome sequencing (MGI, Illumina, and PacBio platforms) was used, with de novo assemblies performed using Unicycler v0.4.8 on the BV-BRC platform. The databases used for the resistome were CARD and NDARO, and for the virulome, VFDB. Phylogenetic reconstruction was based on core-genome alignments generated with Roary v3.13.0, with maximum likelihood reconstruction performed in IQ-TREE v2.1.2. The statistical significance of the segregation of resistance and virulence patterns was evaluated using PERMANOVA analysis. The results revealed a significant clonal prevalence of sequence types (ST) 307 and ST 167. Phylogenomic analysis grouped the isolates into three main clades; Clade 1 stood out for having the highest resistance gene load (mean of 75 genes/genome), establishing itself as the main reservoir of multidrug-resistant profiles. Genotype-phenotype concordance reached 65.5% overall, with high accuracy for aminoglycosides (87.8%) and fluoroquinolones (82.9%). Furthermore, virulome analysis identified 67 distinct patterns that were significantly segregated among the clades (PERMANOVA: R2=0.31, p=0.001). These findings demonstrate that the evolution of P. aeruginosa lineages in the pediatric clinical setting involves parallel and coordinated adaptations in both their resistance potential and their virulence arsenal. This study underscores the need to adopt a multidisciplinary approach to the clinical management of chronic P. aeruginosa infections in pediatric patients. The persistence of extensively drug-resistant (XDR) strains calls for the integration of genomic surveillance and functional diagnostics, as well as the search for therapeutic alternatives for the clinical management of patients with cystic fibrosis.

Enterococcus faecalis is an opportunistic pathogen and facultative anaerobe that primarily relies on fermentative metabolism to colonize a wide range of aerobic and anaerobic environments. In the presence of exogenous heme, E. faecalis can assemble a minimal electron transport chain consisting of membrane-associated primary dehydrogenases, demethylmenaquinone, and the terminal cytochrome bd oxidase (CydAB). This respiratory chain is thought to generate a proton motive force to drive ATP synthesis via the F-type ATP synthase, thereby improving energy conservation under aerobic conditions. However, a cytosolic NADH oxidase (Nox) also consumes NADH and oxygen, potentially competing with the electron transport chain for reducing equivalents and terminal electron acceptors; but the relative physiological contributions of these two oxygen-reducing pathways remain poorly understood. To define the roles of CydAB and Nox under normoxic and hypoxic conditions, we constructed {Delta}cydAB and {Delta}nox mutants. Real-time, in situ measurements revealed {Delta}cydAB had no significant effect on oxygen utilization while in the {Delta}nox it was significantly reduced; revealing Nox as the major consumer of oxygen. Semi-untargeted metabolomic analysis further revealed oxidase-specific alterations in central metabolism with the {Delta}nox causing pronounced shifts in the ATP and NADH ratios; highlighting Nox as a key determinant of intracellular redox and energy homeostasis. Finally, single-cell fluorescence microscopy showed that membrane potential, a component of proton motive force, was substantially diminished only in the absence of both CydAB and Nox, or the F-type ATP synthase. These findings indicate that the F-type ATP synthase is a major generator of proton motive force, even upon aerobic growth, and demonstrate a complementary role for the electron transport chain and Nox in the bioenergetics of E. faecalis.

Pancreatic ductal adenocarcinoma (PDAC) harbors a distinct intratumoral microbiome. Yet rigorous characterization of its composition is hampered by pervasive environmental and procedural contamination. Sources of contamination have not been thoroughly explored, and the methods of decontamination have not been sufficiently evaluated in a benchmarking manner. We systematically collected >300 negative control (NCT) samples comprising paraffin from formalin-fixed paraffin-embedded (FFPE) samples, lysis buffer and sterile water over a period of four years processed by different laboratory persons (LP). All samples were sequenced using full-length 16S rRNA gene sequencing with Oxford-Nanopore Technologies. We benchmarked four decontamination methods (restrictive filtering, decontam, SCRuB, and the Nejman et al.-derived (Nj) pipeline) against fresh-frozen tumor samples (FF) from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, using the abovementioned contamination assessment to calculate a composite score for the assessment. Further, we validated those methods via technical replicates. Microbial profiles of NCT samples were significantly determined by control type, LP, year and season reflecting complex batch effects. The 15 most abundant contaminants spanned well-characterized environmental taxa and human commensals from the oral cavity. The LP processing samples left a significant microbial trace highly contributing to the batch effect. Decontamination benchmarking demonstrated that the Nj method consistently outperformed alternatives in both composite score and inter-replicate concordance. Application of Nj to fresh frozen PDAC samples substantially reduced contaminant burden while preserving putative tumor-associated signals in FF but not FFPE samples. Our results support the adoption of the Nj decontamination approach for future intratumoral microbiome studies in fresh frozen tumor samples.

The Bacillus cereus group comprises bacteria of biotechnological interest, but also raises health concerns. Some bacteria in this group are opportunistic human pathogens, mainly causing foodborne gastrointestinal infections. As of today, the presence, sequence variability, or expression of genes encoding toxins or other virulence factors are insufficient to predict the potential of a given isolate to cause the diarrheal form of the disease. To address this limitation, we developed a sandwich ELISA to quantify the NheA and Sphingomyelinase (SMase) proteins in culture supernatants to test them as markers of pathogenic potential. Application of the assay to a collection of B. cereus group isolates revealed that strains associated with food poisoning outbreaks produce significantly more NheA and SMase than those isolated from the environment or from commercial products. Statistical analyses show that the combined quantification of NheA and SMase provides robust discrimination between pathogenic and non-pathogenic (environmental and commercial) profiles. These results demonstrate that the quantitative assessment of both NheA and SMase production can serve as a reliable biomarker for distinguishing diarrheic food poisoning isolates from harmless strains.

Candida auris (currently Candidozyma auris) is an emerging fungal pathogen responsible for dramatic global increase in invasive candidiasis with high mortality. Most concerning, C. auris has a high propensity to colonize patients and persist and develop multidrug resistance to main classes of antifungals. In this study, we investigated the genetic and phenotypic diversity and resistance mechanisms of C. auris clinical isolates recovered from hospitalized infected patients. A total of 53 isolates from 38 unique patients were recovered from various clinical sources and evaluated for susceptibility to routine antifungal drugs. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis were performed to generate a phylogenetic network to infer population structure and identify mutations associated with drug resistance development. Isolates were also phenotypically evaluated for ability to form biofilms and aggregate, and cell wall adhesins gene expression studies were performed to provide mechanistic insights into C. auris phenotypic plasticity. Except for one clade III isolate, all isolates belonged to clade I and all were resistant to fluconazole with incidence of resistance to amphotericin B, echinocandins or both. Non-synonymous SNPs were found in genes associated with antifungal resistance including ERG11, TAC1B, CDR1 and FKS1. Phenotypically, isolates varied in their ability to form biofilm and aggregate which correlated with expression of the Scf1 and Als4112 cell wall adhesins genes highlighting C. auris phenotypic plasticity in circulating clinical strains. These findings underscore the growing clinical threat posed by C. auris and reinforce the need for optimized surveillance and treatment strategies for controlling its spread.

Amplicon sequencing of the 16S rRNA gene is the most widely used approach for profiling bacterial communities, but its taxonomic resolution is typically limited to the genus level. Many species carry multiple divergent 16S rRNA alleles that overlap across species boundaries, an ambiguity that even full-length, long-read sequencing cannot fully resolve. Shotgun metagenomics achieves species-level resolution but remains costly, particularly when only a single genus is of interest. Amplicon sequencing of rapidly evolving, protein-coding housekeeping genes offers a cost-effective alternative, yet no tool exists to identify suitable primer sets for a given target taxon. Here we present AmPair, a Snakemake pipeline that, given a target genus and one or more candidate housekeeping genes, designs and ranks primer pairs binding conserved regions while flanking a variable region capable of species-level discrimination, and validates them in silico across all available genomes. Using the genus Bacillus and the housekeeping gene tuf as a case study, the primer set recommended by AmPair amplified 99% of 2,392 genomes; only 0.04% carried multiple alleles and none showed inter-species allele overlap, compared with 91.41% and 69.49%, respectively, for the standard 16S rRNA V1-V9 region. Applied to a Bacillus community profiled by Nanopore sequencing, the same primers resolved closely related species. AmPair thus offers a generalizable and accessible route to species-level community profiling.

Rinsing-dependent dental hygiene presents a significant public health challenge in water-scarce environments. This study investigated combinations of xylitol (Xyl), chitosan (Chi), glycyrrhizin (Gly), epigallocatechin gallate (EGCG), dicalcium phosphate (DCP), and nano-hydroxyapatite (nHA) on the primary bacteria behind dental caries, S. mutans. These combinations were assessed for markers of dental caries by biofilm reduction, bacterial killing, and acid buffering against S. mutans when applied to an in vitro simulated enamel model using glass bead surfaces for biofilm formation, and gene expression was subsequently examined via RT-qPCR. Separately, mineral retention was also quantified. The EGCG-DCP-Xyl film demonstrated the highest overall efficacy, achieving a significant reduction in biofilm concentration compared to the untreated control and performing similarly in magnitude to the positive toothpaste control. Dead fluorescence staining confirmed that the EGCG-DCP-Xyl film induced the highest rate of non-viable cells, followed by the Chi-Gly film and the Gly-Xyl film. During 10-day pH cycling, the EGCG-DCP-Xyl and DCP-Xyl formulations buffered pH the most, consistently maintaining mean pH levels safely above the demineralization threshold of pH 5.5. The EGCG-DCP-Xyl also optimized mineral stability with the highest retained calcium concentration, significantly outperforming the Chi-Xyl film. At the transcript level, the EGCG-DCP-Xyl film induced substantial downregulation of key virulence genes, yielding decreases in expression for glucosyltransferase B (gtfB), associated with biofilm synthesis, collagen-binding protein (cnm), associated with tissue invasion, and lactate dehydrogenase (ldh), associated with lactic acid production, compared to the untreated control, with effects comparable in magnitude to the positive toothpaste control. This research suggests that targeting bacterial pathways and mineral loss through a portable film may have potential for preventing dental caries, especially in environments where water is limited. However, additional studies are necessary to evaluate real-world effectiveness.

The global prevalence of Extended-spectrum {beta}-lactamase-producing Enterobacterales (ESBL-E) colonization is increasing. However, it is unclear whether ESBL-E persist and if that is associated with an altered gut microbial ecology especially in early life where the developing microbiome may not provide the same colonization resistance as in adults. In this study, we collected longitudinal infant gut microbiome samples at delivery and in the nonclinical home setting in Chicago, Illinois, U.S.A, aiming to disentangle how genetic factors pertaining to the ESBL-E, as well as the surrounding gut ecology, influences persistence in the infant gut microbiome. We observed not only a higher-than-expected prevalence of ESBL-E in healthy infant gut microbiomes, but also a trend of ESBL-E persistence once colonized. Microbial communities showed higher dissimilarity between ESBL-E positive and negative infant gut microbiome at earlier time points. Although dissimilarity decreased over time, we present evidence that ESBL-E persist even when traditional detection methods are negative.

Edible mushrooms have been reported to have antimicrobial properties and other health benefits. This study aims to test the antimicrobial activities of several edible mushrooms from markets and test if co-culturing them with bacteria could induce stronger anti-bacterial properties. Commercial mushrooms, Hericium erinaceus (lions mane), Pleurotus ostreatus (oyster mushroom), Lentinula edodes (Shiitake) and Agaricus bisporus (button mushroom), were grown from strictly controlled/sterile substrates. Ethanol and water extracts from the mushrooms were prepared and tested against the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, and the fungus Candida albicans for antimicrobial activities. Shiitake water extract (SWE) showed strong antibacterial effects against all tested bacterial species, inhibitory effects on their biofilms, and antifungal activity. The antimicrobials in SWE seem to damage the cell wall and cell membrane of the bacteria, prefer weak acidic conditions, and are heat labile. Some antimicrobials are likely proteins and polysaccharides. In contrast, 3 other mushrooms displayed only weak antimicrobial effects. The fast-growing lions mane and oyster mushroom were co-cultured with different bacteria. The co-cultivation promoted the fruiting body development of lions mane. Co-culturing with S. aureus increased the anti-bacterial effects of lions mane against S. aureus, E. coli and particularly B. subtilis. Co-culturing the oyster mushroom with bacteria, especially B. subtilis and P. aeruginosa, boosted the mushroom growth. All tested bacteria, especially S. aureus, increased oyster mushroom anti-bacterial effect against E. coli and B. subtilis. The findings indicate that mushroom-bacteria co-culturing could have benefits both agriculturally and medicinally.

Organosulfur compounds are the predominant sulfur source in terrestrial environments, requiring bacteria to use enzymes for their assimilation. Most described organosulfur-assimilating enzymes require oxygen, and enzymes that function under anoxic conditions remain poorly understood. Recently, methylthio-alkane reductase (Mar), a nitrogenase-like enzyme that reduces the volatile organic sulfur compounds (VOSCs) methylthio ethanol (MT-EtOH), dimethyl sulfide (DMS), and ethyl methyl sulfide (EMS) under anoxic conditions, was identified in the purple nonsulfur bacterium Rhodospirillum rubrum. However, another purple nonsulfur bacterium, Rhodopseudomonas palustris, has three loci of nitrogen fixation-like (NFL) genes with high sequence similarity to Mar, suggesting additional Mar-like enzymes with distinct roles. Here, we tested whether these NFL genes are required for VOSC assimilation in R. palustris. RNA-seq analysis revealed that all three NFL loci are upregulated under sulfur limitation, supporting a role in sulfur assimilation. Only disruption of the NFL genes encoded by RPA2634-37, renamed marBHDK1, caused fitness defects with EMS, DMS, and dimethylsulfoniopropionate (DMSP) as sulfur sources, indicating a functional Mar enzyme. The NFL genes RPA2347-48 and RPA2353-54, renamed marKD2 and marHB2, were required for activity with MT-EtOH or ethanethiol but not DMS, EMS, or DMSP. No activity was observed for the third locus, RPA2363-64, renamed nflDK. Overall, two Mar homologs in R. palustris are capable of VOSC reduction, one specialized for simple VOSCs and the other preferring a substrate with an additional functional group.

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.

Metabolic dysfunction-associated steatotic liver disease (MASLD), the leading cause of chronic liver pathologies worldwide, represents a growing clinical burden. Its diagnosis remains reliant on liver biopsy that limits early detection and the ability to capture molecular changes across disease progression. A systematic understanding of stage-dependent gene expression changes is essential to identify biomarkers and effectively characterize disease mechanisms. Therefore recent studies provided databases for searching genes as well as prediction of multi-gene signatures for disease progression. However, there is still a need for interactive and comprehensive meta-analysis of datasets of MASLD patients with available histological metadata. Herein, we performed a meta-analysis of RNA-seq datasets using NAFLD Activity Score (NAS; n = 897) and fibrosis stage (n = 856) upon conducting pairwise comparisons across histological stages and identified differentially expressed genes associated with disease progression. Most importantly, we provide our findings via a dedicated web server, the MASLD-META NETWORK (https://masld.scilicium.com), enabling users to interactively explore meta-analysis results across diverse network modalities. In addition, we characterized gene expression dynamics across increasing disease stages to identify consistent progression-associated pathways using Louvain clustering. Network-based parameters such as centrality in combination with meta-analysis scores further highlighted central genes and pathways implicated in disease mechanisms. Accordingly, MASLD-META NETWORK enabled an integrative reassessment of recently published gene signatures, identifying COL1A1, COL3A1, THBS2, FBLN5, and PDGFA as the most central genes, and SULF2, MMP14, IL32, GPNMB, and COL3A1 as candidate markers of earlier transcriptional alterations. Network analysis of MASLD associated biological modules further identified LAMA2 and LAMA3 as previously unrecognized central candidate targets.

Background. Urine-based testing offers a promising non-sputum approach for diagnosing paediatric tuberculosis. However, the currently available lipoarabinomannan (LAM) assay shows limited sensitivity in children and is primarily indicated for those living with HIV. Co-detection of LAM with Mycobacterium tuberculosis (Mtb) proteins in urine could provide complementary pathogen-derived biomarkers that improve diagnostic performance. Methods. We developed an ultrasensitive multiplex electrochemiluminescence (ECL) immunoassay to measure Ag85B, CFP-10, ESAT-6, MPT32, and MPT64 in urine. We determined the analytical limits of detection and evaluated the diagnostic performance of individual proteins and LAM using urine samples from children with Confirmed, Unconfirmed, and Unlikely pulmonary tuberculosis enrolled across five high-burden countries (The Gambia, India, Peru, South Africa, and Uganda). Performance was assessed overall, by HIV and nutritional status, and across biomarker combinations. Findings. Urine samples from 630 children were analysed (median age was 4 years [IQR 2-8]; 44% female, 15% living with HIV, 19% underweight, 24% with Confirmed tuberculosis). The ECL assay achieved femtomolar limits of detection (1.5 to 4.0 fM). The sensitivity and specificity of individual Mtb proteins were 12-33% and 98-100%, respectively. Ag85B had the highest sensitivity (33%, 95% CI 26-41) for Confirmed tuberculosis and was similar to LAM. A four-antigen signature (Ag85B, MPT64, MPT32, LAM) was 50% sensitive (95% CI 42-58) and 94% specific (95% CI 90-96), and was significantly more sensitive than LAM alone, in particular among those without HIV. An additional sixteen (10%) of children with Unconfirmed TB had at least one Mtb protein or LAM detected. Interpretation. Multiple Mtb proteins are detectable in paediatric urine with high specificity, and multi-antigen signatures can augment sensitivity versus LAM alone. These findings demonstrate the potential of multi-antigen urine detection for childhood TB and define analytical targets for the development of future point-of-care diagnostics. Funding. National Institutes of Health.

Diatoms are major contributors to marine primary production, yet current approaches for monitoring their abundance and function rely on coarse satellite chlorophyll estimates or sparse cell count and carbon fixation measurements. Molecular markers are a promising approach for high-resolution measurement of both abundance and metabolic activity through analysis of environmental DNA (eDNA) and RNA (eRNA). We present an isothermal quantitative recombinase polymerase amplification (qRPA) assay targeting rbcL gene copies and transcripts of marine diatoms, operating at low temperature and producing results in less than 15 min. We demonstrate specificity and calibration across diverse diatom taxa, then apply the assay to eDNA and eRNA samples from the Mare Chiara Long-Term Ecological Research site in the Bay of Naples, Italy, alongside microscopy, chlorophyll, physicochemical, and carbon-fixation data. Diatom rbcL DNA tracked abundance across five orders of magnitude despite seasonal shifts in community composition. Combining molecular and optical data revealed increased cellular rbcL copies and chlorophyll in low-light winter populations, suggesting enhanced photosynthetic capacity despite lower abundance. Furthermore, rbcL RNA reflected total carbon fixation rates and identified populations with differing carbon fixation activity. These results support rapid, RPA-based rbcL quantification as a robust approach for biomolecular ocean observing.

Genetic variation among microbial strains of the same species can profoundly influence their phenotypes, ecological functions, and impacts on human health. Traditionally, the relative abundance of a species has been used to identify associations between the microbiome and disease. However, this approach overlooks intra-species genetic variation and is susceptible to spurious correlations arising from the compositional nature of abundance data and microbial load. Fast, k-mer-based algorithms can now accurately estimate strain-level Average Nucleotide Identity (ANI) in metagenomes. Despite its value as an orthogonal metric for strain-level analysis, methods for conducting ANI-based association studies remain limited. To address this, we developed StrainSpy, a statistical algorithm that identifies associations between containment ANI and variables of interest across a wide range of study designs, including longitudinal and multi-cohort designs. Re-analysis of a study examining gut microbiota recovery in 12 healthy adults following antibiotic exposure revealed novel strain-level associations, including a reduction in strain-level diversity despite species persistence. Applying StrainSpy to a multi-cohort analysis of 3,414 colorectal cancer metagenomes identified novel strain-level associations with colorectal cancer. However, in a separate collection of microbiome-immunotherapy studies, no individual strain was consistently associated across cohorts. Importantly, across both datasets, StrainSpy informed containment ANI-based machine learning models achieved comparable accuracy to traditional abundance-based methods. StrainSpy is publicly available as an R package github.com/gtonkinhill/strainspy.

Antimicrobial resistance is a public health challenge, driving the need for rapid, cost-effective diagnostic support tools. Artificial intelligence (AI) may enable prediction of susceptibility to untested antibiotics from known susceptibility results, but prospective clinical validation is required before routine use. We evaluated an AI-based decision support method, trained on invasive isolates from the European Surveillance System (TESSy), for prediction of antibiotic susceptibility in clinical Escherichia coli urine isolates. The evaluation included 99 E. coli isolates from urine samples with diversity in age, sex, and antibiotic susceptibility. Predictions were evaluated for 14 antibiotics using patient metadata and susceptibility results for 4-8 antibiotics as input. Prediction uncertainty was handled using conformal prediction, allowing abstention when confidence was insufficient. EUCAST disk diffusion test results were used as reference and genomic sequence data was used to explore mechanisms of the AI performance. Without conformal prediction, 84% of predictions were correct when susceptibility results of six antibiotics were used to predict susceptibility to eight additional antibiotics. Across all predictions generated using susceptibility results for six antibiotics as input, the major and very major error rates were 19% and 12%, respectively. Prediction errors varied between antibiotics and were associated with certain phenotypic and genotypic resistance patterns. Conformal prediction reduced errors but increased abstentions; at confidence levels of 90%, 95%, and 97.5%, the model abstained in 9.6%, 14%, and 22% of instances. The method showed promising performance, but its clinical use remains limited and may require diagnostic data beyond susceptibility test results and demographic variables.

Cutaneotrichosporon dermatis (formerly Trichosporon dermatis) is a basidiomycetous yeast-like fungus known to cause summer-type hypersensitivity pneumonitis, although its virulence in humans remains poorly understood. We performed morphological and molecular identification of an isolate from the sputum and blood cultures of an immunocompromised patient, together with pathogenicity assessment using a Galleria mellonella model, biofilm formation/eradication assays, antifungal susceptibility testing, drug combination effects, and the post-antifungal effect (PAFE), compared with Trichosporon asahii. The isolate was identified as C. dermatis by ITS/IGS1 sequencing, supported by phylogenetic analysis. Growth of C. dermatis increased more at 37 than at 25. In the Galleria mellonella assay, C. dermatis, T. asahii, and Candida albicans each showed dose-dependent pathogenicity at sufficiently high inocula, although Rhizopus oryzae was the most potent pathogen on a per-CFU basis. C. dermatis formed biofilms that were more completely inhibited by terbinafine (TRB) and amphotericin B (AmB) than azole agents, which showed only partial inhibitory activity even at high concentrations. Susceptibility testing showed relatively strong susceptibility to AmB and azole agents. In the TRB and azole combination assay, the fractional inhibitory concentration index (FICI) was below 0.5, indicating synergy. Isavuconazole (ISC) showed a markedly stronger PAFE than the other azole agents tested. These findings indicate that although azoles show only partial efficacy against its biofilm, C. dermatis can still cause invasive infection, and that azole monotherapy or TRB and azole combination therapy, aided by the potent PAFE of ISC, may represent effective treatment options.

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico, conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A-G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.

Phage tail-like elements (PTEs) -- tailocins, bacterial type VI secretion systems (T6SS), and extracellular contractile injection systems (eCIS) -- are contractile nanomachines that bacteria use to kill their neighbors and compete within their micro-ecosystems. PTEs help shape microbial community composition. Most PTE detection tools only detect a single PTE class. Moreover, most tailocin detection methods are largely restricted to Pseudomonas, leaving a key part of tailocin diversity uncharacterized. In this work, we present PhageTAILor (https://github.com/hjcho-bio/PhageTAILor), an integrative and fully automated pipeline that detects and classifies prophages and 3 PTE classes from bacterial genomes. PhageTAILor combines a 6-detector homology-based candidate search (geNomad, tail-gene, PHROGs-tail, SecReT6, eCIStem, and a divergence-tolerant tail-HMM detector) with a LightGBM classifier comprising 1 multiclass and 3 binary heads, trained on 6,501 bacterial genomes carrying 13,082 prophages and PTEs. A phylogeny-free feature matrix used in our model keeps predictions reproducible between model construction and user inference. PhageTAILor performs strongly at the genome level and generalizes beyond its Pseudomonas-rich training set. On a 76-strain cross-clade benchmark, PhageTAILor detected tailocins at F1 = 0.955. Furthermore, it identified 12 of 13 experimentally validated tailocins spanning five genera versus 2 of 13 for a Pseudomonas-restricted tool TattleTail. PhageTAILor also demonstrated sensitivity equivalent to viral detection tool geNomad while avoiding its higher false-positive rate. Applied to 7,925 plant- and soil-associated bacterial isolates, PhageTAILor showed that prophages in the phyllosphere and tailocins in plant-associated bacteria, whereas eCIS are enriched in soil. PhageTAILor is distributed as an open-source, modular pipeline with a command-line interface.

Abstract Rationale: Host genotypes can predict subtherapeutic anti-tubercular drug exposures and treatment-associated toxicities. Screening for these variants could enable personalized dosing, but scalable assays for second-line drugs are lacking. Objectives: We developed a nanopore sequencing panel to detect host variants affecting anti-tuberculosis drug troughs and toxicities, and evaluated its performance as a saliva-based screening tool. Methods: We designed a 16-plex panel targeting 23 variants (21 clinically validated, 2 predicted actionable) relevant to linezolid, bedaquiline, clofazimine, moxifloxacin, and ethambutol exposure. We first sequenced 50 Coriell DNA (1000 Genomes Project) to benchmark accuracy against Illumina, then sequenced saliva from 202 individuals treated for drug-resistant tuberculosis in India using MinION Mk1C (R10.4). Plasma trough concentrations and toxicity frequencies were stratified by genotype. Data were analyzed using in-house pipelines. Measurements and Main Results: The panel showed high coverage in saliva (median 3,609X). Several suggestive genotype-phenotype trends reached nominal significance in distinct subsets. Among patients on high-dose moxifloxacin (800mg daily), UGT1A1 rs3755319 A>C was associated with higher troughs in heterozygotes (6/14, p<0.01) and homozygous alternates (4/14, p<0.05). Among patients with linezolid-associated toxicity dose-reduced to 300mg, ABCB1 rs2032582 A>C homozygous alternates (7/98) had significantly lower Cmin versus wild-type (p<0.05) and heterozygotes (p<0.01); neither association held at standard dosing. Linezolid toxicity was more frequent among ABCB1 rs1128503 A>G heterozygotes versus homozygous reference (58.3% vs. 29.1%), and UGT1A1 rs4148323 G>A heterozygotes showed higher moxifloxacin toxicity rates than wild-type (42.9% vs. 14.3%). Conclusions: Portable, saliva-based sequencing reliably detects pharmacogenetic variants and could inform pre-treatment screening for drug exposure or toxicity.