Microbiology Spectrum

Background Metagenomic next-generation sequencing (mNGS) is increasingly adopted as a rapid and unbiased alternative to culture-based pathogen detection. However, standardized approaches for analytical validation of mNGS workflows are lacking. Validation strategies based solely on comparison with culture are inherently limited due to low diagnostic yield of culture, and the inability to derive analytical parameters such as limit of detection (LoD) from clinical samples with unknown microbial loads. Systematic analytical approaches are therefore required to define mNGS workflow performance independently of clinical comparators. In addition to diagnostics, mNGS is widely applied to microbiome profiling, where preservation of microbial diversity at low DNA inputs is a key performance criterion. Materials and Methods The analytical performance of the PaRTI-Seq mNGS workflow (Micronbrane, Taiwan), comprising host depletion, DNA extraction, library preparation, and bioinformatics analysis, was evaluated. Library-level analytical sensitivity was assessed using quantified genomic DNA (gDNA) from Enterobacter hormaechei, Candida albicans, and Mycobacterium smegmatis. Workflow-level sensitivity was evaluated using enumerated suspensions of E. hormaechei, Staphylococcus aureus, M. smegmatis, C. albicans and Aspergillus brasiliensis. Microbiome performance at low DNA input was evaluated using stool, saliva and gut mock community samples at 50ng, 5ng, and 0.5ng gDNA input and compared with an alternative low-input library preparation workflow. Results The library preparation module demonstrated analytical sensitivity down to 0.1 pg of E. hormaechei and C. albicans gDNA, and 1pg for M. smegmatis gDNA. Workflow-level LoDs were determined to be 1 CFU for E. hormaechei, 272 CFUs for S. aureus, 39 CFUs for A. brasiliensis, 36 CFUs for C. albicans and 3,702 CFUs for M. smegmatis. Differences in extraction efficiencies and extracted gDNA qualities appeared to be important determinants of workflow-level LoDs. For microbiome applications, Micronbrane Unison Ultra-low DNA input library preparation kit maintained more consistent bacterial diversity indices down to 5ng DNA input for stool and saliva samples, and the gut mock community than the comparator workflow. Conclusion This study outlines practical and generalizable analytical validation approaches for mNGS library preparation and complete workflows using defined spike-in models, providing a foundation for informed clinical interpretation and implementation of mNGS in diagnostic and microbiome applications.

Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between PnimBG (MTZ-resistant) and PnimBT (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.

Objective: To evaluate the specimen-level performance of a multichannel electronic nose (e-nose) sensor array in classifying stool specimens into relevant Clostridioides difficile categories. Design: Retrospective single-center validation study with reference to polymerase chain reaction (PCR) and C. difficile toxin testing. Participants: A convenience sample of 31 discarded stool specimens with PCR and confirmatory toxin testing. Categorized into PCR-positive (+)/toxin+, PCR+/toxin-negative (-), and PCR-. Methods: Specimens were sampled in controlled headspace by two e-nose sensor arrays in 10-minute sessions with 5-second sampling intervals. The primary endpoint was one-versus rest classification performance (sensitivity, specificity, positive predictive value, and accuracy) into three classes: PCR+/toxin+, PCR+/toxin-, and PCR-. Data were normalized and the region of interest was extracted for training and testing. Random forests were utilized for classification of normalized data with a test-train split at the specimen-level. Results: There were 457 unique 10-minute sampling events (147 PCR+/toxin+, 183 PCR+/toxin-, 127 PCR-). Sensors 1 and 2 captured 20,331 and 23,538 unique 5-second exposures within the region of interest, respectively. Testing data classification accuracy was 0.79 [0.78, 0.81] and 0.84 [0.83, 0.85] for Sensors 1 and 2. Sensitivity for PCR+/toxin+ exposures was 0.82 [0.79, 0.84] and 0.94 [0.92, 0.95], and specificity was 0.91[0.90, 0.93] and 0.93 [0.92, 0.94] for Sensors 1 and 2. Conclusion: An e-nose sensor array partially differentiated PCR-classified C. difficile specimen classes at the exposure level under headspace-controlled conditions. Performance was strongest for PCR+/toxin+ exposures. Findings support further prospective validation in the hospital room setting.

Heme metabolism regulates the availability of heme, an iron-containing molecule essential for oxygen transport, mitochondrial respiration, electron transfer, and enzymatic reactions. Increasing evidence implicates heme metabolism in regulating viral infection, potentially representing an important innate antiviral mechanism. Components of both heme biosynthesis and degradation pathways exhibit antiviral activity against different viruses; however, their contributions and conservation across human coronaviruses remain incompletely defined. We investigated the impact of modulating heme metabolism on infection by seasonal human coronaviruses HCoV-OC43 and HCoV-229E. Treatment with 5-aminolevulinic acid (5-ALA) produced cell line-dependent antiviral effects, with robust inhibition in H1299 cells and selective HCoV sensitivity in MRC-5 cells. These differences correlated with intracellular accumulation of protoporphyrin IX (PPIX), suggesting that cellular metabolic capacity influences antiviral efficacy. In contrast, components of the heme degradation pathway, including heme oxygenase-1 (HO-1), carbon monoxide, and biliverdin, did not exhibit antiviral effects against HCoV-OC43 or HCoV-229E. PPIX and synthetic porphyrin CoPPIX inhibited early infection through direct viral inactivation. Collectively, these findings indicate that antiviral effects of heme metabolism against seasonal human coronaviruses are primarily driven by direct inactivation by PPIX rather than cellular antiviral responses induced by the heme degradation pathway

GeneCapture is developing a novel, point-of-care, rapid AST technique that, when fully automated, can accomplish AST within 2 hours directly from a raw sample, representing a significant advancement in diagnostic capabilities. For this study, a panel of uropathogen isolates was collected from clinical specimens positive for urinary tract infection from various laboratories in the southern US. A selection of these isolates were then spiked into fresh clean-catch urine samples to produce mock samples covering a variety of antibiotic responses. Testing was performed on 20 strains of Escherichia coli, the cause of over 80% of UTIs, and 6 strains each of Klebsiella pneumoniae and Proteus mirabilis, the next most common causes. Each strain was tested against the susceptible, intermediate and resistant (S-I-R) breakpoints of up to 5 appropriate antibiotics including Amoxicillin-Clavulanate, Cefpodoxime, Ciprofloxacin, Nitrofurantoin and Trimethoprim-Sulfamethoxazole. The new method uses a phenotypic assessment of bacterial growth, comparing responses from bacteria treated with antibiotics at S-I-R breakpoint concentrations to untreated controls as the metric for determining susceptibility. Exposure to the drugs for only 90 minutes gave a Categorical Agreement of 98% to gold standard culture results across the resulting 247 bacteria/antibiotic pairs. Testing on separate days of nine of these strains in triplicate gave a reproducibility of 97%. The sequential steps of this novel AST - sample processing, growth, labeling, and counting - can readily be performed on an automated cartridge that is currently under development. The presented work, which took place in a laboratory setting, demonstrates the efficacy of the assay.

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

Respiratory viruses pose a major threat to human health. Environmental virus persistence in respiratory secretions is essential for successful spread, yet studies have focused largely on a select few viruses in non-physiological matrices. Here, we compared the stability of structurally distinct human respiratory viruses, adenovirus, influenza virus, and rhinovirus, in deposited particles of pooled human saliva under indoor-relevant relative humidity (RH) conditions. Adenovirus exhibited <2.2-log10 decay over 6 h regardless of RH, while rhinovirus exhibited significantly greater degradation (>3.8-log10, at 50% and 80% RH). Influenza virus showed minimal decay at 20% and 50% RH but degraded significantly more (3-log10) at 80% RH. To distinguish aspects of human saliva that are key determinants in driving virus decay, we recapitulated saliva composition using the inorganic solute and protein concentrations determined via ion-coupled plasma mass spectrometry and proteomics, respectively. Rhinovirus was significantly more stable in simulated saliva than in human saliva at 50% RH, while influenza virus and adenovirus behaved similarly in both matrices. These findings emphasize virus-specific environmental persistence and highlight limitations of relying on a single virus and particle matrix for defining public health strategies. Ultimately, these insights will contribute to approaches for mitigating environmental transmission of distinct respiratory pathogens.

Antimicrobial resistance (AMR) is a critical One Health challenge at the interface of agriculture, the environment and human health. In Ireland, cattle production systems generate large volumes of manure and slurry, which are routinely applied to land. However, data on the prevalence and environmental persistence of clinically important resistance phenotypes remain limited. This study investigated the occurrence, characterisation and persistence of extended-spectrum {beta}-lactamase (ESBL) and carbapenemase-producing Enterobacterales (CPE), as well as fluoroquinolone-resistant bacteria on Irish cattle farms. A total of 450 pooled bovine faecal samples were collected from farms across 25 counties, screened using selective culture, antimicrobial susceptibility testing, and MALDI-TOF MS identification. Carbapenemase activity in presumptive CPE isolates was confirmed using phenotypic and biochemical assays, including MALDI-TOF MBT STAR analysis to demonstrate carbapenem hydrolysis. In parallel, a controlled persistence study evaluated the survival of phenotypically resistant ESBL and CPE bacteria in bovine slurry under simulated seasonal conditions. Overall, 29.6% of farms were positive for ESBL and/or CPE, with ESBL only resistance most prevalent (13.7%), followed by combined ESBL and CPE phenotypes (7.5%). Fluoroquinolone resistance was frequently detected and commonly co-occurred with {beta}-lactam resistance, indicating multidrug resistance. Escherichia coli was the dominant carrier of both ESBL and carbapenemase phenotypes. Persistence experiments demonstrated sustained phenotypic resistance in slurry for up to nine months under simulated winter conditions and up to three months during simulated summer conditions, with resistance becoming undetectable following extended verification. These findings demonstrate that clinically important AMR phenotypes are embedded within Irish cattle production systems and can persist in agricultural waste streams, highlighting slurry as a significant environmental reservoir with implications for environmental dissemination and One Health surveillance.

Background: Glioblastoma, isocitrate dehydrogenase wildtype, central nervous system World Health Organization grade 4, is the most common primary malignant brain tumor in adults and remains associated with poor prognosis despite aggressive multimodal treatment. The gut-brain axis has been proposed as a modulator of glioblastoma biology, but human data are scarce and the gut mycobiome has not been characterized in these patients. We characterized both bacteria and fungi at diagnosis as well as the intratumoral myeloid compartment and survival. Methods: In a prospective case-control study, we sequenced bacterial (16S rRNA gene) and fungal (internal transcribed spacer, ITS) communities from rectal swabs of 23 patients with newly diagnosed glioblastoma and 23 healthy controls. Additionally, we quantified the intratumoral myeloid compartment by immunofluorescence and performed survival analysis. Results: Both bacterial and fungal communities were more diverse in patients than in controls (Shannon, 16S p < 0.001; ITS p = 0.004), and even more diverse in patients with shorter survival. Community structure itself was unchanged, locating the difference at the level of individual taxa. Fourteen of 218 bacterial and archaeal taxa and four of 102 fungal species differed at nominal, uncorrected p [&le;] 0.05. Members of the Lachnospiraceae and Ruminococcaceae were reduced in glioblastoma patients, while the methanogenic archaeon Methanobrevibacter and Nakaseomyces glabratus (formerly Candida glabrata) were enriched. Survival was also addressed at the tissue level. In the tumor, a higher density of ionized calcium-binding adapter molecule 1-positive myeloid cells was associated with longer survival, independently of age, performance status and extent of resection (hazard ratio 0.82 per percentage point, 95% confidence interval 0.67-0.99). Conclusions: This study characterizes the gut bacteriome and mycobiome of newly diagnosed glioblastoma patients and identifies candidate taxa, among them several butyrate- and propionate producers within the depleted Lachnospiraceae and Ruminococcaceae. Combining these observations with the established role of short-chain fatty acids in microglial maturation, we hypothesize that the myeloid compartment is the interface at which the gut-level and tissue-level findings converge.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

The growth and performance of cattle is dependent on efficient feed conversion, facilitated by the gut microbiome, including both prokaryotic and eukaryotic organisms. Extrinsic factors such as management practices and intrinsic factors like age and genetics can shape these communities. To characterize the relative effects of such factors on the prokaryotic and eukaryotic microbiomes, fresh fecal samples from dairy heifers (n = 8), beef heifers (n = 8), bred beef heifers (n = 8), and adult beef cows (n = 8) were subjected to paired 16S rRNA (V4) and a novel 18S rRNA (V4-V5) metabarcoding assay, and 18S rRNA amplicons were annotated using two databases in parallel. Taxonomic resolution of 18S rRNA amplicons was excellent, with SILVA and EUKARYOME databases each providing better resolution of different groups of organisms. While no differences in eukaryotic richness or alpha diversity were detected, beta diversity differed in all pairwise comparisons except between beef heifers and bred beef heifers. Prokaryotic richness was lower in adult beef cows than the other groups, and beta diversity differed in all pairwise comparisons. Adult beef cows were the most distinct, characterized by replacement of fungal organisms with protozoa and loss of thermophilic bacteria in the eukaryotic and prokaryotic compartments, respectively. Among numerous differentially abundant features, dairy heifers harbored greater abundance of Cooperia oncophora while beef heifers contained greater abundance of Buxtonella sulcata and Mucor. Correlation network analysis revealed multiple significant positive and negative associations between bacterial, fungal, and protozoal features during maturation of the fecal microbiome. Collectively, these data highlight the richness and diversity of both the prokaryotic and eukaryotic compartments of the bovine fecal microbiome, and the influence of management practices and age on both communities. From a technical standpoint, these data demonstrate the ability to resolve fungal, protozoal, and metazoan organisms, including potential parasites and pathogens, with a single metabarcoding assay. They also highlight the nuanced differences between databases and the value of parallel analyses using more than one database.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

Shigella flexneri is an enteric pathogen responsible for causing shigellosis, a type of bacillary dysentery of significant global health concern. The increasing prevalence of antibiotic resistance has driven the search for alternative treatments, including bacteriophage (phage) therapy. However, the effectiveness of orally administered phages for enteric pathogens depends on their ability to survive passage through the gastrointestinal tract. In this study, the ability of bacteriophage Sf14 to maintain infectivity was evaluated under simulated gastrointestinal conditions using a modified INFOGEST 2.0 in vitro digestion model. This model includes salivary, gastric, and intestinal fluids with physiologically relevant compositions of electrolytes, bile salts, and enzymes. Phages were incubated in each simulated phase at standard physiological temperature, then tested for infectivity using quantitative plaque assays. Results show that Sf14 remained stable during the short duration of the oral phase and for the duration of the intestinal phase. By contrast, survival in simulated gastric fluid required a pH of 2.75 or higher, with rapid and substantial loss of infectivity at pH 2.5 and below. These findings demonstrate that while Sf14 is stable in oral and intestinal environments, the gastric phase poses a critical barrier to phage survival. With in vivo fasting gastric pH ranging from 1.5 to 3, encapsulation would enable the most flexible oral phage therapy against enteric pathogens, but ingestion after eating or alongside antacids may be sufficient.

Staphylococcus epidermidis is a common member of the healthy skin microbiome that contributes to barrier function and protection against pathogen colonization. In contrast, Staphylococcus aureus is a major human pathogen and the leading cause of skin and soft tissue infections, owing in part to its robust biofilm-forming capacity and increasing antimicrobial resistance. We previously demonstrated that S. epidermidis cell-free conditioned media (CFCM) inhibit S. aureus biofilm formation by altering bacterial gene expression without affecting growth. Here, we identify the metabolite responsible for this activity. Bioactivity-guided fractionation of S. epidermidis CFCM revealed pyroglutamic acid (PCA) as the active antibiofilm compound. Commercially sourced PCA inhibited S. aureus biofilm formation and reduced adhesion to epithelial cells. In a murine skin infection model, PCA treatment significantly attenuated disease severity, accelerated wound healing, and reduced bacterial burden. Analysis of S. epidermidis CFCM by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry confirmed the presence of PCA and identified it as the bioactive constituent responsible for antibiofilm activity. Together, these findings uncover a previously unrecognized mechanism by which S. epidermidis suppresses S. aureus virulence and highlight the potential of microbiome-derived metabolites as therapeutics for the prevention and treatment of S. aureus skin infections.

Background Group B Streptococcus (GBS) is a leading cause of bacterial sepsis globally. Late-onset (LO) GBS disease is preceded by colonization within the gastrointestinal tract in vulnerable neonates. Previous studies demonstrate that Candida albicans (C. albicans) promotes GBS colonization of host tissues including the bladder and vagina. However, its impact on GBS colonization of the intestinal tract is unknown. Methods We established a new animal model to study GBS and C. albicans co-colonization model in the context of the newborn host to examine C. albicans impact on GBS and further evaluated fluconazole prophylactic treatment to these same outcomes. Physical interactions between organisms in vivo were imaged and GBS gene expression was evaluated. Results C. albicans increased GBS burden in the neonatal gut and GBS was physically colocalized with C. albicans hyphae as evidenced by the colocalization analysis. Fluconazole treatment of co-colonized mice decreased extraintestinal GBS dissemination. C. albicans presence upregulated genes encoding proteins responsible for GBS adhesion and invasion of intestinal epithelial cells. Conclusion C. albicans enhances GBS burden and pathogenicity in the neonatal gut. These findings highlight the need for further investigation into C. albicans as a potential risk factor for GBS colonization and LO disease in human infants.

Purpose: Vancomycin-resistant Gram-positive organisms represent a potential therapeutic challenge in endophthalmitis. To evaluate alternative treatment options, the FDA-approved oxazolidinones linezolid (LZD) and tedizolid (TZD) were tested against a collection of Gram-positive bacterial endophthalmitis isolates. Methods: Minimum inhibitory concentrations (MICs) were determined using epsilometers (Etest strips) for coagulase negative staphylococci (CoNS, n=20), Staphylococcus aureus (n=18), Bacillus species (n=10), Enterococcus species (n=5), and Streptococcus pneumoniae (n=1). Available CLSI and EUCAST breakpoints were applied to determine susceptibility. Results: TZD exhibited a >5-fold reduction in median MIC compared with LZD across all organisms tested (p<0.0001). MIC90 values for CoNS were 2.0 /ml for LZD and 0.25 /ml for TZD. For S. aureus, MIC90 values were 1.5 /ml (LZD) and 0.19 /ml (TZD). For Bacillus spp., MIC90 values were 1.0 /ml (LZD) and 0.25 /ml (TZD). Among Enterococcus species, the MIC range was 0.5-4.0 /ml (LZD) and 0.064-0.75 /ml (TDZ). The single S. pneumoniae strain had a MIC of 0.5 for LZD and 0.064 for TZD. All isolates were susceptible to TZD. One Enterococcus isolate was non-susceptible to LZD; the remaining isolates were susceptible. Conclusions: LZD and TZD demonstrated in vitro activity against Gram-positive bacterial endophthalmitis isolates, with TZD displaying consistently lower MICs than LZD. These findings suggest that oxazolidinones may represent potential alternatives when vancomycin cannot be used, including infections caused by vancomycin-resistant Gram-positive organisms. Given their excellent oral bioavailability, favorable safety profile, and reported anti-virulence effects in certain pathogens, further evaluation of oxazolidinones for the treatment of intraocular infections is warranted.

Histomonosis, caused by Histomonas meleagridis, is a severe disease in poultry with main pathological lesions in ceca and liver, leading to significant animal suffering and economic losses, with no licensed treatments available. While cecal pathology and microbiota disruption have been documented, the metabolic consequences of infection and vaccination remain poorly understood. Here, we used untargeted LC-MS metabolomics to profile cecal tissue and luminal digesta in a controlled vaccination-challenge chicken trial (n=100, 25 birds/group), including uninfected and unvaccinated but challenged controls, together with birds vaccinated either cloacally, or orally via an edible gel following challenge, across five post-challenge timepoints. Results showed that H. meleagridis infection caused extensive, time-dependent disruption of the cecal metabolome, especially at 7-14 days post-infection (DPI). At DPI 14, 325 tissue and 359 digesta metabolites were significantly altered in unvaccinated challenged birds. Infection was associated with depletion of microbiota-related metabolites, including dicarboxylic acids, tryptophan indoles, phytochemical derivatives, bilirubin catabolites, phenolics, vitamins, and secondary bile acids, alongside accumulation of microbial substrates and fermentation products. Changes in the host-associated metabolites indicated mucosal inflammation, epithelial injury, mitochondrial and amino acid metabolic disturbance, oxidative stress, and membrane lipid remodeling. Cloacal vaccination provided greater metabolic protection than oral vaccination, with 78.3% of disrupted metabolites preserved at DPI 14, compared with 60.7% vaccination by oral gel delivery, pointing towards the importance of vaccine take. By DPI 21, cloacally vaccinated birds showed nearly complete metabolic recovery across most pathway groups, whereas gel-vaccinated birds retained broader metabolic disturbance. These findings reveal histomonosis as a disruption of the cecal host-microbiota metabolic interface and provide a metabolite-level framework for future studies of pathogenesis, vaccine development, and prevention strategies.

This study developed a streamlined, sensitive and rapid method for co-detection of low concentrations of Salmonella enterica, Shigella flexneri, Listeria monocytogenes, and pathogenic Escherichia coli from soil. First, autoclaved soil from Iowa with serial dilutions of bacteria to confirm individual species recovery rates on selective agar by qPCR under varying primary enrichment times was used. All pathogens were detected by qPCR of colonies recovered from replicate sterile soils spiked with 10^1 or greater bacteria within 5 hours of enrichment. Shiga toxin expressing E. coli (STEC) and L. monocytogenes were consistently co-recovered in autoclaved and natural soil, pathogen spiking concentration, and secondary enrichment time. S.enterica was consistently recovered at autoclaved and natural concentrations, while S. flexneri detection rates fluctuated. Twenty-three soil samples from Kenyan neighborhoods, with prior evidence of contamination, was compared to qPCR detection at each protocol stage: processing, primary enrichment, secondary enrichment, and selective colony isolation. Detection rates substantially increased for S. enterica, STEC and other pathogenic E. coli after enrichment, with optimal detection rates occurring after secondary enrichment. Testing colonies from selective agar resulted in high false negative rates, possibly reflecting competition from native soil microbiota that was absent in spiked sterile soil experiment. Evaluation of the method with other environmental sample types yielded consistent detection patterns. Evidence suggests a protocol including 24-hour primary enrichment, followed by 24-hour secondary enrichment, and qPCR testing of soil DNA at primary and secondary steps is a reproducible and more expedient method for co-detecting common Enterobacteriaceae and L. monocytogenes pathogens in soil.

Background: Amplicon-based next-generation sequencing is a cornerstone of viral genomic surveillance, but it often produces datasets with highly uneven read coverage due to PCR amplification bias. This leads to excessive data generation and substantial computational overhead for downstream bioinformatic analysis. Results: We evaluated the efficacy of two data reduction strategies on 220 SARS-CoV-2 clinical specimens and 12 wastewater samples: random subsampling with SeqKit and read depth normalization with BBNorm. FASTQ files were reduced to target depths from 10X to 10,000X to compare genome coverage, lineage assignment concordance, and computational resource usage. Both strategies markedly reduced total processing time. For clinical isolates, read depth normalization corrected for amplification bias resulting in highly concordant lineage assignments at a target depth of just 200X. Random subsampling preserved inherent bias but produced reliable assignments at depths over 500X. Reducing target depths below 500X in complex wastewater mixtures severely degraded variant deconvolution accuracy and heavily skewed lineage abundance profiles for both methods. Conclusion: Both random subsampling and read depth normalization successfully reduce computational overhead while preserving key clinical sequencing metrics at appropriate reduction settings. The optimal method depends on the analytical goal, ranging from computationally lightweight random subsampling for rapid lineage assignment, to read depth normalization for generating high-quality consensus sequences. However, aggressive data reduction should be avoided in environmental surveillance, where maintaining a high baseline sequencing depth is critical for accurate mixed-variant deconvolution.

Bacterial cultures entering stationary phase (SP) undergo complex physiological alterations, helping the cells to survive when medium resources are exhausted. The onset of various stress responses underlying SP physiology is generally believed to be due to reactions of individual cells to the environmental cues such as starvation, toxic metabolites, etc. The SP physiological state makes bacteria unsuitable for the replication of most bacteriophages; however, some phages are able to infect and multiply in them. We investigated Escherichia coli phage DH23 growth in SP cultures of E. coli MG1655 infected at different hours post inoculation (ages). Under our conditions the cells enter SP at about 6 h, but the phage replication was possible till 11h and then dropped abruptly by 13h of culture aging, indicating an abrupt physiological change to deeper dormancy during SP. The onset of this phage-inhibiting middle-SP dormancy (MSPD) turned out to be mediated by intercellular communication mediated by the middle-SP signal particles (MSP) which are larger than 100 kDa and contain both RNA and DNA. These MSP are inactivated by RNAse or by DNAse, enabling phage growth in 14h-old cultures and lifts the tolerance of such cultures to kanamycin. Moreover, the RNAse or DNAse treatment makes 24h culture spent media suitable for additional cell growth, and cultures initiated in fresh LB supplemented with RNAse reach an OD600 about 1.5 times higher compared to the untreated control. This indicates that MSP signaling influences cell physiology well before the MSPD onset and helps the population cease growth pre-emptively to save about 1/3 of medium resources to support the viability during SP.