The identification of infection from gastric biopsy samples requires PCR or bacterial cultures. However, it is difficult to culture because it is a fragile bacterium. Next-generation sequencing (NGS) allows direct assessment of the resistome and virulome. Here we describe a new NGS method for studying the resistome and virulome of directly from gastric biopsies, based on enrichment analyses and targeted sequencing of DNA. In all, 19 DNA samples from human gastric biopsies that tested positive for were analyzed. The Agilent SureSelectXT target-enrichment protocol was used with a custom bait library prior to sequencing using the Agilent MagnisDx NGS Library Prep System. NGS sequencing was performed on the Illumina iSeq 100 sequencer using RNA probes for virulence, resistance, and molecular typing genes. The method yielded significant results with a limit of detection of around 1.8e CFU per mL . Mutations in the sequence associated with macrolide resistance and in the quinolone resistance-determining region of associated with levofloxacin resistance were correctly identified. The results of MLST phylogeny analyses performed after target-enrichment were consistent with those obtained via conventional Sanger sequencing. Among the -positive isolates, the gene was detected correctly, and the genotype was determined. In conclusion, our enrichment method enables rapid assessment of the resistome and virulome of directly from fresh gastric biopsies.IMPORTANCE, a bacterium that infects at least 50% of the world population, is often treated by probabilistic antimicrobial therapies due to the lack of antimicrobial resistance data provided by clinical laboratories to clinicians. However, targeted antimicrobial therapies are increasingly recommended to achieve efficient eradication with a limited impact on the gut microbiota and with fewer adverse events for the patient. Recent advancements in next-generation sequencing strategies have opened new opportunities in the diagnosis of infection. The significance of our research is the development of a novel next-generation sequencing strategy based on target-enrichment. This approach enables the identification of the resistome and the virulome of directly from gastric biopsies, providing clinicians with a broad overview of therapeutic options.

Broad-range polymerase chain reaction (BR-PCR) identifies molecular signatures of microorganisms directly from clinical specimens without requiring microbial growth in culture. BR-PCR may be a powerful tool to reveal microbial causes of infectious diseases, but the impact on diagnosis and clinical management has yet to be fully defined. Consequently, the aims here were to investigate how bacterial, fungal, and mycobacterial (AFB) BR-PCR perform compared to microbiology culture methods in detecting microorganisms and to assess clinical utility, defined as the ability of the results to change antimicrobial therapy or treatment duration. Between 2018 and 2021, 348 unique specimens were sent from 327 patients seen within the University of Maryland Medical System (UMMS). Patient charts were reviewed retrospectively. Organisms identified by BR-PCR were compared to bacterial ( = 302), fungal ( = 137), and AFB ( = 111) cultures to determine concordance and were evaluated to determine if they led to a change in clinical management. Agreement in organism(s) reported by BR-PCR and culture was considered concordant for calculating performance data. Sensitivity of BR-PCR compared to concordant culture results was 30.9% for bacteria (25/81; 95% CI: 21.8-41.6%), 18.8% for fungi (3/16; 95% CI: 5.8-43.8%), and 33.3% for AFB (1/3; 95% CI: 5.6-79.8%) detection. The bacterial negative percent agreement of 80.1% (165/206) may reflect antibiotic pretreatment or detection of fastidious organisms. Despite longer turnaround times, BR-PCR results changed clinical care in 6% of cases. Based on these findings herein, the clinical use of BR-PCR would be best utilized when fastidious organisms are suspected, or specimens remain culture negative, but should not replace routine culture methods at this time.IMPORTANCEDetermining infectious etiology can be challenging in patients with chronic presentation and in those receiving empiric therapy. In addition to the standard of care (microbiology cultures), providers can order a broad-range polymerase chain reaction and sequencing (BR-PCR) test to identify microorganisms directly from clinical specimens and independently from culture. While studies have been done from individual hospitals or systems, there is a lack of broadly applicable clinical evidence detailing clinical scenarios in which BR-PCR should be utilized. This study adds to the growing body of literature surrounding BR-PCR clinical usage, examining assay performance and clinical utility of BR-PCR test results. Although BR-PCR and culture had low concordance among organisms identified, it was shown to complement the standard of care for uncommonly isolated and fastidious organisms. Overall, BR-PCR results changed clinical management in 6% of cases, which is similar to other studies that include a broad representation of specimen types.

Coronavirus disease 2019 (COVID-19) has complicated the management of acute respiratory infections and impacted antibiotic use. We assessed the relationship between nirmatrelvir/ritonavir (NMV/r) receipt and outpatient antibiotic prescribing among patients with COVID-19 in a large national health system. We conducted a retrospective cohort study among outpatients enrolled in the Veterans Affairs Healthcare System who had a positive severe acute respiratory syndrome coronavirus 2 test or COVID-19 diagnosis and were eligible for NMV/r treatment between 1 April 2022 and 31 March 2024. NMV/r-treated patients were compared with those who did not receive NMV/r and were considered unexposed until NMV/r was dispensed. We assessed the relationship between NMV/r receipt and being prescribed an outpatient antibiotic in the 30 days after a COVID-19 diagnosis using adjusted Cox proportional hazards regression. We included 302,600 NMV/r-eligible outpatients with COVID-19, of whom 67,649 received NMV/r and 234,951 did not receive NMV/r. NMV/r-treated patients were less likely to receive outpatient antibiotics compared to those who did not receive NMV/r (7.2% [4,901/67,649] vs 9.2% [21,533/234,951], respectively; adjusted hazard ratio [HR] 0.65, 95% CI: 0.63‒0.68). After excluding patients who received an antibiotic prescription upon COVID-19 diagnosis (i.e., likely empiric therapy), this relationship was attenuated (HR: 0.91, 95% CI: 0.87‒0.95). NMV/r-eligible patients with COVID-19 who received NMV/r were 35% less likely to be prescribed outpatient antibiotics compared to patients who did not receive NMV/r, possibly driven by a diminished perceived need for empiric antibiotic therapy. Treatment with NMV/r may reduce unnecessary outpatient antibiotic use. Antibiotics should be reserved for patients with a high suspicion of bacterial co-infection.IMPORTANCEAntimicrobial resistance, driven by the overuse of antibiotics, is a major global health threat. The coronavirus disease 2019 (COVID-19) pandemic has complicated this issue, with antibiotics often prescribed to patients with COVID-19 despite being ineffective against viruses. These practices, typically aimed at preventing or empirically treating rare bacterial co-infections, have raised concerns about accelerating resistance. The antiviral nirmatrelvir/ritonavir (NMV/r), widely used in high-risk patients with COVID-19 to prevent severe illness, offers an opportunity to reassess antibiotic use in patients with respiratory infections. Our study of over 300,000 patients in a national healthcare system found that those treated with NMV/r for COVID-19 were 35% less likely to receive antibiotics than those who did not receive the antiviral. Lower antibiotic use among patients treated with NMV/r may reflect a reduction in unnecessary outpatient antibiotic use. These findings highlight the potential role of antivirals in supporting antibiotic stewardship and addressing a critical public health challenge.

Viability testing for anaerobes is a time-consuming and expensive process, posing challenges for research and public health settings. Here, we present a rapid, economical, and reliable method for testing anaerobe viability using the Geometric Viability Assay (GVA) with as our model, a bacterium known for causing toxin-related systemic and enteric diseases. This method is efficient and cost-effective, requiring one pipette tip per sample, and is compatible with the economical anaerobic jar system. The results align with traditional plate-based assays in terms of colony-forming unit (CFU) measurements. Anaerobic GVA has low technical bias and a dynamic range extending over 5 orders of magnitude. In addition, our method determined the bactericidal activity of antibiotics in a dose-dependent manner, when an antibiotic sensitivity testing (AST) was performed with a panel of four antibiotics (ampicillin, gentamicin, meropenem, and tetracycline). Furthermore, the minimum concentrations for complete bactericidal activity (MBC) of four clinical isolates were determined and the MBC concentration for tetracycline was up to 8× higher than the concentration for complete growth inhibition (MIC). Additional tests involving and demonstrated the generality of our method for other anaerobic species. Beyond viability testing, the GVA measured spore concentrations of various isolates, showing consistency with classical plating methods. Our study confirms that the anaerobic GVA is a valuable tool for rapid, accurate viability screening in anaerobic settings and is compatible with routine assays, such as AST and spore screening. This method enhances the scalability and utility of anaerobic viability-based assays.

Gram-negative bacteria are a major concern for public health, particularly due to the continuous rise of antibiotic resistance. A major factor that helps the development of resistance is the outer membrane that is essential since it acts as a strong permeability barrier to many antibiotics that are effective against other bacteria. In this study, we determine the specific permeability coefficients for various antibiotics in strains, which differ from each other for their porin expressions. We showed that OprD and OpdP porins contribute both to internalize meropenem and biapenem. Using qRT-PCR, we demonstrated that their expression is dependent of the various phases of cellular growth. We were able to show how the OpdP porin is less expressed in exponential growth phases, while it tends to be produced when the bacterial culture enters into the latent phase, in an inversely proportional way compared to the OprD porin. The deletion of the OpdP porin, in the presence of meropenem at concentrations equivalent to the MIC values, contributes to the selection of carbapenem-resistant strains. Therefore, the presence of mutations/deletions of the OpdP porin should receive greater consideration from a clinical point of view as the use of meropenem at nonoptimal concentrations could lead to the appearance of resistance phenotypes.IMPORTANCECarbapenem-resistant strains of are among the major threats to public health. The permeability of the outer membrane for the β-lactam antibiotics is one of the major factors that reduce the activity of the antibiotics. In this study, we measure the low permeability coefficient of the outer membrane to β-lactams. The methodology we develop to determine the permeability can be applied to other antibiotic families and/or pathogens.

Pine wilt disease (PWD), caused by pinewood nematodes, is highly destructive to pine forests in Asia and Europe, including Korean white pine (). The microbiome in the needles and trunk of spp. are recognized to play key roles in resistance against PWD. However, the role of root and soil microbiomes in the resistance remains unclear. This study compares bacterial and fungal communities in the root endosphere, rhizosphere soil, and bulk soil of diseased versus healthy . Results showed that PWD increased the α-diversity of fungi in rhizosphere soil but did not affect the microbial diversity in the root endosphere or bulk soil. The composition of bacterial and fungal communities in rhizosphere and bulk soils was significantly altered by PWD. Specifically, the relative abundance of Planctomycetes decreased, and the relative abundance of Tremellomycetes increased, while Agaricomycetes decreased in both rhizosphere and bulk soils after infestation with PWD, respectively. Relative abundances of Chloroflexi and Verrucomicrobia increased, while Proteobacteria decreased in bulk soil following PWD. Relative abundances of Leotiomycetes and Eurotiomycetes increased in the rhizosphere soil and bulk soil following PWD, respectively. Furthermore, with the host plant infestation by PWD, the relative abundance of ectomycorrhizal fungi decreases, while that of saprotrophic fungi increases in both rhizosphere and bulk soils. Our results revealed that PWD significantly affects the soil microbiomes of , with varying impacts across different plant-soil compartments. This study provides insights into how root and soil microbiomes respond to PWD, enhancing our understanding of the disease's ecological consequences.IMPORTANCEThe belowground microbiome is often sensitive to infection of forest diseases and is also recognized as a potential reservoir for selection of microbial agents against PWD. Our study demonstrates that the dynamics of belowground microbiome following natural infection of PWD are compartment and taxa specific, with varying degrees of responses in both diversity and composition of bacterial or fungal communities across the root endosphere, rhizosphere soil, and bulk soil. The results highlight the importance of utilizing appropriate plant-soil compartments and microbial taxa to understand the ecological consequences of the destructive PWD.

is a bacterial pathogen that causes soft rot disease in many plant species worldwide, including temperate, subtropical, and tropical regions. This bacterium employs the type III secretion system (T3SS) to manipulate host immune responses. Although cyclic-di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, negatively regulates the expression of T3SS genes in , the underlying mechanism remains unclear. In this study, we identified a potential transcriptional regulator, CdeR, which regulates the T3SS involving c-di-GMP. Through transposon mutagenesis, we discovered that deletion of in a mutant background restored T3SS gene expression. GcpD is a diguanylate cyclase responsible for c-di-GMP synthesis, and its deletion led to high T3SS gene expression due to low c-di-GMP. Further analysis revealed that, in the mutant background, CdeR regulates T3SS by manipulating intracellular c-di-GMP levels, involving another diguanylate cyclase, GcpL, whose expression is upregulated by CdeR. Additionally, we found that removing helical regions within the Helix-Turn-Helix DNA-binding domain of CdeR completely disrupted its regulation of the T3SS, underscoring the essential role of this domain in CdeR's functional activity. This study is the first to identify CdeR as a potential transcriptional regulator involved in T3SS regulation. Our findings provide significant insights into the regulatory mechanisms of T3SS and highlight the complex interactions between bacterial second messengers and transcriptional regulators in pathogenic bacteria.IMPORTANCEBacterial pathogens, such as , must adapt to diverse environmental and host conditions by utilizing intricate regulatory networks to control virulence. This study identifies CdeR, a novel transcriptional regulator, as a crucial factor in modulating the expression of the type III secretion system (T3SS), a key virulence mechanism. Importantly, we show that CdeR operates in a cyclic-di-GMP (c-di-GMP)-dependent manner, linking this second messenger to T3SS regulation in for the first time. Our findings reveal a sophisticated interaction between c-di-GMP signaling and transcriptional regulation, highlighting how these systems collectively drive bacterial virulence. This work advances our understanding of bacterial pathogenesis and opens new avenues for developing targeted strategies to mitigate soft rot disease in crops, potentially improving agricultural productivity and plant health.

Clinical studies of vaccines generally require collections of microbial isolates obtained from various body sites over multiple years. Further, large microbe collections are needed for research due to increasing appreciation for the phenotypic and genotypic diversity among a single microbial species. However, large collections are not generally available due to method limitations. We show a new way to create, recover, and manage microbe collections in 96- or 384-well plates using 50% glycerol at -20°C. Fifty percent glycerol remains liquid at -20°C and permits only the chosen isolates to be reliably sampled without first thawing all other isolates in the plate. Consequently, the glycerol sampling allows integration of microbe collection, labeling, recovery, and storage steps. Creating a microbe collection as an array in microplates reduces physical storage space by 6- or 23-fold with 96-well or 384-well plates, respectively. The array permits direct analysis of the collection with high-throughput assay systems. Further, we show that could be stored for 11 years as a microplate array. Standardized microbe arrays created in microplates with the new method could be easily distributed for studies of microbial structures, genetic diversity, antibiotics, and microbe-host interactions. This integrated method suggests how automated microbe management systems can be created.IMPORTANCEEpidemiologic and microbiology studies require large microbial collections, and the use of microplates could facilitate the creation and management of these collections. However, recovering individual isolates from microplates is manual and tedious. In this study, we demonstrate a simple method for recovering a selected individual isolate from a microplate at -20°C using 50% glycerol. Additionally, we found that could be revived for more than 10 years in microplates. This new method of recovering microbes from frozen microplates could greatly streamline many large-scale epidemiologic studies, particularly those related to pneumococcal vaccine studies. This new method may ultimately automate the collection, management, and storage of microbial isolates.

The efficient carbon source utilization in dynamic environments, including anoxic subsurface contaminated by aromatic compounds is a challenge for anaerobic bacteria such as strain FRC-32. The aim of this study was to elucidate the metabolic pathways employed by FRC-32 during anaerobic benzoate oxidation in the presence of acetate, a key intermediate in anaerobic organic matter degradation, to predict carbon source transport and utilization strategies. Simultaneous carbon source oxidation and monoauxic growth were observed in FRC-32 cultures grown on 1 mM benzoate + 5 mM acetate, 1 mM benzoate + 2 mM acetate, and 2 mM acetate spiked with 1 mM benzoate. Sequential carbon source oxidation and diauxic growth were observed only in cultures grown on 5 mM acetate spiked with 1 mM benzoate. Benzoate accumulation in FRC-32 whole cell lysates indicated that intracellular benzoate transport occurred during benzoate oxidation in the presence of acetate. Expression analyses of putative benzoate transporter BenK and protein-ligand binding affinity prediction suggested BenK's specificity for transporting benzoate. Relative expression levels for the gene encoding BenK, and the genes involved in the benzoyl-CoA pathway, were significantly higher in cultures grown on both benzoate and acetate than in cultures grown on acetate as sole carbon source, indicating that intracellular benzoate accumulation facilitated the regulation of . Our results demonstrated that FRC-32 can perform differential benzoate oxidation in the presence of acetate, by either simultaneous or sequential carbon source oxidation, which indicated the metabolic plasticity of FRC-32 in response to varying carbon source availability.IMPORTANCEThe contamination of anaerobic subsurface environments by crude oil derivatives including aromatic compounds is a global concern due to the persistence and toxicity of these pollutants. Anaerobic bacteria play a crucial role in the degradation of aromatic hydrocarbons under anoxic conditions; however, the potential mechanisms involved in metabolic regulation of aromatic degradation pathways are not well understood. This study contributed to elucidating how strain FRC-32 efficiently utilizes benzoate as a carbon source in the presence of acetate. Findings of intracellular benzoate accumulation and regulation of key genes associated with benzoate oxidation contributed to the understanding of FRC-32's aromatic degradation pathways, provided significant insights into potential mechanisms that modulate anaerobic benzoate oxidation in the presence of the energetically favorable carbon source acetate, and indicated metabolic strategies of FRC-32 in response to dynamic environmental conditions.

Amazonian soil microbial communities are known to be altered by land-use change. However, attempts to understand these impacts have focused on broader community alterations or the response of specific microbial groups. Here, we recovered and characterized 69 soil bacterial and archaeal metagenome-assembled genomes (MAGs) from three forests and three pastures of the Eastern Brazilian Amazon and evaluated the impacts of land conversion on their genomic features. Pasture MAGs had significantly higher GC content (64.9% vs 60.2%), genome size (4.0 vs 3.1 Mbp), and number of coding sequences (4,058 vs 3,306) compared to forest genomes. Taxonomically, MAGs belonged to eight phyla; however, most (90%) had low similarity to previously known species, indicating potentially novel taxa at multiple levels. We also observed that the functional profiles associated with biogeochemical cycling and carbohydrate-active enzyme genes were impacted by forest conversion, with pasture MAGs exhibiting a notably higher number of both gene groups. Together, these data constitute the largest single-sourced genomic data set from upland soils of the Brazilian Amazon to date and increase the known MAG richness in these soils by 78%. Our data, therefore, not only add to a neglected yet emerging field but, importantly, highlight that land-use change has drastic impacts on the genomic characteristics and functional traits of dominant soil microbes.IMPORTANCEThe Brazilian Amazon is facing unprecedented threats, including increasing deforestation and degradation, which together impact half of the original forest area. Soil microorganisms are sensitive indicators of land-use change, linked to a rise in microbial methane emissions and antibiotic-resistance genes in the Amazon. However, most Amazonian soil microbes remain unknown, and little attention has been given to their genomes. Using sequencing and bioinformatics, we recovered and characterized 69 soil bacterial and archaeal genomes (metagenome-assembled genomes). These abundant members of the microbial communities diverged across forests and pastures in terms of taxonomic and functional traits. Forest conversion favors organisms with specific genomic features - increased GC content, genome size, and gene number - selecting for microorganisms that can thrive under altered conditions. Our paper helps us understand the intricate relationships between microbes and the environment, which are crucial pieces of information for comprehensive soil health assessments and future policy formulation.

Marek's disease virus (MDV) is an avian alphaherpesvirus associated with Marek's disease, an immunosuppressive and lymphoproliferative disease in chickens. The DNA sensing pathway mediates innate immune defense against infection by many DNA-containing pathogens, while viruses have evolved multiple strategies to evade the host immune response to survive in host cells. This study found that ectopic expression of MDV protein kinase US3 inhibited beta interferon (IFN-β) and interleukin-6 (IL-6) production induced by interferon-stimulatory and viral DNA. US3 was further shown to abolish the nuclear factor κB (NF-κB) activation. The US3 kinase activity was indispensable for its inhibitory function, as the kinase-dead US3 mutant (US3K220A) did not inhibit NF-κB activation. Further studies showed that US3 interacted with the Rel homology domains of the NF-κB subunits p65 and p50, which phosphorylated these transcription factors and blocked their nuclear translocation. Finally, US3 deficiency promoted IFN-β and IL-6 production, resulting in reduced viral replication and lower MDV-specific lesion incidence during MDV infection in chickens. Altogether, these findings reveal a novel mechanism for MDV to evade host antiviral immunity.IMPORTANCEMarek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes an economically important disease affecting the health and welfare of poultry worldwide. Whereas human herpesviruses have been shown to evolve various strategies to inhibit the DNA sensing signaling for the evasion of the host's innate immunity, little is known regarding the mechanism for MDV to regulate this pathway. In this study, MDV US3 protein kinase was demonstrated to inhibit the activation of NF-κB in the DNA sensing pathway via binding to the Rel homology domains of the NF-κB subunits p65 and p50, which hyperphosphorylated these transcription factors and abolished their nuclear translocation. This is an important finding toward a better understanding of the functions of avian alphaherpesviruses encoded US3 protein kinase.

Bacteria that adhere to epithelial cells, form intracellular bacterial communities (IBCs), or transition to filamentous forms are referred to as morphotype-positive bacteria. () with this morphotype plays a critical role in urinary tract infections (UTIs), yet its impact on urine culture outcomes and molecular epidemiological characteristics remains unclear. In this retrospective study, we assessed the effect of bacterial morphotype on urine culture results and investigated the molecular differences between strains with and without this morphotype, using PCR and whole genome sequencing (WGS). We observed that with the morphotype-positive phenotype frequently appeared in urine sediments, leading to fewer colony-forming units (CFUs) in culture and contributing to false-negative results. However, vortexing the urine samples significantly increased CFUs, improving culture sensitivity. Additionally, with the positive morphotype carried more adhesion-related virulence genes (VGs), with the majority belonging to phylogenetic group B2. Whole genome sequencing further revealed a broader array of virulence genes in these strains. Our findings demonstrate that vortexing is an effective method to enhance urine culture positivity by releasing intracellular bacteria, and that morphotype-positive harbors a diverse set of virulence factors, indicating their potential high pathogenicity. These results highlight the importance of detecting bacterial morphotypes in urine samples for accurate UTI diagnosis and emphasize the need for increased attention to these highly virulent strains.

Urinary tract infections (UTIs) are the most common bacterial infections in humans. They are mainly caused by and other Enterobacterales for which increasing resistance to antibiotics and in particular to β-lactams is extensively reported. The detection of β-lactam resistance phenotypes is currently time-consuming (18 h). Hence, most treatments are given without any results of antibiotic susceptibility testing and may involve broad-spectrum antibiotics. A biochemical diagnostic test has been developed to rapidly evaluate the production of β-lactamases (and consequently the β-lactam resistance) from cultures (10 and 10 CFU/mL) of Gram-positive and Gram-negative isolates representative of bacterial species as a source of UTIs ( = 112). It relies on a centrifugation step after a 90 min preliminary culture and the detection of β-lactamase activity with nitrocefin as substrate using a special filter. Overall, the test gave a positive result for 87.6% of the tested resistant strains with a bacterial load of 10 CFU/mL, and a positive result for 100% of tested extended-spectrum β-lactamases and for carbapenemase producers at the same load. This cost-effective test can be performed in any laboratory and ultimately shall be tested at the patient side and at the general practitioner. Its turn-around-time to get results is less than 2 h. After further improvements, the results obtained with this proof-of-concept test suggest that its use may contribute to rapidly guide the treatment of non-complicated UTIs and, therefore, limit the use of broad-spectrum antibiotics and the emergence of antibiotic resistance.

We aim to define the optimal white cell count threshold that correlates with a clinically significant urinary tract infection, as there is insufficient data exploring this in the adult population. We conducted a retrospective cohort study at the Royal Melbourne Hospital analyzing urine samples collected over 6 months in 2022. Urinary tract infection (UTI) was defined as the presence of symptoms (dysuria, urgency, frequency, flank pain, or loin to groin pain) and isolation of a uropathogen with colony counts greater than 10 CFU/L. The relationship between urinary white cell count, growth of uropathogen, and likelihood of UTI was estimated using locally weighted scatterplot smoothing. Of the 6,328 samples included, at a urinary white cell count of less than 10 per microliter, 38% grew a microorganism, while 7% of the total grew a uropathogen. For our sub-analysis part C, at the same WBC count, 2% of samples fulfilled our criteria for UTI. The optimal WBC range for identifying a UTI was 30-50 WBC/µL, demonstrating the most pragmatic balance of sensitivity (92.3%-94.9%, 95% CI 85.9-98.1) and specificity (41.6-47.2, 95% CI 38.6%-50.3%) for a UTI. A lower-than-expected number of UTIs were confirmed in our study, likely due to inappropriate indications for culture collection and the types of urine specimens collected. However, the optimal white cell cutoff for identifying a UTI was higher than the defined pyuria cutoff of 10 WBC/µL. Utilizing a higher urinary WBC cutoff could improve urine culture processing protocols.

is a primary cariogenic pathogen involved in dental biofilm formation, a major virulence factor in the development of dental caries. In , the competence-stimulating peptide (CSP), encoded by , plays a critical role in environmental stress response, growth regulation, and virulence expression. In this study, we performed transcriptome analysis to investigate the role of SMU.1147, a unique core gene in , in biological pathways related to transport, defense responses, and environmental sensing. The deletion of SMU.1147 led to the upregulation of genes involved in carbohydrate uptake and metabolism, particularly phosphotransferase system (PTS) transporters, thereby enhancing the sugar transport capacity. However, despite increased sugar uptake, the mutant strain did not show significant changes in growth rate or ATP production and displayed slightly reduced organic acid production. Additionally, the mutant exhibited significantly reduced cell viability after an 8-h incubation compared to the parental strain. Notably, genes associated with CSP-dependent signal transduction and stress defense, such as , , , , and , were downregulated in the mutant strain. Furthermore, stress-related genes, including , , and , were significantly downregulated, suggesting compromised protein quality control and oxidative stress responses. Our findings suggest that SMU.1147 plays a critical role in regulating peptide-mediated signaling, metabolic coordination, and environmental adaptation in , positioning it as a key integrator of the metabolic and stress response networks that are essential for pathogenicity and survival.

Our previous studies constructed a strategic system for testing antibiotics against specific resistance mechanisms using and . However, it lacked resistance mechanisms specifically expressed only in species. In this study, we constructed this system using . In-frame deletion, site-directed mutagenesis, and plasmid transformation were used to generate genetically engineered strains with various resistance mechanisms from two fully susceptible strains. Antimicrobial susceptibility testing was used to test the efficacy of antibiotics against these strains in vitro. A total of 31 engineered strains with various antimicrobial resistance mechanisms from KPA888 and ATCC 27853 were constructed, and the same antibiotic resistance mechanism showed a similar effect on the MICs of the two strains. Compared to the parental strains, the engineered strains lacking porin OprD or lacking the regulator genes of efflux pumps all showed a ≥4-fold increase on the MICs of some of the 19 antibiotics tested. Mechanisms due to GyrA/ParC mutations and β-lactamases also contributed to their corresponding resistance as previously published. The strains constructed in this study possess well-defined resistance mechanisms and can be used to screen and evaluate the effectiveness of antibiotics against specific resistance mechanisms in . Building upon our previous studies on and , this strategic system, including a panel, has been expanded to cover almost all the important antibiotic resistance mechanisms of gram-negative bacteria that are in urgent need of new antibiotics.IMPORTANCEIn this study, an antibiotic assessment system for was developed, and the system can be expanded to include other key pathogens and resistance mechanisms. This system offers several benefits: (i) compound design: aid in the development of compounds that can bypass or counteract resistance mechanisms, leading to more effective treatments against specific resistant strains; (ii) combination therapies: facilitate the exploration of combination therapies, where multiple antibiotics may work synergistically to overcome resistance and enhance treatment efficacy; and (iii) targeted treatments: enable healthcare providers to prescribe more targeted treatments, reducing unnecessary antibiotic use and helping to slow the spread of antibiotic resistance. In summary, this system could streamline the development process, reduce costs, increase the success rate of new antibiotics, and help prevent and control antimicrobial resistance.

Post-chikungunya viral arthritis may persist for months to years after infection and is characterized by relapsing and remitting symptoms. This study investigates the relationship between autoantibodies and chikungunya arthritis severity, providing insights into arthritis pathogenesis. We assessed arthritis measures in a cohort of serologically confirmed chikungunya cases from Colombia between 2019 and 2021 ( = 144). We measured arthritis disease severity, flare intensity, pain, and disability, then plasma antibody levels of rheumatoid factor IgM, anti-cyclic citrullinated peptide (CCP), anti-citrullinated α-enolase peptide 1 (CEP-1), anti-nuclear antibody (ANA), anti-citrullinated vimentin (Sa), and immunoglobulins produced in response to chikungunya, Zika and Mayaro. Finally, we examined the correlation between the arthritis measures with the titers of antibodies hypothesized to play a potential role in arthritis pathogenesis. Cases were characterized by moderate disease severity (Disease Activity Score-28 mean, 3.66 ± 1.23) in current arthritis flare with moderate intensity (Flare Score, 25.42 ± 12.38), moderate pain (61.47 ± 27.23 on visual analog scale 0-100), and some disability (Health Assessment Questionnaire 0.77 ± 0.58). After Bonferroni adjustment, there were no statistically significant correlations between the levels of antibodies and arthritis measures. Weak correlations between rheumatoid factor IgM with arthritis severity and pain ( < 0.01) and anti-CEP1 with disability ( < 0.05) were observed when unadjusted for multiple comparisons. The data suggest that autoantibodies, such as RF, anti-CCP, and anti-CEP-1, do not correlate with post-chikungunya arthritis disease severity, thus unlikely to significantly contribute to pathogenesis. Exposure to other arboviral infections was not related to worse post-chikungunya arthritis. This suggests that other pathways for arthritis disease pathogenesis should be examined.IMPORTANCEThis cohort study describes the correlation between levels of autoantibodies, viral antibodies, and arthritis outcomes, suggesting that autoantibodies known to play an important role in other autoimmune diseases do not correlate with chikungunya arthritis relapse disease severity and are unlikely to contribute significantly to arthritis pathogenesis. This suggests that other pathways for arthritis disease pathogenesis should be examined to identify diagnostic and prognostic markers of alphaviral arthritis.

The plaque reduction neutralization test (PRNT) has been used widely for the detection and quantitation of yellow fever (YF) virus-neutralizing antibodies in human serum; however, it is labor-intensive and challenging to adapt to high-throughput clinical testing needed for vaccine licensure. Here, we describe the development and validation of a new Vero cell-based YF microneutralization (MN) assay, with immunostaining readout, for the detection and quantification of YF virus-neutralizing antibodies in human serum. Comparison of neutralizing antibody titers measured with the YF MN assay versus the historical YF PRNT, based on a 50% reduction in plaque counts (PRNT), demonstrated 100% serostatus agreement at a titer of 10 (1/dil) in participants with a history of YF vaccination. For validation, intra-assay precision (repeatability), intermediate precision, dilutional accuracy, linearity, specificity, upper limit of quantitation (ULOQ), and lower limit of quantitation (LLOQ) were assessed. The YF MN assay demonstrated suitable intra-assay precision (repeatability) and intermediate precision of 36% and 54%, respectively, with an ULOQ of 10,240. At the lower end of detection, repeatability and intermediate precision were 38% and 41%, respectively, with a LLOQ of 10 (1/dil). Suitable dilutional accuracy, linearity, and specificity across orthoflaviviruses (dengue virus, Japanese encephalitis virus, and Zika virus) and serum matrices (hemolytic, lipemic, and icteric) were also demonstrated. Overall, these promising results led the Center for Biologics Evaluation and Research to confirm the suitability of the validated YF MN assay for the detection and quantification of YF virus-neutralizing antibodies.

The microbiome inhabiting animal skin plays a crucial role in host fitness by influencing both the composition and function of microbial communities. Environmental factors, including climate, significantly impact microbial diversity and the functional attributes of these communities. However, it remains unclear how specific climatic factors affect amphibian skin microbial composition, community function, and the relationship between these two aspects. Understanding these effects is particularly important because amphibians are poikilotherms and, thus, more susceptible to temperature fluctuations. Here, we investigated the skin microbiome of the rhacophorid tree frog across different climatic regimes using 16S rRNA gene sequencing. Skin swab samples were collected from nine populations of adults in the Guangxi region, China. The majority of the core microbiota were found to belong to the genus . Our findings indicate that microbial community diversity, composition, and function are associated with changes in climatic conditions. Specifically, the taxonomic and functional diversity of the skin microbiome increased in response to higher climate variability, particularly in temperature fluctuations. Additionally, the functional traits of microbial communities changed in parallel with shifts in community diversity and composition. The significant correlations of the functional redundancy index with climatic factors suggest that environmental filtering driven by climate change impacts microbial community functional stability. These results highlight the critical influence of climatic factors on amphibian skin microbiomes and offer new insights into how microbial composition and function contribute to host adaptation in varying environmental conditions.IMPORTANCEThis study is important in understanding the association between climate variability, microbial diversity, and host adaptation in amphibians, which are particularly vulnerable to environmental changes due to their poikilothermic nature. Amphibians rely on their skin microbiome for key functions like disease resistance, yet little is known about how climate fluctuations impact these microbial communities. By analyzing the microbiome of across different climatic regimes, our analysis reveals that warmer climates could reduce the microbial diversity and community functional redundancy, indicating the functional stability of skin microbiome could be susceptible to climate variability, particularly in hosts adapted to relatively cooler conditions. These findings highlight the potential ecological consequences of climate change and emphasize the need to integrate microbiome health into amphibian conservation strategies.

Zinc (Zn) is essential for all bacteria, but excessive Zn levels are toxic. Bacteria maintain zinc homeostasis through regulators, such as Zur, AdcR, and ZntR. is a significant pathogen causing acute serositis in ducks and other birds. In this study, we identified a homolog of ZntR, a regulator for zinc homeostasis, and demonstrated its contribution to the pathogenicity of . Deletion of makes the bacteria hypersensitive to excess Zn but not to other metals like manganese (Mn), copper (Cu), cobalt (Co), and nickel (Ni). Deletion of also leads to intracellular zinc accumulation but not of other metals. Additionally, compared to the wild type, the deletion of increases resistance to oxidants hydrogen peroxide (HO) and sodium hypochlorite (NaOCl), respectively. The deletion of causes significant changes in transcriptional and protein expression levels, revealing 35 genes with potential zinc metabolism functions. Among them, , which is inhibited by ZntR, is required for zinc transport and resistance to oxidative stress. Finally, deletion of leads to attenuation of colonization in ducklings. In summary, ZntR is a crucial regulator for zinc homeostasis and contributes to the pathogenicity of .IMPORTANCEZinc homeostasis plays a critical role in the environmental adaptability of bacteria. is a significant pathogen in poultry with the potential to encounter zinc-deficient or zinc-excess environment. The mechanism of zinc homeostasis in this bacterium remains largely unexplored. In this study, we showed that the transcriptional regulator ZntR of is critical for zinc homeostasis by altering the transcription and expression of a number of genes. Importantly, ZntR inhibits the transcription of zinc transporter ZupT and contributes to colonization in . The results are significant for understanding zinc homeostasis and the pathogenic mechanisms in .

Dietary fibers play a significant role in shaping the composition and function of microbial communities in the human colon. Our understanding of the specific chemical traits of dietary fibers that influence microbial diversity, interactions, and function remains limited. Toward filling this knowledge gap, we developed a novel measure, termed Chemical Subunits and Linkages (CheSL) Shannon diversity, to characterize the effects of carbohydrate complexity on human fecal bacteria cultured under controlled, continuous flow conditions using media that systematically varied in carbohydrate composition. Our analysis revealed that CheSL Shannon diversity demonstrated a strong Pearson correlation with microbial richness across multiple fecal samples and study designs. Additionally, we observed that microbial communities in media with higher CheSL Shannon diversity scores exhibited greater peptide utilization and more connected, reproducible structures in computationally inferred microbial interaction networks. Taken together, these findings demonstrate that CheSL Shannon diversity can be a useful tool to quantify the effects of carbohydrate complexity on microbial diversity, metabolic potential, and interactions. Furthermore, our work highlights how robust and stable community data can be generated by engineering media composition and structure. These studies provide a valuable framework for future research on microbial community interactions and their potential impacts on host health.IMPORTANCEFor the human adult gut microbiota, higher microbial diversity strongly correlates with positive health outcomes. This correlation is likely due to increased community resilience that results from functional redundancy that can occur within diverse communities. While previous studies have shown that dietary fibers influence microbiota composition and function, we lack a complete mechanistic understanding of how differences in the composition of fibers are likely to functionally impact microbiota diversity. To address this need, we developed Chemical Subunits and Linkages Shannon diversity, a novel measure that describes carbohydrate complexity. Using this measure, we were able to correlate changes in carbohydrate complexity with alterations in microbial diversity and interspecies interactions. Overall, these analyses provide new perspectives on dietary optimization strategies to improve human health.