Microbiology spectrum最新文献

Staphylococcus aureus exhibits remarkable tolerance to antibiotic stress, facilitated by a complex network of cellular responses and metabolism controlled by numerous gene expression patterns that can be rapidly remodeled. This tolerance can lead to treatment failure and the emergence of antibiotic resistance. However, the expression patterns of these genes caused by metabolic alterations driving antibiotic tolerance remain poorly understood. Our objective was to identify the core metabolic genes involved in the development of tolerance. Using proteomic analysis and gene complementation assays, we found that seven tolerant isolates shared similar protein expression profiles and mechanisms for tolerance. Seven metabolic genes, including NWMN_0676-0677, opuCB, gltD, adhE, clpP, and rarA, were confirmed as major contributors to tolerance. Notably, these genes were linked to elevated intracellular reactive oxygen species (ROS) levels in drug-tolerant strains. Treatment with ROS scavengers increased the sensitivity of these strains to antibiotics. These results demonstrate that changes in the expression of metabolic genes play a crucial role in the development of drug tolerance, and the regulation of ROS metabolism may be central to the broader metabolic alterations in drug-tolerant bacteria.

Importance: S. aureus poses a major public health threat due to its remarkable ability to develop antibiotic tolerance, often leading to treatment failure and resistance emergence. This study provides critical insights into the underlying metabolic mechanisms. Proteomic analysis revealed that different genetic mutations in tolerant isolates converged on similar gene expression changes, which directly impacted the tolerance phenotype. Notably, the tolerant strains exhibited elevated intracellular reactive oxygen species (ROS) levels, and ROS scavenger treatment increased their antibiotic susceptibility. These findings demonstrate that shifts in core metabolic gene expression are pivotal for S. aureus to withstand antibiotic stress, with ROS metabolism regulation being a central component of the broader metabolic adaptations conferring drug tolerance. Understanding these metabolic underpinnings is crucial for developing more effective treatments against persistent, tolerant S. aureus infections. The identified metabolic targets and ROS-modulating approaches offer promising strategies to combat escalating antibiotic resistance.

Endophytes play essential roles in protecting plants against abiotic stresses. However, whether and how they enhance waterlogging resilience in mulberry through changes in host-associated microbiota and metabolites remains unclear. Here, an endophytic bacterium strain HLG18, with plant growth promotion potential, was selected and identified as Pseudomonas koreensis HLG18. Genome analysis revealed that it possessed multiple genes involved in phytohormone biosynthesis, mineral dissolution, and stress adaptation. Greenhouse experiments consistently indicated that P. koreensis HLG18 significantly stimulated mulberry growth under waterlogging stress, accompanied by enhanced antioxidant enzyme activities and osmoprotectants. Amplicon sequencing revealed distinct endospheric microbiome profiles following HLG18 treatment, with notable changes in genera, such as Rhizorhapis, Bacillus, Caulobacter, and Rhodococcus. Meanwhile, soil potassium, phosphorus, and iron levels also differed. Correlation analyses indicated that the relative abundances of Rhizorhapis, Bacillus, Caulobacter, and Rhodococcus were significantly associated with soil properties and mulberry performance. Concurrently, metabolomic profiling revealed distinct metabolic signatures between treatments, including higher levels of stress-related metabolites (e.g., L-arginine, L-isoleucine) and differences in key metabolic pathways, such as tryptophan and purine metabolism. Overall, this study uncovers that P. koreensis HLG18 is linked to altered microenvironmental features and host metabolic patterns under waterlogging, providing new insights into endophyte-assisted plant stress adaptation.IMPORTANCEWaterlogging severely threatens the riparian zone of the Three Gorges Reservoir in China, causing extensive plant mortality and hindering restoration efforts. Mulberry is a promising candidate for ecological restoration, yet its growth is severely constrained under such conditions. Endophytes have emerged as key mediators of plant stress tolerance; however, their potential role in supporting mulberry adaptation to waterlogging in riparian zones remains largely unexplored. Our results show that the endophytic bacterium Pseudomonas koreensis HLG18 significantly promotes mulberry growth and enhances waterlogging tolerance. HLG18 inoculation is associated with distinct shifts in the host's endophytic microbiome, soil properties, and metabolite profiles, suggesting potential links to mulberry performance under waterlogging. Our findings highlight the potential of endophytes as bioinoculants to enhance mulberry waterlogging tolerance for ecological restoration in fragile riparian ecosystems and provide a valuable reference for harnessing beneficial microbial resources in sustainable agriculture under waterlogged conditions.

Metallo-β-lactamases (MBLs) hydrolyze a broad range of β-lactams, including carbapenems. VIM-28, an MBL identified in Pseudomonas aeruginosa, is an H224L/S228R variant of VIM-1 and H224L variant of VIM-4. Compared with VIM-26 (R228S), VIM-28 displayed decreased K_m (12.5 for VIM-28 vs 513 μM for VIM-26; 9.66 vs 150 μM) and increased k_cat/K_m(15.3 vs 1.81 μM^-1s^-1; 28.6 vs 5.89 μM^-1s^-1) for ampicillin and cephalothin, respectively. VIM-1, which has a His in position 224 and Ser in position 228, displayed intermediate kinetic values (K_m 215 and 77.0 μM; k_cat/K_m 2.63 and 8.61 μM^-1s^-1) for ampicillin and cephalothin, respectively, indicating that the presence of a positively charged residue at either position 224 or 228 enhanced substrate interactions. The combined L224H/R228S substitutions in VIM-1 increased the catalytic efficiency of the enzyme for ceftazidime by more than one order of magnitude. These kinetic trends were consistent with the minimum inhibitory concentration (MIC) data, with an eightfold increase in ceftazidime MIC for VIM-1-producing cells. Moreover, relative MIC assay showed that VIM-26 (R228S)-producing cells were more refractory to the addition of chelators than cells producing VIM-28, whereas VIM-4 (L224H)-producing cells showed reduced resistance, suggesting that the residues at positions 224 and 228 influence the metal-binding affinity of the enzyme. Differential scanning fluorimetry assay revealed that the R228S substitution increased the melting temperature of the enzyme, whereas the L224H substitution reduced its thermal stability. VIM-28 exhibited high catalytic efficiency for substrates other than ceftazidime, and the H224L substitution conferred higher zinc-binding affinity and thermal stability compared with VIM-4.IMPORTANCEβ-Lactam-resistant bacteria, especially carbapenem-resistant strains, pose a major global health threat, often through metallo-β-lactamases (MBLs). To anticipate resistance evolution, we characterized VIM-28, a variant of the widespread VIM-1/VIM-4-type enzymes, focusing on the roles of two variable L10 loop residues. Substitutions at positions 224 and 228 strongly affected substrate specificity, enzyme stability, and zinc affinity. Arg228 was important for carbapenem recognition, while combined substitutions at positions 224 and 228 could enhance activity toward ceftazidime. Notably, the R228S substitution improved zinc binding and thermal stability, supporting enzyme function under zinc-limited host conditions. These findings reveal mechanisms driving MBL diversity and highlight evolutionary strategies sustaining antibiotic resistance.

Searching publicly archived sequence data for emerging aquatic animal pathogens is a powerful but challenging approach for increasing our understanding of newly identified or poorly characterized organisms. However, searching for target sequences within the sequence read archive (SRA) database requires significant time, data storage, and computing power, limiting its accessibility. Utilizing a new database, Logan, we undertook a meta-analysis of SRA data sets to investigate the presence of an emerging virus, Macrobrachium rosenbergii golda virus (MrGV). MrGV was first characterized in M. rosenbergii larvae in 2020, associated with repeated mass mortalities in Bangladesh hatcheries. MrGV has since been detected in two separate reports from the Jiangsu Province of central, coastal China, and during a larval mortality event in India. Here, we discovered that MrGV is present in two additional provinces in southern China, Thailand, and India. We also found molecular evidence to confirm, as previously suspected, the circulation of the virus within Southern Asian populations of M. rosenbergii as far back as 2011, and that, based on relative abundance, MrGV is mostly associated with larvae. Overall, the identification of MrGV sequences in data sets that are largely unpublished within the scientific literature has provided novel insights into the pathogen's biology, including the prevalence of MrGV globally and the life stages of prawns that should be screened to prevent the spread of the virus. This work illustrates how mining public sequencing data, supported by databases like Logan and standardized metadata submissions, can support cost-effective epidemiological studies of pathogens and strengthen One Health approaches to global disease monitoring.IMPORTANCESearching for target sequences within the sequence read archive (SRA) database requires significant time, data storage, and computing power, limiting its accessibility. This study demonstrates how the Logan database, constructed from an SRA-wide genome assembly, can be utilized to rapidly and efficiently find target sequences within the SRA database, expanding the use of these publicly available data sets outside of their original intended purposes. Here, we searched for an emerging virus, Macrobrachium rosenbergii golda virus, in prawns to reveal insights into its geographic distribution, host range, and relative abundance, without the need for additional sampling. We demonstrate how, with careful application of this approach, alongside improvements in metadata quality and accessibility, sequencing data sets can be used to uncover critical insights into pathogen biology. This type of data mining could add otherwise unknown data to epidemiological studies of emerging, re-emerging, and rare pathogens globally, allowing the determination of the spread of agents within and between populations.

Cholera, caused by Vibrio cholerae, remains a significant diarrheal disease, especially in coastal regions of developing countries. In Malaysia, cholera is largely non-endemic except in Sabah, which has had recurrent outbreaks accounting for ∼75% of national cases between 2004 and 2014. To understand the origin and transmission of the disease, we sequenced the genomes of clinical isolates of V. cholerae O1 collected during an outbreak in 2019 and 2020. Genotypic analyses revealed that all Sabah isolates were atypical El Tor biotype harboring Classical CTX prophage elements. In particular, the strains carried two tandem CTX prophage copies in chromosome 2 and three tandem RS1 sequences on chromosome 1, including a Classical type rstR, which is atypical for canonical El Tor. Genome comparisons revealed conserved seventh-pandemic genomic islands (VSP1 and VSP2) and variably arranged biotype-specific loci, suggesting pandemic-lineage markers and mobile elements linked to environmental adaptation. Phylogenetic reconstruction placed the Sabah strains within wave 2 of the seventh-pandemic clade, forming a distinct subclade with two genotypes, consistent with regional endemicity over the last few decades. Although wave 3 strains have largely replaced wave 2 globally, an established population of wave 2 strains in Southeast Asia suggests that they are more resilient than previously thought.IMPORTANCEThis study addresses a critical public health concern by investigating the genomic characteristics of Vibrio cholerae O1 strains responsible for recurrent cholera outbreaks in Sabah, Malaysia. Although cholera is largely non-endemic in most parts of Malaysia, Sabah remains an exception, contributing disproportionately to national case counts. By sequencing clinical isolations from the 2019 and 2020 outbreaks, this research provides essential insights into the origins, evolutionary dynamics, and transmission patterns of V. cholerae in a region with persistent endemicity. These findings underscore the importance of continuous genomic surveillance in geographically distinct settings and offer valuable data for informing public health strategies aimed at cholera control and prevention in Southeast Asia.

The tumor immune microenvironment and intratumoral microbiota play critical roles in cancer progression and immunotherapy response, yet their integrated functions in stomach adenocarcinoma (STAD) are not well understood. This study conducted a multi-omics analysis of transcriptomic and microbiome data from 348 patients with STAD. Using the ImmuCellAI algorithm, immune cell infiltration (ICI) was estimated, and non-negative matrix factorization classified samples into three immune subtypes (INC-1, INC-2, and INC-3). Differential expression analysis identified immune-related signature genes enriched in immune signaling pathways. Tumor mutational burden, microsatellite instability, immune checkpoint gene expression, and drug sensitivity were compared across subtypes. Microbiome clustering identified three subtypes (MC-1, MC-2, and MC-3), with associations to immune infiltration and microbial composition. The immune subtypes showed distinct patterns of ICI, clinical stage, and gene expression, with differentially expressed genes enriched in immune and tumor-related pathways. Microbiome subtypes exhibited unique diversity metrics and associations with the immune microenvironment. Integration of immune and microbial data improved immune checkpoint blockade (ICB) prediction, with genera like Staphylococcus and Ralstonia correlating with immune genes such as CD22, VIPR2, and FLT3. These findings provide insights into ICB response and support more precise immunotherapy strategies for STAD.IMPORTANCEDeciphering the interactions between the tumor immune microenvironment and the intratumoral microbiota is crucial for advancing precision immunotherapy in stomach adenocarcinoma (STAD). In this study, we present an integrative multi-omics framework that stratifies patients into distinct immune and microbial subtypes, uncovering their associations with immunogenomic profiles, immune cell infiltration patterns, and clinical features. Notably, we identify specific microbial genera correlated with immune-related gene expression and immune checkpoint blockade responsiveness. These findings provide novel insights into the immune-microbiome axis in STAD and underscore the potential of integrative multi-omics approaches to enhance patient stratification and guide more effective immunotherapeutic strategies.

Rhinoviruses (RVs) and enteroviruses (EVs) are important respiratory pathogens. Although numerous molecular assays have been developed for detection of RVs and EVs, their genetic similarities pose challenges for molecular differentiation. In this study, we described a real-time nested reverse transcription (RT)-PCR assay using SYBR green and RV- and EV-specific reverse primers to differentially detect RVs and EVs. The primers were designed so that the numbers and locations of mismatches should be the most adequate for objective viruses and the least adequate for opposite viruses using all EV and RV sequences in GenBank. The assay was validated using nasopharyngeal swab specimens from pediatric patients who have fever and/or respiratory symptoms at Keio University Hospital from November 2021 to January 2023 and tested positive for Human Rhinovirus/Enterovirus by the FilmArray Respiratory Panel 2.1. The species and serotypes were identified by analyzing sequences of PCR products using the BLAST program. The results of the present RT-PCR assay and the BLAST analysis were completely consistent with each other. Furthermore, the current assay revealed the cases of dual infections of RV and EV. No significant differences were observed in patient demographics or clinical courses among viral species. The assay presented here may be the most suitable for routine diagnosis and surveillance of RV and EV infections.

Importance: We describe a real-time nested reverse transcription-PCR assay that enables us to differentially detect rhinoviruses and enteroviruses. Differential diagnosis of rhinovirus and enterovirus infections has not been succeeded because of their genetic diversities and similarities. We resolved this problem by using specific PCR primers that were designed by in silico analysis of all rhinovirus and enterovirus sequences obtained from GenBank. The developed method was validated by applying to more than 100 nasopharyngeal swab specimens from pediatric patients in Keio University Hospital in Japan and analyzing with the BLAST algorithm. The assay may be suitable for routine diagnosis and surveillance of rhinovirus and enterovirus infections.