mSystems®

The development and severity of metabolic dysfunction-associated steatotic liver disease (MASLD) in children are closely related to alterations of gut microbiota. This study aims to investigate changes in the gut microbiota signature and microbial metabolites in children with MASLD. We collected fecal samples from children and adolescents aged 6–16 years, and the presence of MASLD was diagnosed by ultrasound. We performed 16S ribosomal DNA sequencing and targeted metabolomics in 36 and 25 subjects, consisting of healthy controls, children with obesity, and children with MASLD. The α-diversity was significantly lower in children with obesity and MASLD compared with healthy controls. Linear discriminant analysis of effect size analysis identified Anaerostipes and A. hadrus as the top biomarkers differentiating the obesity group from the MASLD group. In MASLD patients with high alanine aminotransferase values (≥50 U/L for boys and 44 U/L for girls), we observed a decrease in the gut microbiota health index. MASLD patients with high shear wave elastography ( E ) values (≥6.2 kPa) showed an increased abundance of Ruminococcus torques , which was positively correlated with the levels of deoxycholic acid (DCA) and E values. Importantly, the mediation analysis identified positive associations between R. torques and clinical indicators of MASLD that were mediated by DCA. Overall, our study suggests that gut microbiota and metabolites are significantly altered in children with MASLD, and targeting R. torques may offer potential benefits for disease management. IMPORTANCE This study investigated alterations in the gut microbiota signature and microbial metabolites in children with metabolic dysfunction-associated steatotic liver disease (MASLD). We found that an increased abundance of Ruminococcus torques was associated with increased levels of deoxycholic acid and the progression of MASLD, suggesting that R. torques may serve as a novel clinical target in pediatric MASLD.

Inflammatory bowel disease (IBD) is an immune-mediated gastrointestinal disorder that significantly impacts the life quality of people worldwide. Genetic factors play crucial roles in the development of IBD. Tas2rs, members of the G protein-coupled receptor (GPCR) superfamily, are known for their roles in bitter taste perception. However, Tas2rs have also been identified in the gut, where they help sense luminal contents and regulate gastrointestinal hormones. Periodontal Tas2r105 has been shown to modulate innate immunity by interacting with metabolites produced by oral bacteria. In this study, we observed increased Tas2r105 in the inflammatory colons induced by dextran sulfate sodium salt (DSS). We also noted that α-gustducin, the α-subunit of GPCRs, is present in the intestine, and that α-gustducin knockout mice exhibit aggravated colitis. Based on these findings, we hypothesize that Tas2r105 may play a role in immune regulation during IBD pathogenesis. To test this hypothesis, we used Tas2r105 knockout (KO) mice in a colitis model. Our results show that the KO mice had significantly shorter colon length, more severe colon inflammation, and greater destruction of the gut barrier compared with control mice. We also observed increased recruitment of macrophages to the lamina propria mucosa in the KO mice. Microbiological analysis revealed a significant increase in Proteobacteria and Bacteroidota , with a concomitant decrease in Firmicutes after Tas2r105 knockout. Metabolomic analysis showed a significant reduction in lysophosphatidylethanolamine (LPE) levels in the KO mice, which is known to have anti-inflammatory effects. Based on these findings, we speculate that Tas2r105 may help protect the intestine from inflammation by influencing the gut microbiota composition and LPE production. IMPORTANCE Increased Tas2r105 was detected in the inflamed colon of mice outside the tongue. Tas2r105 deletion aggravated mice colon colitis. Tas2r105 might alleviate mice colitis by downregulating the Proteobacteria and the Bacteroidota abundance in the colon. Lysophosphatidylethanolamine (LPE) might be the key metabolite that mediated the intestinal protection of Tas2r105.

Fosfomycin represents a last-line reserve antibiotic for the treatment of infections caused by multidrug-resistant (MDR) bacteria. Nevertheless, the advent of plasmid-mediated fosfomycin resistance among bacteria from humans and food animals incurs great concern. This study reports the detection and genomic portrait of the plasmid-mediated fosfomycin resistance gene, fosA4 , amid Escherichia coli from wild birds co-harboring plasmid-mediated tigecycline resistance gene , tet (X4), and colistin resistance gene, mcr-1 . A total of 100 samples from fecal droppings of wild birds in the urban parks in Faisalabad, Pakistan were subjected for the isolation and characterization of fosfomycin-resistant E. coli . The fosA4 gene was identified in 11 (11%) of the E. coli isolates, and all exhibited an MDR phenotype. Genome sequencing confirmed that all the fosA4 -positive isolates also co-harbored the mobile tigecycline resistance tet (X4) gene on a large MDR IncFII plasmid. One isolate PKF8 belonging to ST48 also co-carried the colistin resistance gene mcr-1 on the IncHI2 plasmid. To the extent of our knowledge, this is the first discovery of E. coli isolates in wild birds co-harboring the mcr-1 , fosA4 , and tet (X4) genes. The emergence of these pivotal antimicrobial resistance genes in wild birds native to South Asia with their close association to humans and animals is alarming. Our findings highlight the urgent need for further surveillance of bacterial resistance to last-resort antibiotics in the clinics, animal farming, and environment with the One Health approach. IMPORTANCE The global spread of the plasmid-mediated fosfomycin resistance gene fosA4 bearing Escherichia coli strains incurs a public health concern. However, research focusing on the pervasiveness of fosA4 -positive isolates in wild birds is still rare, and to the best of our knowledge, this is the first documentation from South Asia highlighting the concurrent presence of the fosA4 , mcr-1 , and tet (X4) genes within E. coli isolates recovered from fecal samples of wild birds in Pakistan. This co-existence of ARGs along with phylogenetic analysis revealed that MDR plasmids carried by E. coli isolates have the ability to spread horizontally between wild birds, food animals, and humans. Co-existence of fosA4 , tet (X4), and mcr-1 -carrying plasmids is worrying and warrants further investigation.

Klebsiella pneumoniae carbapenemases (KPCs) have evolved into over 245 distinct variants, with over one-third of variants exhibiting reduced susceptibility to ceftazidime-avibactam, while the underlying selection mechanisms remain elusive. To better elucidate these resistant phenotypes, we cloned 33 clinically described KPC variants (from KPC-2 to KPC-36) and 8 artificially created variants into a common plasmid vector and assessed their impact on β-lactam susceptibility. Strains expressing KPC-14, KPC-28, and KPC-31 exhibited increased resistance to ceftazidime and ceftazidime-avibactam but decreased resistance to carbapenems. We further studied the catalytic mechanism of β-lactam hydrolysis by KPC-4, KPC-14, KPC-15, KPC-16, KPC-21, KPC-25, KPC-28, KPC-31, and the ancestral KPC-2 and KPC-3 enzymes. Antimicrobial susceptibility test, enzyme kinetics, and molecular modeling revealed diverse selective pressures, including but not limited to aztreonam and ceftriaxone, driving KPC evolution, with ceftazidime playing a central role. Substitutions within the KPC hydrolytic active sites notably reduced the inhibitory effect of avibactam on KPC, demonstrated by isothermal titration calorimetry analysis, resulting in enhanced hydrolysis of ceftazidime by enzyme kinetics. This highlights that avibactam may serve as an additional driving force in KPC evolution. IMPORTANCE The rapid evolution of KPC carbapenemases, including resistance to ceftazidime-avibactam, threatens the effectiveness of last-resort antibiotics against Klebsiella pneumoniae infections, necessitating understanding of of the underlying selection pressures. This study investigates the evolutionary mechanisms driving KPC diversification and resistance to ceftazidime-avibactam, providing crucial information for developing effective strategies to combat carbapenem-resistant Klebsiella pneumoniae (CRKP) infections and preserve antibiotic efficacy.

Selenoproteins are a special group of proteins with major roles in cellular antioxidant defense. They contain the 21st amino acid selenocysteine (Sec) in the active sites, which is encoded by an in-frame UGA codon. Compared to eukaryotes, identification of selenoprotein genes in bacteria remains challenging due to the absence of an effective strategy for distinguishing the Sec-encoding UGA codon from a normal stop signal. In this study, we have developed a deep learning-based algorithm, deep-Sep, for quickly and precisely identifying selenoprotein genes in bacterial genomic sequences. This algorithm uses a Transformer-based neural network architecture to construct an optimal model for detecting Sec-encoding UGA codons and a homology search-based strategy to remove additional false positives. During the training and testing stages, deep-Sep has demonstrated commendable performance, including an F 1 score of 0.939 and an area under the receiver operating characteristic curve of 0.987. Furthermore, when applied to 20 bacterial genomes as independent test data sets, deep-Sep exhibited remarkable capability in identifying both known and new selenoprotein genes, which significantly outperforms the existing state-of-the-art method. Our algorithm has proved to be a powerful tool for comprehensively characterizing selenoprotein genes in bacterial genomes, which should not only assist in accurate annotation of selenoprotein genes in genome sequencing projects but also provide new insights for a deeper understanding of the roles of selenium in bacteria. IMPORTANCE Selenium is an essential micronutrient present in selenoproteins in the form of Sec, which is a rare amino acid encoded by the opal stop codon UGA. Identification of all selenoproteins is of vital importance for investigating the functions of selenium in nature. Previous strategies for predicting selenoprotein genes mainly relied on the identification of a special cis -acting Sec insertion sequence (SECIS) element within mRNAs. However, due to the complexity and variability of SECIS elements, recognition of all selenoprotein genes in bacteria is still a major challenge in the annotation of bacterial genomes. We have developed a deep learning-based algorithm to predict selenoprotein genes in bacterial genomic sequences, which demonstrates superior performance compared to currently available methods. This algorithm can be utilized in either web-based or local (standalone) modes, serving as a promising tool for identifying the complete set of selenoprotein genes in bacteria.

Acute gastrointestinal injury (AGI) is known for its poor long-term prognosis and the associated increase in mortality among intensive care unit (ICU) patients. As the role of the gut microbiome and resistome in AGI remains unclear, the present study aimed to explore the possible associations between dysbacteriosis and in-hospital mortality in ICU patients with gastrointestinal dysfunction. Fecal samples were collected from a prospective cohort of 210 ICU patients with AGI, and shotgun metagenomic sequencing was used to determine the taxonomic composition of gut microbiota and the differences of antibiotic resistance genes (ARGs) between the Death and Survival groups. Compared to the Survival group, patients in the Death group shifted from strict anaerobes to facultative anaerobes in the fecal microbial community, with more Klebsiella but less Prevotella . The co-occurrence patterns revealed that more ARG subtypes were enriched in microbial taxa in the Death group, especially for Clostridium and Methanobrevibacter . Furthermore, the ARG type had large area under the curve (AUCs) in receiver operating characteristic for predicting the disease severity, and a combined gut microbiota-ARG subtype classifiers showed better performance than either of them. Thus, comparative metagenome-associated analysis can help to obtain valuable information about gut microbiota and gene coding for antibiotic resistance in AGI patients. IMPORTANCE A metagenomic-related strategy was conducted to obtain a highly valuable resource to improve understanding of intestinal microbiota dysbiosis and antibiotic resistance genes (ARGs) profiles. The results indicate that intestinal microbiota, including Klebsiella and Prevotella , changed dramatically in intensive care unit (ICU) patients with acute gastrointestinal injury (AGI). Due to longer ICU stays and receiving more antibiotic treatment, the types and correlations of ARGs in the Death group were significantly higher than those in the Survival group. The findings of this study are expected to expand our knowledge of gut microbiota and resistome profiles reflecting gastrointestinal status, accelerate the identification of disease biomarkers, and provide new insights into the prevention and treatment of AGI-related diseases.

The global crisis of antimicrobial resistance poses a major threat to human health, underscoring the urgency of developing new antibacterial strategies. Antimicrobial peptides (AMPs) are promising alternatives to antibiotic therapy, yet potential microbial resistance is of great concern. Resistance is often accompanied by fitness costs, which may in turn influence the spread of drug-resistant bacteria and their susceptibility to other antimicrobial agents. Herein, we investigate the evolutionary trajectory of bacterial resistance to antibiotics and AMPs in Escherichia coli , and evaluate the fitness costs and collateral sensitivity of drug-resistant strains. We reveal that E. coli develops resistance to antibiotics, particularly ciprofloxacin and kanamycin, at a notably faster rate than to AMPs. Moreover, antibiotic-evolved strains exhibit slightly higher fitness costs than AMP-evolved bacteria, primarily manifested in reduced bacterial growth and swimming motility. Notably, we demonstrate that trimethoprim-resistant E. coli, with mutations in thyA gene, displays enhanced susceptibility to pexiganan, as evidenced by both in vitro and in vivo studies. Overall, our findings shed new insights for the clinical deployment of AMPs and propose innovative therapeutic strategies for combating antibiotic-resistant bacterial infections. IMPORTANCE The global spread of antimicrobial resistance necessitates the development of innovative anti-infective strategies. Antimicrobial peptides (AMPs) represent promising alternatives in the post-antibiotic era. By monitoring the evolutionary trajectory of bacterial resistance to eight antibiotics and ten AMPs in Escherichia coli , we demonstrate that E. coli exhibits slower emergence of resistance against AMPs compared with antibiotics. Additionally, these antibiotic-resistant strains incur significant fitness costs, particularly in bacterial growth and motility. Most importantly, we find that some antibiotic-resistant strains show collateral sensitivity to specific AMPs in both in vitro and animal infection models, which is conducive to accelerating the development of AMP-based antibacterial treatment.

Industrial anaerobic digestion (AD) represents a relevant energy source beyond today’s fossil fuels, wherein organic matter is recycled to methane gas via an intricate and complex microbial food web. Despite its potential, anaerobic reactors often undergo process instability over time, which is frequently caused by substrate composition perturbations, making the system unreliable for stable energy production. To ensure the reliability of AD technologies, it is crucial to identify microbial and system responses to better understand the effect of such perturbations and ultimately detect signatures indicative of process failure. Here, we investigate the effect of the microalgal organic loading rate (OLR) on the fermentation product profile, microbiome dynamics, and disruption/recovery of major microbial metabolisms. Reactors subjected to low- and high-OLR disturbances were operated and monitored for fermentation products and biogas production over time, while microbial responses were investigated via 16S rRNA gene amplicon data, shotgun metagenomics, and metagenome-centric metaproteomics. Both low- and high-ORL fed systems encountered a sudden decline in methane production during OLR disturbances, followed by a recovery of the methanogenic activity within the microbiome. In the high-OLR disturbances, system failure triggered an upregulation of hydrolytic enzymes, an accumulation of fermentation products, and a shift in the methanogenic population from hydrogenotrophic to acetoclastic methanogens, with the latter being essential for recovery of the system after collapse. IMPORTANCE Anaerobic digestion (AD) with microalgae holds great potential for sustainable energy production, but process instability caused by substrate disturbances remains a significant barrier. This study highlights the importance of understanding the microbial dynamics and system responses during organic loading rate perturbations. By identifying key shifts in microbial populations and enzyme activity, particularly the transition from hydrogenotrophic to acetoclastic methanogens during recovery, this research provides critical insights for improving AD system stability and can contribute to optimizing microalgae-based AD processes for more reliable and efficient methane production.

Clostridioides difficile is a gram-positive spore-forming pathogen that commonly causes diarrheal infections in the developed world. Although C. difficile is a genetically diverse species, certain ribotypes are overrepresented in human infections, and it is unclear if metabolic adaptations are essential for the emergence of these epidemic ribotypes. To identify ribotype-specific metabolic differences, we therefore tested carbon substrate utilization by 88 C . difficile isolates and looked for differences in growth between 22 ribotypes. As expected, C. difficile was capable of growing on a variety of carbon substrates. Further, C. difficile strains clustered by phylogenetic relationship and displayed ribotype-specific and clade-specific metabolic capabilities. Surprisingly, we observed that two emerging lineages, ribotypes 023 and 255, have divergent metabolic phenotypes. In addition, although C. difficile Clade 5 is the most evolutionary distant clade and often detected in animals, it displayed robust growth on simple sugars similar to Clades 1–4. Altogether, our results corroborate the generalist metabolic strategy of C. difficile but also demonstrate lineage-specific metabolic capabilities. IMPORTANCE The gut pathogen Clostridioides difficile utilizes a wide range of carbon sources. Microbial communities can be rationally designed to combat C. difficile by depleting its preferred nutrients in the gut. However, C. difficile is genetically diverse with hundreds of identified ribotypes, and most of its metabolic studies were performed with lab-adapted strains. To identify ribotype-specific metabolic differences, we profiled carbon metabolism by a myriad of C. difficile clinical isolates. While the metabolic capabilities of these isolates clustered by their genetic lineage, we observed surprising metabolic divergence between two emerging lineages. We also found that genetically newer and older clades grew to a similar level on simple sugars, which contrasts with recent findings that newer clades experienced positive selection on genes involved in simple sugar metabolism. Altogether, our results underscore the importance of considering the metabolic diversity of pathogens in the study of their evolution and the rational design of therapeutic interventions.

The incidence of colorectal cancer (CRC) has been increasing in recent decades. Current methods for CRC screening have their own drawbacks, thus there is an urgent need to identify the key microbes that drive the development of CRC for wider application in the early detection and prevention of CRC. To address this issue, we performed fecal microbiome analysis by high-throughput sequencing of 16S rRNA gene combined with blood biochemical indicators in patients with CRC stages I, II, III, and IV, healthy people, and patients with polyps. Fecal microbiota of patients with CRC was disturbed, as evidenced by significantly reduced α-diversity in patients with CRC stage IV and markedly different β-diversity. The random forest model identified the top 25 genera from 174 training data, resulting in a diagnostic accuracy of 87.95%. Further, by combining with differential genera analysis, we screened out 11 biomarkers that significantly changed in different groups. Peptostreptococcus , Parvimonas , Shewanella , Oscillibacter , Eggerthella , and Gemella associated with the development of CRC were significantly enriched, while Fenollaria , Staphylococcus , Ezakiella , Finegoldia, and Neisseria associated with the remission of CRC were significantly suppressed in patients with CRC. Importantly, carcinoembryonic antigen (CEA) was significantly correlated with these 11 microbial biomarkers, and carbohydrate antigen 19-9 (CA 19-9) was markedly correlated with Oscillibacter . Notably, co-occurrence network analysis at the genus level exhibited that the microbial co-occurrence network of CRC IV was the most complex and stable. These results suggested that CEA, CA 19-9 and 11 microbial biomarkers may be co-biomarkers for the disease occurrence and development, and non-invasive diagnosis of CRC. IMPORTANCE Identifying the key microbes that drive the development of colorectal cancer (CRC) has been important in this field. We delved into the research on the association between CRC and fecal microbiota in this study, providing a detailed analysis of the characteristics of fecal microbiota during the transition from normal intestine to polyps to cancer. Fecal bacterial biomarkers and blood biochemical indicators may be co-biomarkers in the development of CRC.

Colon cancer (CC) is one of the most common cancers globally, which is associated with the gut microbiota intimately. In current research, exploring the complex interaction between microbiomes and CC is a hotspot. However, the information on microbiomes in most previous studies is based on fecal, which does not fully display the microbial environment of CC. Herein, we collected mucosal and tissue samples from both the tumor and normal regions of 19 CC patients and clarified the composition of mucosal microbiota by 16S rRNA and metagenomic sequencing. Additionally, RNA-Seq was also conducted to identify the different expression genes between tumor and normal tissue samples. We revealed significantly different microbial community structures and expression profiles to CC. Depending on correlation analysis, we demonstrated that 1,472 genes were significantly correlated with CC tumor microbiota. Our study reveals a significant enrichment of Campylobacter jejuni in the mucosa of CC, which correlates with bile secretion. Additionally, we observe a negative correlation between C. jejuni and immune cells CD4+ Tem and mast cells. Finally, we discovered that metabolic bacterial endosymbiont of Bathymodiolus sp., Bacillus wiedmannii , and Mycobacterium tuberculosis had a significant survival value for CC, which was ignored by previous research. Overall, our study expands the understanding of the complex interplay between microbiota and CC and provides new targets for the treatment of CC. IMPORTANCE This study contributes to our understanding of the interaction between microbiota and colon cancer (CC). By examining mucosal and tissue samples rather than solely relying on fecal samples, we have uncovered previously unknown aspects of CC-associated microbiota. Our findings reveal distinct microbial community structures and gene expression profiles correlated with CC progression. Notably, the enrichment of Campylobacter jejuni in CC mucosa, linked to bile secretion, underscores potential mechanisms in CC pathogenesis. Additionally, observed correlations between microbial taxa and immune cell populations offer new avenues for immunotherapy research in CC. Importantly, this study introduces CC-associated microbiota with survival implications for CC, expanding therapeutic targets beyond conventional strategies. By elucidating these correlations, our study not only contributes to uncovering the potential role of gut microbiota in colon cancer but also establishes a foundation for mechanistic studies of gut microbiota in colon cancer, emphasizing the broader impact of microbiota research on cancer biology.

During infection, Histoplasma capsulatum yeasts interact with a variety of phagocytic cells, where macrophages represent an important niche for long-term intracellular fungal survival and replication. In the phagosomes of macrophages, a hostile environment where most microorganisms are killed, Histoplasma not only survives but overcomes several biological challenges and proliferates intracellularly. To better understand the characteristics of intracellular Histoplasma and the phagosomal environment, a metabolomic platform was used to analyze Histoplasma yeasts cultured on different carbon sources and yeasts extracted from macrophages, identifying metabolites associated with pathogenesis. Metabolomic results of in vitro -grown yeasts were further characterized with available transcriptomic data, informing underlying gene expression patterns in response to contrasting milieus. These approaches revealed that Histoplasma yeasts, unlike many other yeasts, do not ferment sugars to ethanol, and, when cultivated on glycolytic versus gluconeogenic carbon sources, produce distinct metabolomes with altered intracellular amino acid, lipid, and sugar contents. Furthermore, analysis of Histoplasma -inoculated media illustrated that Histoplasma secretes mannitol and anthranilates. Lastly, a comparison of the metabolomes derived from in vitro cultivation versus intracellular growth highlighted leucine and cysteine/cystine as amino acids, which may serve as sources of carbon, nitrogen, and sulfur to yeasts within macrophages. These results detail metabolites linked to Histoplasma metabolism during macrophage infection, identifying potential candidates to target for novel histoplasmosis therapeutics. IMPORTANCE Intracellular pathogens reside within host cells, surviving against innate immune responses while exploiting host resources to proliferate. Understanding the mechanisms that underlie their survival and proliferation is critical for developing novel treatments and therapeutics for the diseases these pathogens cause. While Histoplasma is a unique example of a true intra-phagosomal pathogen, insights into its pathogenesis may still inform the study of other intracellular pathogens.

Caviid gammaherpesvirus 1 (CaGHV-1), formerly known as the guinea pig herpes-like virus, is an oncogenic gammaherpesvirus with a sequenced genome but an as-yet uncharacterized transcriptome. Using nanopore long-read RNA sequencing, we annotated the CaGHV-1 genome and constructed a detailed transcriptomic atlas. Our findings reveal diverse viral mRNAs and non-coding RNAs, along with mapped promoter elements for each viral gene. We demonstrated that the CaGHV-1 RTA lytic cycle transcription factor activates its own promoter, similar to Kaposi’s sarcoma-associated herpesvirus (KSHV), and that the CaGHV-1 ORF50 promoter responds to RTA proteins from other gammaherpesviruses, highlighting the evolutionary conservation of RTA-mediated transcriptional mechanisms. Additionally, our analysis uncovered extensive transcriptional overlap within the viral genome, suggesting a role in regulating global gene expression. Given its tumorigenic properties, broad host range, and non-human pathogenicity, this work establishes CaGHV-1 as a promising small animal model for investigating human gammaherpesvirus pathogenesis. IMPORTANCE The molecular underpinnings of gammaherpesvirus pathogenesis remain poorly understood, partly due to limited animal models. This study provides the first comprehensive transcriptomic atlas of CaGHV-1, highlighting both coding and non-coding RNAs and revealing regulatory elements that drive viral gene expression. Functional studies of the CaGHV-1 RTA transcription factor demonstrated its ability to self-activate and cross-activate promoters from homologous gammaherpesviruses, reflecting conserved mechanisms of transcriptional control. These findings solidify CaGHV-1 as a unique and versatile small animal model, offering new opportunities to investigate gammaherpesvirus replication, transcriptional regulation, and tumorigenesis in a controlled experimental system.

Toxin–antitoxin (TA) systems consist of toxic proteins and their inhibitors, and were originally shown to ensure plasmid maintenance in bacterial populations. Over time, however, TA systems have also been identified on bacterial chromosomes, raising questions about their roles unrelated to plasmid stability. Among the eight currently recognized types of TA systems, type II has been the most extensively investigated. Type II systems are often found in pathogenic bacterial species, including staphylococci. Staphylococcus aureus , a notorious human pathogen, harbors multiple type II TA systems, both plasmid- and chromosome-encoded, while their potential relation to virulence remains to be addressed. Here, we investigate the co-occurrence of TA systems and antibiotic resistance (AR) determinants in S. aureus , focusing on the potential negative impact of type II toxin RNases on antibiotic resistance. We considered both well-characterized and newly characterized TA loci of S. aureus . Our findings demonstrate a relationship between TA systems and AR determinants, wherein TA systems negatively affect antibiotic resistance. Due to substantial selective pressure, the migration of TA systems from plasmids to chromosomes results in their inactivation. This observation may be an important factor shaping the spread and evolution of both TA systems and AR determinants in bacteria. We exemplify this phenomenon in detail using the well-known PemIK-Sa1 system and a newly identified SCCmec-related PemIK-Sa6 system characterized in this study. IMPORTANCE Toxin–antitoxin (TA) systems are entities unique to bacteria. They are involved in the maintenance of mobile genetic elements (MGEs), regulation of gene expression and bacterial virulence. Staphylococcus aureus is a dangerous human pathogen with increasing antibiotic resistance (AR). The maintenance and dissemination of AR determinants is often driven by MGEs, which link AR and TA systems. Our study identified a negative correlation between TA systems and AR determinants in S. aureus . Furthermore, we have shown that the expression of a toxic component of an exemplary TA system negatively affects antibiotic resistance. We argue that in particular strains, a selective pressure maintains either the TA system or AR determinant. Alternatively, TA systems are inactivated by mutations when present together with AR determinants to maintain the functionality of the latter. Our observations uncover an important factor shaping the spread and evolution of both TA systems and AR determinants in bacteria, which is especially relevant to pathogenic species.

The pancreas tumor microbiota may influence tumor microenvironment and influence survival in early-stage pancreatic ductal adenocarcinoma (PDAC); however, current studies are limited and small. We investigated the relationship of tumor microbiota to survival in 201 surgically resected patients with localized PDAC (Stages I–II), from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. We characterized the tumor microbiome using RNA-sequencing data. We examined the association of the tumor microbiome with overall survival (OS), via meta-analysis with the Cox PH model. A microbial risk score (MRS) was calculated from the OS-associated microbiota. We further explored whether the OS-associated microbiota is related to host tumor immune infiltration. PDAC tumor microbiome α- and β-diversities were not associated with OS; however, 11 bacterial species, including species of Gammaproteobacteria , confirmed by extensive resampling, were significantly associated with OS (all Q < 0.05). The MRS summarizing these bacteria was related to a threefold change in OS (hazard ratio = 2.96 per standard deviation change in the MRS, 95% confidence interval = 2.26–3.86). This result was consistent across the two cohorts and in stratified analyses by adjuvant therapy (chemotherapy/radiation). Identified microbiota and the MRS also exhibited association with memory B cells and naïve CD4 ⁺ T cells, which may be related to the immune landscape through BCR and TCR signaling pathways. Our study shows that a unique tumor microbiome structure, potentially affecting the tumor immune microenvironment, is associated with poorer survival in resected early-stage PDAC. These findings suggest that microbial mechanisms may be involved in PDAC survival, potentially informing PDAC prognosis and guiding personalized treatment strategies. IMPORTANCE Much of the available data on the PDAC tumor microbiome and survival are derived from relatively small and heterogeneous studies, including those involving patients with advanced stages of pancreatic cancer. There is a critical knowledge gap in terms of the tumor microbiome and survival in early-stage patients treated by surgical resection; we expect that advancements in survival may initially be best achieved in these patients who are treated with curative intent.

Flavonoids, a major component of plant root exudates, play a crucial role in mediating plant-microbe interactions. However, the mechanisms by which flavonoids are perceived and trigger downstream signaling events in microbes remain largely unknown. In this study, we characterized AefR, a flavonoid-sensing transcriptional regulator from Pseudomonas fluorescens 2P24, a plant growth-promoting rhizobacterium (PGPR) known for its biocontrol properties. AefR was found to repress the expression of the mexEF-oprN efflux pump, which putatively exports N-acylhomoserine lactones (AHLs). This repression attenuates the PcoR/PcoI quorum-sensing system, leading to decreased production of the antibiotic mupirocin in P. fluorescens 2P24. Furthermore, quantitative proteomic analysis revealed that the PcoR/PcoI quorum-sensing system regulates a diverse range of physiological processes, including mupirocin production and denitrification. Collectively, these findings demonstrate a quorum-quenching role of flavonoids in a PGPR strain, establishing that flavonoids can disrupt quorum-sensing by enhancing the efflux of quorum-sensing signaling molecules. These findings have practical implications for the development of sustainable biocontrol strategies, where leveraging natural plant-microbe interactions could enhance the suppression of plant pathogens without the use of synthetic chemicals. IMPORTANCE Flavonoids are key mediators of plant-microbe interactions; however, their role in regulating microbial signaling remains poorly understood. This study identifies AefR as a flavonoid-sensing regulator in Pseudomonas fluorescens 2P24, revealing a novel quorum-quenching mechanism where flavonoids enhance the efflux of quorum-sensing signals. These findings shed light on the molecular basis of flavonoid-mediated microbial regulation and offer new strategies for sustainable plant health management.

This study aimed to elucidate the complement protein C3-mediated host-pathogen interaction in the brain abscess caused by Staphylococcus aureus infection. Dual RNA-seq was employed to analyze the transcriptomic differences between C3 deficiency and wild-type mice of S. aureus- induced brain abscess model, and then we investigated the potential regulatory pathways of S. aureus- host interaction mediated by C3 and S. aureus genes associated with the pathogenesis of brain abscess. Finally, C3 deficient-mice and hla mutants of S. aureus were used to verify the specific pathogen-host interaction. In the S. aureus- induced brain abscess mouse model, the transcriptomic analysis revealed significant changes in bacterial virulence factors, such as hemolysin. Based on these data, we predicted a regulatory network formed by genes like hrcA and dnaK , which represent a possible regulation mechanism of S. aureus responding to the host. Furthermore, we identified that hla was the C3 response gene in S. aureus . From the host perspective, we observed that the absence of C3 significantly impacted the host’s inflammatory response, primarily by altering the gene expression of several key immune and inflammatory pathways. These findings suggest that C3 deficiency may impair the host’s ability to recognize and respond to external pathogens. To the best of our knowledge, this study proposed that S. aureus may affect host immune response through C3, and C3 plays a critical role in regulating inflammation and immune signaling pathways in the brain abscess caused by S. aureus infection. IMPORTANCE In this work, we employed immunofluorescence and Western blot analysis to reveal a significant upregulation of microglia-derived C3 in the brain abscess mice model caused by S. aureus infection. By integrating the individual RNA sequencing data of S. aureus and the dual RNA-seq data of S. aureus infection brain abscess mice model, the potential regulatory pathways between S. aureus and host were identified, and host C3 not only affects the immune response but also mediates the regulation network of S. aureus . This study provided the potential novel targets for therapeutic strategies in mitigating the effects of S. aureus infections and improving treatment outcomes.

Soil moisture and porosity regulate microbial metabolism by influencing factors, such as system chemistry, substrate availability, and soil connectivity. However, accurately representing the soil environment and establishing a tractable microbial community that limits confounding variables is difficult. Here, we use a reduced-complexity microbial consortium grown in a glass bead porous media amended with chitin to test the effects of moisture and a structural matrix on microbial phenotypes. Leveraging metagenomes, metatranscriptomes, metaproteomes, and metabolomes, we saw that our porous media system significantly altered microbial phenotypes compared with the liquid incubations, denoting the importance of incorporating pores and surfaces for understanding microbial phenotypes in soils. These phenotypic shifts were mainly driven by differences in expression of Streptomyces and Ensifer , which included a significant decrease in overall chitin degradation between porous media and liquid. Our findings suggest that the success of Ensifer in porous media is likely related to its ability to repurpose carbon via the glyoxylate shunt amidst a lack of chitin degradation byproducts while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits expressed by Ensifer and others, including motility, stress resistance, and carbon conservation, that likely influence the metabolic profiles observed across treatments. Together, these results demonstrate that porous media incubations promote structure-induced microbial phenotypes and are likely a better proxy for soil conditions than liquid culture systems. Furthermore, they emphasize that microbial phenotypes encompass not only the multi-enzyme pathways involved in metabolism but also include the complex interactions with the environment and other community members. IMPORTANCE Soil moisture and porosity are critical in shaping microbial metabolism. However, accurately representing the soil environment in tractable laboratory experiments remains a challenging frontier. Through our reduced complexity microbial consortium experiment in porous media, we reveal that predicting microbial metabolism from gene-based pathways alone often falls short of capturing the intricate phenotypes driven by cellular interactions. Our findings highlight that porosity and moisture significantly affect chitin decomposition, with environmental matrix (i.e., glass beads) shifting community metabolism towards stress tolerance, reduced resource acquisition, and increased carbon conservation, ultimately invoking unique microbial strategies not evident in liquid cultures. Moreover, we find evidence that changes in moisture relate to community shifts regarding motility, transporters, and biofilm formation, which likely influence chitin degradation. Ultimately, our incubations showcase how reduced complexity communities can be informative of microbial metabolism and present a useful alternative to liquid cultures for studying soil microbial phenotypes.

Natural products are a critical source of novel chemotypes for drug discovery. However, the implementation of natural product extract libraries in high throughput screening is hampered by natural product structural redundancy and potential for bioactive re-discovery. This challenge and large library sizes drastically increase the time and cost during initial high throughput screens. To address these limitations, we developed a method that leverages liquid chromatography-tandem mass spectrometry spectral similarity to dramatically reduce natural product library size, with minimal bioactive loss, and applied this to a collection of fungal extracts. Importantly, this method also afforded increased bioassay hit rates against microbial targets, with broad applicability across assays and natural product sources. Thus, this method offers a broadly applicable strategy for accelerated and cost-effective natural product drug discovery. IMPORTANCE Natural product libraries are large collections of extracts derived from fungi, plants, bacteria, or any other natural sources. These libraries play an important role in the initial phases of drug discovery, providing the basis for bioassays against a target of interest. However, these collections often comprise thousands of extracts with sometimes overlapping chemical structures, which can result in a bottleneck in both time and costs for the initial phases of drug discovery. Here, we have developed a method that uses mass spectrometry to dramatically reduce the size of these libraries, with minimal tradeoffs and improved success rates in bioassays. Ultimately, this will speed up the process of bioactive candidate identification and isolation, and drug development overall.

Encoded within many microbial genomes, biosynthetic gene clusters (BGCs) underlie the synthesis of various secondary metabolites that often mediate ecologically important functions. Several studies and bioinformatics methods developed over the past decade have advanced our understanding of both microbial pangenomes and BGC evolution. In this minireview, we first highlight challenges in broad evolutionary analysis of BGCs, including delineation of BGC boundaries and clustering of BGCs across genomes. We further summarize key findings from microbial comparative genomics studies on BGC conservation across taxa and habitats and discuss the potential fitness effects of BGCs in different settings. Afterward, recent research showing the importance of genomic context on the production of secondary metabolites and the evolution of BGCs is highlighted. These studies draw parallels to recent, broader, investigations on gene-to-gene associations within microbial pangenomes. Finally, we describe mechanisms by which microbial pangenomes and BGCs evolve, ranging from the acquisition or origination of entire BGCs to micro-evolutionary trends of individual biosynthetic genes. An outlook on how expansions in the biosynthetic capabilities of some taxa might support theories that open pangenomes are the result of adaptive evolution is also discussed. We conclude with remarks about how future work leveraging longitudinal metagenomics across diverse ecosystems is likely to significantly improve our understanding on the evolution of microbial genomes and BGCs.

A multidrug-resistant clinical Acinetobacter baumannii isolate with resistance to most antibiotics was isolated from a patient at an intensive care unit. The genetic environment, transcriptome, mobile, and resistome were characterized. The MicroScan system, disc diffusion, and broth microdilution were used to determine the resistance profile of the isolate. A multiplex PCR assay was also used to screen for carbapenemases and mcr -1 to -5 resistance genes. Efflux-pump inhibitors were used to evaluate efflux activity. The resistome, mobilome, epigenome, and transcriptome were characterized. There was phenotypic resistance to 22 of the 25 antibiotics tested, intermediate resistance to levofloxacin and nalidixic acid, and susceptibility to tigecycline, which corresponded to the 27 resistance genes found in the genome, most of which occurred in multiple copies through replicative transposition. A plasmid-borne (pR-B2.MM_C3) mcr- 1 and chromosomal bla PER-7 , bla OXA-69 , bla OXA-23 (three copies), bla ADC-25 , bla TEM-1B , and bla NDM-1 were found within composite transposons, ISs, and/or class 1 and 2 integrons on genomic islands. Types I and II methylases and restriction endonucleases were in close synteny to these resistance genes within the genomic islands; chromosomal genomic islands aligned with known plasmids. There was a closer evolutionary relationship between the strain and global strains but not local or regional strains; the resistomes also differed. Significantly expressed/repressed genes (6.2%) included resistance genes, hypothetical proteins, mobile elements, methyltransferases, transcription factors, and membrane and efflux proteins. The genomic evolution observed in this strain explains its adaptability and pandrug resistance and shows its genomic plasticity on exposure to antibiotics. IMPORTANCE A pandrug-resistant pathogen that was susceptible to only a single antibiotic, tigecycline, was isolated from a middle-aged patient in an ICU. This pathogen contained two plasmids and had a chromosome that contained portions that were integrated externally from plasmids. These genomic islands were rich with resistance genes, mobile genetic elements, and restriction-modification systems that protected the pathogen and facilitated gene regulation. The strain contained 35 resistance genes and 12 virulence genes. The strain was of closer evolutionary distance to several international strains suggesting that it was imported into South Africa. However, its resistome was unique, suggesting an independent evolution on exposure to antibiotic therapy mediated by epigenomic factors and MGE transposition events. The varied mechanisms available to this strain to overcome antibiotic resistance and spread to other areas and/or transfer its resistance determinants are worrying. This is ultimately a risk to public health, evincing the need for antibiotic stewardship.