The escalating antibiotic resistance crisis poses a significant challenge to global public health, threatening the efficacy of current treatments and driving the emergence of multidrug-resistant pathogens. Among the various factors associated with bacterial antibiotic resistance, small regulatory RNAs (sRNAs) have emerged as pivotal post-transcriptional regulators which orchestrate bacterial adaptation to antibiotic pressure via diverse mechanisms. This review consolidates the current knowledge on sRNA-mediated mechanisms, focusing on drug uptake, drug efflux systems, lipopolysaccharides, cell wall modification, biofilm formation, and mutagenesis. Recent advances in transcriptomics and functional analyses have revealed novel sRNAs and their regulatory networks, expanding our understanding of resistance mechanisms. These findings highlight the potential of targeting sRNA-mediated pathways as an innovative therapeutic strategy to combat antibiotic resistance, and offer promising avenues for managing challenging bacterial infections.

Streptomyces are a crucial source of bioactive secondary metabolites with significant clinical applications. Recent studies of bacterial and metagenome-assembled genomes have revealed that Streptomyces harbors a substantial number of uncharacterized silent secondary metabolite biosynthetic gene clusters (BGCs). These BGCs represent a vast diversity of biosynthetic pathways for natural product synthesis, indicating significant untapped potential for discovering new metabolites. To exploit this potential, genome mining using comprehensive strategies that leverage extensive genomic databases can be conducted. By linking BGCs to their encoded products and integrating genetic manipulation techniques, researchers can greatly enhance the identification of new secondary metabolites with therapeutic relevance. In this context, we present a step-by-step guide for using the antiSMASH pipeline to identify secondary metabolite-coding BGCs within the complete genome of a novel Streptomyces strain. This protocol also outlines gene manipulation methods that can be applied to Streptomyces to activate cryptic clusters of interest and validate the functions of biosynthetic genes. By following these guidelines, researchers can pave the way for discovering and characterizing valuable natural products.

Strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2, identified as coagulase-negative, Gram-stain-positive, halotolerant, non-motile coccoid bacteria, were isolated from traditional Korean soybean foods. Strains Mo2-6^T and S9 were both catalase- and oxidase-negative, whereas KG4-3^T and 50Mo3-2 were catalase-positive but oxidase-negative. The optimal growth conditions for Mo2-6^T and S9 were 30°C, 2% NaCl, and pH 7.0, while KG4-3^T and 50Mo3-2 grew best at 35°C, 2% NaCl, and pH 7.0. All strains contained menaquinone-7 as the predominant isoprenoid quinone, with anteiso-C_15:0 and iso-C_15:0 as the major cellular fatty acids (> 10%). Additionally, anteiso-C_13:0 was a major fatty acid in strain KG4-3^T. The DNA G + C contents of strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2 were 33.4%, 33.3%, 32.5%, and 32.7%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, formed distinct lineages within the genus Staphylococcus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, belonged to the same species. Meanwhile, dDDH and ANI values between strains Mo2-6^T and KG4-3^T, as well as comparisons with other Staphylococcus type strains, were below the species delineation thresholds, indicating they represent novel species. Based on phenotypic, chemotaxonomic, and molecular data, we propose strain Mo2-6^T as the type strain of Staphylococcus parequorum sp. nov. (=KACC 23685^T =JCM 37038^T) and strain KG4-3^T as the type strain of Staphylococcus halotolerans sp. nov. (=KACC 23684^T =JCM 37037^T).

Extracellular vesicles derived from probiotics have received considerable attention for their pivotal role in bacterial‒host communication. These nanosized, bilayer-encapsulated vesicles carry diverse bioactive molecules, such as proteins, lipids, nucleic acids, and metabolites. Currently, ample evidence has emerged that probiotic extracellular vesicles may modulate several processes of host physiological hemostasis and offer therapeutic benefits. This review examines the biogenesis, composition, and immunomodulatory functions of probiotic-derived extracellular vesicles in probiotic–host interactions, highlighting the therapeutic potential of probiotic extracellular vesicles in the diagnosis and treatment of conditions such as cancer and inflammatory bowel disease. We further summarize the techniques for the separation and purification of extracellular vesicles, providing a methodological foundation for future research and applications. Although the field of probiotic extracellular vesicle research is still in its infancy, the prospects for their application in the biomedical field are broad, potentially emerging as a novel therapeutic approach.

The increase of sequence data in public nucleotide databases has made DNA sequence-based identification an indispensable tool for fungal identification. However, the large proportion of mislabeled sequence data in public databases leads to frequent misidentifications. Inaccurate identification is causing severe problems, especially for industrial and clinical fungi, and edible mushrooms. Existing species identification pipelines require separate validation of a dataset obtained from public databases containing mislabeled taxonomic identifications. To address this issue, we developed FunVIP, a fully automated phylogeny-based fungal validation and identification pipeline (https://github.com/Changwanseo/FunVIP). FunVIP employs phylogeny-based identification with validation, where the result is achievable only with a query, database, and a single command. FunVIP command comprises nine steps within a workflow: input management, sequence-set organization, alignment, trimming, concatenation, model selection, tree inference, tree interpretation, and report generation. Users may acquire identification results, phylogenetic tree evidence, and reports of conflicts and issues detected in multiple checkpoints during the analysis. The conflicting sample validation performance of FunVIP was demonstrated by re-iterating the manual revision of a fungal genus with a database with mislabeled sequences, Fuscoporia. We also compared the identification performance of FunVIP with BLAST and q2-feature-classifier with two mass double-revised fungal datasets, Sanghuangporus and Aspergillus section Terrei. Therefore, with its automatic validation ability and high identification performance, FunVIP proves to be a highly promising tool for achieving easy and accurate fungal identification.

This study aimed to provide a taxonomic description of two bacterial strains, NKC19-3^T and NKC19-16^T, isolated from commercially produced kimchi obtained from various regions within the Republic of Korea. Both strains were rod-shaped, gram-stain-positive, facultatively anaerobic, and displayed positive reactions for oxidase and catalase. Additionally, these bacteria were motile, halophilic (salt-tolerant), and proliferated under alkaline conditions. Genetically, both strains showed 98.0% similarity in their 16S rRNA gene sequences and were most closely related to Virgibacillus natechei FarD^T, with 96.5 and 96.8% sequence similarity, respectively. ANI values indicated that the two novel strains were distinct from V. natechei FarD^T, as they were below the species demarcation threshold. The ANI value between strains NKC19-3ᵀ and NKC19-16ᵀ was 84.64–84.75%, and the values between these strains and other related strains did not exceed 80.0%, further supporting their classification as novel species. Phylogenetic analysis revealed that strains NKC19-3^T and NKC19-16^T formed a distinct branch within the genus Virgibacillus, clearly distinguishing them from other species in the same genus. Regarding genomic characteristics, the GC content was 38.9% for strain NKC19-3^T and 39.5% for strain NKC19-16^T. The genome of strain NKC19-3^T had a size of approximately 4.1 Mb and contained 3,785 protein-coding genes (CDSs). Strain NKC19-16^T had a slightly smaller genome, approximately 3.9 Mb in size and harbored 3,726 CDSs. The polar lipid profiles of strains NKC19-3ᵀ and NKC19-16ᵀ included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), glycolipids (GL), and an unidentified lipid (L). The predominant fatty acids of both strains were anteiso-C_15:0 and anteiso-C_17:0. Considering the comprehensive analysis encompassing phenotypic, genomic, phylogenetic, and chemotaxonomic data, strains NKC19-3^T and NKC19-16^T are proposed to represent two novel species within the genus Virgibacillus. The suggested names for these species are Virgibacillus saliphilus sp. nov. (type strain NKC19-3^T, also referred to as KACC 22326^T and DSM 112707^T) and Virgibacillus salidurans sp. nov. (type strain NKC19-16^T, also referred to as KACC 22327^T and DSM 112708^T).

Synbiotics have become a new-age treatment tool for limiting the progression of metabolic dysfunction-associated steatotic liver disease; however, inclusive comparisons of various synbiotic treatments are still lacking. Here, we have explored and evaluated multiple synbiotic combinations incorporating three distinctive prebiotics, lactitol, lactulose and fructooligosaccharides. Of the synbiotic treatments evaluated, a combination of fructooligosaccharides and probiotics (FOS+Pro) exhibited superior protection against western diet-induced liver degeneration. This synbiotic (FOS+Pro) combination resulted in the lowest body weight gains, liver weights and liver/body weight ratios. The FOS+Pro synbiotic combination substantially alleviated liver histopathological markers and reduced serum AST and cholesterol levels. FOS+Pro ameliorated hepatic inflammation by lowering expression of proinflammatory markers including TNF-α, IL-1β, IL-6, and CCL2. FOS+Pro significantly improved steatosis by restricting the expression of lipid metabolic regulators (ACC1, FAS) and lipid transporters (CD36) in the liver. These findings are critical in suggesting that synbiotic treatments are capable of restraining western diet-induced metabolic dysfunction in the liver. Additionally, this study demonstrated that adding probiotic strains amplified the effectiveness of fructooligosaccharides but not all prebiotics.

Streptococcus pneumoniae is a conditionally pathogenic bacteria that colonizes the nasopharynx of 27% to 65% of children and 10% of adults. Capsular polysaccharides are the most critical virulence factor of S. pneumoniae, and nonencapsulated strains are usually non-pathogenic. Previous studies have shown that glucose regulates capsule synthesis. To investigate the mechanism of carbon metabolism regulatory factors CcpA and HPr regulating capsule synthesis in the presence of glucose as the sole carbon source, we constructed deletion mutants (D39ΔccpA and ΔptsH) and complemented strains (D39ΔccpA::ccpA and ΔptsH::ptsH). In this study, we found that the promoting effect of capsule synthesis by glucose disappeared after the deletion of ccpA and ptsH, and demonstrated that the protein CcpA regulates capsule synthesis by binding to the cps promoter and altering the transcription level of the cps gene cluster. Increased glucose concentration up-regulated the level of HPr-Ser46~P, which enhanced the binding ability of CcpA to the DNA sequence of the cps promoter, thus promoting capsule synthesis. HPr also has a regulatory effect on capsule synthesis. These insights reveal a new synthesis mechanism of capsular polysaccharide and provide a new strategy of antibacterial drugs for S. pneumoniae.

Mycobacterium avium complex (MAC) organisms are widespread environmental pathogens associated with chronic pulmonary infections. Although M. avium is known to invade epithelial cells, the molecular mechanisms underlying this process remain incompletely understood. In this study, we identified a novel role for MAVRS09815 (formerly MAV2054), a family 2A encapsulin nanocompartment shell protein, in mediating bacterial adhesion, epithelial cell invasion, and in vivo virulence. We engineered a recombinant M. smegmatis strain expressing MAV2054 (Ms_2054) and an M. avium MAV2054 deletion mutant (Δ2054). Ms_2054 exhibited enhanced epithelial invasion, whereas Δ2054 showed reduced intracellular survival. Recombinant MAV2054 protein was bound directly to human epithelial cells in a dose-dependent manner. Pretreatment of host cells with cytochalasin D or vinblastine significantly inhibited bacterial internalization, indicating that MAV2054-mediated invasion is cytoskeleton-dependent. Confocal and scanning electron microscopy revealed MAV2054-dependent membrane rearrangements during infection. Pull-down assays demonstrated that MAV2054 activates Cdc42, a key regulator of actin polymerization, with reduced activation observed in Δ2054-infected cells. In a murine intratracheal infection model, the Δ2054 exhibited significantly reduced bacterial burdens and lung inflammation compared to the wild type. These findings demonstrate that MAV2054 enhances M. avium virulence by promoting epithelial cell invasion through Cdc42-dependent cytoskeletal remodeling. This study reveals a previously unrecognized role for an encapsulin-like protein in host-pathogen interactions and highlights its potential as a therapeutic target in MAC infections.

Nosocomial infections caused by Pseudomonas aeruginosa (P. aeruginosa) have become increasingly common, particularly among immunocompromised individuals, who experience high mortality rates and prolonged treatment durations due to the limited availability of effective therapies. In this study, we screened for anti-ExoS compounds targeting P. aeruginosa and identified pycnogenol (PYC) as a potent inhibitor of the type III secretion system (T3SS), a major virulence mechanism responsible for the translocation of effectors such as ExoS. Using ELISA, western blotting, and real-time PCR analyses in both P. aeruginosa and infected H292 cells, we found that PYC significantly reduced T3SS activity. Mechanistically, PYC suppressed the transcription of T3SS-related genes by downregulating exsA expression in P. aeruginosa. Furthermore, pretreatment with PYC attenuated the cytotoxic effects and reduced the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18), in P. aeruginosa-infected H292 cells. These effects were associated with the inhibition of NF-κB signaling and inflammasome activation. Taken together, our findings suggest that PYC may serve as a promising therapeutic candidate against P. aeruginosa infections by targeting T3SS-mediated virulence and modulating host inflammatory responses.

Salmonella enterica is a clinically significant oro-fecal pathogen that causes a wide variety of illnesses and can lead to epidemics. S. enterica expresses a lot of virulence factors that enhance its pathogenesis in host. For instance, S. enterica employs a type three secretion system (T3SS) to translocate a wide array of effector proteins that could change the surrounding niche ensuring suitable conditions for the thrive of Salmonella infection. Many antimicrobials have been recently introduced to overcome the annoying bacterial resistance to antibiotics. Enoxacin is member of the second-generation quinolones that possesses a considerable activity against S. enterica. The present study aimed to evaluate the effect of enoxacin at sub-minimum inhibitory concentration (sub-MIC) on S. enterica virulence capability and pathogenesis in host. Enoxacin at sub-MIC significantly diminished both Salmonella invasion and intracellular replication within the host cells. The observed inhibitory effect of enoxacin on S. enterica internalization could be attributed to its ability to interfere with translocation of the T3SS effector proteins. These results were further confirmed by the finding that enoxacin at sub-MIC down-regulated the expression of the genes encoding for T3SS-type II (T3SS-II). Moreover, enoxacin at sub-MIC lessened bacterial adhesion to abiotic surface and biofilm formation which indicates a potential anti-virulence activity. Importantly, in vivo results showed a significant ability of enoxacin to protect mice against S. enterica infection and decreased bacterial colonization within animal tissues. In nutshell, current findings shed light on an additional mechanism of enoxacin at sub-MIC by interfering with Salmonella intracellular replication. The outcomes presented herein could be further invested in conquering bacterial resistance and open the door for additional effective clinical applications.

Pseudomonas aeruginosa (P. aeruginosa) is resistant to several drugs as well as antibiotics and is thus classified as multidrug resistant and extensively drug resistant. These bacteria have a secretion system called the "type 3 secretion system (T3SS)", which facilitates infection by delivering an effector protein. ExoenzymeS (ExoS) is known to induce cell death and activate caspase-1. In particular, patients infected with P. aeruginosa develop diseases associated with high mortality, such as pneumonia, because no drug targets an ExoS or T3SS. We selected natural compounds to treat T3SS-mediated pneumonia and chose alizarin, a red dye. We confirmed the effects of alizarin on T3SS by bacterial PCR and ELISA. It was confirmed that alizarin regulates ExoS by inhibiting exsA but also popD and pscF. Furthermore, in infected H292 cells, it not only attenuates inflammation by inhibiting lipopolysaccharide (LPS)-induced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 but also interferes with the level of ExoS delivered into the host and modulates caspase-1. We confirmed this result and determined that it led to decreases in proinflammatory cytokines such as Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), and Interleukin-18 (IL-18). Therefore, we suggest that alizarin is a suitable drug for treating pneumonia caused by P. aeruginosa because it helps to attenuate inflammation by regulating T3SS and NF-κB signaling.

Tella is a traditional beverage widely accepted by consumers, despite the lack of product consistency owing to its reliance on natural fermentation. This study aimed to identify potential industrial lactic acid bacteria (LAB) starter cultures based on their technological properties. Seven LAB strains isolated from Tella were characterized for their carbohydrate utilization, salt content, temperature, and acid tolerances, growth and acidification rates, and metabolite profiles. Most strains efficiently utilized various carbohydrates, with Lactiplantibacillus plantarum TDM41 showing exceptional versatility. The strains exhibited similar growth characteristics. Principal component analysis of stress tolerance properties revealed that L. plantarum TDM41, Pediococcus pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 exhibited superior tolerance ability. Strong acidification properties were detected in the L. plantarum TDM41, P. pentosaceus TAA01, and Leuconostoc mesenteroides TDB22 strains after 24 h incubation at 30°C. L. plantarum TDM41 displayed the fastest acidification rate throughout the analysis period. All LAB strains produced significant amounts of diverse organic acids, including lactic acid, citric acid, acetic acid, malic acid, and succinic acid, with lactic acid being the primary acid produced by each strain. Overall, strains L. plantarum TDM41 and P. pentosaceus TAA01 prove to be potential candidates for Tella industrial starter cultures and similar cereal products owing to their robust technological properties.

Chronic toxoplasmosis is caused by Toxoplasma gondii bradyzoites. This study assessed six candidate small molecule kinase inhibitors (SMKIs) against bradyzoites (ME49 strain), the reactivated form of the parasite resulting from the rupture of brain cysts. Bradyzoites were obtained from mouse brain cysts, cultured in ARPE-19 cells, and treated with afatinib and neratinib (HER2/HER4 inhibitors), ACTB-1003 and regorafenib (VEGFR-2 inhibitors), or altiratinib and foretinib (c-MET inhibitors). The effects on the growth of T. gondii were analyzed by western blot and immunofluorescence assay. Changes in the host cells were assessed using markers for cell viability, apoptosis, necrosis, and autophagy. All inhibitors blocked the growth of bradyzoites, although afatinib was less effective. Afatinib enhanced autophagy signals, while ACTB-1003 and neratinib affected mitochondrial biosynthesis and mitophagy. Altiratinib demonstrated an effect against bradyzoites at the lowest concentration with minimal impact on the host cells. It may be effective in blocking the reactivation of brain cysts in immunodeficiency patients caused by bradyzoites.

Antimicrobial resistance (AMR) poses a serious threat to public health, with the emergence of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae, particularly Escherichia coli, raising significant concerns. This study aims to elucidate the drivers of antimicrobial resistance, and the global spread of cefotaxime-resistant E. coli (CREC) strains. Whole-genome sequencing (WGS) was performed to explore genome-level characteristics, and phylogenetic analysis was conducted to compare twenty CREC strains from this study, which were isolated from broiler chicken farms in Bangladesh, with a global collection (n = 456) of CREC strains from multiple countries and hosts. The MIC analysis showed over 70% of strains isolated from broiler chickens exhibiting MIC values ≥ 256 mg/L for cefotaxime. Notably, 85% of the studied farms (17/20) tested positive for CREC by the end of the production cycle, with CREC counts increasing from 0.83 ± 1.75 log10 CFU/g feces on day 1 to 5.24 ± 0.72 log10 CFU/g feces by day 28. WGS revealed the presence of multiple resistance genes, including bla_CTX-M, which was found in 30% of the strains. Phylogenetic comparison showed that the Bangladeshi strains were closely related to strains from diverse geographical regions and host species. This study provides a comprehensive understanding of the molecular epidemiology of CREC. The close phylogenetic relationships between Bangladeshi and global strains demonstrate the widespread presence of cefotaxime-resistant bacteria and emphasize the importance of monitoring AMR in food-producing animals to mitigate the spread of resistant strains.

This study aimed to determine if the microbiota in four different oral sites and the oral health status differ between patients with primary Sjögren’s syndrome (pSS), non-pSS sicca symptoms, and healthy controls. All participants underwent an interview and clinical oral examination. Stimulated whole saliva (SWS), supragingival plaque (SGP), buccal mucosa tissue (BLM), and tongue scrape (TGS) samples from 23 pSS patients, 36 patients with sicca symptoms, not fulfilling the classification criteria for pSS (non-pSS sicca), and 21 age-matched healthy controls (HC) were analyzed using V3–V4 16S rRNA gene amplicon sequencing, and determination of amplicon sequence variants (ASVs). PSS and non-pSS sicca patients did not differ with respect to oral health status, saliva flow rates, abundance of predominant genera, relative abundance on genus level or bacterial diversity in any of the oral sites. Both patient groups differed significantly from the healthy control group in the abundance of 61 ASVs across all sites. The alpha-diversity was lower in SGP from non-pSS sicca patients (p = 0.019), and in TGS from pSS patients (p = 0.04). The proportion of variation in the beta-diversity across all four sites could be explained by the diagnosis (pSS, non-pSS sicca, and HC). However, subgrouping of patients according to their stimulated salivary flow rates (SWS > 0.7 ml/min versus SWS ≤ 0.7 ml/min), revealed significantly different abundance of three ASVs in SWS, 11 in SGP, and six in TGS. Our findings suggest that hyposalivation rather than pSS itself modifies the microbial composition in oral site-specific patterns leading to oral diseases.

The widespread use of antibiotics in aquaculture has led to the emergence of multidrug-resistant pathogens and environmental concerns, highlighting the need for sustainable, eco-friendly alternatives. In this study, we isolated and characterized three novel bacteriophages from aquaculture effluents in Korean shrimp farms that target the key Vibrio pathogens, Vibrio harveyi, and Vibrio parahaemolyticus. Bacteriophages were isolated through environmental enrichment and serial purification using double-layer agar assays. Transmission electron microscopy revealed that the phages infecting V. harveyi, designated as vB_VhaS-MS01 and vB_VhaS-MS03, exhibited typical Siphoviridae morphology with long contractile tails and icosahedral heads, whereas the phage isolated from V. parahaemolyticus (vB_VpaP-MS02) displayed Podoviridae characteristics with an icosahedral head and short tail.

Whole-genome sequencing produced complete, circularized genomes of 81,710 bp for vB_VhaS-MS01, 81,874 bp for vB_VhaS-MS03, and 76,865 bp for vB_VpaP-MS02, each showing a modular genome organization typical of Caudoviricetes. Genomic and phylogenetic analyses based on the terminase large subunit gene revealed that although vB_VhaS-MS01 and vB_VhaS-MS03 were closely related, vB_VpaP-MS02 exhibited a distinct genomic architecture that reflects its unique morphology and host specificity. Collectively, these comparative analyses demonstrated that all three phages possess genetic sequences markedly different from those of previously reported bacteriophages, thereby establishing their novelty. One-step growth and multiplicity of infection (MOI) experiments demonstrated significant differences in replication kinetics, such as burst size and lytic efficiency, among the phages, with vB_VhaS-MS03 maintaining the most effective bacterial control, even at an MOI of 0.01. Additionally, host range assays showed that vB_VhaS-MS03 possessed a broader spectrum of activity, supporting its potential use as a stand-alone agent or key component of phage cocktails. These findings highlight the potential of region-specific phage therapy as a targeted and sustainable alternative to antibiotics for controlling Vibrio infections in aquaculture.