Northern Russia is characterized by socio-environmental conditions contributing to the spread of tuberculosis (TB) and a high ~30% rate of primary multidrug-resistant (MDR) TB. We applied high-resolution molecular methods to study the Mycobacterium tuberculosis population in the Arkhangelsk region of Northern Russia. All available M. tuberculosis isolates recovered from newly diagnosed patients from January to December 2018 (n = 88) were genotyped using 24-loci MIRU-VNTR, spoligotyping, and, partly, by whole-genome sequencing (WGS). The population structure revealed a predominance of the Beijing genotype and Euro-American lineage, with significant drug resistance burden associated with Beijing and its B0/W148 strain. Beijing strains showed a significantly higher association with MDR and pre-extensively drug-resistant (pre-XDR) TB compared to non-Beijing strains (P = 0.0013). All Beijing B0/W148 isolates were MDR, whereas the majority (71.4%) of Beijing Central Asian/Russian subtype strains were drug-sensitive. WGS analysis of newly discovered Beijing clusters 3828-32 and 10167-32 in this area indicated a historical transmission over several decades, reflecting long-term endemic circulation. The presence of compensatory mutations in rpoC among MDR strains suggests enhanced fitness facilitating their ongoing transmission. An intriguing cluster of recent transmission of a non-Beijing strain (spoligotype SIT53, L4.8 sublineage) was identified through combined epidemiological and genomic investigation. To conclude, the prevalence of Beijing strains rose from 39.3% in 1998 to 67.0% (P < 0.001), and Russian epidemic MDR strain B0/W148 increased its rate from 11.2% in 1998 to 20.5% (P = 0.097). This highlights the key role of MDR Beijing strains, including new resistant clusters, in disseminating MDR-TB in the region and the importance of continuous surveillance using high-resolution genotyping.IMPORTANCEThe Arkhangelsk region is the largest province of northern European Russia. One-third of newly diagnosed tuberculosis patients are infected with multidrug-resistant (MDR) Mycobacterium tuberculosis strains. We assessed the molecular population structure of M. tuberculosis in the Arkhangelsk region in the COVID-19 pre-pandemic year 2018. We identified important MDR clusters and elucidated tuberculosis transmission patterns. An intriguing cluster of recent transmission was identified through the combined use of epidemiological investigation and whole-genome sequencing. The prevalence of Beijing genotype strains increased from 39.3% in 1998 to 67.0%, and the Russian epidemic MDR strain B0/W148 doubled from 11.2% in 1998 to 20.5%. Furthermore, we described new MDR clusters emerging within the Beijing genotype. This highlights the key impact of the MDR Beijing strains and the importance of continuous surveillance using high-resolution genotyping. This study of the pre-pandemic strain collection provides an indispensable intermediate time point between earlier studies carried out 25 years ago and ongoing surveillance.

Antimicrobial resistance in gram-negative bacteria has renewed reliance on colistin, yet resistance to this last-line agent is increasing. In Klebsiella pneumoniae, activation of the PmrA/PmrB two-component system remodels lipid A and reduces colistin binding, motivating the sensor kinase PmrB as an adjuvant target. Here, we combined computational drug repurposing with experimental validation to identify colistin potentiators. High-throughput virtual screening of DrugBank using integrated ligand- and structure-based approaches shortlisted five approved drugs (mebendazole, flurbiprofen, tirbanibulin, flufenamic acid, and netarsudil) for experimental evaluation. In vitro assays against colistin-resistant K. pneumoniae showed flufenamic acid most robustly resensitized resistant isolates and suppressed the emergence of additional resistance under combination exposure. Mechanistic studies indicated that the colistin-flufenamic acid combination increased membrane permeability and significantly downregulated PmrA/PmrB-regulated genes (pmrC and arnT). Molecular docking and molecular dynamics simulations further supported a plausible interaction of flufenamic acid with the PmrB ATPase region, and representative PmrB substitutions (D150Y, T157P, and R256G) did not alter the predicted binding mode. Together, these data suggest that modulation of the PmrA/PmrB pathway may contribute to colistin potentiation and demonstrate a target-guided repurposing-to-validation framework for antibiotic adjuvant discovery in colistin-resistant K. pneumoniae.IMPORTANCEColistin is one of the last remaining treatment options for multidrug-resistant Klebsiella pneumoniae infections, but resistance to this drug is rising worldwide. In this study, we used a PrmB-informed, computer-guided drug repurposing workflow followed by in vitro validation to identify approved compounds that can potentiate colistin activity against colistin-resistant K. pneumoniae. This approach independently prioritized flufenamic acid as the most robust colistin enhancer among the screened candidates, restoring colistin activity and reducing the emergence of additional resistance under combination exposure. Mechanistically, our findings are consistent with modulation of the PmrB/PmrA resistance pathway, supporting bacterial histidine-kinase signaling as a promising direction for antibiotic adjuvant development. Overall, this study highlights a practical and scalable framework for discovering resistance-targeted adjuvants to help protect last-resort antibiotics and improve treatment options for difficult-to-treat infections.

This study elucidates the critical molecular mechanism and evolutionary pathway of a novel KPC variant, KPC-194, that confers resistance to the last-resort antibiotic combination ceftazidime-avibactam in a high-risk Klebsiella pneumoniae strain. We identified that two amino acid substitutions (D179Y/P183L) in KPC-194 are responsible for ceftazidime-avibactam resistance. Crucially, our work reveals a dual-threat dynamic: the resistance phenotype is not only caused by the KPC mutation but also profoundly exacerbated by horizontal gene transfer. blaKPC-194 mobilized from a low-risk IncR plasmid to a highly transmissible IncFII plasmid via IS26-mediated replicative transposition. This event dramatically enhances the potential for widespread dissemination among clinical pathogens.

The influence of microbially and plant-synthesized compounds on colonization of plant growth-promoting rhizobacteria (PGPR) on various regions of the plant root is underexplored. Here, we examine the influence of surfactin and glutamate on the level and specificity of colonization of Bacillus subtilis along different regions of tomato root by adding exogenous compounds and testing mutated bacterial strains. First, B. subtilis PY79 was modified to express a full-length phosphopantetheinyl transferase (sfp) native to other surfactin-producing B. subtilis strains, and surfactin biosynthesis was observed under static incubation at 25°C in plant culturing media. Microscopy was performed using PY79 sfp- (surfactin null), PY79 sfp+ (surfactin overproducing), and the wild isolate B. subtilis UD1022 to map the colonization of each strain along the entire root of young tomato plants incubated at 25°C. In addition, experiments involving supplementation of surfactin or glutamate were also performed to evaluate the root colonization of sfp+ and sfp- strains. Root mapping of a tomato seedling shows that B. subtilis prefers to colonize near the mature region of the root and that colonization patterns vary based on exogenous metabolite concentration. Inclusion of glutamate in the media or through transient priming of the plant prior to bacterial inoculation strongly promoted root colonization by B. subtilis strains (both sfp+ and sfp-). In addition, the data shows that the lab strains were less efficient in colonization compared to the wild-type B. subtilis strain. Interestingly, in the presence of glutamate, microbes lost their preference for colonization at the mature region, instead colonizing along the entire root. Overall, our work reveals a preference for colonization of these B. subtilis strains to the mature region of tomato in the absence of glutamate supplementation and demonstrates a smaller than anticipated role of biosynthesized or supplemented surfactin on root colonization, at least in a hydroponic culturing format.IMPORTANCEPlants are associated with large communities of microbes across the rhizosphere. However, comparatively little is known about the drivers underpinning the diversity of microbes, especially regarding how they are recruited by plants. Growing evidence indicates that the rhizospheric microbiome supports plant growth in response to biotic and abiotic stresses. Of late, the usage of a synthetic community of plant growth-promoting rhizobacteria (PGPR), especially Bacillus subtilis, has been recognized for its role as a potential biofertilizer and bio-fungicide agent. The role of PGPR-derived metabolites has been debated as a driver for enhanced root colonization. However, the knowledge pertaining to where and how PGPR colonize on the root surface is currently unknown. Therefore, it is prudent to elucidate the role of bacterially derived compounds and other carbon sources in the rhizosphere that drive root colonization.

Acinetobacter baumannii, a major pathogen that causes nosocomial infections, exhibits drug resistance. Efflux pumps are critical in developing multidrug resistance. Berberine hydrochloride exhibited inhibitory effects against bacteria and toxins, demonstrating antibacterial efficacy. However, there is limited research on berberine hydrochloride combined with multiple antibiotics through regulation of efflux pumps in multidrug-resistant A. baumannii. We evaluated the antibacterial effect of berberine hydrochloride in combination with multiple antibiotics, explored the resistance mechanisms regulated by efflux pumps, and analyzed the differential expression of active efflux pump genes (adeR, adeS, adeJ, adeM, adeG, and adeB) before and after berberine hydrochloride exposure, to provide new therapeutic strategies for managing multidrug-resistant A. baumannii infections. One hundred non-repetitive A. baumannii strains were isolated from patients with respiratory infections. The microdilution method was used for the determination of minimal inhibitory concentration (MIC) and the checkerboard method for the determination of the fractional inhibitory concentration (FIC) index. Reverse transcription polymerase chain reaction quantified efflux pump gene expression in sensitive and resistant strains before and after berberine hydrochloride treatment. Strains showing significant differences were selected for transcriptomic sequencing. Berberine hydrochloride showed synergistic effects with nine antibiotics, the strongest being amikacin, meropenem, and cefepime. Resistance reversal occurred especially with levofloxacin. adeJ and adeB expression differed significantly between sensitive and resistant strains. Berberine hydrochloride induced notable changes in adeS, adeG, adeB, and adeR. Transcriptome sequencing revealed the significant expression of adeA, adeN, and adeT2. Berberine hydrochloride enhances antibiotic sensitivity and reverses resistance by regulating efflux pump gene expression, providing a theoretical foundation for improved treatment.IMPORTANCEThis study investigates multidrug-resistant Acinetobacter baumannii (MDRAB) strains isolated from clinical infections using the microplate dilution method to evaluate the antibacterial efficacy of berberine in combination with various antibiotics. The research simultaneously analyzes the differential expression levels of active efflux pump genes before and after berberine treatment. The findings aim to identify novel therapeutic strategies for MDRAB, explore the drug resistance mechanisms mediated by efflux pumps in AB, and elucidate berberine's role in reversing AB resistance through efflux pump regulation. These insights provide theoretical foundations for improving cure rates in MDRAB-infected patients and advancing drug target design.

Lactobacin A represents a unique addition to the Class IId bacteriocins, characterized by its non-pediocin-like structure and novel functional properties. This study not only introduces the first bacteriocin-producing strain closely related to Liquorilactobacillus mali but also underscores the potential of lactobacin A as a stable and effective antimicrobial agent under diverse environmental conditions. Its robust stability and narrow-spectrum activity against gram-positive bacteria make it a valuable candidate for applications in food preservation and therapeutic development.

Chronic pulmonary aspergillosis (CPA) is an important sequela of pulmonary tuberculosis (PTB), particularly in high-burden countries such as India. This study evaluated the diagnostic performance of the LDBio Aspergillus ICT lateral flow assay and western blot (WB) for CPA among post-TB patients in South India. In this prospective study (July 2023 to June 2025) conducted in Bengaluru, India, we enrolled adults with a history of PTB and persistent respiratory symptoms. CPA was diagnosed using the modified European Society for Clinical Microbiology and Infectious Diseases (ESCMID)-European Confederation of Medical Mycology (ECMM)-European Respiratory Society (ERS) criteria. All participants underwent comprehensive clinical evaluation; direct microscopy and culture of respiratory secretions; and testing with serum Aspergillus-specific IgG ELISA, ICT, WB, and galactomannan (GM). Of 132 post-TB patients, 107 (81%) were classified as CPA and 25 as non-CPA; 70 control sera were also included. ICT and WB were positive in 74% and 84% of CPA cases, respectively, outperforming ELISA (68%) and GM (30%). WB showed the highest sensitivity (84%, 95% CI: 75.8-90.5), whereas ICT demonstrated the highest specificity (94.7%, 95% CI: 88.1-98.3). Combining ICT and WB improved diagnostic accuracy (87.1%, 95% CI: 81.7-91.4) and yielded the highest discriminative power (Youden's index 0.805). WB displayed robust band-based differentiation, with strong reactivity in seropositive CPA. The agreement between ICT and WB was substantial (κ = 0.82). Latent class analysis estimated sensitivities of 92% (95% CI: 84-97) for ICT and 87% (95% CI: 77-94) for WB. These findings indicate that LDBio ICT and WB are robust diagnostic tools for CPA in post-TB patients and that their combined use offers optimal accuracy in resource-limited, high-burden settings.IMPORTANCEChronic pulmonary aspergillosis (CPA) is a serious but often overlooked complication in people who have previously been treated for tuberculosis. Many of these patients continue to struggle with cough, breathlessness, and lung damage; however, routine tests often fail to explain their symptoms. This study shows that CPA is extremely common in this high-risk group and demonstrates that two simple blood tests, the LDBio Aspergillus immunochromatographic test and western blot, can reliably identify the disease. Western blot detects CPA even in patients who typically test negative on standard methods, and using both tests together markedly improves accuracy. Because these tools are quick, inexpensive, and easy to perform, they offer a practical way to diagnose CPA earlier, especially in settings where advanced testing is unavailable. Early diagnosis allows timely antifungal treatment, which can greatly reduce illness and prevent unnecessary suffering among the growing number of TB survivors.

Current methods used for the Mycobacterium kansasii complex (MKC) species identification lack the resolution to differentiate between individual MKC species and require time-demanding multiple steps and costly analyses. Herein, we present a novel one-step multiplex PCR assay that enables rapid and cost-effective species-level identification of MKC members, suitable for implementation in both clinical diagnostics and epidemiological investigations. The method was evaluated using 143 Mycobacterium strains, including 17 reference strains and 126 clinical or environmental isolates. It accurately identified the vast majority (98%) of strains, with only two atypical M. kansasii subtype IIB isolates misidentified as M. persicum. The calculated sensitivity and specificity of the assay were 98% and 100%, respectively.

Clinical metagenomic next-generation sequencing (mNGS) is a diagnostic tool allowing near-universal pathogen detection directly from clinical specimens. Despite promising clinical data, broad adoption of mNGS has been hindered by high cost and reduced sensitivity relative to targeted nucleic acid amplification tests (NAATs). Recently, Ultima Genomics introduced the UG 100 NGS platform which advertises 10 billion reads per $2,400 sequencing wafer. By lowering costs and improving sequencing depth, the historical value proposition of mNGS may be improved. This study evaluates the UG 100 sequencer's ability to generate reads for metagenomic pathogen detection from cerebrospinal fluid specimens. Ultima reads demonstrated 93% (26/28) positive agreement with orthogonal test results and 63% (10/16) negative agreement against a syndromic panel for meningitis and encephalitis. Near full-length genomes were recovered for three organisms (human herpesvirus-1 [HSV-1], Streptococcus pneumoniae, and Haemophilus influenzae), with the ability to detect putative antimicrobial resistance genes for H. influenzae. Recovery of Borrelia burgdorferi reads (6.1 reads per million [RPM] and 9.03 RPM) was achieved from clinical samples with late cycle threshold values (39.7 and 43.0, respectively). Limit of detection (LoD) studies demonstrated detection of HSV-1 and S. pneumoniae reads at concentrations of 50 genomes/mL each, which is below the reported LoD for the orthogonal NAATs used in this study. Reducing sequencing costs and improving the analytical sensitivity remove two major hurdles for mNGS adoption by clinical laboratories. While these results are preliminary, they demonstrate a future in which mNGS may be more widely implemented.IMPORTANCEClinical metagenomic next-generation sequencing has struggled to gain wider adoption for nearly a decade, due in part to its high cost and reduced performance versus targeted molecular assays. This study demonstrates the ability of the UG100 sequencing platform to reduce per-base metagenomic sequencing costs while producing reads that maintain high positive agreement with existing molecular assays. Further improvements to cost and analytical performance may shift clinical metagenomics from an expensive test of last resort to a front-line diagnostic for identifying infections.

Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin that hijacks bacterial iron uptake pathways to traverse the outer membrane, offering potent activity against carbapenem-resistant Acinetobacter baumannii (CRAB). Although mutations in pirA, a TonB-dependent siderophore receptor, have been linked to CFDC resistance, the broader genetic basis remains poorly defined. Using Himar1 transposon mutagenesis in a CFDC-susceptible sequence type 2 (ST2) CRAB strain, we identified ten genes whose disruption reduced CFDC susceptibility, spanning siderophore-mediated uptake (pirA and puiA), oxidative and redox stress responses (oxyR, nfuA, aarF, cyoA, and bfmRS), and cell envelope morphogenesis (mreB). Most mutants retained wild-type susceptibility to other β-lactams, indicating that reduced CFDC susceptibility can arise independently of target modification. Quantification of cellular iron revealed modest reductions in several mutants, with the largest decreases observed in strains with disruptions in TonB-dependent receptors. Inactivation of pirA or puiA altered the expression of several alternative TonB-dependent siderophore receptors. Whole-genome sequencing of ST2 clinical isolates with reduced CFDC susceptibility uncovered mutations in TonB-dependent receptors, porins, and PBP3, along with increased β-lactamase expression. Importantly, the β-lactamase inhibitor avibactam restored CFDC susceptibility in isolates with β-lactamase upregulation and intact uptake pathways, whereas strains with concurrent uptake defects remained resistant, underscoring the interplay between permeability and enzymatic drug inactivation. These findings define a multifactorial resistance landscape integrating impaired uptake, redox and envelope stress adaptation, and β-lactamase-mediated drug inactivation.IMPORTANCECefiderocol (CFDC) is one of the few remaining antibiotics with activity against carbapenem-resistant Acinetobacter baumannii (CRAB), an urgent global health threat. Yet, resistance to CFDC is increasingly reported, and the underlying mechanisms remain incompletely defined. Most prior studies have examined single pathways, such as loss of TonB-dependent receptors. Here, we used genome-wide transposon mutagenesis together with genomic and phenotypic analysis of CFDC-resistant clinical isolates to generate a more comprehensive view of how resistance emerges. Our findings show that CFDC resistance is multifactorial, involving disrupted siderophore uptake, alterations in oxidative and envelope-stress responses, porin and cell-wall changes, and β-lactamase activity. By defining how these pathways converge, this work provides a broader mechanistic framework for interpreting emerging resistance in clinical settings. These insights underscore the need for integrated surveillance strategies and highlight the biological complexity that must be considered to preserve the effectiveness of this last-line antibiotic.