Microbiology Spectrum

Tuberculosis screening is not mandatory for prospective tissue donors. In 2021 and 2023, two different bone allograft products caused nationwide tuberculosis outbreaks. We assessed the morbidity and mortality of the second outbreak and reviewed donor and tissue screening to identify deficiencies. Thirty-six people residing in nine states received the product during spinal and dental procedures. Twenty-seven recipients had tuberculosis infection, 11 had microbiologic or imaging evidence of tuberculosis disease, and two died from tuberculosis within 12 months of outbreak detection. Another recipient died from tuberculosis nearly 3 years after product implantation. The bone donor died of pneumonia and septic shock. Polymerase chain reaction testing of the product before and after distribution did not detect Mycobacterium tuberculosis. Mycobacterial culture was not performed until after outbreak detection, when M. tuberculosis was isolated from 2 of 6 unused product units. This outbreak demonstrates persistent gaps in tissue transplant safety. Appropriate selection of donors and mycobacterial culture of donated tissues could reduce but not eliminate the risk of M. tuberculosis transmission. Therefore, it is important that clinicians monitor tissue recipients and promptly report adverse events to tissue establishments and health authorities.

Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the hosts immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNAs susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. IMPORTANCEmNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. Key PointsO_LIThe choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. C_LIO_LIThe combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. C_LIO_LIRNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics. C_LI

ObjectivesListeria monocytogenes is an opportunistic pathogen, associated with foodborne infections that disproportionately affect newborns, elderly and immunocompromised patients. L. monocytogenes can be classified on the antigenic and related structural variation of cell-associated wall teichoic acid (WTA) molecules through conventional serotyping techniques. The WTA structure of serovars (SV) 1/2, 1/2*, 3 and 7 consists of a linear poly-ribitolphosphate (RboP) polymer either with or without decoration with rhamnose (Rha) and/or N-acetylglucosamine (GlcNAc). Of these four SVs, SV1/2 (WTA with GlcNAc and Rha) causes [~] 99% of all listeriosis cases. However, conventional serotyping cannot accurately discriminate between these four SVs, particularly SVs1/2* (WTA with Rha). MethodsHere we applied two identified monoclonal antibodies (mAb), with specificity for the RboP backbone or GlcNAc modification to develop a discriminatory serotyping scheme for SV1/2, 1/2*, 3 and 7. Isogenic mutants for the different SVs were created in L. monocytogenes SV1/2 strain EGD-e. The typing scheme was then adapted to an immnoblot assay and applied to a collection of 317 previously classified listeriosis isolates from the Netherlands Reference Laboratory for Bacterial Meningitis. ResultsBinding of the RboP-specific mAb was limited to EGD-e wild type (SV1/2), but increased significantly for isogenic EGD-e mutants representing SV1/2*, 3 and 7. In contrast, the GlcNAc-specific mAb only recognized EGD-e mutants representing SVs 1/2 and 3. The combined staining profiles of the two mAbs allowed accurate discrimination of the four SVs as verified on clinical isolates. Applying this typing scheme to 317 listeriosis isolates previously typed as SV1/2, we confirmed SV designation in >90% of isolates, but also identified SV1/2* (5.4%), SV3 (0.6%) and SV7 (0.3%) isolates. SV1/2* isolates were also identified among meningitis patients. ConclusionThe increased discriminatory capacity of L. monocytogenes serotyping provides a more detailed insight of the epidemiological landscape and the critical factors for L. monocytogenes infections.

Bacteria require rapid adaptation under fluctuating environmental conditions. Commonly recognized global regulators enable bacteria to respond promptly to external changes, though they are either restricted to specific bacterial taxonomies or physiological statuses, suggesting that additional regulators are required for adaptation. DNA methylation is a reversible modification affecting bacterial gene regulation. However, conventional methods can only detect one DNA methylation form each round, leaving the understanding of DNA methylation in bacterial adaptation mostly unknown. This study aimed to identify genome-wide DNA methylation variation (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) in Escherichia coli under different culture conditions using Oxford Nanopore sequencing. DNA samples from six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure) during the exponential and stationary phases were extracted. When culture conditions were compared to the normal condition, E. coli exhibited more differentially methylated sites during the exponential phase than in the stationary phase. During the exponential phase, the genes differentially methylated in all conditions were involved in cellular activities, such as cellular and metabolic processes. During the stationary phase, universally differentially methylated genes were associated with oxidation responses. Subsequent analysis found that although DNA methylation analysis was affected by batch effects, some genes (e.g. rpoS) showed consistently differential methylation across datasets. Our findings suggest that the E. coli DNA methylation profile was affected by growth phases and conditions, and DNA methylation profiling by Oxford Nanopore sequencing could be a potential approach for gene activity estimation in environmental samples. ImportanceBacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. Three major forms in bacteria are N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we characterized genome-wide variation in these methylation types in Escherichia coli under six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure). DNA methylation signatures in E. coli varied with growth conditions. Using the normal condition as a baseline, E. coli during the exponential phase exhibited more differentially methylated genomic loci under stress conditions compared to the stationary phase. Under stress conditions, genes with differential methylation were associated with cellular processes or oxidative responses, depending on the growth phase. Our findings reveal that the DNA methylation signature in E. coli was affected by growth phases and conditions, and Oxford Nanopore-based DNA methylation profiling could be a potential approach for gene activity estimation in environmental samples.

Lacritin is a tear, saliva, plasma and cerebrospinal fluid glycoprotein with broad polypharmacology. Selective deficiency of its bioactive monomeric form appears to be deleterious for ocular surface health for which replacement therapy is beneficial. Its cleavage-potentiated C-terminus represented by the N-104 proteoform in tears is bactericidal and synergizes with the tear thrombin peptide GKY20. In the pathogenic and multidrug resistant PA14 strain of P. aeruginosa, we recently discovered that N-104 binds to the outer-membrane lipoprotein YaiW to gain access to the periplasm where it targets and inhibits the inner-membrane ferrous iron transporter FeoB (of FeoABC) as well as PotH, a subunit of the polyamine transporter PotFGHI. Further, PA14 gene expression shifts toward anaerobic respiratory pathways. Here we explore N-104-associated transcriptional changes over a broader range of functional categories pointing to a reduction in: (i) virulence by suppressed gene expression of virulence factors AprA and LasA; and Hcp1 and PsrA necessary for the respective assembly of type VI and III secretion systems, (ii) fitness (less AtsC, MgtA), (iii) metabolism (less AdhA, AtsC, GcvH2, GcvP2, FadE1, SsuD, SsuF, TauB, TauD, UspK, UspN), (iv) stress response (less UnG, PfpI, RmF), (v) proteostasis (less ClpB, GrpE, HtpG), (vii) quorum sensing (less CifR, GcvH2, GcvP2, PsrA, QuiP), and (viii) survival under anaerobic conditions (less AdhA, MhR, ModA, UspKLNO). Upregulated genes are directed towards enhancing PA14: (i) multidrug (more OprJ of MexCD-OprJ) and (ii) tellurite (more TerC) efflux, coupled with a seemingly PA14 survival attempt at (iii) anaerobic respiration (more NosR), (iv) translational fidelity (more QueE, RimP, TrmD) and (v) metabolism (CysT, MoaA1, Sbp, SsuA, SsuE). The overlap with aminoglycosides (4.3%), {beta}-lactams (0%), cyclic peptides (2.5%), fluoroquinilones (0%) and macrolide (1.9%) classes of antibiotics in P. aeruginosa was minimal. Thus, N-104 appears to widely perturb PA14 fundamental processes in a distinctive manner.

Background: External Quality Assurance (EQA) is an essential component of modern laboratory medicine. Current scientific evidence on EQA focuses primarily on the analyses carried out by EQA providers while relatively little research has been conducted in individual clinical laboratories. Methods: In this retrospective single-center observational study in a clinical laboratory, EQA results were analyzed over a period of four years (2021-2024). The evaluation was based on EQA action reports documented in the institutes internal quality management system. Deviations were classified according to department, type of discrepancy, root cause category (analytical, preanalytical, systemic, unidentifiable), and measures taken. Results: A total of 7226 EQA participations were evaluated during the observation period. The overall error rate remained consistently low, ranging between 0.8% and 1.6%, with no significant change over time (p = 0.87). Most deviations occurred in the departments of clinical chemistry and immuno/autoimmune diagnostics (p < 0.001). These were predominantly quantitative discrepancies (false low/false negative or false high/false positive). Root cause analysis showed a clear dominance of analytical causes (p < 0.001), while preanalytical and systemic causes were identified less frequently. In most cases, corrective measures, such as re-analyses, recalibrations, process adjustments, or staff training, were implemented promptly. Hard structural measures, such as changing methods or discontinuing tests, were rarely necessary. Conclusion: In a clinical laboratory, EQA is an important tool for structured error analysis and continuous quality improvement. Consistent processing of deviating EQA results goes hand in hand with stable analytical performance and a low error rate.

Serratia marcescens is an opportunistic pathogen that causes severe hospital-acquired infections, notable for its biofilm formation abilities and development of extensive antibiotic resistance. Here we evaluated the efficacy of bacteriophages, antibiotics, and antimicrobial peptides (BAP), alone and in combination, against fourteen multi-drug-resistant (MDR) S. marcescens isolates sourced from hospitals and other environmental settings in an in vitro biofilm model. Phage combination with a cocktail of sub-minimal inhibitory concentration (MIC) of penicillin-streptomycin, kanamycin, and ciprofloxacin, reduced biofilm biomass, however, complete decolonization was not achieved. Incorporating an antimicrobial peptide cocktail into this regimen eradicated 99.99% of multi-drug-resistant isolates grown planktonically or in surface-associated biofilms. Microscopy and viability assays confirmed extensive biofilm disruption and bacterial clearance without regrowth. These findings reveal that simultaneous interference of cell wall synthesis, protein translation, DNA replication, and membrane integrity can overcome S. marcescens antimicrobial defenses, establishing a multifaceted therapeutic framework for managing device-associated infections caused by MDR pathogens.

BackgroundVirtual colony count is a kinetic, 96-well turbidimetric assay that has been used since 2003 to determine the antimicrobial activity of antimicrobial peptides including the defensin HNP1. Virtual colony count results differed from traditional colony counting results in studies of the antimicrobial activity of the human cathelicidin LL-37 and related peptides. The difference could possibly have been caused by an inoculum effect. MethodsThe virtual colony count assay was conducted using inocula that varied from 1250 to 1x108 virtual colony forming units (CFUv) per milliliter. ResultsThe virtual colony count assay demonstrated a pronounced inoculum effect of HNP1 against Staphylococcus aureus ATCC 29213, accompanied by biofilm formation observed in the wells of the 96 well plates at all inocula. The S. aureus inoculum effect was not as drastic as previously reported for Escherichia coli. ConclusionsThe inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1.

Salmonella is a major zoonotic foodborne pathogen, and antimicrobial resistance (AMR) in Salmonella presents a significant public health challenge. Whole-genome sequencing (WGS) offers a more rapid and comprehensive method for AMR characterization compared to conventional antimicrobial susceptibility testing (AST), supporting antimicrobial therapy and surveillance efforts. In this study, Oxford Nanopore Technology (ONT)-based WGS was performed on 1,490 Salmonella isolates collected through nationwide surveillance in Taiwan in 2025. Genotypic resistance inferred from WGS data was compared with phenotypic AST results to assess the performance of ONT-WGS. Overall, WGS-inferred resistance showed high concordance with phenotypic resistance for most antimicrobials. However, major genotype- phenotype discordance was observed, attributed to four categories: (i) breakpoint-dependent classification, (ii) reduced or absent phenotypic expression of resistance genes, (iii) MIC modulation by ramAp, and (iv) absence of known AMR determinants. Notable discrepancies included tigecycline resistance without known genetic determinants, nalidixic acid resistance linked to ramAp-mediated MIC elevation, and a high prevalence of colistin resistance (35.4%) in S. Enteritidis without identifiable AMR determinants. Additionally, a significant proportion of ESBL- and AmpC-producing isolates were classified as susceptible or intermediate to cefotaxime and ceftazidime under CLSI criteria, highlighting the potential for misclassification and treatment failure. These findings demonstrate that ONT-WGS enables accurate, comprehensive AMR characterization, offering direct identification of AMR determinants and minimizing misclassification due to breakpoint-based AST interpretations. When interpreted appropriately, WGS can support better antimicrobial selection and serve as a valuable alternative to conventional susceptibility testing.

Noble rot, caused by Botrytis cinerea, profoundly alters grape berry physiology and is essential to produce botrytized wines. In this study, we profiled bacterial and fungal communities associated with Vitis vinifera cv. Semillon berries across four stages of noble rot development and four consecutive vintages using 16S rRNA gene and ITS1 amplicon sequencing. Noble rot stage significantly impacted the structure of bacterial communities across vintages, while fungal communities showed more variable vintage-dependent responses. Bacterial alpha diversity increased consistently at advanced stages of infection (S3), coinciding with a marked shift from Pseudomonas-dominated communities toward acetic acid bacteria, particularly Gluconobacter, which was significantly enriched at S3 in all vintages. Fungal communities remained dominated by Sclerotiniaceae throughout infection, consistent with pervasive B. cinerea colonization, while non-Botrytis fungal taxa shifted from filamentous fungi such as Cladosporium and Alternaria toward fermentative yeasts including Hanseniaspora and Lachancea. Co-occurrence network analysis revealed a positive association between Gluconobacter and these fermentative yeasts, suggesting coordinated enrichment of oxidative and fermentative microorganisms at advanced noble rot stages. Together, these results reveal a reproducible stage-associated microbial succession during noble rot progression and identify acetic acid bacteria as consistent markers of advanced infection.

Ethambutol (EMB) is a vital drug for treating Mycobacterium avium-intracellulare (MAI) infections; however, the genomic mutations underlying EMB resistance in MAI remain unclear. Herein, we evaluated eight sets of MAI clinical isolates, each containing at least two serial isolates collected from the same patient who received EMB in Japan. In four sets, the isolates independently increased EMB MIC by 4-fold, coinciding with mutations in the upstream region of embA or those corresponding to Mycobacterium tuberculosis (Mtb) embB Met306Val and Gln497Arg. Based on the increased EMB MIC values, we defined normal and elevated EMB MICs as [&le;]8 {micro}g/mL and [&ge;]16 {micro}g/mL, respectively. In the other four sets, all of the isolates had elevated EMB MICs. In silico promoter prediction and expression analysis indicated that the upstream region of embA corresponds to the embA-embB promoter region, and mutations in this region increased the transcription of embA and embB, increasing EMB MICs. Furthermore, the analysis of 60 epidemiologically unrelated strains revealed that isolates with mutations in the embA-embB promoter and at embB codons 306/497 exhibited significantly higher EMB MICs compared with those without mutations. Publicly available genomic data demonstrate the worldwide occurrence of these mutations in clinical isolates. These results establish an association between elevated EMB MICs and mutations at embB codons 306/497 and the embA-embB promoter and are expected to predict EMB resistance.

Salmonella spp. remains one of the leading foodborne pathogens worldwide, and the circulation of multidrug-resistant strains in the poultry industry poses a significant challenge. In this study, five isolates from poultry litter swabs (commercial broiler chickens) belonging to the Salmonella Heidelberg and Salmonella Minnesota serovars were characterized using an integrated approach involving phenotypic resistance profiling, whole-genome sequencing, structural prioritization of molecular targets, and in silico screening of ligands. All isolates exhibited multidrug resistance phenotypes and genetic repertoires consistent with resistance to {beta}-lactams, sulfonamides, and tetracyclines, as well as determinants linked to efflux systems, virulence, and persistence. Genomic analysis allowed for the prioritization of five proteins for structural investigation: CTX-M-2, CMY-2, Sul2, AcrB, and SpvC. Sequence-structure validation revealed high correspondence between the proteins of the isolates and the experimental structures selected for CMY-2, Sul2, AcrB, and SpvC, while CTX-M-2 was modeled with high structural confidence. Molecular docking analyses with GNINA revealed distinct behaviors among the targets. Sul2 showed biological relevance but a more conservative structural response, with no significant gain after analog generation. In contrast, AcrB stood out as the most promising target, with analogs generated by BRICS yielding better scores and, in some cases, coherent international networks identified by PLIP. The results demonstrate that the integration of phenotype, comparative genomics, and structural prioritization constitutes a rational strategy for selecting targets and molecular candidates in multidrug-resistant avian strains of S. Heidelberg and S. Minnesota.

AimsPseudomonas aeruginosa biofilm-associated infections pose a significant clinical challenge due to their inherent antibiotic tolerance. This study aimed to evaluate the antibacterial and antibiofilm activity of Placentrex, a standardised aqueous placental extract, against P. aeruginosa and to elucidate its molecular mechanism of action using RNA sequencing (RNA-seq). Methods and ResultsPlacentrex exhibited potent bactericidal activity against P. aeruginosa at 50 mg/mL. Biofilm formation was significantly inhibited by [~]87% at 50mg/mL after 72 hours. Preformed biofilms were eradicated by [~]93% and [~]89% at 50 and 25 mg/mL, respectively. Interestingly, biofilm viability was reduced by [~]93% and [~]87% upon treatment with 50 mg/mL and 25 mg/mL of Placentrex, respectively. EPS characterisation revealed that the EPS contain a single large polysaccharide, and chromatography data suggested that it is made up of glucose as a monomer. RNA-seq identified coordinated downregulation of seven key genes, namely, flp major pilin (surface attachment), extracellular solute binding protein (ABC transporter-mediated nutrient sensing and biofilm maintenance), gntP permease (carbon metabolism), AraC family transcriptional regulator (quorum sensing and polysaccharide biosynthesis), ureE (urease nickel metallochaperone), aromatic amino acid permease (pyoverdine and PQS biosynthesis), and MFS transporter (efflux and autoinducer export). ConclusionsPlacentrex exerts comprehensive antibiofilm and antibacterial activity through simultaneous disruption of surface attachment, nutrient-sensing-driven biofilm maintenance, quorum sensing, carbon metabolism, urease virulence maturation, and efflux-mediated persistence. This polypharmacological mechanism supports Placentrex as a promising multi-target antibacterial agent against P. aeruginosa biofilm-associated infections. Impact statementPlacentrex is a potential anti-biofilm agent against Pseudomonas aeruginosa.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen, with antibiotic resistance mediated by the vanA and vanB operons. Rapid and accurate detection of antibiotic resistance is critical for the timely treatment of bacteremia and sepsis. Although imaging-based approaches using fluorescence in situ hybridization (FISH) provide a potential diagnostic solution, detecting mRNAs of antibiotic resistance genes (ARGs) in individual cells remains particularly challenging due to their low copy number and transient expression. Here, we present a peptide nucleic acid (PNA)-FISH method for direct detection of vanA- and vanB-associated resistance in individual VREfm cells. A universal probe targeting the conserved region across vancomycin resistance genes and a set of probes exclusively targeting the vanB gene were designed. The universal probe showed increased fluorescence in the vanA-genotype strain upon vancomycin or teicoplanin treatment, and in the vanB-genotype strain upon vancomycin treatment. In contrast, vanB-specific probes showed increased fluorescence exclusively from the vanB-genotype strain upon vancomycin treatment, confirming their specificity to the vanB gene. Efficient cellular penetration and strong hybridization of PNA probes enabled efficient and accurate detection of antibiotic-resistant bacterial cells, even under a wide-field fluorescence microscope. No detectable signals above background were observed in other major bacterial species associated with bacteremia and sepsis. These findings demonstrate robust detection of antibiotic-resistant cells in mixed microbial populations. When integrated with microbe-capturing techniques, this method may support culture-free detection of antibiotic resistance without nucleic acid amplification or sequencing, with the potential to reduce diagnostic turnaround time.

Acinetobacter spp. represents critical opportunistic pathogens driving severe bloodstream infections (BSIs) in intensive care unit (ICU) and neonatal intensive care unit (NICU) settings. The convergence of carbapenem resistance and emerging biocide tolerance, often mediated by mobile genetic elements, has intensified concerns regarding co-selection and persistence in clinical environments. A total of 90 molecularly confirmed Acinetobacter isolates (ICU = 44; NICU = 46) from bloodstream infections were analyzed. Antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method in accordance with CLSI M100 (2024) guidelines and extended-spectrum {beta}-lactamase production was assessed by combined disc diffusion. Polymerase chain reaction (PCR) was employed to detect carbapenemase genes (blaVIM, blaNDM, blaIMP, blaOXA-23, blaOXA-58), biocide resistance determinants (qacE, qac{Delta}E1), and the class 1 integron-integrase gene (intI1). Multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes were identified in 71.1% (64/90) and 22.2% (20/90) of isolates, respectively. High resistance (>71%) was observed against meropenem and cephalosporins, whereas colistin (51.1%, 46/90) and amikacin (47.8%, 43/90) showed moderate susceptibility. The most prevalent genotypes were qac{Delta}E1 (76.6%, 69/90) and blaVIM (56.6%, 51/90). Statistical and network analyses revealed significant correlations between biocide and carbapenemase genes, identifying IntI1 as a primary driver of co-resistance. The findings demonstrate that integron-mediated co-carriage of carbapenemase and biocide resistance genes is a major driver of MDR and XDR phenotypes in Acinetobacter BSIs. This co-selection dynamic highlights the urgent need to reassess disinfection strategies alongside antimicrobial stewardship to curb the persistence and spread of highly resistant strains in critical care settings.

Biofilms pose a significant challenge to antimicrobial therapy. Bacteria in biofilms differ from planktonic counterpart in their altered metabolism, collective behavior, protective role of extracellular matrix and diversified microbial subpopulations. These attributions significantly influence bioavailability and activity of antibiotics. The presence of bacterial aggregates during acute infections expands the problem to many other conditions previously not discussed in the biofilm context. Klebsiella pneumoniae is a leading cause of life-threatening hospital-acquired infections and is included in the WHO Bacterial Priority Pathogens List due to increasing antimicrobial resistance. The combination of antimicrobial resistance and the ability to form biofilms severely limits the efficacy of antibiotic treatments. In this study, we investigated the in vitro susceptibility of mature biofilms to 13 antimicrobials of K. pneumoniae clinical isolates from a single hospital. The resistance profiles of the local clinical isolates were consistent with the global epidemiology of K. pneumoniae. Minimal biofilm eradication concentrations (MBEC) for mature biofilms were defined with two assays (biomass and metabolic activity measurements) and brought into relation with susceptibility breakpoints and plasma (Cmax). Colistin sulfate, tigecycline, cephalosporins and combination of imipenem with cilastatin were the most potent biomass eradicators, while suppression of metabolic activity was barely reachable. Moreover, we observed a notable increase in metabolic activity upon exposure to sub-MBEC concentrations of antibiotics. Finally, our data broach a subject of antibiotic prioritization with respect to biofilm tolerance. IMPORTANCEThis study addresses the critical gap between standard antibiotic susceptibility testing and the tolerance of biofilm and microbial aggregates during infections caused by K. pneumoniae. By systematically evaluating mature biofilms from a significant number of clinical isolates, we demonstrate that colistin and tigecycline show potent activity against both biofilm biomass and metabolic activity, whereas cephalosporins primarily reduce biomass without effectively suppressing bacterial metabolism, and other drugs have only weak effects on biofilms at clinically achievable concentrations. Furthermore, the alarming observation that sub-inhibitory biofilm eradication concentration (sub-MBEC) of antibiotic can paradoxically increase the metabolic activity of biofilms highlights a potential risk factor for therapy failure and resistance development. Our findings contribute to the necessary evidence base for prioritizing existing antibiotics in the limited armamentarium against biofilm-forming K. pneumoniae.

The high prevalence of aerotolerant human Campylobacter jejuni isolates suggests a correlation between the ability to survive in aerobic conditions, virulence and resistance to harsh stress conditions. However, the mechanisms are still unclear. Here, we investigated the role of bacterial extracellular vesicles (bEVs) in the adaptation of the clinical aerotolerant C. jejuni Bf strain to thermal and oxidative stress. We show that C. jejuni Bf survives and actively multiplies under this combined stress. Stress exposure induced cell rounding and loss of motility, remodeling of membrane composition, decreased membrane fluidity, and metabolic reprogramming with increased intracellular ATP levels. Lipidomic analyses further revealed that bEVs composition is markedly different from that of the parent membranes indicating that vesicle formation is selective and regulated. Although bEVs were produced in similar amounts under both microaerophilic and stress conditions, stress exposure generated significantly larger vesicles with greater diameter and dry mass, and altered their protein and lipid profiles. bEVs derived from stressed cells showed increased toxicity toward the epithelial barrier of Caco-2 cells. Taken together, these results indicate that C. jejuni bEV secretion is part of a survival strategy that connects environmental adaptation with pathogenicity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/714464v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@17aa2aforg.highwire.dtl.DTLVardef@4eab9dorg.highwire.dtl.DTLVardef@e4fba8org.highwire.dtl.DTLVardef@146109a_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectivesAntimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil. MethodsA total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05. ResultsK. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting {beta}-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors. ConclusionsMDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies. What is known?O_LIAntimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality. C_LIO_LIKlebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are on the World Health Organizations (WHO) priority list as major causes of morbidity and mortality worldwide. C_LIO_LIClassical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria. C_LI What is new?O_LIThe study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae, A. baumannii, and P. aeruginosa circulating in hospital ICUs in Brazil. C_LIO_LIMDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes (bla_KPC, bla_NDM, bla_CTX-M, and OXA) and RND-type (MEXs) and acrAB-tolC efflux pumps, without changes in virulence genes. C_LIO_LIProteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation. C_LI

Phage therapy offers promise to combat antimicrobial resistance, including drug-resistant tuberculosis (TB). Understanding phage activity against Mycobacterium tuberculosis (Mtb) adapted to physiologic microenvironments, such as hypoxia and acidity in granulomas, is essential since these conditions induce non-replicating states. We evaluated a phage combination against Mtb under hypoxic, acidic (pH 5.5), and stationary-phase conditions in vitro. In planktonic Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in Mtb H37Rv load, which was maintained over 31 days. Phage addition prevented regrowth was observed with rifampicin and isoniazid alone. Individual phage stability was differentially affected by acidic media conditions, resulting in variability of antimycobacterial activity. In hypoxic conditions and stationary growth experiments, phage titers remained stable over time with no change in mycobacterial load compared to controls. Model-based predictions were able to adequately capture phage-mycobacterial interactions with and without rifampicin. The lack of antimycobacterial activity in assays with non-replicating mycobacteria suggest that phages need actively replicating mycobacteria to exert lytic activity. Stable phage concentrations in assays with non-replicating mycobacteria suggests low grade phage replication in these conditions. Established models can support future study design through simulations of different experimental scenarios.

Emerging infectious diseases and antimicrobial resistance (AMR) have surfaced as two major public health threats over the past two decades. Consequently, integrative surveillance systems capable of detecting both emerging pathogens and resistance-carrying bacteria are crucial. With advances in next-generation sequencing, simultaneous detection of pathogens and AMR is increasingly feasible. In this study, we used short-read metatranscriptomics complemented by total 16S rRNA metagenomic long-read sequencing to analyze paired oral and plasma samples from a cohort of febrile individuals at two locations in Senegal. Oral microbiomes differed in community composition between locations, and reduced diversity and richness were significantly associated with high fever. We identified at least one known pathogen in 15.33 % (23/150) of samples, with Borrelia crocidurae as the most frequently detected pathogen. We detected both pathogenic and non-pathogenic viruses in oral (10/72) and plasma (09/78) samples. Finally, we observed a high frequency of genes associated with resistance and virulence: 10% of samples expressed at least one AMR gene (ARG), and 24% expressed virulence factor genes. Resistance to widely used beta-lactam antibiotics was the most prevalent. Our findings provide critical data on oral and plasma microbiomes in the context of acute febrile illness in Senegal while expanding understanding of circulating ARGs.