Microbiology Spectrum

ObjectiveUrine cultures are frequently requested at an early stage in primary care and outpatient settings, often without a comprehensive clinical assessment. This practice increases healthcare costs and laboratory workload and may lead to misleading results due to asymptomatic bacteriuria and specimen contamination. This study aimed to evaluate whether routinely reported microscopic urinary leukocyte findings can predict urine culture positivity under real-world clinical conditions. The distribution of isolated microorganisms and the frequency of mixed or contaminated growth were assessed. MethodsThis retrospective, laboratory-based diagnostic accuracy study included all urine samples sent for culture over a one-year period at a tertiary care hospital, provided concurrent microscopic urinalysis was available. No additional clinical exclusion criteria were applied to reflect the routine practice. Leukocyte findings were reported semi-quantitatively and analyzed both categorically and as approximate numerical values. The urine culture results were classified as positive, negative, or mixed/contaminated growth. The diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. ResultsA total of 8,478 urine samples were analyzed in this study. Urine culture positivity was detected in 2,666 (31.4%) samples, whereas 5, 812 (68.6%) showed no growth. Culture positivity increased significantly with higher leukocyte levels (p < 0.001), ranging from 13.1% in the lowest category to 83.1% in samples with abundant leukocytes. ROC analysis demonstrated an acceptable discriminative performance (AUC = 0.747). The Youden index identified an optimal threshold of approximately 5.5 leukocytes per high-power field, with a sensitivity of 60.4% and a specificity of 77.8%. Mixed or contaminated growth was the most common finding among culture-positive samples (43.5%), followed by Escherichia coli (29.5%). ConclusionMicroscopic urinary leukocyte findings were significantly associated with urine culture positivity and demonstrated acceptable predictive performance in real-world clinical practice settings. Although leukocyte microscopy alone is not diagnostic, it may support more selective urine culture ordering, reduce contamination, and contribute to rational diagnosis and antimicrobial management in primary care.

Parenteral drugs must meet strict release criteria to ensure patient safety upon administration. Sterility is a critical requirement, and is typically assessed using the compendial U.S. Pharmacopoeia (USP) <71> sterility test. However, with the growing demand to reduce batch release times, the tests 14-day incubation period is becoming a concern, emphasizing the need for more rapid alternatives. Here, we compared the performance of the calScreener+ isothermal microcalorimetry (IMC) device (Symcel) to that of the compendial USP <71> sterility test, using a panel of sixteen microorganisms (six USP <71> reference strains and ten field isolates) in two inoculum sizes (100 and 5 CFU). The IMC-based method detected a higher number of positive samples compared to the compendial method (95.8% vs 87.5%; p < 0.05). Furthermore, IMC was consistently faster, reducing mean detection times from 43 hours to 19 hours at 100 CFU and from 46 hours to 28 hours at 5 CFU (p < 0.001). In conclusion, the calScreener+ IMC device shows promise as a rapid and sensitive alternative to the compendial sterility test, with the potential to speed up batch releases without compromising patient safety.

Validated inactivation procedures are required for the safe handling and downstream analysis of highly pathogenic organisms, particularly those categorized as biological select agents and toxins (BSATs). TRIzol-based extraction methods are widely used for nucleic acid and protein isolation, yet their reliability for bacterial inactivation has not been comprehensively evaluated. In this study, we assessed TRIzol-based extraction methods for sample quality and inactivation reliability across a series of mock failure scenarios using five attenuated bacterial isolates: Francisella tularensis holarctica LVS, Bacillus anthracis Sterne, Yersinia enterocolitica, Mycobacterium marinum, and Burkholderia cepacia. Dilution of TRIzol to induce incomplete cell lysis for the initial extraction step, including 0% TRIzol, consistently inactivated all surrogate organisms, suggesting that downstream precipitation and sample washing reagents, including isopropanol and 70% ethanol, were sufficient to inactivate organisms in the absence of TRIzol. Several protocol failure scenarios were then evaluated to simulate human error by omitting extraction, precipitation, and washing steps individually or in combination for the most resistant organism, B. anthracis Sterne strain. Failure-scenario testing demonstrated that reliable inactivation of B. anthracis required strict adherence to the complete protocol due to the spore-forming ability of B. anthracis. Collectively, this work provides a reference with experimental evidence supporting the use of TRIzol-based extraction as a bacterial inactivation strategy for a wide range of bacterial pathogens.

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

Streptococcus constellatus is an opportunistic pathogen frequently associated with abscess formation in various body sites. While the species has been shown to acquire exogenous DNA through natural transformation, functional analyses of its underlying mechanisms and optimized genetic editing protocols remain limited. Thus, our aim was to characterize the natural transformation system in S. constellatus and investigate environmental factors regulating its natural transformation system. In addition, we sought to develop an optimized protocol for genome editing. Genomic analysis revealed that 73% of analyzed strains possess orthologs for essential competence regulon genes, with 55% harboring both a complete ComCDE-based operon and the necessary transformation machinery. While all complete genomes harbored three copies of the master regulator sigX, the accessory regulator comW was seemingly absent. Lacking the peptide exporter comAB, we demonstrated that S. constellatus utilizes the bacteriocin transporter silED for competence-stimulating peptide export. Gene expression assays indicated system activation at peptide concentrations as low as 4 nM, with peak sigX expression obtained over 60 nM. With the goal of optimizing gene editing strategies, we developed a protocol utilizing rich media supplemented with bovine serum albumin and calcium chloride, substantially increasing transformation frequencies. Furthermore, we observed that environmental stressors can upregulate the system, including hydrogen peroxide and subinhibitory concentrations of the antibiotics erythromycin, chloramphenicol, and ampicillin. Given the increasing clinical relevance of the anginosus group, elucidating horizontal gene transfer mechanisms can provide critical insights into the evolutionary success and pathogenic potential of these species.

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.

AimsBacteriophage (phage) propagation has traditionally relied on bacterial culture media containing animal-derived ingredients; however, safety concerns with animal-derived materials for production of phages for therapeutic use limit their acceptability. We compared animal-free and traditional media formulations, and evaluated their effects on phage yield, bactericidal activity, and genomic characteristics, hypothesizing no significant differences would be observed. Methods and ResultsPhages targeting Pseudomonas aeruginosa (n=8) and Staphylococcus aureus (n=1) were propagated in solid and liquid media containing animal-free (AF) or animal-derived (LB) peptones. Kinetic assays were used to assess phage suppression of host bacterial growth. In a mock therapeutic phage screen, spot tests, Efficiency of Plating (EOP) and kinetic assays were performed against novel bacterial targets. Whole genome sequencing of phages and their bacterial hosts propagated in AF or LB broth was used to observe genomic differences between formulations. Animal-free peptone did not impact phage yield, with both AF and LB phage stocks growing to high titers ([&ge;]108 PFU/mL). Kinetic assay results showed similar suppression indices for AF and LB-grown phages. Likewise, phage screen spot test, EOP, and kinetic assay results were similar between AF and LB phages. Comparisons of phage and bacterial genome annotations showed no major differences arising from media formulation. ConclusionsFindings suggest animal-free peptones do not significantly alter phage yield, bactericidal activity, or genomic characteristics, supporting use of animal-free medium for medicinal phage manufacture. This is one of the first studies to systematically combine phenotypic and genomic assessment of phages and hosts across animal-free and traditional media. Impact StatementPhage therapy is increasingly used to treat antimicrobial resistance infections. Emerging guidelines and regulations for the manufacture of phage therapeutics will impact laboratory processes and materials used for phage production. Here, we explored the use of an animal-free medium for medicinal phage propagation, providing data on phage yield and metrics of phage activity.

BackgroundBiofilms are central to Salmonella pathogenesis, and targeting their formation is believed to produce less evolutionary pressure of growth inhibition than traditional antibacterials. In this study, we screened the Medicines for Malaria Venture (MMV) Pathogen Box library to identify anti-biofilm agents against S. enterica that possess drug-like properties. Methodology/ Principal FindingsA crystal-violet-based medium-throughput antibiofilm screen of Salmonella enterica serovar Typhimurium ATCC 14028 and a clinical Salmonella enterica serovar Elisabethville isolate was performed on polystyrene surfaces using the 400-compound Pathogen Box library. Compounds that inhibited biofilm formation by >30% and growth by <10% were identified as hits. Salmonella red-dry-rough and motility phenotypes were explored in mechanism of action studies on one hit compound. The Salmonella antibiofilm hit rate was 0.75% for this library. MMV688371 (benzamide) inhibited biofilm formation of S. Typhimurium ATCC 14028 by 33% without inhibiting growth. An ethambutol analogue (MMV687273) and auranofin (MMV688978) met the hit criteria against S. Elisabethville LLD035X. Auranofin showed concentration-dependent, growth-inhibition-independent antibiofilm activity against typhoidal and non-typhoidal Salmonella from Nigeria, and inhibited the motility of S. Elisabethville LLD035X at 5 {micro}M. At 5 {micro}M aurothioglucose, an auranofin gold (I) analogue, and non-gold analogue 1-Thio-beta-D-glucose tetraacetate, inhibited biofilm formation by 61.30% and 11.39%, respectively, pointing to essentiality of the gold (I) moiety for activity. Conclusions/ SignificanceStructurally diverse small molecules can inhibit biofilm formation by Salmonella, and motility inhibition is an important mechanism for this activity. Auranofin inhibits typhoidal and non-typhoidal Salmonella biofilm formation, with its gold content being required for these activities.

We genotyped 1189 multidrug-resistant Mycobacterium tuberculosis isolates identified during 2013-2022 in Argentina, through a mixed strategy using PCR-based methods and whole-genome sequencing. Epidemiological, geographic distribution and microbiological data were integrated. Most cases belonged to a cluster (75.7%). The proportion of orphan and clustered cases varied across regions. The Euro-American lineage4 was virtually predominant. The most important clusters, M, Ra, Rb and Callao2 strains, comprised 45.9% of the newly diagnosed cases, and their relative importance varied over time. A preliminary genomic analysis showed that several local transmission chains due to the Callao2 strain, imported from Peru, were active, including a superspreading event that occurred circa 2020. A good performance of the current second-line regimes can be expected for most of the cases. Heightening suspicion of drug-resistance and enhancing timely and active surveillance in specific risk groups could contribute to the tuberculosis management in Argentina, tackling transmission and resistance amplification. BiosketchBiochemist Federico Lorenzo is a professional of the Servicio de Micobacterias, Departamento de Bacteriologia, INEI, ANLIS "C. G. Malbran" and is specialized in the microbiological diagnosis of mycobacterial diseases using next-generation sequencing technologies. His research interests are drug-resistant tuberculosis, non-tuberculous mycobacteria and bioinformatic analysis applied to diagnostics. One-sentence summaryWe evaluated the genotypes associated with multidrug-resistant tuberculosis in Argentina, 2013-2022.

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

1.1Antimicrobial resistant infections present a concerning and expanding healthcare problem that is compounded by the reduction of antimicrobial research by the pharmaceutical industry. Additionally, the currently used antimicrobials consistently present less than ideal clinical treatment outcomes. This contention is supported by in vitro analysis in appropriate models. Here, in mature methicillin resistant Staphylococcus aureus (MRSA) in vitro cultures, we tested multiple antimicrobials and showed that for a biofilm grown for 72 hours, no antimicrobial tested was able to completely eradicate the biofilm even after 24 hours of exposure. However, the addition of an enzymatic biofilm-dispersal agent (DNase I or Proteinase K), greatly improved the performance of vancomycin and tigecycline in this in vitro model. Despite the improved performance in the presence of the dispersal agent, a high concentration of antimicrobial, 2000 {micro}g/mL, was needed to completely eradicate the infection as demonstrated by analyses using both a traditional XTT assay as well as a subculture assay to account for persister cells. It was shown that the addition of DNase I improved the diffusion of vancomycin through the biofilm. This suggests vancomycin efficacy is limited by the biofilm. The presented work provides a potential avenue for future treatments of MRSA lung infections by utilizing a traditional antibiotic combined with a passive dispersive agent.

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.

To evaluate the diagnostic performance of clinical shotgun metagenomic sequencing (SMg) for detecting medically relevant fungi and parasites compared with standard of care (SoC), and to define read-based thresholds for interpretation, we retrospectively analyzed 198 clinical samples from 187 patients across four university hospitals (2018-2022): blood (n=37), faeces (n=63), respiratory fluids (n=54), other biological fluids (n=24), and tissue biopsies (n=20). Total nucleic acids were sequenced ([&ge;]10 million reads per library) and processed with MetaMIC v2.2.1. Data were normalized as reads per million (RPM). Receiver operating characteristic analyses were used to derive optimal RPM thresholds by sample type. SoC identified microorganisms in 152/198 samples (76.8%). All 46 SoC-negative samples were also negative by SMg. At the genus level, SoC identified 187 taxa and SMg 175. Of these, 147 (78.6%) were detected by both methods, 40 (21.4%) by SoC only, and 28 (14.9%) by SMg only. The overall genus-level F1-score was 0.84. Quantification cycle (Cq) values (n=57) correlated inversely with RPM (p<0.001), and no false negatives occurred with Cq<28.6. Optimal thresholds were 0.06 RPM for faeces (AUC 0.89), 0.07 for respiratory fluids (AUC 0.93; sensitivity 88.9%, specificity 90.7%), 0.09 for blood (AUC 0.99), 0.19 for other fluids (AUC 0.94), and 0.57 for biopsies (AUC 0.89). A global threshold of 0.06 RPM yielded an AUC of 0.92 (sensitivity 88.9%, specificity 88.5%). A pragmatic uniform 0.1 RPM threshold maintained performance, while sample-type specific thresholds further improved accuracy, supporting standardized implementation of clinical metagenomics for fungal and parasitic diagnostics.

AbstractO_ST_ABSBackgroundC_ST_ABSShort-chain fatty acids (SCFAs) are widely used as functional readouts of gut microbial activity in vivo. The growing adoption of decentralised study designs and self-collection protocols has amplified the need for reliable room-temperature storage and shipment strategies. However, SCFAs volatility and the persistence of post-collection microbial metabolism raise concerns regarding pre-analytical stability and the interpretability of measured concentrations. MethodsWe assessed the temporal stability of fatty acids (FAs) across intestinal and systemic matrices under room-temperature storage. Untreated stool was compared with two nucleic acid stabilisation devices (eNAT and OMNIgene-GUT), while whole blood, plasma and dried blood spots (DBS) were evaluated as minimally invasive systemic sampling strategies. Profiles were quantified using complementary GC-MS and LC-MS/MS workflows. ResultsUntreated stool showed fermentation-driven increases in major SCFAs, whereas immediate freezing preserved baseline profiles. eNAT maintained faecal FA stability for up to 21 days, while OMNIgene-GUT exhibited baseline and time-dependent alterations. In systemic matrices, plasma and whole blood showed upward drift, whereas DBS declined initially before stabilising after approximately 14 days. ConclusionsFA measurements are highly matrix- and device-dependent. Our findings provide practical guidance for the selection of sampling strategies in microbiome-associated FA studies and emphasise the need for controlled pre-analytical conditions in decentralised microbiome studies.

Bacillus paranthracis was formally defined as a species in 2017, after decades of carrying the name "emetic B. cereus" based on cereulide production and clustering within B. cereus sensu lato phylogenetic group III. Commonly associated with foodborne intoxication, reports rarely link B. paranthracis to non-foodborne clinical illness. As such, the new taxonomy and close resemblance of the name to the biothreat pathogen Bacillus anthracis cause confusion in diagnostic and public health settings. To address this issue, B. paranthracis clinical strains (n=20) from the CDC collection were tested with microbiological methods used for identification of B. anthracis and antimicrobial susceptibility testing. Some B. paranthracis phenotypes were similar to B. anthracis, however others were inconsistent across strains. Like B. anthracis: 3 strains tested capsule positive, 5 were non-hemolytic on blood agar, and 9 non-motile. All B. paranthracis strains were resistant to gamma phage lysis, which differentiated them from B. anthracis. Treatment regimens for B. paranthracis infections are not well established, as antimicrobial therapy is not indicated for emetic intoxication caused by B. paranthracis. Notably, six B. paranthracis strains had elevated minimal inhibitory concentrations to anthrax-recommended antibiotics: one for ciprofloxacin, three for doxycycline and tetracycline, and two for clindamycin. Rapid MinION sequencing was assessed for antimicrobial resistance detection prediction but had limited value when using PiMA v.1. These microbiological observations and susceptibility profiles of B. paranthracis expand our understanding of this pathogen, strengthening our ability to differentiate this bacterium from B. anthracis to improve diagnosis and patient outcomes. IMPORTANCEThis study describes in vitro characterization of 20 archived clinical strains of B. paranthracis, an opportunistic pathogen identified more frequently in recent reports. Our findings highlight phenotypic differences and similarities between B. paranthracis and B. anthracis using standard microbiological methods and drug susceptibility profiling. We also assess a rapid B. anthracis specific MinION long read genome sequencing workflow with B. paranthracis. This report highlights the overlapping morphological features shared by B. paranthracis and B. anthracis to improve future laboratory diagnosis and strengthen anthrax preparedness. This article will effectively reach an audience of public health professionals and microbiologists strengthening anthrax preparedness.

Aminoglycoside drugs, amikacin, streptomycin, and amikacin liposome inhalation suspension are crucial for treating refractory Mycobacterium avium-intracellulare complex pulmonary disease. In Mycobacterium tuberculosis, cross-resistance occurs between amikacin and kanamycin, but not between amikacin and streptomycin in genetic drug susceptibility testing. However, the occurrence of cross-resistance among aminoglycosides remains unclear in M. avium-intracellulare complex. We aimed to evaluate cross-resistance among aminoglycosides to determine whether streptomycin or kanamycin remains effective after the development of amikacin resistance. This single-center retrospective study included 20 patients with amikacin-resistant M. avium-intracellulare complex harboring rrs mutations. Paired analyses of streptomycin and kanamycin minimum inhibitory concentration values before and after amikacin resistance development were performed. In addition, streptomycin- and kanamycin-resistant strains were generated in vitro and resistance-associated mutations were identified using whole-genome sequencing. No significant increase was observed in streptomycin minimum inhibitory concentration values following amikacin resistance. In contrast, kanamycin values uniformly increased to >256 g/mL after the acquisition of amikacin resistance. Furthermore, amikacin- and kanamycin-resistant isolates shared mutations at position 1408 in the rrs gene, whereas streptomycin-resistant isolates exhibited mutations at position 20 in the rrs gene. These results suggest that amikacin and kanamycin exhibit cross-resistance in M. avium-intracellulare complex, whereas amikacin and streptomycin may not. Two cases in our cohort in which streptomycin treatment was effective after the acquisition of amikacin resistance further support these findings. In conclusion, streptomycin may be a potential therapeutic alternative for amikacin-resistant M. avium-intracellulare complex pulmonary disease. Future studies correlating streptomycin minimum inhibitory concentration values with clinical outcomes are required.