Microbiology Spectrum

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

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.

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

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.

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.

Recent advances in high-throughput sequencing and novel culture techniques have revolutionized our understanding of the human microbiota. However, most studies primarily focused on bacterial communities, often overlooking the fungal component. Building upon our previous metagenomic analysis of the Inuit oropharyngeal microbiome 1, this study used culturomics to provide a more comprehensive view of both bacterial and fungal communities. We analyzed oropharyngeal swabs from the Qanuilirpitaa? 2017 Inuit Health Survey 2, demonstrating the complementarity of metagenomic and culturomic approaches. Our findings highlight the importance of culturomics in revealing low-abundance microorganisms, particularly fungi, which are often underrepresented in metagenomics data. Moreover, we designed an approach to isolate previously uncultivated species. We described two Pauljensenia sp., and provided insights into the phylogenetic relationship between Schaalia and Pauljensenia genera. This study underscores the necessity of a holistic approach to microbiome research, combining multiple techniques to fully elucidate microbial diversity in unique populations like the Inuit.

The lack of a reliable chronic murine model limits drugs evaluation against Mycobacterium abscessus. Models show discrepancies, especially regarding host factors (mouse strain, sex and age). Using beads-model, we compared BALB/cJRJ and C57BL/6NCrl across sexes and ages. BALB/cJRJ showed more sustained infection and lower variability, with no significant sex- or age-related differences. Considering these results and the higher prevalence of NTM pulmonary infections in female patients, 5-6 weeks-old female BALB/cJRJ are appropriate for M. abscessus beads-model.

Cercospora species associated with soybean cause Cercospora leaf spot and purple seed stain, which are major diseases affecting soybean production worldwide and can lead to significant yield and seed quality losses. However, unstable and poor sporulation under laboratory conditions remains a critical challenge, hindering the recovery of genetically homogeneous isolates and the establishment of standardized experimental protocols. These limitations further restrict our understanding of the biology, epidemiology, and pathogenicity of these pathogens. In this study, we developed specialized legume-based culture media derived from soybean and pea tissues to mimic host-associated environmental conditions. We compared the sporulation efficacy of these media with commonly used artificial media, including potato dextrose agar (PDA) and V8 juice agar. Our results demonstrated that legume-based media consistently supported higher levels of sporulation than PDA and V8 across multiple strains, although conidial yields varied depending on the strain and medium concentration. Transcriptional analysis of sporulation-related genes revealed that while abaA, wetA, and steA did not show significant differential expression among media, velB exhibited distinct medium-dependent expression patterns. Further evaluation using additional field isolates confirmed that legume-based media provide a more reliable method for inducing sporulation than PDA. Overall, legume-based media represent a practical and effective approach for promoting sporulation in soybean-associated Cercospora species under laboratory conditions.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of persistent diarrhea in children in low- and middle-income countries, and the emergence of multidrug-resistant (MDR) strains necessitates non-antibiotic therapeutic strategies. This study evaluates Lactobacillus johnsonii, previously characterized by our group, as a probiotic candidate against a clinically confirmed MDR EAEC isolate resistant to ampicillin, ciprofloxacin, azithromycin, amoxicillin, and gentamicin. L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, phenol tolerance, and rapid autoaggregation reaching 80.4 {+/-} 2.3% by 4 hours, collectively supporting mucosal colonization potential. In antimicrobial assays, L. johnsonii produced zones of inhibition against MDR EAEC substantially exceeding those of gentamicin, reduced viable biofilm-associated EAEC by over 80%, and displaced pre-adhered EAEC from HCT-116 intestinal epithelial cells in a time-dependent manner. L. johnsonii also attenuated MDR EAEC-induced gas production and reduced nitric oxide levels by 67.7% in infected RAW 264.7 macrophages, suggesting immunomodulatory activity. Nutrient competition did not appear to contribute to EAEC suppression under tested conditions, indicating inhibition is predominantly secretome-dependent. Fractionation of the L. johnsonii cell-free supernatant by fast protein liquid chromatography yielded five fractions below 75 kDa; fractions S5 and S6 exhibited sustained bactericidal activity at 6 hours. Gram staining confirmed that both fractions reduced viable EAEC cell numbers, with S6 producing a greater reduction than S5, indicating quantitatively distinct bactericidal potencies. These in vitro findings support the potential of L. johnsonii as a biotherapeutic candidate for antibiotic-resistant enteric infections. In vivo validation and chemical characterization of active fractions remain important next steps.

Epitranscriptomics has recently gained significant momentum due to technological advances and translational applications, however, studies on bacterial RNA modifications remain limited. Bacterial RNA remains notoriously prone to degradation and methodologies to investigate the epitranscriptome are challenging. Prior research has shown RNA modifications modulate antimicrobial resistance, virulence and pathogenicity. This research employed CRISPR interference to knock down five known Escherichia coli rRNA modification genes (rlmF, rlmJ, rluD, rsmF and rsmG) in three E. coli strains. These isolates underwent growth curves, proteome analysis and native RNA sequencing CRISPRi adequately silenced the majority of RNA modification genes in E. coli (>80% reduction). Significant growth delays were associated with rlmF, rsmF and rsmG repression. Unique protein pathways corresponding with RNA modification loss were found for rlmJ (TreB, XylF), rluD (CysH, HycB, PutP, TrpB), rsmF (EvgA) and rsmG (OppC). Known rRNA modification sites for rluD ({Psi}) and rsmG (m7G) were detected from analysis of nanopore electrical signal, however, only a weak signal was apparent for m6A (rlmF, rlmJ) and m5C (rsmF) modifications. The inhibition of rRNA modifications resulted in mRNA modification changes including for genes ompC, cspC, dbhA, dbhB and secY. Our work provides an approach for unravelling the epitranscriptome of E. coli and gain insight into its functional role.

The Klebsiella pneumoniae species complex (KpSC) is a clinically important group of closely related pathogens associated with invasive infections. The complex comprises seven closely related members, which are often reported as K. pneumoniae, particularly in resource-limited settings. Accurate differentiation of KpSC members remains challenging because routine laboratory methods lack sufficient resolution, and approaches like mass spectrometry and whole genome sequencing (WGS) are not widely available. Consequently, the epidemiology and clinical significance of non-K. pneumoniae members of the KpSC remain underrecognized. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR targeting species- and subspecies-specific single-nucleotide polymorphisms in the housekeeping gene rpoB, with six primer sets for differentiation of common KpSC members. The assay was validated against 49 genomically characterized clinical isolates, after which 179 wastewater-derived isolates provisionally identified as Klebsiella spp. by standard microbiological methods were tested. Of these, 174 were assigned to specific KpSC members by the assay, while 5 produced inconclusive amplification patterns. A subset of 16 environmental isolates was selected for WGS, including four of the five inconclusive isolates. All environmental isolates with interpretable MAMA PCR patterns were concordant with WGS. The four inconclusive environmental isolates were identified as Enterobacter spp. Overall, comparison of MAMA PCR with WGS showed 100% sensitivity and 100% specificity for all tested targets, and the total cost was approximately US$1. This rpoB-based multiplex MAMA PCR provides a simple, accurate, and low-cost approach for differentiation of KpSC members in routine laboratories and may support improved identification and surveillance in resource-limited settings. ImportanceThe Klebsiella pneumoniae species complex (KpSC) has seven members but is often reported as a single organism in routine laboratories, masking clinically and epidemiologically important diversity. As a result, the contribution of non-K. pneumoniae KpSC members to human and environmental microbiology remains poorly defined, especially in low-resource settings. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR based on discriminatory rpoB single nucleotide polymorphisms for differentiation of common KpSC members using standard PCR and agarose gel electrophoresis. The assay demonstrated 100% sensitivity and 100% specificity against whole-genome sequencing and excluded non-Klebsiella environmental isolates initially identified as Klebsiella pneumoniae using standard microbiological procedures. With an estimated per-test cost of about US$1, this method offers an affordable and scalable option for laboratories seeking more accurate KpSC identification and improved surveillance.

Gingival inflammation is associated with dysbiotic oral biofilms characterized by reduced nitrate-reducing capacity and diminished nitric oxide (NO) bioavailability. While dietary nitrate has been shown to influence oral microbial activity, the effects of sustained, localized nitrate delivery on oral biofilm ecology and gingival inflammation remain incompletely defined. In this randomized, double-blind, placebo-controlled trial, 30 adults with gingival bleeding were assigned to receive localized prebiotic nitrate (~0.989 mmol per dose) or placebo for 21 days. The primary outcome was mean bleeding on probing (mBOP). Secondary outcomes included modified Gingival Index (mGI), Quigley-Hein plaque index (QHPI), salivary nitrite (as a proxy for NO bioavailability), oral pH, and microbiome composition assessed by 16S rRNA gene sequencing. Prebiotic nitrate supplementation formulation delivered in a slow-release chewing gum significantly reduced mBOP (25.7% to 15.3%; p = 0.0002) compared to placebo chewing gum. Salivary nitrite levels and oral pH increased, indicating enhanced nitrate metabolism. Microbiome analysis demonstrated enrichment of nitrate-reducing taxa, including Rothia mucilaginosa and Neisseria spp., and a relative reduction in inflammation-associated genera such as Prevotella and Porphyromonas. Localized prebiotic nitrate formula delivered in a functional chewing gum was associated with reduced gingival inflammation and shifts in oral microbiome composition consistent with enhanced nitrate-reducing capacity critical in nitric oxide formation. These findings support a role for biofilm-directed nutritional modulation as a non-antimicrobial approach for managing gingival inflammation and improving nitric oxide bioavailability.

The tobacco hornworm moth (Manduca sexta) is evaluated as a model of bacterial virulence and host-pathogen dynamics. Infections of Pseudomonas aeruginosa were established by injection of 5th-instar larvae, and multiple assays of virulence were evaluated. Infected larvae exhibited dose-dependent mortality, reduced growth, melanization, behavioral changes, and altered frass constitution. Even low-dose infections that were not fatal exhibited impaired growth, but individual growth trajectories revealed considerable heterogeneity among worms given the same dose. Twice-daily antibiotic treatment with gentamicin or cefepime improved survival four- to five-fold but did not rescue 100%. Heat-killed cells and filtered culture supernatant alone induced significant morbidity and mortality, suggesting secreted bacterial products are important to pathogenesis. Bacterial burden analysis revealed a shifting bacterial distribution over time, with decreasing hemolymph titers and increasing localization in fat body, gut, and carcass. Hornworms thus offer a more sensitive analysis of bacterial infection dynamics and consequences than do larvae of the more commonly used wax moth.

1Canine atopic dermatitis (CAD) is a chronic inflammatory skin condition sometimes associated with microbial dysbiosis, including alterations in colonization by the lipophilic yeast Malassezia pachydermatis. This study investigated the population diversity of M. pachydermatis in the ear canals of healthy and CAD-affected dogs using Fourier-transform infrared (FTIR) spectroscopy and whole genome sequencing (WGS). Among 60 dogs, M. pachydermatis prevalence was significantly higher in CAD cases than in healthy controls. FTIR spectroscopy revealed greater strain heterogeneity in CAD-affected dogs, often with distinct genotypes in each ear, while healthy dogs exhibited more homogeneous populations. Using a previously developed FTIR-based artificial neural network classifier, we assigned strains to three phylogroups. Strains from phylogroups I and III were significantly enriched in CAD-affected dogs, while phylogroup II was most prevalent overall and the dominant phylogroup in healthy controls. This suggests that CAD-associated inflammation may favor specific M. pachydermatis phylogroups and sub-clusters within phylogroups, shaping colonization dynamics. FTIR-based typing showed full concordance with WGS across 35 sequenced isolates, recapitulating relationships among phylogenetically related isolates and their similar phenotypic profiles. Overall, our findings reveal strain-level shifts in M. pachydermatis populations associated with CAD and establish FTIR spectroscopy as a rapid, cost-effective tool for large-scale epidemiological studies.

Measurement of antibody responses to viral infection is essential for surveillance, diagnostics, epidemiological research, and natural history of infection studies. However, current methods to detect virus-specific antibodies are often resource-intensive and impractical for deployment in outbreak settings or in field-based studies. This manuscript presents two economical, high-throughput immunoassays--the cytoblot immunoassay (CBA) and strip immunoblot assay (SIA)--for detecting and quantifying anti-lymphocytic choriomeningitis mammarenavirus (LCMV) antibodies in mouse serum. To validate, we tested serum from acutely or persistently experimentally infected mice. Both assays detected LCMV-specific IgG and IgM antibodies with high sensitivity and specificity across multiple timepoints. By facilitating the study of immune responses in rodent reservoirs, these tools can enhance our understanding of zoonotic viral transmission, provide scalable platforms for outbreak preparedness, and serve as adaptable models for the development of rapid serological assays for other viral pathogens.

Antimicrobial resistance (AMR) presents a pressing need to ensure that the right antimicrobials are used to target the right microbes at the right time. Ideally, the appropriate antimicrobial is selected after patient samples have been cultured and assessed with antimicrobial sensitivity testing (AST). However, the time needed for culture-based diagnosis leads to immediate empirical treatment, often with broad-spectrum and/or high-tier antimicrobials. Direct nanopore metagenomic whole genome sequencing to identify pathogens and predict their antimicrobial resistance is a rapid and patient-side alternative. A limitation of this approach is potential inconsistencies in in silico predicted AMR phenotypes. Here, we benchmarked the current performance of in silico AMR prediction strategies for nanopore-generated long read data. Using nanopore data paired with AST phenotyping for 201 samples, we assessed the impact of basecalling mode, data volume, and assembly strategy, and compared the performance of eight in silico AMR prediction tools with seven AMR databases. We found that basecalling accuracy mode does not affect the overall accuracy of in silico AMR predictions, but assembly strategy and data volume both do. Prediction tools using the ResFinder database scored best for balanced accuracy (0.80 {+/-} 0.02 for both ResFinder and ABRicate), whilst DeepARG scored best for sensitivity (0.65 {+/-} 0.03). However, even the best performing in silico AMR prediction strategy missed some resistance identified by lab-based AST. In silico AMR prediction can therefore supplement lab-based AST, but cannot yet replace it. Impact statementAntimicrobial resistance (AMR) is threatening modern standards of human and veterinary healthcare. Rapid and patient-side diagnostic tests are needed to diagnose bacterial infections and allow clinicians to select effective antibiotics. Current tests based on bacterial cultures take several days, which may delay diagnosis and treatment, or lead to inappropriate "just in case" treatment while waiting for the results. In contrast, nanopore metagenomic whole genome sequencing can identify bacterial infections and predict which antibiotics will be effective in minutes to hours. However, the accuracy of these tests is uncertain. We therefore compared the performance of eight AMR prediction tools and seven databases of AMR determinants, using 201 bacterial samples with known antibiotic susceptibility and resistance. We found that the sensitivity (i.e. false negative rate), specificity (i.e. false positive rare) and overall accuracy of the tools and databases varied. In particular, even the best performing AMR prediction methods missed some AMR. Therefore, while these tools are useful for rapid and patient-side diagnosis and treatment decisions, they still have limitations and should be used alongside bacterial cultures and antibiotic sensitivity testing. Data summarySequencing data for the samples sequenced for this study are available at the NCBI under BioProject ID PRJNA1292816 (SRA accessions for all datasets used here are available in Supplementary Table S1). All commands and code used can be found at: https://github.com/nataliering/nanopore_AMR_tools/ The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Despite multiple treatment strategies and extensive research on resistance mechanisms, tuberculosis (TB) remains a major global health threat, largely because of the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Among various mechanisms complicating the situation, active antibiotic export via efflux pumps is particularly significant, yet largely unexplored. Mycobacterium sp. encodes numerous transporters, many of which are overexpressed in clinical isolates or under drug stress. Here, we examined the possible role of Rv0783c, a putative transporter that is reportedly overexpressed in drug-stressed conditions. Rv0783c conferred resistance to multiple structurally diverse antibiotics, fluoroquinolones and anti-TB drugs in the heterologous hosts, namely, Escherichia coli and Mycobacterium smegmatis. Reduced drug accumulation and active efflux of ethidium bromide (EtBr) confirmed its transport activity, which in turn gets nullified upon using the proton-motive force blocker, CCCP. On the other hand, its expression enhanced biofilm formation, linking antibiotic resistance to persistence-associated phenotype. Furthermore, site-directed mutagenesis confirmed the presence of crucial interacting residues with antibiotics that were identified by in silico analysis. Overall, we demonstrate the role of Rv0783c in the extrusion of first and second-line anti-TB drugs and enhancing biofilm formation.