BMC Microbiology

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

The human gut, containing 100 trillion microbes, is also considered the "second brain," having control over the different functions of the physiological system. With advancements in bioinformatics and the development of sequencing technologies, researchers are able to explore the diversity and functional implications of gut microbiota (GM), which have become strongly associated with a variety of diseases. Microbial imbalance, or dysbiosis, acts as a biomarker for early detection and prognosis of a disease. Artificial Intelligence and Machine Learning (AI/ML) methods, although extensively used in predicting GM associated diseases, are seldom translated to having practical real-world outcomes, necessitating the design of robust AI/ML models applicable in real-world scenario. We have therefore come up with designing stacking-based ensemble architectures (EM1 and EM2), developed by integrating multiple ML-based learning algorithms for improving disease prediction accuracy. The GM datasets, after split into training and test sets, were eventually fed into the proposed two-layer ensemble models, which combines the output from standardized base learners via a meta-classifier, strengthening classification robustness as well as ensuring consistency in optimized performance across diverse datasets. Both the proposed hybrid ensemble models have emerged to be superior performers over all baseline and deep learning models, with an average accuracy of 0.87 and 0.84 respectively. By combining multiple learners, the proposed ensemble models outperform traditional single-algorithm-based approaches to attain higher accuracy and robustness on complex GM datasets. Key messagesO_LIDevelopment of stacking-based hybrid ensemble models (EM), which can be employed to integrate different AI/ML algorithms with better prediction accuracy of gut microbiome (GM)-associated diseases. C_LIO_LIUse of independent GM datasets with preprocessing methods such as SMOTE and PCA to address class imbalance and high dimensionality. C_LIO_LIAll the proposed EM architectures are mostly superior to the existing state-of-the-art AI/ML methods (highest prediction accuracy: 0.87 and 0.84 with EM1 and EM2 models respectively) for GM diseases predictions. C_LIO_LIThe cross-cohort validation demonstrates high prediction accuracy and robustness, (AUC values close to 0.98 and 0.99, for EM1 and EM2). C_LIO_LIThese therefore demonstrate the effectiveness of EM frameworks for GM associated disease prediction, paving the way for corresponding applications in precision medicine. C_LI

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

The aim of this study was to evaluate the impact of two sustainable dietary protein sources on the structure and composition of the gut microbiota in European sea bass (Dicentrarchus labrax) juveniles. These protein sources were incorporated to the aquafeeds containing (a) Lupinus albus meal, treated with either exogenous enzymes (Solid state hydrolysis-SSH) or fermented with Saccharomyces cerevisiae (Solid state fermentation, SSF) and (b) Lagocephalus sceleratus meal. In the first case (a), the control aquafeed simulated a standard commercial diet, containing soybean meal whereas in the rest of the diets soybean meal was partially or totally replaced by hydrolysed or fermented Lupin meal. In the second case (b) the fish were fed Lagocephalus sceleratus unprocessed fishmeal as well as treated at different temperatures to deactivate tetrodotoxin (TTX). A control diet with 30% commercial fish meal was also fed as a reference diet. Both diets in all inclusion levels did not cause any significant gut microbiota change, suggesting their neutral role in this aspect. However, the gut bacterial communities of the fish fed with 12.5% lupin meal inclusion, had increased amino acid biosynthetic pathways suggesting a beneficial effect.

Background: Urinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. Results: We present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. Conclusions. The catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Accurate taxonomic profiling of human microbiomes is essential for advancing research and understanding the complex role microbial communities play in human health. When using shotgun metagenomics, the sequencing data is analyzed through metagenomic pipelines, which incorporate various open-source tools and classify microbes based on matched paired-end DNA reads. However, differences in sequencing and computational approaches can produce substantially different microbiome profiles from the same sample, making validation critical. One approach for validation is benchmarking with realistic mock communities, but this remains relatively rare. Additionally, existing benchmarks often overlook microbiome variability across life stages and body sites, limiting their clinical and research utility. Here, we developed age- and body site-stratified synthetic metagenomes, enabling context-aware benchmarking of microbiome pipelines. Using novelty-based sampling to prioritize microbial diversity and minimize redundancy among selected samples, we selected 300 representative, real biological samples spanning six categories: adult, child, toddler, and infant (>6 months and <6 months) gut samples, as well as adult vaginal samples. We validated three pipelines, Tiny Health's proprietary Metagenomic Classifier v2 (THMCv2), MetaPhlAn4, and Kraken2+Bracken, using precision, recall, F1 score, and area under the precision-recall curve (AUPR) across age groups and sample types. THMCv2 demonstrated higher recall and F1 scores, detecting more taxa across sample types and ages, while MetaPhlAn4 achieved the highest precision. THMCv2 also achieved the highest area under the precision-recall curve, reflecting peak performance across both abundant and rare species. When analyses were weighted by abundance, THMCv2 and MetaPhlAn4 each characterized the mock community nearly perfectly. Errors for THMCv2 were largely restricted to very low-abundance taxa (<0.001%), whereas MetaPhlAn4 occasionally produced false positives for higher-abundance taxa. Species-level analyses of clinically relevant microbes confirmed these patterns, with THMCv2 demonstrating higher sensitivity, MetaPhlAn4 higher specificity, and Kraken2 lower overall performance. These results demonstrate clear precision-recall trade-offs in metagenomic profiling. This benchmarking framework provides a reproducible approach for evaluating pipeline performance across diverse microbiome contexts and life stages.

Iron is an essential micronutrient that shapes host-pathogen interactions during infection. However, the contribution of iron to the virulence adaptation of the Enterobacter cloacae complex (ECC) remain poorly characterized. This study profiled the effects of iron on E. roggenkampii and E. asburiae clinical isolates. Growth kinetics were assessed in Luria-Bertani broth supplemented with varying iron concentrations and 5% sheep blood, and EDTA. Recovered strains were used for motility and antibiotic susceptibility assays. Phenotypic virulence trait of iron-naive and iron-recovered strains was determined using biofilm formation assays. Whole-genome sequencing was conducted to identify genetic determinants associated with iron acquisition and metabolism. Presence of iron increased bacterial growth, reduced antibiotic susceptibility, and enhanced biofilm formation. At higher iron concentrations, iron-recovered strains exhibited increased biofilm biomass, while there was a high biofilm formation with iron-naive strains at lower iron levels. Genomic analysis identified genes associated with ferrous and ferric iron transport, heme uptake, siderophore biosynthesis, and virulence-related functions, including adhesion and biofilm formation. These findings demonstrate that iron availability and prior exposure modulate ECC physiology and phenotypic traits associated with virulence, supporting a role for iron in shaping adaptive pathogenic potential. Graphical AbstractThe influence of iron metabolism on virulence adaptation of Enterobacter cloacae complex O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737523v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@aa351eorg.highwire.dtl.DTLVardef@855345org.highwire.dtl.DTLVardef@11e0da5org.highwire.dtl.DTLVardef@11f851_HPS_FORMAT_FIGEXP M_FIG C_FIG

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

The human gut microbiome harbours a diverse community of microeukaryotes, predominantly fungi, which may potentially play important roles in gut ecology and homeostasis. Despite their potential, the study of gut microeukaryotes has been hampered by the limited sensitivity of standard sequencing approaches, which struggle to capture DNA from low-abundance microorganisms against the overwhelming background of bacterial biomass. To address this, we developed a method to selectively enrich for microeukaryotic cells in human faecal samples by depleting bacterial cells prior to metagenomic sequencing. Through systematic comparison and optimisation at each processing step, we established a robust standard operating procedure (SOP) for microeukaryotic cell enrichment. By benchmarking this SOP across eight human faecal samples with three technical replicates each, we showed that it consistently increased microeukaryote representation in metagenomic libraries, greater microeukaryotic taxonomic diversity, and a reduced proportion of unclassified taxa. Together, these improvements enabled substantially deeper characterisation of the microeukaryotic fraction of the human gut microbiome.

Hand eczema has been described as having an increased prevalence in persons with increased frequency of hand washing. This study investigated the differences in the hand microbiome of persons with and without a history of eczema and secondly the sensitivity of these microbes to commercial liquid soap as a potential trigger for eczema flares. The study identified Staphylococcus to be the most populus genus on the hands in both groups, but the distribution of species was different. Additionally, there was no difference in the number of soaps that produced zones of inhibition but there were some differences in the overall sensitivity to the different soaps tested. Overall, it was determined that liquid soap can cause bactericidal effects on some species of the commensal microbiome, but further work is required to determine if this could be the cause of hand eczema.

Probiotics produce antimicrobial peptides and small molecules that are secreted into the medium. Antimicrobial activity of cell-free supernatants can be tested using various qualitative and quantitative methods. Many of these techniques employ methods which introduce uncontrolled variables, impacting reproducibility and making comparison of reported results difficult. Here, we present a simple procedure for quantitative estimation of antimicrobial activity of cell-free supernatants which overcomes drawbacks of commonly used methods.

Background: Phenotypic antibiotic susceptibility testing (AST) for Helicobacter pylori (H. pylori) has relied on bacterial culture for three decades, requiring 5-7 days to yield results. Genotypic rapid tests can only detect known resistance mutations and fail to reliably identify amoxicillin resistance. To our knowledge, no culture-free rapid phenotypic AST method for H. pylori has been previously reported. Methods: We developed a phenotypic AST method based on fluorescence rapid on-site evaluation (ROSE) technology that completely bypasses bacterial culture. Gastric mucosal biopsy specimens from 40 H. pylori-positive patients were homogenized and co-incubated with an acridine orange/ethidium bromide (AO/EB)-based viability staining reagent and three first-line antibiotics (amoxicillin, clarithromycin, and levofloxacin) at concentrations corresponding to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for H. pylori, at 37C for 1 hour. Fluorescence intensity was measured using a microplate reader. A reduction in fluorescence relative to an antibiotic-free control indicated susceptibility, whereas no significant reduction indicated resistance. Conventional culture-based AST (E-test) served as the reference method. The overall concordance rate, sensitivity, specificity, and Cohen's kappa coefficient were calculated. Results: Fourteen of the 40 samples had unsuccessful culture and were excluded, leaving 26 samples for statistical analysis of each antibiotic. The overall concordance rates between the ROSE method and culture-based AST were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohen's kappa coefficients indicated moderate agreement for all three antibiotics ({kappa} = 0.523, 0.539, and 0.412, respectively). Unlike genotypic methods, the ROSE method successfully assessed amoxicillin susceptibility in all 40 patients, a critical first-line antibiotic for which no reliable genetic resistance marker currently exists. The turnaround time was approximately 1 hour (55-65 minutes), compared with 5-7 days for culture-based methods; preliminary estimates indicated a cost reduction of approximately 3,000-5,000 Chinese yuan (CNY) per patient, mainly attributable to the elimination of culture media, prolonged incubation, and repeat clinic visits. Conclusions: This study reports, for the first time, a culture-free 1-hour phenotypic AST for H. pylori. The method enables same-day, susceptibility-guided treatment decisions, addressing an unmet clinical need spanning three decades. Algorithm optimization and a prospective randomized controlled trial are currently underway to further improve diagnostic accuracy and validate clinical utility.

Nakaseomyces glabratus (Candida glabrata) is a WHO high-priority fungal pathogen associated with fungal antimicrobial resistance (fAMR). Given nosocomial transmission occurs sporadically, resistant strains could be transmitted, a concern for critically ill patients. We conducted a genomic investigation and retrospective observational study of N. glabratus to identify any nosocomial transmission of fAMR and understand resistance mechanisms and clinical and demiological factors among patients at a quaternary hospital in Melbourne, Australia. We selected stored N. glabratus with and without fAMR associated with similar patient clinical characteristics and performed whole genome sequencing. Clinical and epidemiological data were extracted from medical records. Phylogenetic, mutational, copy-number variation (CNV) and mitochondrial genomic analyses were performed, with a focus on the fAMR gene PDR1. Of 54 isolates collected over seven years, 20 (37%) were fluconazole-resistant and four (7%) had elevated flucytosine minimum inhibitory concentrations (MICs) (range 2-32 g/ml). There were no significant clinical differences between patients with and without fluconazole resistance. Most (55%) fluconazole-resistant isolates carried PDR1 mutations. Resistance was distributed throughout the phylogeny suggesting predominantly independent acquisition. However, a cluster of four resistant isolates with the same PDR1 mutation suggested nosocomial transmission. One probable ERG11 gene duplication, and two petite variants with apparent mitochondrial genomic deletions, were seen in association with fluconazole resistance. In this study, we identified a small probable nosocomial fAMR transmission cluster, and novel variants in PDR1, ERG11 and FCY2 associated with fAMR phenotypes. Future study should confirm functional impacts and systematically investigate for nosocomial transmission of resistance, including colonisation states.

Horizontal gene transfer (HGT) is a driving force in microbial evolution that allows community members to rapidly evolve to cope with environmental stressors and competition. Despite the importance of HGT for the generation of genetic diversity, little is known about the specific mechanisms or dynamics of transfer in complex communities. Transductomics is a sequencing based technique which identifies potential HGT by bacteriophages (transduction) through sequencing of the transductome - the DNA carried by bacteriophages and other virus-like particles in a sample. We analyzed the murine gut transductome before and after perturbations with antibiotics and Clostridioides difficile infection (CDI). We found that several bacterial families - the Oscillospiraceae, Butyricoccaceae, and Turicibactericeae - disproportionally contributed to the transductome. Some families, like the Butyricicoccaceae, were frequent transducers in both the baseline and perturbed murine gut microbiome while other taxa displayed condition-specific transduction indicating that there may be specific transducing subpopulations or regulatory mechanisms controlling transduction frequency. Additionally, we found a diversity of highly abundant and enriched mobile genetic elements (MGEs) in the transductome including plasmids, integrative conjugative elements, phage satellites and transposons. The detection of MGEs containing conjugative elements suggest that some MGEs may spread through both transduction and conjugation. Overall, our work reveals a complex network of gene exchange occurring through transduction in the gut microbiome.

Early-life wheezing in children has been associated with microbial alterations along the gut-airway axis, yet studies simultaneously investigating bacterial communities in both compartments remain scarce. The aim of this cross-sectional exploratory pilot study (n=25) was to characterize and compare nasal and stool bacterial communities in preschool children aged 1-4 years with recurrent wheezing and healthy controls using 16S rRNA gene metabarcoding. Across participants, nasal and stool bacteriomes were highly individualized and taxonomically diverse. Overall richness, evenness, and community composition did not differ significantly between healthy children and wheezers in either compartment. However, wheezers displayed markedly higher within-group variability, particularly in nasal communities. Stratification based on microbiome similarity to healthy samples revealed increased Moraxella and reduced commensal genera including Prevotella spp. and Veillonella, along with lower richness and evenness (all p<0.001) in nasal samples with divergent bacterial communities. Stool alterations were more subtle but included trends toward reduced Bacteroides, Faecalibacterium, and Alistipes in wheezers more divergent from healthy controls. Community assembly in both compartments was largely governed by stochastic processes but accompanied by less complex and more fragmented bacterial interaction networks in wheezing children. Cross-compartment correlations were also altered, most prominently involving stool Lactococcus showing stronger and more numerous correlations with nasal taxa in wheezers than in healthy controls. Divergent wheezers exhibited distinct modular network structure and cross-compartment profiles, consistent with a differentiated microbial organization. Together, these findings suggest compartment-specific differences in microbial interaction patterns across the gut-airway axis in early-life wheezing, despite limited differences in overall community diversity. Take home messagePreschool wheezers showed fragmented gut-airway microbial networks and Moraxella-associated airway community stratification despite limited differences in overall diversity.

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72.

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 microg/mL were determined as temperature (32.5 degreeC), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin production was obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated.

Biofilm represents a complex aggregation of bacteria embedded within a self-produced extracellular polymeric substance (EPS). We investigated the characteristics of mycobacterial biofilm using Mycobacterium smegmatis (Msm) as model organism. By combining transcriptomic (RNA-seq) and proteomic (LC-MS) analyses, the research captures dynamic changes during the establishment and maturation of the biofilm. Transcriptomics analysis showed a distinct gene expression profile as compared to its planktonic form. Interestingly, clear differences were seen between initial (~2-day old) and mature (~5-day old) biofilm stages, highlighting phasic gene expression throughout biofilm development. Marked alteration in oxidative stress-related genes and energy metabolism from ATP to NADH was observed. Furthermore, quantitative mass spectrometry-based proteome examination of EPS showed an abundance of cytoplasmic proteins present differentially between initial and mature biofilm stages. Pathway enrichment revealed enhanced oxidative stress responses and metabolic shifts in mature biofilms, including upregulation of NADH dehydrogenase and downregulation of ATP synthase, indicating altered energy metabolism. Our findings thus provide insights into the molecular adaptations, including production of mycofactocin, occurring during mycobacterial biofilm establishment and maturation, and advance our understanding of mycobacterial biofilm physiology.

Background: Monitoring Pseudomonas aeruginosa (P. aeruginosa) infection in people with cystic fibrosis (pwCF) is essential for early detection, targeted treatment, and prevention of chronification. Sputum culture is the current standard, yet many patients, particularly those receiving CFTR modulator therapy, struggle to expectorate sputum. Microbial aerosols from the respiratory tract offer a non-invasive alternative. This proof-of-principle study assessed the accuracy and feasibility of the AveloMask, a novel breath aerosol collection kit paired with qPCR detection. Methods: Adult pwCF and bronchiectasis patients attending routine monitoring visits and healthy controls were enrolled in a cross-sectional study. Participants wore the mask for 30 minutes, followed by 20 instructed coughs. Mask filters were tested with a triplex qPCR assay targeting P. aeruginosa specific ecfX and gyrB, and human RPP30 as an endogenous control. Accuracy was evaluated using a composite reference standard (sputum culture and PCR). Results: Of 25 patients enrolled, 23 were included in the analyses. Sensitivity was 12/19 (63.2%) for breath qPCR versus 15/19 (78.9%) for sputum culture. Breath qPCR missed 5 cases detected by sputum culture but detected 2 sputum culture-negative/qPCR-positive cases. Specificity of breath qPCR was 100% in 4 patients and 15 healthy controls. RPP30 was detected in all mask samples. AveloMask was perceived as easy to use, with many patients preferring it over sputum collection. Discussion: Mask-based breath collection demonstrated promising diagnostic accuracy for detection of P. aeruginosa. Breath sampling may complement or partially substitute sputum-based diagnostics, especially in patients unable to expectorate. Further studies are needed to define its clinical role.