Microbiological Research

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential

Aspergillus fumigatus, an opportunistic human fungal pathogen, encodes numerous secondary metabolite biosynthetic gene clusters (BGCs) that are tightly regulated and often remain silent under standard conditions. Co-cultivation with Streptomyces rapamycinicus or treatment with the secondary metabolite from this species, the arginoketide azalomycin F, induce the otherwise silent fumicycline (fcc) BGC of A. fumigatus. To elucidate the underlying regulatory circuitry, we performed transcriptome analyses of A. fumigatus exposed to azalomycin F or co-cultured with S. rapamycinicus. Both conditions triggered a coordinated antibacterial response, characterized by induction of specific secondary metabolites and antibacterial effectors, alongside repression of other BGCs, including those for fusarinine C, pyripyropene A, and fumagillin. Among the most strongly induced genes was a zinc cluster transcription factor, designated SmpR for secondary metabolite multiple pathway regulator, which is conserved within Ascomycota. SmpR expression was selectively induced by azalomycin F, specific Streptomyces species and other bacteria isolated from soil such as Kribbella spp. and Arthrobacter spp.. Functional analyses revealed that SmpR is required for activation of the fumicycline BGC: its deletion reduced, whereas its overexpression enhanced fumicycline production independently of external stimuli. We further demonstrate that SmpR acts upstream of the pathway-specific regulator FccR and additionally controls multiple antibacterial BGCs, including those for hexadehydroastechrome, helvolic acid and xanthocillin. Together, our data identify SmpR as a key regulator coordinating antibacterial secondary metabolism in response to bacterial signals in A. fumigatus.

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathobiont associated with bacteremia, infective endocarditis, and colorectal cancer. However, the genomic diversity of this subspecies and the distribution of key virulence determinants, particularly the type VII secretion system (T7SS), remain poorly characterized. Here, we performed genomic analyses of 76 Sgg strains from diverse geographic and host origins. Core- and pan-genome analyses, multi locus sequence typing, and phylogenetic reconstruction revealed dominant sequence types (STs) that correlate with geographic origin or source of isolation. Furthermore, systematic characterization of the T7SS locus identified five new T7SS subtypes and demonstrated a strong association between T7SS subtype and ST. We further expanded the known repertoire of T7SS LXG domain-containing polymorphic toxins (LXG toxins) in Sgg substantially through genome-wide searches. Distinct distribution patterns were observed for the LXG toxins across the strains. Lastly, our data indicated that T7SS subtype was significantly associated with biofilm formation capacity of Sgg strains. Together, these findings advance our understanding of Sgg genomic diversity, reveal substantial lineage-associated variation in T7SS architecture and effector repertoires, and suggest a previously unrecognized connection between T7SS and biofilm formation in Sgg.

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.

Abstract Introduction: Streptococcus is the second genus involved in bone and joint infections (BJIs) after Staphylococcus. Streptococcus agalactiae is the predominant Streptococcus species implicated in BJIs. However, unlike Staphylococcus-related BJIs, data on S. agalactiae infections remain scarce. Methods: We conducted a retrospective cohort study from the West Region cohort of the CRIOAc registry among six university hospitals including all microbiologically confirmed streptococcal BJI in adults between 2014 and 2023. Results: 1454 patients were included, with a median age of 67 years and 65% male. S. agalactiae was the predominant streptococcal species involved 423/1454(29%). The most prevalent comorbidities identified were obesity (378/1454;26%) and diabetes mellitus (343/1454;24%). Prosthetic joint infections (PJIs) were the most common (653/1454;45%), although diabetic foot osteitis was less prevalent overall, it was significantly more associated with S. agalactiae infections (48/423;11% versus 70/1031;7%, p=0.05). S. agalactiae BJIs were more frequently lower-limb infections and chronic infections (240/423;57% versus 502/1031;49%, p=0.04). Half of the cohort had a polymicrobial infection and were slightly more frequent with S. agalactiae BJIs (235/423;56% versus 498/1031;48%, p=0.1). These results were consistent with a sensitivity analysis excluding diabetic foot related osteitis. Logistic regression analysis identified arteriopathy (OR: 4.16; IC95:1.64-11.24, p=0.003), and obesity (OR: 2.57; IC95: 1.41-4.78, p=0.002) as specific risk factors for S. agalactiae BJIs. Conclusion: S. agalactiae emerges as a prominent and distinct pathogen in complex streptococcal BJIs, with specific risk factors such as arteriopathy, obesity and diabetes mellitus, and more chronic infections.

Tailocins are phage tail-like bacteriocins (PTLBs) thought to be remnants of prophages that have lost the ability to package viral genomes while retaining the ability to kill closely-related bacterial strains, thereby mediating bacterial competition. Tailocins produced by Pseudomonas aeruginosa are referred to as pyocins. Apart from their contribution to ecological fitness, they also have the potential to be harnessed as highly-specific antimicrobials to treat antibiotic resistant bacterial infections. Although pyocins lack the genetic components to package viral genomes, pyocin-encoding gene clusters share a high degree of genetic homology to phage tail genes, attributed to their shared ancestry. This poses a significant annotation-based challenge, as current prophage prediction tools, which rely on phage homology for prediction, can misclassify pyocins or tailocins as prophages. Pyocins unknowingly being misannotated as prophages is not only a bioinformatic issue, but can certainly confound experiments examining bacterial competition and prophage induction, if the experimental setup is based on this unintentional misannotation. In this study, we present "TattleTail", the first version of a bioinformatic tool designed to accurately identify tailocins in genome sequences, with a focus on identifying phage tail-derived pyocin-encoding gene clusters in P. aeruginosa in its first iteration. The tool leverages conserved pyocin gene cluster markers and accounts for the absence of canonical phage features, such as capsid, terminase and integrase genes, thereby distinguishing pyocins from intact and cryptic prophages. Validation in P. aeruginosa and non-P. aeruginosa genomes confirmed the presence of pyocin regions in all P. aeruginosa genomes, while none were detected in any non-P. aeruginosa genomes. Notably, TattleTail enabled the identification of representative pyocin-encoding gene clusters in clinical P. aeruginosa isolates. The identified pyocins in the clinical isolates were induced using mitomycin C, visualized via transmission electron microscopy, processed via tangential flow filtration and demonstrated bactericidal activity, thereby confirming TattleTail predictions. TattleTail aims to complement existing prophage prediction tools during genomic analyses involving phage-derived elements in bacterial genomes, allowing more accurate identification of these elements facilitated by robust discrimination between prophages and tailocins.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

Botrytis cinerea is a necrotrophic fungal pathogen that infects thousands of plant species. During infection, these diverse plant hosts produce different specialized metabolites that can inhibit pathogen growth and shape pathogen fitness. However, the genetic architecture of pathogen resistance toward individual host defense metabolites remains poorly understood. To address this question, we exposed 83 B. cinerea isolates to the metabolite linalool and quantified metabolic and structural responses. Exposure revealed extensive phenotypic diversity across isolates. Genome-wide association identified 101 genes of interest associated with membrane transport and stress response regulation. Genetic associations were stronger for morphological traits than for metabolic traits, suggesting that hyphal architecture may have a complex genetic architecture contributing to linalool resistance. Together, these results establish natural variation in linalool response and provide candidate loci for understanding how generalist pathogens respond to host-derived chemical defenses. Article SummaryTo understand how a generalist pathogen responds to host defenses, we asked how Botrytis cinerea responds to linalool, a widespread monoterpene involved in plant defense. We exposed 83 B. cinerea isolates to 1000 {micro}M of linalool for 72 hours and quantified metabolic traits (growth curves and growth dynamics over time) and morphological traits (hyphal network features). Using GWA, we linked phenotypic variation to genetic variants. Results indicate substantial natural variation in linalool resistance and distinct genetic architectures across trait classes: metabolic responses are driven by a relatively small number of loci with larger effects, whereas structural/morphological responses appear more polygenic.

Biofilm formation is a major cause of chronic implant-related bone infections and is associated with impaired immune responses. In a previous study, we identified the cGAS-STING pathway as a potential therapeutic target, as its activation--observed in response to planktonic Staphylococcus aureus (SA)--was absent in the corresponding biofilm setting. The present study aimed to identify potential mechanisms underlying the lack of cGAS activation in the biofilm environment. As biofilm-derived nucleases might degrade cGAS ligands, we assessed presence and activity of micrococcal nuclease in conditioned media from planktonic and biofilm-grown SA and evaluated the impact of extracellular DNases on cGAS pathway activation in macrophages. In addition, we examined altered cGAS expression, the requirement for continuous biofilm exposure and potential downstream inhibition resulting from degradation of the cGAS product. Biofilm formation was associated with dynamic nuclease expression, and exposure to the biofilm environment led to reduced cGAS levels in macrophages, accompanied by a lack of interferon response. Exogenous cGAS activation by G3-YSD failed to restore signaling, independent of nuclease activity or continuous biofilm exposure. In contrast, supplementation with the cGAS product and STING ligand 2'3'-cGAMP fully restored interferon responses and enhanced macrophage activation, indicating that increased degradation of the second messenger in the biofilm environment is not responsible for impaired pathway activation. Similar effects observed with Staphylococcus epidermidis and primary macrophages suggest a broader mechanism that is not SA- or cell line-specific. In conclusion, our data provide novel mechanistic insight into biofilm-mediated impairment of cGAS-STING signaling, revealing a previously unrecognized mechanism of immune evasion in staphylococcal biofilms. These findings extend our previous work and support the therapeutic potential of targeting STING as promising strategy to restore immune responses in chronic implant-related bone infections. HighlightsO_LIBiofilm-derived factors impair cGAS-STING pathway activation and suppress interferon responses in macrophages. C_LIO_LIImpaired signaling is not primarily explained by extracellular micrococcal nuclease-mediated degradation of potential cGAS ligands. C_LIO_LIBiofilm exposure reduces cGAS expression levels and inhibits exogenous cGAS activation independently of continuous presence. C_LIO_LIExogenous 2'3'-cGAMP fully restores interferon responses, indicating that impaired signaling is not due to degradation of the cGAS product. C_LIO_LIDirect activation of STING broadly enhances macrophage activation and by this could amplify overall immune responses. C_LIO_LIBypassing cGAS via direct STING targeting represents a potential therapeutic strategy to overcome immune evasion in chronic implant-related bone infections. C_LI

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

IntroductionCaves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we took advantage of a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the worlds deepest vertical systems, which had remained untouched by humans for 19 years. MethodsWe performed a high-resolution vertical profiling of the caves microbiome by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation using various media and temperatures, we established a biobank of 820 bacterial isolates. ResultsTaxonomic identification of a 362-isolate subset revealed a diverse community spanning 25 genera and 4 phyla, dominated by Pseudomonas, Bacillus, and Stenotrophomonas. Beyond characterizing diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum, Verticillium dahliae, and Phytophthora nicotianae, uncovered a significant group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria (Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs); notably, over half of these showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Finally, ex vivo trials showed that the Pseudomonas sp. SRL917 isolate, significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. DiscussionCollectively, our results demonstrate that deep karstic systems are not just geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.

In response to the comparatively sudden application of industrial scale levels of antibiotics to an ecosystem where naturally produced antibiotics are scarce, namely the ecologies within and around agricultural settings, animal-associated microbes have had to rapidly adapt, mostly relying on mobile genetic elements (MGEs) taken up due to loss of CRISPR functionality. Due to selection for resistance and other adaptive traits carried by dynamic and rapidly recombining MGEs, plasmids and transposons have rapidly accumulated in this human-proximal environment. Because of the occurrence of Enterococcus faecalis in a wide range of hosts up and down the food chain, and the fact that this species represents the greatest generalist of the genus, we comprehensively examined the mobilome of multidrug-resistant E. faecalis (ST330, ST591, ST710, and ST711) from healthy piglets raised on dispersed Brazilian farms, using long-read sequencing, analysis of plasmid pangenomes, and conjugation assays with these strains serving as donors. Genomes ranged from [~]2.8-3.1 Mb, with diverse MGEs constituting [~]7-15% of those genomes. Large modular antimicrobial resistance-encoding gene blocks ([~]40 Kb) were observed to be integrated into a [~]67 Kb chromosomal segment of the pathogenicity island AF454824. Prophages contributed up to 70% of the chromosomal mobile element content, integrating into both CRISPR-deficient genomes and those with intact type II-A CRISPR1 arrays, which were enriched with Caudoviricetes phage-targeting spacers across all strains. Plasmid content showed pronounced mosaicism driven by diverse insertion sequences, transposons, and related mobile elements, many directly implicated in AMR gene cluster acquisitions. RepA_N, Inc18, and Rep3 plasmids, mostly conjugative, also carried various persistence-related traits, suggesting they may actively enhance agricultural fitness rather than passively accumulate due to loss of CRISPR protection.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) are designated by the WHO as a top-priority pathogen due to their antibiotic resistance profiles, capacity to disseminate resistance, and associated mortality. The prototypical CRKp clade, CG258, is associated with acute respiratory infections; however, urinary tract infections (UTIs) caused by CRKp are increasing, and frequently linked to the emergent clade CG307. Notably, CG307 isolates have extensive accessory genomes that may drive adaptation to the urinary tract, including a novel capsule gene cluster and high-affinity urea transporter. In this study, we show that UTIs caused by Kp are increasing across the Southern US and that in addition to CG307s circulating within Houston, TX hospital systems, the lineage was also detected in healthcare systems in the broader Gulf Coast region. Characterization of CG307 isolates demonstrate that while these strains exhibit similar mucoviscosity compared to the reference UTI strain TOP52, the lineage displays significantly higher i) growth in artificial urine, ii) urease activity, and iii) UTI in a mouse model. These results suggest that CG307 is spreading across the Southern US and encodes distinct pathogenic features that promote urinary tract tropism, underscoring a need for targeted surveillance and future studies that mechanistically examine the factors that promote UTI.

BackgroundThe virulence-persistence trade-off is considered a fundamental organizing principle of Listeria monocytogenes population biology: hypervirulent clonal complexes dominate clinical cases but are rarely found in processing environments, while hypovirulent lineages dominate industrial niches but are underrepresented in severe disease. However, whether this dichotomy operates as an absolute paradigm has not been quantitatively evaluated at the population scale. Here we develop a multi-dimensional genomic scoring approach that simultaneously quantifies virulence potential (V), environmental persistence capacity (P), clonal epidemiological context (C), and antimicrobial resistance (R) across 903 genomes from four independent datasets spanning five countries, and apply it to test the universality of the trade-off and to characterize the ecological strategies of L. monocytogenes at the population level. MethodsThe scoring approach integrates four components into a composite 0-100 score through empirically calibrated weights (V: 30%, P: 40%, C: 20%, R: 10%). Validation employed 903 L. monocytogenes genomes from four public BioProjects: longitudinal industrial surveillance in Norway (Fagerlund et al. 2022, n = 513, PRJNA689484), retail environments in the United States (Stasiewicz et al. 2015, n = 191, PRJNA245909), clinical-environmental context in China (Wang et al. 2021, n = 151, PRJNA759341), and meat processing in Poland (Kurpas et al. 2020, n = 48, PRJNA629756). ResultsThe composite score achieved excellent discriminatory performance for identifying persistent clones (AUC = 0.933; 95% CI: 0.910-0.954) with perfect specificity (1.000; zero false positives). The inverse V-P correlation was statistically significant across all four datasets (Spearman {rho} from -0.144 to -0.713; p < 0.01), providing the first cross-dataset quantitative confirmation of the trade-off. However, simultaneous evaluation of V-P profiles at the population scale revealed that the species does not conform to a binary dichotomy but rather exhibits three quantitatively distinguishable ecological strategies, for which we propose a functional trophic taxonomy: nosotrophic lineages (22.7%; V > 65, P < 35), specialized in the pathogenic niche; saprotrophic lineages (5.8%; V < 30, P > 45), with irreversible virulence attenuation and industrial specialization; and, as the central finding, amphitrophic lineages (39.1%; V [&ge;] 35, P [&ge;] 40), which simultaneously retain functional inlA and stress tolerance determinants (SSI-1) without detectable genomic sacrifice. The three strategies differed significantly (Kruskal-Wallis H = 138.7; p = 7.6 x 10-3{superscript 1}). The correspondence between trophic strategy and CC was predominant but not absolute, demonstrating that this phenotypic classification captures intra-CC functional heterogeneity inaccessible through conventional typing. Furthermore, comparison between genome-based and surveillance-informed classifications revealed that 60 hypervirulent isolates (CC1/CC14), genetically classified as nosotrophic, persisted for up to 8 years in industrial facilities despite lacking any recognized persistence markers -- indicating that their prolonged survival reflects environmental opportunity rather than intrinsic genomic adaptation. ConclusionsMulti-dimensional genomic profiling reveals that the virulence-persistence trade-off, while statistically robust, does not operate as an absolute paradigm. The amphitrophic strategy -- documented here for the first time as a quantitatively distinguishable category encompassing 39.1% of the analyzed population -- challenges the prevailing dichotomous model and identifies a previously unrecognized combined ecological niche. The ability to discriminate between genome-encoded persistence capacity and environmentally facilitated persistence provides a biological framework for understanding the ecological determinants of L. monocytogenes population dynamics in anthropogenic environments.

Skin infections are among the most common types of infections in the United States and pose a serious health threat. Effective resolution requires both bacterial clearance and tissue repair, however the mechanisms underlying tissue repair remain poorly understood. Here, we investigate the cellular responses in a murine model of S. aureus dermonecrotic skin infection using single cell transcriptomics. Within the infected lesions, we identified a major influx of heterogeneous neutrophils, including a distinct subtype expressing high levels of Spp1, the gene encoding osteopontin (OPN). Cell-cell interaction analysis identified Spp1 signaling as the most enriched pathway in infected tissue. In OPN knockout (KO) mice, transcriptomic profiling revealed a lower proportion of fibroblast and keratinocyte cells at the infection site, and a reduction in associated repair signaling pathways. In vivo, OPN KO mice developed significantly larger lesions during skin infection, whereas WT mice treated with recombinant OPN showed accelerated healing relative to untreated controls. Together, these findings highlight the importance of specific neutrophil subtypes and OPN signaling as critical mediators in infected tissue repair. Further our study suggests that modulating OPN may represent a promising therapeutic strategy to accelerate infected tissue repair.

Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable the efficient conversion of poorly water-soluble gaseous substrates, thereby offering significant potential to advance bioremediation and bioproduction. However, microorganisms in the gas phase are exposed to various environmental stresses, mainly due to the absence of bulk water. While survival strategies of microorganisms in gaseous environments have been studied in environmental microbiology, the metabolic adaptations that sustain bacterial cell activity remain poorly understood. In this study, we elucidated the comprehensive metabolic alterations of a highly adhesive bacterium Acinetobacter sp. Tol 5 degrading toluene under gas- and aqueous-phase conditions. An integrated approach combining metabolomics, lipidomics, and transcriptomics revealed significant differences in metabolic profiles between cells under these conditions. Under the gas-phase condition, the degradation of amino acids and nucleic acids was significantly promoted, and the intracellular glutamate pool was maintained at high levels. Notably, citrulline was found to accumulate specifically under the gas-phase condition, representing a stress response similar to that reported in Cucurbitaceae plants during drought. Furthermore, lipidomics revealed the lipid composition of Tol 5 and demonstrated a shift in response to environmental conditions. Specifically, the degradation of intracellular storage lipids was promoted under gas-phase conditions, suggesting a crucial link to bacterial survival in water-limited environments. These findings provide critical insights into the adaptation strategies of bacteria adapting to gaseous environments, offering fundamental information for the rational design of robust gas-phase bioprocesses and a deeper understanding of environmental microbiology.