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PubMed2025

Antimicrobial resistance among Gram-positive agents of bacteraemia in the UK and Ireland: trends from 2001 to 2019.

OBJECTIVES: The BSAC Bacteraemia Resistance Surveillance collected isolates from UK and Irish hospitals for central testing. Concurrent UKHSA surveillance collated English hospitals' own susceptibility data. Results were collated and compared. METHODS: BSAC Surveillance collected quotas of isolates per site annually from 2001 to 2019. MIC testing was by BSAC agar dilution, with resistance mechanisms identified by synergy tests, interpretive reading and PCR. The UKHSA sought hospitals' data on all bacteraemia isolates. RESULTS: Both surveillance systems recorded dramatic falls in MRSA, from c. 40% of bloodstream Staphylococcus aureus in 2001 to <10% by 2019. Both noted rises in the proportion of MRSA (especially) and MSSA resistant to fusidic acid, along with declines of ciprofloxacin and macrolide resistance amongst MRSA. Methicillin resistance also fell among coagulase-negative staphylococci, albeit only modestly; fusidic acid resistance rose. Shifts for pneumococci were complex, reflecting vaccine-contingent serotype displacements; resistance rates remained low, with high-dose penicillin almost universally active. Enterococcus faecium became more prevalent relative to Enterococcus faecalis; vancomycin resistance averaged 29% among E. faecium versus 2% in E. faecalis, without trend. Erythromycin resistance rose among groups B, C and G (but not group A) streptococci. Oxazolidinones, tigecycline, daptomycin and anti-PBP2' cephalosporins retained near-universal activity against target species, except that tigecycline has been compromised by breakpoint reductions for streptococci. CONCLUSIONS: Gram-positive pathogens were the dominant historical pathogens of bacteraemia. The trends seen here-with many near-universally active antibiotics-indicate little hazard of this situation returning. Nevertheless, few treatments exist in some settings, notably multi-resistant E. faecium endocarditis.

Humans↗

PubMed2026

Noncanonical bactericidal activity of teleost type I interferon is conferred by a membrane-targeting C-terminal peptide.

Type I interferons (IFNs) are indispensable antiviral cytokines in nonspecific immunity, yet they play dual roles in bacterial infections in mammals. Recent studies have revealed a subset of strongly cationic type I IFNs possessing potent antimicrobial properties across nonmammalian vertebrates. In this study, we identified a type I IFN gene, CaIFNi, from Cromileptes altivelis that is characterized by a unique triple-disulfide bond architecture. In Vibrio harveyi-challenged models, overexpression of CaIFNi potentiated bacterial clearance capacity in tissues, whereas its knockdown exacerbated bacterial colonization, highlighting its ability to protect the host against bacterial infection in vivo. In vitro assays further confirmed that CaIFNi directly binds to and kills both gram-negative (G-) and gram-positive (G+) bacteria, which first revealed the antibacterial function of new subgroup IFNi within teleost type I IFNs. Furthermore, the α-helical peptide CaIFNi-18 derived from CaIFNi was identified as a novel antimicrobial peptide (AMP) that has broad-spectrum antibacterial efficacy against G- and G+ bacteria and membrane-targeting ability. Further mechanistic studies revealed that CaIFNi has bactericidal effects on both G- and G+ bacteria through membrane depolarization and disruption, alteration of the bacterial ultrastructure, and in vitro binding to genomic DNA. In addition, CaIFNi-18 also has significant in vivo therapeutic efficacy against bacterial infection, highlighting its great potential as an antibacterial agent. Encouragingly, the loss of antibacterial activity in the truncation mutant (rCaIFNiΔ148-165) lacking the CaIFNi-18 segment suggests that this region is essential for the bactericidal function of the full-length protein and likely acts as its core domain. Further computational simulations revealed that the deletion of the CaIFNi-18 region attenuated the interaction between the protein and the bacterial membrane. These findings not only expand the functional scope of type I IFNs beyond their canonical antiviral role but also identify their derivative CaIFNi-18 as both a promising antimicrobial candidate and the essential bactericidal domain of CaIFNi, thereby offering novel therapeutic strategies against bacterial infections in the aquaculture industry and beyond.

Animals↗

PubMed2026

Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of Clostridioides difficile and Clostridium perfringens.

UNLABELLED: Clostridioides difficile and Clostridium perfringens are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two C. difficile and two C. perfringens strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for C. difficile, achieving >100× coverage and enabling reliable circularization of chromosomes and plasmids, including a C. difficile metronidazole resistance plasmid and C. perfringens plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for C. difficile, highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of C. difficile and C. perfringens, supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens. IMPORTANCE: High-quality genomic data are essential for accurate characterization of Clostridioides difficile and Clostridium perfringens, two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of C. difficile and C. perfringens for genomic surveillance or outbreak investigations.

Clostridioides difficile↗

PubMed2026

Broad-Spectrum, Cell Envelope-Active Marinocyclin Antibiotics From a Coral-Derived Bacterium Are Effective Against Colistin-Resistant Bacteria.

The marine bacterial genus Aquimarina comprises diverse members with numerous natural product biosynthetic gene clusters but few characterized compounds. Here we report a novel class of lipopeptides with exceptional antibiotic activity, named marinocyclins, isolated from Aquimarina megaterium EL43 associated with the octocoral Eunicella labiata. The major congener marinocyclin A exhibited potent and uniform activity against a broad panel of drug-resistant gram-negative and gram-positive pathogens, including ESKAPE bacteria. The natural product efficiently compromised the outer and inner bacterial membranes, leading to rapid cell permeabilization and lysis. This activity profile was mediated by the ability to bind lipopolysaccharides, anionic phospholipids enriched in bacterial membranes, and peptidoglycan precursors. Eukaryotic cytotoxicity required higher doses than antibacterial activity. Genomic data suggest a nonribosomal biosynthetic origin for marinocyclins. These findings position marinocyclins as a promising new scaffold for antibiotic development and highlight the potential of Aquimarina spp. as a source of novel antibiotics. Further medicinal chemistry optimization of marinocyclins could enhance their prokaryotic selectivity to generate leads for treating infections caused by drug-resistant pathogens.

antibacterial activity↗

PubMed2025

Streptococcus pyogenes EVs induce the alternative inflammasome via caspase-4/-5 in human monocytes.

The sensing of Gram-negative Extracellular Vesicles (EVs) by the innate immune system has been extensively studied in the past decade. In contrast, recognition of Gram-positive EVs by innate immune cells remains poorly understood. Comparative genome-wide transcriptional analysis in human monocytes uncovered that S. pyogenes EVs induce proinflammatory signatures that are markedly distinct from those of their parental cells. Among the 209 genes exclusively upregulated by EVs, caspase-5 prompted us to study inflammasome signaling pathways in depth. We show that lipoteichoic acid (LTA), a structural component of Gram-positive bacterial membranes present on EVs from S. pyogenes and other Gram-positive species, is sensed by TLR2 which triggers the alternative inflammasome composed of NLRP3 and the inflammatory caspases-4/-5 to mount an IL-1β response without inducing cell death. For S. pyogenes, we identify TLR8 as a sensor to mediate caspase-4/-5-dependent IL-1β secretion. Notably, inflammasome activation by intact bacteria is independent of the global virulence regulator CovS in monocytes. Overall, our study highlights a new role for TLR2 and caspase-4/-5 in the recognition of Gram-positive EVs in human monocytes.

Humans↗

PubMed2024

Comparison of the antibiotic resistance mechanisms in a gram-positive and a gram-negative bacterium by gene networks analysis.

Nowadays, the emergence of some microbial species resistant to antibiotics, both gram-positive and gram-negative bacteria, is due to changes in molecular activities, biological processes and their cellular structure in order to survive. The aim of the gene network analysis for the drug-resistant Enterococcus faecium as gram-positive and Salmonella Typhimurium as gram-negative bacteria was to gain insights into the important interactions between hub genes involved in key molecular pathways associated with cellular adaptations and the comparison of survival mechanisms of these two bacteria exposed to ciprofloxacin. To identify the gene clusters and hub genes, the gene networks in drug-resistant E. faecium and S. Typhimurium were analyzed using Cytoscape. Subsequently, the putative regulatory elements were found by examining the promoter regions of the hub genes and their gene ontology (GO) was determined. In addition, the interaction between milRNAs and up-regulated genes was predicted. RcsC and D920_01853 have been identified as the most important of the hub genes in S. Typhimurium and E. faecium, respectively. The enrichment analysis of hub genes revealed the importance of efflux pumps, and different enzymatic and binding activities in both bacteria. However, E. faecium specifically increases phospholipid biosynthesis and isopentenyl diphosphate biosynthesis, whereas S. Typhimurium focuses on phosphorelay signal transduction, transcriptional regulation, and protein autophosphorylation. The similarities in the GO findings of the promoters suggest common pathways for survival and basic physiological functions of both bacteria, including peptidoglycan production, glucose transport and cellular homeostasis. The genes with the most interactions with milRNAs include dpiB, rcsC and kdpD in S. Typhimurium and EFAU004_01228, EFAU004_02016 and EFAU004_00870 in E. faecium, respectively. The results showed that gram-positive and gram-negative bacteria have different mechanisms to survive under antibiotic stress. By deciphering their intricate adaptations, we can develop more effective therapeutic approaches and combat the challenges posed by multidrug-resistant bacteria.

Anti-Bacterial Agents↗

PubMed2020

Natural brominated phenoxyphenols kill persistent and biofilm-incorporated cells of MRSA and other pathogenic bacteria.

Due to a high unresponsiveness to chemotherapy, biofilm formation is an important medical problem that frequently occurs during infection with many bacterial pathogens. In this study, the marine sponge-derived natural compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol and 3,4,6-tribromo-2-(2',4'-dibromophenoxy)phenol were found to exhibit broad antibacterial activity against medically relevant gram-positive and gram-negative pathogens. The compounds were not only bactericidal against both replicating and stationary phase-persistent planktonic cells of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa; they also killed biofilm-incorporated cells of both species while not affecting biofilm structural integrity. Moreover, these compounds were active against carbapenemase-producing Enterobacter sp. This simultaneous activity of compounds against different growth forms of both gram-positive and gram-negative bacteria is rare. Genome sequencing of spontaneous resistant mutants and proteome analysis suggest that resistance is mediated by downregulation of the bacterial EIIBC phosphotransferase components scrA and mtlA in MRSA likely leading to a lower uptake of the molecules. Due to their only moderate cytotoxicity against human cell lines, phenoxyphenols provide an interesting new scaffold for development of antimicrobial agents with activity against planktonic cells, persisters and biofilm-incoporated cells of ESKAPE pathogens. KEY POINTS: • Brominated phenoxyphenols kill actively replicating and biofilm-incorporated bacteria. • Phosphotransferase systems mediate uptake of brominated phenoxyphenols. • Downregulation of phosphotransferase systems mediate resistance.

Animals↗

PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗

PubMed2024

Rapid inference of antibiotic susceptibility phenotype of uropathogens using metagenomic sequencing with neighbor typing.

UNLABELLED: Timely diagnostic tools are needed to improve antibiotic treatment. Pairing metagenomic sequencing with genomic neighbor typing algorithms may support rapid clinically actionable results. We created resistance-associated sequence elements (RASE) databases for Escherichia coli and Klebsiella spp. and used them to predict antibiotic susceptibility in directly sequenced (Oxford Nanopore) urine specimens from critically ill patients. RASE analysis was performed on pathogen-specific reads from metagenomic sequencing. We evaluated the ability to predict (i) multi-locus sequence type (MLST) and (ii) susceptibility profiles. We used neighbor typing to predict MLST and susceptibility phenotype of E. coli (64/80) and Klebsiella spp. (16/80) from urine samples. When optimized by lineage score, MLST predictions were concordant for 73% of samples. Similarly, a RASE-susceptible prediction for a given isolate was associated with a specificity and a positive likelihood ratio (LR+) for susceptibility of 0.65 (95% CI, 0.54-0.76) and 2.26 (95% CI, 1.75-2.92), respectively, with an increase in the probability of susceptibility of 10%. A RASE-non-susceptible prediction was associated with a sensitivity and a negative likelihood ratio (LR-) for susceptibility of 0.79 (95% CI, 0.74-0.84) and 0.32 (95% CI, 0.24-0.43) respectively, with a decrease in the probability of susceptibility of 20%. Numerous antibiotic classes could reasonably be reconsidered empiric therapy by shifting empiric probabilities of susceptibility across relevant treatment thresholds. Moreover, these predictions can be available within 6 h. Metagenomic sequencing of urine specimens with neighbor typing provides rapid and informative predictions of lineage and antibiotic susceptibility with the potential to impact clinical decision-making. IMPORTANCE: Urinary tract infections (UTIs) are a common diagnosis in hospitals and are often treated empirically with broad-spectrum antibiotics. These broad-spectrum agents can select for resistance in these bacteria and co-colonizing organisms. The use of narrow-spectrum agents is desirable as an antibiotic stewardship measure; however, it is counterbalanced by the need for adequate therapy. Identification of causative organisms and their antibiotic susceptibility can help direct treatment; however, conventional testing requires days to produce actionable results. Methods to quickly and accurately predict susceptibility phenotypes for pathogens causing UTI could thus improve both patient outcomes and antibiotic stewardship. Here, expanding on previous work showing accurate prediction for certain Gram-positive pathogens, we demonstrate how the use of RASE from metagenomic sequencing can provide informative and rapid phenotype prediction results for common Gram-negative pathogens in UTI, highlighting the future potential of this method to be used in clinical settings to guide empiric antibiotic selection.

Humans↗

PubMed2026

Genome-wide Identification and Expression Profiling Reveal the Galectin Gene Family Diversity and their Possible Role in Antibacterial Mucosal Immunity in Japanese Flounder (Paralichthys olivaceus).

Galectins are a family of proteins that bind specifically to β-galactosides. Their importance in innate immunity of mammals has been well-documented. However, the systematic identification and characterization of galectin gene family remain limited in teleost. In this study, we identified 13 galectin genes (lgals2, lgals2a, lgals2b, lgals3, lgals3a, lgals3b, lgals4, lgals8, lgals8a, lgals9, grp, grp-b, grp-c) from Paralichthys olivaceus genome and analyzed their tissue expressions and expressions in response to Gram-negative and Gram-positive bacterial infections in mucosal tissues (gills, intestine and skin). The P. olivaceus galections were classified into three distinct types based on carbohydrate recognition domains (CRDs). Phylogenetic and syntenic analyses revealed that these galectins are closely related to their counterparts in turbot and zebrafish. Moreover, the transcripts of the 13 galectins were widespread across all tested tissues of healthy fish and regulated following challenge with Vibrio anguillarum or Streptococcus iniae in mucosal tissues, indicating their involvement in P. olivaceus immune response to bacterial infections. The lgals2a was significantly upregulated in the three mucosal tissues by either bacterial infection, whereas lgals9 and grp were basically downregulated in these tissues by either infection. On the other hand, the lgals3b and lgals4 exhibited a bacteria-specific responsive expression as they were upregulated by V. anguillarum whereas remained stable upon S. iniae infection in the gills. We also observed a positive correlation between expression level and bacterial load for the upregulated galectin genes and a negative correlation for the downregulated galectin genes. These results suggest a functional divergence among galectin members in mucosal immunity against bacterial infection in P. olivaceus.

Animals↗

PubMed2025

Characterization of the novel Cutibacterium acnes phage KIT08 and its associated pseudolysogenic bacterial isolate.

Cutibacterium acnes, formerly Propionibacterium acnes, is a Gram-positive bacterium commonly recognized as an important factor in acne vulgaris and infections associated with prosthetic medical devices. With the rise in antibiotic resistance, phage therapy has gained renewed attention as a promising alternative to antibiotics. In addition to a strict lytic cycle, some virulent phages may enter a pseudolysogenic state and exclude superinfections, thereby significantly limiting the applicability of these potential antimicrobial agents. However, the trade-off induced by phage infection of bacterial cells during this state and its molecular mechanism are yet to be confirmed, especially for C. acnes phages. In this study, a novel Cutibacterium acnes phage, KIT08, was isolated and characterized. It demonstrated rapid infectivity and moderately strong bacteriolysis. After infection of C. acnes NBRC 107,605, pseudolysogenic bacteria were collected and examined for physiological tradeoffs. The pseudolysogenic isolate exhibited slower growth and downregulation of the transcriptional levels of biofilm-producing genes, such as lipase 2 and hyaluronate lyase, leading to a decrease in biofilm formation. Additionally, a genomic study of phage KIT08 revealed that open reading frames 23 and 34 encode putative proteins homologous to repressor C and LTP proteins, which may play an important role in the induction of pseudolysogeny and superinfection exclusion in C. acnes.

Propionibacterium acnes↗

PubMed2026

Necrotizing fasciitis associated with Acinetobacter lactucae and Bacillus paramobilis infections in 2 dogs.

Two immunocompetent dogs from separate households were presented to a tertiary referral hospital with soft tissue wounds attributable to severe bacterial infection. A 5-y-old, castrated male Golden Retriever dog (case 1) was presented with extensive hemorrhagic cellulitis of the forelimbs and neck that developed over 48-h and was attributable to Acinetobacter lactucae infection. Additionally, an 8-y-old, spayed female Labrador Retriever-mix dog (case 2) sustained a deep penetrating wound over the left hip that progressed over 24 h to necrotizing fasciitis, from which Bacillus paramobilis was isolated. Histopathologic findings in both cases included severe acute, skeletal muscle necrosis and necrosuppurative myofasciitis, dermatitis, and panniculitis with intralesional rod-shaped bacteria. Whole-genome sequencing and phylogenetic analysis of the bacterial isolates revealed numerous cytolytic toxins and other virulence genes carried by the isolates, expanding the profile of these 2 bacteria. To our knowledge, canine cases of A. lactucae and B. paramobilis associated with necrotizing fasciitis have not been reported previously. In both cases, progression from an inciting incident to septic disease was extremely rapid, occurring within 48 h in case 1 and 24 h in case 2, underscoring the fulminant nature of necrotizing fasciitis.

Animals↗

PubMed2026

Narasin used as a feed additive in conventional rearing of broilers can co-select for vancomycin-resistant Enterococcus faecium through the NarAB ionophore resistance mechanisms.

OBJECTIVES: To investigate the role of the NarAB resistance mechanism in the selection of vancomycin-resistant Enterococcus faecium (VREfm) and assess the impact of ionophore feed additives, particularly narasin, on the emergence of VREfm in broiler chickens. MATERIALS AND METHODS: Three isogenic E. faecium strains with different antimicrobial resistance determinants were created by mutagenesis and conjugation and used in a controlled animal experiment. Ross 308 broiler chickens were inoculated with either a rifampicin-resistant, a rifampicin- and vancomycin-resistant or a rifampicin-, vancomycin- and narasin-resistant strain and fed diets supplemented with selected ionophores. Bacterial populations were analysed on selective Slanetz and Bartley agar to determine the presence and selection of VREfm and other vancomycin-resistant species. Bacterial inoculation strains and isolates were whole genome sequenced for species identification and to identify genetic resistance mechanisms. RESULTS: Narasin was shown to select for VREfm in broilers, with NarAB being essential for co-selection. Intrinsically vancomycin-resistant Pediococcus acidilactici and Enterococcus gallinarum were identified as part of the broilers' vancomycin-resistant resident microbiota. Notably, among the P. acidilactici isolates that were susceptibility tested, strains resistant to both vancomycin and narasin were only found in broilers fed narasin, supporting that narasin promotes the growth of narasin-resistant populations. CONCLUSION: Narasin use in broiler feed can co-select for vancomycin-resistant bacteria, including VREfm, through the NarAB mechanism. These findings emphasize the concerns associated with the use of particular ionophores in poultry and suggest that vancomycin and narasin resistance may be more widespread in the broiler microbiota than previously recognized. Further research is needed to understand the implications for antimicrobial resistance and human health.

Animals↗

PubMed2026

Predicting bloodstream infection by plasma cell-free metagenomic sequencing: a prospective cohort study.

BACKGROUND: Patients receiving myelosuppressive chemotherapy or haematopoietic cell transplantation are at high risk for life-threatening bloodstream infections. A novel pre-emptive treatment paradigm guided by pathogen detection before symptoms appear might reduce this risk, but no validated screening test is available. This study evaluated the sensitivity and specificity of plasma microbial cell-free DNA metagenomic sequencing (mcfDNA-Seq) for predicting bloodstream infections in children and adolescents receiving therapy for high-risk leukaemia. METHODS: In this prospective cohort study, between Aug 9, 2017, and Feb 28, 2022, leftover clinical plasma samples were prospectively collected up to once per day from patients who were younger than 25 years, receiving care for leukaemia at St Jude Children's Research Hospital (Memphis, TN, USA), and at high risk for life-threatening bloodstream infections. mcfDNA-Seq was used to identify pathogen DNA in blood samples obtained during the 7 days before to 1 day after bloodstream infection onset, and in control samples from the same population in the absence of fever or infection. The testing laboratory was masked to sample status. Primary outcomes were predictive sensitivity of mcfDNA-Seq for detecting the expected bloodstream infection pathogen during the 3 days preceding the day of bloodstream infection onset, with a prespecified favourable sensitivity of 50%, and predictive specificity of mcfDNA-Seq in control samples. Exploratory analyses comprised assessing sensitivity and specificity restricted to bacteria or common bloodstream infection pathogens, and after applying a data-derived DNA fragment concentration cutoff; estimating the predictive sensitivity on each of the 7 days before bloodstream infection onset; identifying clinical characteristics that affected predictive sensitivity or specificity; and examining the clinical relevance of additional organisms identified by mcfDNA-Seq during bloodstream infection episodes. Diagnostic sensitivity was also assessed on samples collected on the day of, or day after, diagnosis of bloodstream infection. This study is registered with ClinicalTrials.gov, NCT03226158. FINDINGS: 94 evaluable bloodstream infections occurred in 60 (38%) of 158 enrolled participants; 19 episodes were previously described in the pilot phase of this study. The predictive sensitivity of mcfDNA-Seq was 51·9% (95% CI 40·5-63·1) for all bloodstream infection episodes, 53·8% (42·2-65·2) for bacterial infection only, and 51·9% (40·5-63·1) when applying a DNA fragment concentration cutoff of 140 molecules per μL. Sensitivity was lowest at day -7 and increased daily until the day of diagnosis. Diagnostic sensitivity was 81·3% (95% CI 71·0-89·1) for all bloodstream infection episodes and 83·1% (72·9-90·7) for bacterial infections only. Predictive specificity was 82·7% (95% CI 76·0-88·2), but improved to 88·9% (83·0-93·3) for common bloodstream infection pathogens, and to 93·8% (88·9-97·0) when also applying the DNA fragment concentration cutoff. Predictive sensitivity was higher in participants with acute lymphoblastic leukaemia (adjusted odds ratio [aOR] 11·1 [1·7-74·2] vs those with acute myeloid leukaemia), and it was lower in polymicrobial infections (aOR 0·0 [0·0-0·2] vs monomicrobial Gram-positive infections). Clinical false-positive results were positively associated with gastrointestinal disturbance alone (p=0·037) or combined with recent administration of high-dose cytarabine (p=0·012). Additional organisms identified by mcfDNA-Seq that were not identified by blood culture were less likely than expected organisms to have an increasing DNA concentration during the days preceding bloodstream infection diagnosis. INTERPRETATION: mcfDNA-Seq can detect causative pathogens before the onset of some bloodstream infection episodes in profoundly immunocompromised patients. Predictive specificity might be improved by restricting results to a subgroup of relevant organisms, excluding patients with high risk of false-positive results, or applying a higher concentration cutoff. Clinical trials are needed to evaluate mcfDNA-Seq-guided pre-emptive therapy for preventing life-threatening bloodstream infections in patients with high risk. FUNDING: The National Cancer Institute, American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital, and Karius.

Adolescent↗

PubMed2025

Novel, rapid, and reliable typing of vancomycin-resistant Enterococcus faecium CC17/ST80 strains using MALDI-TOF MS.

Vancomycin-resistant Enterococcus faecium (VREfm) is an important nosocomial pathogen. The recent emergence of the highly virulent clonal complex 17 (CC17) is posing a challenge for both therapeutic interventions and hospital infection control measures. Hence, prompt discrimination of CC17 VREfm from unrelated and less-virulent VREfm strains is essential for preventing its spread in hospitals and beyond. Between January 2022 and November 2024, 340 VREfm primary isolates have been identified in our lab and underwent genotyping by pulsed-field gel electrophoresis (PFGE) to survey a potential outbreak in the Tyrol region. In addition, whole-genome sequencing (WGS) was performed on a selected subset (n = 40). To curtail the lengthy time-to-result (TTR) of these methods, a novel typing protocol using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was established, validated, and optimized for rapid sample processing. PFGE and WGS showed that 61.2% of isolates (n = 208) belonged to a specific VREfm cluster identified as CC17 sequence type (ST) 80 vanA VREfm. A comprehensive MALDI-TOF MS analysis identified a distinct peak pattern specific to this lineage. This phenotypic characterization was used as a novel typing method with excellent performance (sensitivity: 1.00 [0.98-1.00], specificity: 0.89 [0.70-0.97]) and demonstrated a short TTR of 1 day after the cultural growth of VREfm. A rapid and novel MALDI-TOF MS-based typing approach for a specific CC17/ST80 vanA VREfm cluster was developed and enabled real-life application in routine diagnostics to assure accurate infection prevention and control measures. Future outbreak investigations may benefit from adopting this cost- and labor-efficient approach.IMPORTANCEThis study addresses the urgent need for faster ways to detect problematic hospital bacteria. A highly transmissible strain of Enterococcus faecium (CC17) has been spreading in healthcare settings, making infections harder to treat and control. Traditional methods to identify and track outbreaks are accurate but slow and resource-intensive, delaying critical infection control actions. By developing and validating a new method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the researchers demonstrated that this strain can be identified quickly, reliably, and at lower cost. Importantly, the new approach delivers results within a day, compared to the lengthy turnaround times of existing methods. This rapid detection tool provides hospitals with a practical solution to respond to outbreaks more effectively, prevent further spread, and protect vulnerable patients. The findings highlight a valuable step forward in strengthening hospital infection control and improving patient safety.

Enterococcus faecium↗

PubMed2025

Hidden diversity in Enterococcus faecalis revealed by CRISPR2 screening: eco-evolutionary insights into a novel subspecies.

Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals and is a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B." Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly humans, and the lack of clinically relevant resistance determinants indicate that subspecies B currently poses minimal threat to public health compared with the broadly disseminated "subspecies A." Nevertheless, the unclear potential for genetic exchange between these subspecies and the frequent association of subspecies B with food sources calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.IMPORTANCEExploring intraspecific genetic variability in generalist bacteria with pathogenic potential, such as Enterococcus faecalis, is a key to uncovering stable evolutionary trends. By screening the CRISPR2 locus across a representative set of genomes from diverse sources, this study reveals a previously unrecognized lineage within the population structure of E. faecalis, associated with underexplored nonhuman and nonhospital reservoirs. These findings broaden our knowledge of the species' genetic landscape and shed light on its adaptive strategies and patterns of ecological dissemination. By bridging phylogenetic patterns with variation in genetic defense systems and accessory traits, the study generates testable hypotheses about the genomic determinants and corresponding selective pressures that shape the species' behavior and long-term dissemination. This work offers new perspectives on the eco-evolutionary dynamics of E. faecalis and highlights the value of genomic surveillance beyond clinical settings, in alignment with One Health principles.

Enterococcus faecalis↗

PubMed2026

Antibacterial mechanisms and pathogen-dependent protective effects of the golden pompano LEAP2-derived peptide TroLEAP2-21.

Antimicrobial peptides (AMPs) are essential components of the innate immune system, with liver-expressed antimicrobial peptide 2 (LEAP2) playing a pivotal role in fish immunity. This study investigated the antimicrobial activity and mechanisms of TroLEAP2-21, a 21-amino-acid short peptide from golden pompano (Trachinotus ovatus), against Gram-positive (Lactococcus garvieae, Staphylococcus epidermidis) and Gram-negative (Vibrio alginolyticus, Vibrio harveyi) bacteria. The predicted three-dimensional structure and helical wheel projection of TroLEAP2-21 suggested typical AMP-like physicochemical features. troleap2 expression in the liver and intestine of T. ovatus was significantly upregulated post L. garvieae or V. harveyi infection, suggesting its potential involvement in antibacterial defense. In vitro, TroLEAP2-21 exhibited antibacterial activity against the tested bacterial strains, with membrane disruption, increased membrane permeability, cytoplasmic leakage, and membrane depolarization observed after peptide treatment. Gel retardation assays further indicated species-dependent association of TroLEAP2-21 with bacterial genomic DNA. In vivo, under the tested intraperitoneal injection conditions, TroLEAP2-21 was associated with improved survival and reduced tissue damage in V. harveyi-infected T. ovatus, whereas no significant survival benefit was observed against L. garvieae. Transcriptomic analysis at 48 h post-infection showed transcriptional changes in immune-related DEGs (rsad2, mx1/mx2, il-8) and enrichment of TLR and Jak-STAT signaling pathways at the transcriptional level in peptide-treated fish. FISH showed the tissue localization of tnf-α and nf-κb transcripts and revealed treatment-associated changes in fluorescence signals, and qRT-PCR of eight immune genes supported transcriptomic results in tissues at 48 h post-infection. Collectively, these findings characterize TroLEAP2-21 as a short LEAP2-derived peptide with antibacterial and immunomodulatory activities. Its comparative advantages over other LEAP2-related peptides and its practical application potential remain to be further investigated.

AMPs↗

PubMed2026

Do wound protectors reduce contamination in total shoulder arthroplasty? A randomized controlled trial.

HYPOTHESIS: Cutibacterium acnes is the most frequent cause of shoulder prosthetic joint infection with skin edges as a source of wound contamination. The primary purpose of this study was to determine if the use of a wound protector device decreases the deep wound bacterial colonization in primary shoulder arthroplasty. The secondary purpose was to assess the effect of device usage on deltopectoral muscle and cephalic vein injury. METHODS: This was a prospective, randomized controlled trial. A total of 100 patients undergoing primary total shoulder arthroplasty were enrolled and randomized into 2 groups: a wound protector group and a control group. Five patients withdrew from the study, leaving 48 patients in the wound protector group and 47 controls. Three deep wound culture swabs were taken after final arthroplasty implantation. The surgeon also graded deltoid, pectoralis major, and cephalic vein injury on a 0-3 scale based on modification to the Tscherne classification of soft tissue injury. The primary outcome of this study was positive culture results for C acnes. Secondary outcomes included total bacterial culture positivity as well as soft tissue injury grades. A subanalysis removing likely contaminant positive cultures (growth >7 days and 1 colony only) was also performed. Comparisons between groups were made using Fisher exact test for categorical outcomes and t tests and Mann-Whitney U tests for continuous variables. RESULTS: The use of a wound protector did not result in any significant differences compared with controls in the rate of positive cultures for C acnes (15% vs. 21%, P = .593) or all bacteria (15% vs. 26%, P = .304). Removing likely contaminant positive cultures did not demonstrate any significant difference in culture positivity (9% vs. 17%, P = .355, for C acnes; 9% vs. 19%, P = .231, for all bacterial species). The wound protector group had better soft tissue injury scores for the deltoid muscle (P < .001) and pectoralis muscle (P < .001). No difference in cephalic vein injury was noted between the 2 groups (P > .05). No difference in surgical time was noted. CONCLUSION: The use of a surgical wound protector device in total shoulder arthroplasty did not significantly decrease bacterial colonization of the deep wound. However, soft tissue damage to the deltoid and pectoralis muscle was less severe in the wound protector group. These findings suggest that this device reduces iatrogenic soft tissue injury.

Humans↗