Search Results (4,918)

Rapid detection of Pseudomonas aeruginosa and its virulence factor ExoU is essential for improving patient outcomes. In this study, a CRISPR–Cas13a-based diagnostic assay combined with recombinase polymerase amplification (RPA) was developed to detect P. aeruginosa and the exoU gene in blood samples. The assay demonstrated robust amplification, with detection limits of 6 log₁₀ and 8 log₁₀ CFU/mL in Luria–Bertani medium and blood, respectively, and a 100% specificity, without cross-reactivity against four Gram-negative bacilli and Staphylococcus aureus reference strains. The utilisation of a fluorescence-based readout facilitated unambiguous discrimination between P. aeruginosa and P. aeruginosa/exoU⁺ isolates vs. negative controls. In conclusion, these results support the potential of RPA/CRISPR-Cas13a diagnostics for the rapid identification of P. aeruginosa and its ExoU virulence factor. Further optimisation and clinical validation are required to confirm its utility as a bedside diagnostic test, where its application would speed up clinical decisions in the treatment of these infections. Full article

Background/Objectives: Serial passage in embryonated eggs is widely used to attenuate the infectious bronchitis virus (IBV) for vaccine production; however, the evolutionary processes underlying attenuation and immunogenicity remain incompletely understood. Here, we analyzed genome-wide viral evolution during serial passages to investigate how mutations emerge, persist, are lost, or become fixed over time and how these dynamics relate to changes in pathogenicity and immunogenicity. Methods: Deep sequencing was performed on 11 representative serial passages (P2–P79) of the UY/11/CA/18 strain, including two derivative lineages: P7 VIR (virulent) and P53 VAC (attenuated and immunogenic). Results: This study identified an early adaptive phase characterized by a limited set of mutations potentially associated with genome replication, viral RNA processing, and virion assembly, including a key change in non-structural protein 14 and variants in M and 3c (E). This phase was followed by a broader expansion of the variant spectrum across replicase genes and delayed accumulation of Spike protein variants. Most Spike changes emerged during later passages and exhibited transient dynamics, and only a subset reached a high frequency after the establishment of early replicase- and structural-associated changes. Consistent with these dynamics, P7 VIR diverged before the late accumulation of Spike variants and retained a pathogenic phenotype, whereas P53 VAC diverged after the emergence of early high-frequency variants but before the extensive late-stage Spike variation observed in P79, which was associated with reduced immunogenicity. Conclusions: These findings support a multi-step model of IBV attenuation in which progressive filtering of genome-wide variation shapes distinct evolutionary outcomes during serial passages. This evolutionary framework provides insight into the relationship between attenuation and immunogenicity and may help guide the rational design of live attenuated vaccines. Full article

Accurate detection and monitoring of antimicrobial resistance (AMR) in Helicobacter pylori mainly rely on phenotypic methods and culture, which can sometimes fail when bacterial load is low or after recent treatment. We investigated whether gastric biopsies classified as H. pylori-negative by standard diagnostic techniques still contain detectable bacterial DNA, including regions linked to AMR, and assessed whether selected DNA fragments can mediate allelic exchange in vitro. Gastric biopsies from 46 dyspeptic patients in the Democratic Republic of the Congo (including 23 phenotypically positive and 23 phenotypically negative individuals) were analyzed using long-read amplicon sequencing of seven resistance-associated loci, selective whole-genome amplification (sWGA) followed by long-read sequencing of H. pylori-enriched reads, and a proof-of-concept natural transformation assay. Phenotypically negative biopsies exhibited significantly lower sequencing depth across multiple loci (including 23S rRNA, gyrA, gyrB, and pbp1A; p = 0.003–0.014), indicating a reduced H. pylori DNA burden. However, AMR-associated mutations linked to various antibiotic classes were found in both groups. sWGA enabled recovery of fragmentary H. pylori sequence data from phenotypically negative samples, including reads that map to resistance- and virulence-associated genes. In vitro, 23S rRNA A2143G amplicons from both phenotypically positive and negative biopsies produced clarithromycin-resistant transformants in strain 26695. These findings indicate that phenotypically negative gastric biopsies might contain low-abundance and fragmentary H. pylori DNA. Although certain DNA fragments can mediate allelic exchange under controlled in vitro conditions, these results do not confirm bacterial viability, active infection, or clinically relevant in vivo resistance transfer. Therefore, they should be interpreted with caution in molecular AMR surveillance and detection contexts. Full article

Staphylococcus aureus nasal carriage contributes to asymptomatic transmission in both community and healthcare settings. This study aimed to characterize S. aureus strains isolated from students of the Pomeranian Medical University in Szczecin, Poland, using phenotypic and genotypic methods. A total of 175 S. aureus strains were isolated from the nasal vestibules of 800 students between 2014 and 2015. Species identification and antimicrobial susceptibility testing were performed using standard microbiological methods, while virulence-associated genes and agr groups were analyzed using Single-PCR and Multiplex-PCR assays. Genotypic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The prevalence of S. aureus nasal carriage among students was 21.9% and did not differ according to faculty or year of study. Most isolates (84.0%) were susceptible to all tested antibiotics, and no methicillin-resistant S. aureus (MRSA) strains were detected. All strains carried the hla gene, whereas hld and hlg were identified in 93.7% and 93.1% of isolates, respectively. In addition, the tst gene was detected in 22.3% of strains, while the lukS-PV/lukF-PV genes were identified in only one isolate (0.6%). The most prevalent enterotoxin genes were sep (17.1%) and sea (13.7%), whereas genes of the egc cluster, including seg, sei, and seo, were detected in 53.7% of isolates. Significant associations were observed between specific egc gene combinations and superantigen gene profiles, including increased frequencies of sec, sel, and tst genes (p < 0.001). The predominant agr type was agr-1 (49.7%), followed by agr-3 (28.6%) and agr-2 (20.0%). Strains carrying agr-1 more frequently harbored the g i m n o cluster as well as the sec, sel, and sep genes, whereas agr-3-positive isolates were significantly associated with the g i m o u and g i o u clusters and with the presence of tst, sea, and seh genes (p < 0.05). PFGE analysis demonstrated substantial genetic heterogeneity among the isolates, with no evidence of a predominant clonal lineage. These findings indicate a heterogeneous, non-epidemic population structure of S. aureus strains circulating among university students and highlight the considerable diversity and interrelationships of virulence-associated genetic profiles within this population. Full article

Background: Klebsiella pneumoniae is a globally relevant pathogen whose growing association between hypervirulence and antimicrobial resistance represents a major public health challenge. Methods: A systematic review was performed following the PRISMA 2020 guidelines. Studies published between 2005 and 2025 were searched in Google Scholar, Scopus, PubMed and Science Direct that reported the molecular detection of virulence genes in clinical isolates. Results: A total of 676 studies were included, in which 475 virulence genes were reported. A progressive increase in their detection was observed, Hypervirulent strains were associated with a higher proportion of genes associated with capsule and hypermucoviscosity, while classical strains were associated with a higher representation of adhesion and biofilm genes. Conclusions: The virulence of K. pneumoniae is organized into functional modules dominated by iron acquisition and capsular regulation. These findings support the prioritization of key determinants for molecular surveillance and the study of the global distribution and temporal trends of this pathogen. Full article

Autophagy, also referred to as the “self-eating machinery”, is a crucial process where organisms maintain intracellular homeostasis through recycling or degrading non-essential and damaged cellular components. It is important in numerous biological functions such as cellular differentiation, aging, nutrient sensing, stress response, tissue homeostasis, immunity, and programmed cell death. Autophagy induction occurs with the formation of a double-layered membrane structure called “autophagosome”. The autophagosome wraps damaged organelles or proteins and transports them to the vacuole or lysosome for degradation. Autophagy is beneficial to organisms, and it should be optimally regulated because elevated or decreased levels are detrimental for survival. To date, more than 40 autophagy-related genes (ATGs) have been identified in the budding yeast Saccharomyces cerevisiae, with most having homologs in fungi and higher eukaryotes. Majority of the ATGs in industrial and pathogenic fungal species have been characterized and known to play vital roles in growth, development, and virulence. In this review we provide a comprehensive overview of ATGs in various fungal species and highlight how autophagy is regulated and controls various functions in plant, human, and industrial fungal species. Full article

The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) threatens the efficacy of this last-line antibiotic. The GraSR two-component system is frequently mutated in VISA strains. Here, we demonstrate that the GraS(T136I) point mutation, identified in the clinical VISA isolate XN108, is a key determinant of reduced vancomycin susceptibility. Introducing this mutation into the susceptible strain Newman increased the vancomycin MIC from 1.5 to 4 mg/L, while its reversion in XN108 decreased the MIC from 12 to 8 mg/L. The mutation conferred common phenotypes, including thickened cell wall, decreased autolysis, and reduced cell surface negative charge via upregulation of the dltABCD operon and mprF. Notably, the GraS(T136I) mutation also upregulated virulence genes (efb, hlb, sbi, hld) and enhanced hemolytic activity. Interestingly, despite this hypervirulent profile, the mutant showed impaired long-term survival within macrophages. Our study reveals that a single GraSR mutation can co-regulate vancomycin resistance and virulence, offering new insights into the adaptation of S. aureus to antibiotic pressure. Full article

Background/Objectives: The co-production of New Delhi metallo-β-lactamases (NDM) and OXA-48-like carbapenemases in Klebsiella pneumoniae represents a major therapeutic challenge due to extensive drug resistance and limited treatment options. This study aimed to investigate the molecular epidemiology, resistance profiles, and mechanisms associated with reduced susceptibility to cefiderocol in clinical isolates co-producing NDM and OXA-48-like carbapenemases. Methods: A total of 45 clinical K. pneumoniae isolates collected in healthcare settings in Northern Italy were analyzed. Antimicrobial susceptibility testing, including cefiderocol and aztreonam/avibactam, was performed according to EUCAST guidelines. Whole-genome sequencing was used to characterize sequence types, resistance determinants, virulence factors, plasmid replicons, and phylogenetic relationships. Mutations in iron uptake and transport genes were investigated in cefiderocol-resistant isolates. Results: Most isolates belonged to the high-risk clone ST147 (44/45) and were grouped into three main phylogenetic clusters. The isolates exhibited extensive multidrug resistance, with universal susceptibility only for aztreonam/avibactam. Cefiderocol resistance was observed in 42.2% of isolates and was unevenly distributed across the phylogeny. Mutations in iron uptake genes, particularly cirA and chrA, were identified in the majority of resistant isolates, although several strains retained wild-type sequences, indicating heterogeneous resistance mechanisms. Comparative phylogenetic analysis demonstrated close relatedness to international isolates, suggesting the global dissemination of related lineages. Conclusions: NDM- and OXA-48-like carbapenemase co-producing K. pneumoniae isolates are characterized by clonal dissemination, complex resistance profiles, and emerging cefiderocol resistance driven by multifactorial mechanisms. The preserved activity of aztreonam/avibactam highlights its potential as a key therapeutic option against these high-risk pathogens. Full article

Background/Objectives: Escherichia coli (E. coli) spans commensal, intestinal pathogenic, and extraintestinal pathogenic lineages distributed across human, animal, and environmental reservoirs, yet the extent to which virulence architectures are shared across these compartments remains incompletely understood. Using a One Health framework, we profiled putative virulence determinants in pooled multidrug-resistant (MDR) E. coli source groups representing human, animal, and environmental sectors. Methods: Virulence genes were predicted with VirulenceFinder, and presence–absence profiles were integrated to define functional composition, sector overlap, source-group distribution breadth, and pathotype-associated signatures. Predicted pathogenic potential was assessed with PathogenFinder and compared with pathogenic family richness. Results: Overall, 114 putative virulence genes were detected, with adhesion/colonization functions dominating the virulome (33/114), followed by toxin-associated genes (12/114). A conserved core of 50 virulence genes was shared across all three sectors, including determinants linked to serum resistance (iss, ompT, traT), adhesion (csgA, fimH), stress adaptation (terC), and iron acquisition (sitA, iutA, fyuA). ExPEC-associated determinants were most numerous in environmental source groups (n = 52), whereas diarrheagenic E. coli markers were most frequent in animal-associated groups (n = 42). LEE-associated effectors were infrequent and largely absent from human source groups. Despite ecological differences in virulence composition, pathogenicity scores remained consistently high across sectors (0.83–0.92) and showed no significant association with pathogenic family richness (Spearman’s ρ = 0.197, p = 0.392). Conclusions: Within the limits of pooled source-group analysis, these findings suggest that MDR E. coli across One Health compartments shares a broadly distributed, ExPEC-associated virulence repertoire overlaid with sector-specific pathotype signals, underscoring the value of integrated genomic surveillance while highlighting the need for isolate-resolved analysis. Full article

Toxigenic Klebsiella oxytoca strains linked to antibiotic-associated hemorrhagic colitis produce the cytotoxins tilimycin and tilivalline, which contribute to intestinal epithelial damage during infection. Tilimycin and tilivalline are synthesized by enzymes encoded within the nonribosomal peptide synthetase (NRPS) operon, yet the regulatory mechanisms controlling operon expression remain poorly understood. SdiA, an orphan LuxR-type quorum-sensing regulator, detects exogenous N-acyl homoserine lactones (AHLs) produced by neighboring bacterial species and modulates gene expression in response to interspecies communication. Although SdiA has been implicated in virulence regulation in several enteric pathogens, its role in K. oxytoca remains unclear. This study demonstrates that SdiA positively regulates npsA, the first gene in the NRPS operon, and that this regulatory effect is enhanced in the presence of exogenous AHL. Electrophoretic mobility shift assays indicate that SdiA directly binds to the upstream regulatory region of npsA, supporting a direct interaction consistent with positive transcriptional regulation. Furthermore, deletion of sdiA significantly reduces cytotoxicity toward HeLa cells under the conditions tested. Collectively, these findings identify SdiA as a quorum-sensing-responsive activator of npsA expression and support its role in modulating cytotoxicity in toxigenic K. oxytoca strains. These results provide new insight into the influence of interspecies quorum-sensing signals on virulence-associated regulatory pathways in K. oxytoca. Full article

Alternaria blight, caused by fungi of the genus Alternaria, is one of the most common and damaging diseases affecting tomatoes, leading to significant yield losses. The intensive use of chemical fungicides faces the problems of pathogen resistance development and negative environmental impacts. This study investigated the possibility of using RNA interference technology based on exogenous double-stranded RNAs (dsRNAs) to protect tomatoes against the causal agent of early blight (EB), Alternaria alternata. Key genes of the pathogen A. alternata were selected as targets: Alt-a1 (a major allergen and virulence factor), TEF1a (translation elongation factor 1-alpha) and β-Tub (β-tubulin). Specific dsRNAs were synthesized in vitro and applied to tomato plants (Solanum lycopersicum L. cv. Micro-Tom) simultaneously with inoculation of A. alternata strain C7.24-T2-L-F1 spores. Visual assessment, measurement of chlorophyll A and B, and real-time quantitative PCR analysis showed that treatment with dsRNAs targeting the Alt-a1, TEF1a and β-Tub genes significantly suppressed infection development, reducing the amount of pathogen DNA in plant tissues by 7 to 27 times depending on the dsRNA type. The most effective was dsRNA to the Alt-a1 gene. Thus, the obtained results demonstrate the promise of spray-induced gene silencing (SIGS) as a strategy for protecting tomato plants against the pathogen A. alternata. Full article

Recently, a strain of Streptococcus agalactiae serotype Ia sequence type 7 clonal complex 1 (SaIa ST7 CC1) has emerged in Latin American tilapia aquaculture as an international threat. This study evaluated outbreaks of acute streptococcosis occurring between 2021 and 2025 on commercial Nile tilapia (Oreochromis niloticus) farms in six Latin American countries, aiming to integrate molecular, clinical, pathological, and environmental data. In total, 360 moribund or recently dead fish at various production stages (larvae/fry, pre-grow-out, and grow-out) were examined, and 25 S. agalactiae isolates were serotyped and subjected to real-time PCR analysis, multilocus sequence typing (MLST), virulence and antimicrobial resistance gene profiling, and antimicrobial susceptibility testing. All isolates belonged to SaIa and shared the same ST7 CC1 MLST profile, forming a highly homogeneous cluster with reference SaIa ST7 CC1 strains previously isolated from tilapia farms in Asia. These results are consistent with the regional spread of a single clonal line. At the larval and fry stages, SaIa ST7 CC1 was associated with hyperacute septicemia, gastrointestinal hemorrhage, and frequent intestinal intussusception, whereas in pre-grow-out and grow-out fish, neurological signs were more prominent, followed by ocular signs, systemic hemorrhages, and coelomic lesions. Histopathological examination showed profuse colonization of the brain, spleen, liver, and intestine by Gram-positive cocci, accompanied by marked acute circulatory and inflammatory lesions and few chronic granulomatous responses, consistent with a rapidly progressing, highly aggressive infectious process. All outbreaks occurred during extended periods of warm water (>32 °C), with large day–night thermal gradients and reduced dissolved oxygen, suggesting that thermal stress may exacerbate disease expression in affected systems. All SaIa ST7 CC1 strains exhibited phenotypic susceptibility to florfenicol and amoxicillin, whereas 84% (21/25) and 100% (25/25) exhibited intermediate susceptibility to oxytetracycline and enrofloxacin, respectively. In total, 5 of the 21 isolates (23.8%) with intermediate susceptibility to oxytetracycline carried tetracycline resistance genes (tetM, tetO). These findings identify SaIa ST7 CC1 as a clinically significant emerging threat associated with thermally facilitated and geographically expanding streptococcosis in tilapia production in Latin America. Immediate priorities include screening imported broodstock using MLST or whole-genome sequencing (WGS), harmonized regional molecular surveillance, climate-adaptive farm management practices, prudent antimicrobial use, and serotype-matched vaccination and breeding strategies that improve both disease and heat resilience. Full article

Candida albicans is the causal agent of invasive candidiasis, which might be lethal in immunocompromised patients. Biofilm formation is considered a key virulence factor of C. albicans and is associated with its elevated resistance to antifungals. C. albicans and bacteria like E. coli are frequently found to form mixed biofilms on biotic or abiotic surfaces, rendering them more refractory to existing antifungals. To investigate how E. coli endogenous indole interplaying with exogenous IAA exerts modulatory effects on dual-species biofilm with C. albicans, an E. coli strain deficient in the indole biosynthetic gene tnaA was constructed, and the enzyme TnaA inhibitor was administered to block the indole production in E. coli monoculture and/or E. coli–C. albicans dual culture. Phenotypic assay revealed that indole deficiency attenuated E. coli mono-species biofilm by 12% (tnaA∆ versus WT E. coli), and the lack of indole in the E. coli cell-free culture filtrate abolished the ability to promote C. albicans biofilms, evidenced by the fact that the treatment with WT E. coli culture supernatants exhibited a 1.7-fold promotive effect, while treatment with tnaA∆ displayed no significant difference from the broth control towards C. albicans biofilms. Furthermore, impaired E. coli indole production might disrupt E. coli–C. albicans biofilm, as examined by confocal laser scanning microscopy (CLSM). Moreover, indole-3-acetic acid (IAA) was found to exhibit more potent biofilm-modulatory activity than indole by CLSM imaging with dual biofilms of WT E. coli–C. albicans, in contrast to those of E. coli tnaA∆–C. albicans post-supplemented with exogenous IAA. This study provides evidence for indole as a signaling molecule mediating bacterial–fungal communication during mixed-biofilm formation. Indole and its derivatives, particularly in combination with existing antifungals, have potential in the development of anti-biofilm strategies to eradicate refractory fungal infections. Full article

Hospital wastewater treatment efficacy is conventionally assessed by total antibiotic resistance gene (ARG) abundance; however, whether this metric accurately reflects biosafety risk remains poorly defined. Using a one-year longitudinal metagenomic survey (bimonthly sampling; n = 18 per group), we simultaneously profiled the resistome, virulome, and mobilome of hospital wastewater influent and effluent; stratified functional gene abundances by genomic origin; quantified ARG–mobile genetic element (MGE) colocalization; and characterized multicategory gene cocarriage across the 15 most abundant pathogenic species. Although the abundance of total strict ARGs decreased significantly in the effluent (p = 0.038), the abundances of metal resistance genes and virulence factors increased concurrently (both p < 0.01), and 8 of the 20 ARG subtypes were enriched rather than removed. This decline was driven exclusively by a reduction in the number of plasmid-encoded ARGs (p < 0.001), whereas genes encoding chromosomal virulence factors, metal resistance genes, biocide resistance genes, and MGEs were significantly enriched in the effluent (all p < 0.05). The normalized ARG–MGE colocalization rate was significantly greater in the effluent (p = 0.028), with a concurrent shift toward transposase-mediated chromosomal mobilization. Pathogen-associated metagenomic assemblies of clinically relevant species exhibited synchronous multicategory resistance coenrichment in the effluent, which is consistent with coselection under antibiotic, biocide, and metal pressures. Total ARG abundance is fundamentally decoupled from biosafety risk in treated hospital wastewater, warranting integrated surveillance beyond ARG-centric metrics. Full article

Extended-spectrum β-lactamase (ESBL)-producing bacteria are a growing public health concern within the One Health framework. This study aimed to characterize ESBL-producing Enterobacterales in industrial and artisanal organic fertilizers marketed in southwestern Colombia. Five commercial fertilizer brands were analyzed using a selective culture on ceftriaxone supplemented media (4 µg/mL), antimicrobial susceptibility testing by broth microdilution to determine minimum inhibitory concentrations (MICs), phenotypic synergy testing for ESBL confirmation, and polymerase chain reaction (PCR) to detect bla_TEM, bla_SHV, and bla_CTX-M genes. Overall, 18.6% of the samples showed growth of ceftriaxone-resistant Enterobacterales, predominantly Escherichia coli and Klebsiella pneumoniae. ESBL producers accounted for 84% of the isolates, all of which carried at least one bla gene, predominantly bla_CTX-M. Statistically significant differences in bacterial growth frequency were observed among fertilizer types, with higher positivity rates observed in manure-based artisanal formulations (p < 0.05). Whole-genome sequencing of selected isolates identified Klebsiella pneumoniae ST37 and Escherichia coli ST224, both harboring bla_CTX-M-55 and additional resistance and virulence determinants. These findings demonstrate that organic fertilizers, particularly manure-derived products, may act as reservoirs and potential dissemination routes for clinically relevant antimicrobial-resistant bacteria. This is the first study in Colombia documenting the presence of ESBL-producing bacteria in organic fertilizers. These results underscore the need to incorporate surveillance of these products into national policies under a One Health perspective. Full article