Enterococci are nosocomial pathogens that can form biofilms, which contribute to their virulence and antibiotic resistance. Although many genes involved in biofilm formation have been defined, their distribution among enterococci has not been comprehensively studied on a genome scale, and their diagnostic ability to predict biofilm phenotypes is not fully established. Here, we assessed the biofilm-forming ability of 90 enterococcal clinical isolates. Major patterns of virulence gene distribution in enterococcal genomes were identified, and the differentiating virulence genes were screened by polymerase chain reaction (PCR) in 31 of the clinical isolates. We found that detection of gelE in Enterococcus faecalis is not sufficient to predict gelatinase activity unless fsrAB, or fsrB alone, is PCR-positive (P = 0.0026 and 0.0012, respectively). We also found that agg is significantly enriched in isolates with medium and strong biofilm formation ability (P = 0.0026). Additionally, vancomycin, applied at sub minimal inhibitory concentrations, inhibited biofilm in four out of five strong biofilm-forming isolates. In conclusion, we suggest using agg and fsrB genes, together with the previously established gelE, for better prediction of biofilm strength and gelatinase activity, respectively. Future studies should explore the mechanism of biofilm inhibition by vancomycin and its possible use for antivirulence therapy.

Background and Objective: Enterococcus faecalis can cause different nosocomial infections, especially urinary tract infection (UTI). Pathogenicity of E. faecalis is driven by various virulence factors; however, no specific genetic pattern is restricted to a particular type of infection. The current study aimed to investigate the correlation between different virulence factors in E. faecalis clinical isolates causing UTIs. Methods: We phenotypically analyzed 60 urinary isolates, identified as E. faecalis, for biofilm formation, gelatinase, protease and hemolytic activities by Crystal Violet assay, gelatin hydrolysis, casein hydrolysis and blood agar hemolysis assays, respectively. Additionally, we detected different genes associated with species identification, virulence phenotypes, adherence and quorum sensing by the polymerase chain reaction (PCR). The detected genes included D-alanine-D-alanine ligase (ddl), cytolysin (cyl), gelatinase (gelE), serine protease (sprE), faecal streptococci regulator locus genes (fsrA, fsrB, fsrC), pili (pil), adhesin to collagen of E. faecalis (ace) and aggregation substance (agg). Results: All isolates formed biofilms, mostly with strong to moderate ability. Although gelE was detected in 87% of the isolates, only 22% of the isolates had gelatinase activity. Similar phenotype–genotype incongruities were observed with hemolysis and casein hydrolysis activities, as the isolates that expressed these two phenotypes were fewer than those carrying the genes encoding them. Conclusion: A clear variability in virulence gene distribution among the isolates was observed, and no particular pattern was associated with UTI. Whereas all isolates carried at least ace and pil, whose products are involved in adherence, which is a virulence phenotype that is required for urinary colonization, six isolates carried the entire set of investigated genes. Statistical analysis of the results suggests cyl as a biomarker for hemolytic activity, fsrB as a diagnostic biomarker for the gelatinase activity, and gelE-sprE as predictors for biofilm formation strength in E. faecalis.

Background: Enterococci, known for their disturbing involvement in nosocomial infections, possess a diverse set of virulence factors, regulated by multiple genes. A key virulence regulator is the fecal Streptococcus regulator (Fsr) quorum sensing system. Multiple reports describe the involvement of fsr genes in several virulence mechanisms, notably gelatinase production and biofilm formation; however, the presence of fsr genes does not necessarily predict those virulence phenotypes. This study investigates the factors affecting the relation between molecular detection of fsr genes and accurate prediction of gelatinase activity and biofilm formation intensity. Methods: One hundred enterococcal samples were collected from patients suffering from urinary tract infections. The isolates were identified through the use of a polymerase chain reaction (PCR) technique targeting the ddl gene. Biofilm formation was quantified by the crystal violet assay, while gelatinase activity was evaluated on gelatin agar plates. PCR was used to detect the fsrA and fsrB genes, as well as the gelatinase enzyme-encoding gene (gelE). Results: Out of the collected 100 isolates, 93% were identified as Enterococcus faecalis. The isolates formed biofilm with different intensities: 47% were strong biofilm producers, 28% moderate, and 21% weak, while only four isolates (4%) did not form biofilm. Only 14% of all isolates had detectable gelatinase activity. The fsrA and fsrB genes were detected in 26% and 28% of the tested isolates, respectively, while gelE was detected in 57% of the isolates. Whereas no association was found between biofilm formation intensity and fsr locus genes or gelatinase activity, a strong positive correlation (r = 1) was found between the detection of both fsrA and fsrB genes and the gelatinase activity. Conclusion: fsrA and fsrB have a diagnostic value and may be used as biomarkers for gelatinase activity in E. faecalis.

Many gut bacteria degrade polysaccharides, providing nutritional advantages to their hosts. Fucose, a mucin degradation product, was suggested as a communication molecule between the resident microbiota and external pathogens. However, the precise role and variants of the fucose utilization pathway remain to be elucidated. Here, we computationally and experimentally investigated the fucose utilization operon of E. coli. While the operon is conserved among E. coli genomes, a variant pathway, in which an ABC transporter system replaces the fucose permease gene (fucP), was computationally identified in 50 out of 1058 genomes. Comparative genomics and subsystems analysis results were confirmed by polymerase chain reaction-based screening of 40 human E. coli isolates, which indicated the conservation of fucP in 92.5% of the isolates (vs. 7.5% of its suggested alternative, yjfF). The in silico predictions were confirmed by in vitro experiments comparing the growth of E. coli strains K12, BL21, and isogenic fucose-utilization K12 mutants. Additionally, fucP and fucI transcripts were quantified in E. coli K12 and BL21, after in silico analysis of their expression in 483 public transcriptomes. In conclusion, E. coli utilizes fucose by two pathway variants, with measurable transcriptional differences. Future studies will explore this variation’s impact on signaling and virulence.

The textile industry is a significant player in the global economy; however, it is known for its contribution to environmental pollution by discharging untreated wastewater containing synthetic dyes. Crystal violet is a commonly used dye in the textile industry but has a harmful effect on humans and the environment. Therefore, developing a sustainable, environmentally friendly, and cheap method for crystal violet biodegradation like microbial biodegradation is crucial. To isolate bacteria degrading crystal violet, textile effluent wastewater was collected from different dying plants in Egypt. The degradation capacity of the bacteria isolated from textile effluent was measured using a spectrophotometer. The two bacterial isolates showing maximum crystal violet degradation FSL-1 and EBA-2, were identified using 16S rRNA as Klebsiella pneumoniae and Pseudomonas oleovorans, respectively. The effect of different parameters, including incubation time, initial dye concentration, pH, temperature, carbon source, agitation, and media composition, was studied on the ability of the two isolates to degrade crystal violet. The highest degradation of 92% was observed with K. pneumoniae isolate when inoculated in MSM-Y media containing 0.01 g/L crystal violet when incubated at 37 °C for 48 h under agitated conditions and pH 7; however, complete crystal violet degradation was observed when MSM-Y was substituted with brain heart infusion broth under the same conditions. Therefore, the newly identified bacterial strains in this study can be effectively utilized for the treatment of textile effluent containing crystal violet when certain parameters are optimized.

Enterococci are Gram-positive opportunistic pathogens responsible for a wide range of nosocomial infections. One enterococcocal species, Enterococcus faecium , is steadily increasing in prevalence and has been listed among major multidrug-resistant ESKAPE pathogens. To gain systems-level insights into its metabolism and support discovery of potential therapeutic targets, we constructed iDR479, a comprehensive manually curated genome-scale metabolic model (GEM) to serve as a digital twin for E. faecium TX0016 (strain DO). The reconstruction was curated through extensive homology searches and literature evidence, and further refined and gap-filled through experimental validation. Phenotypic profiling using Biolog microarrays enabled assessment of carbon source utilization, while amino acid leave-out growth assays allowed the evaluation of auxotrophies. The final refined model is 100% accurate in predicting amino acid auxotrophy and 85% accurate in predicting growth on sole carbon sources. Discrepancies between model predictions and experimental phenotypes identified specific knowledge gaps across metabolic pathways, including unresolved carbon source utilization phenotypes, e.g., psicose, sorbitol, and palatinose utilization. Those gaps will guide future experimental characterization. Additionally, gene essentiality analysis was conducted to evaluate the predictive capacity of iDR479 model. Since no experimental gene essentiality data are currently available for E. faecium , model predictions were compared against Tn-seq experimental results from E. faecalis MMH594. Under simulated rich medium conditions, iDR479 achieved 86.7% concordance with the experimental essentiality results of E. faecalis MMH594. iDR479 thus provides a framework for studying E. faecium , offers insights into its metabolic network, and serves as a source for guiding future research and identification of therapeutic targets.