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.

Biofilm-formed enterococcal urinary tract clinical isolates (n = 92) were used for studying the antibiofilm activity of cinnamon, ginger, and chemical AgNPs. The average particle sizes of cinnamon, ginger, and chemical AgNPs were 8.7, 41.98, and 55.7 nm, respectively. The results of Fourier transform infrared analysis revealed that phytocompounds, such as cinnamaldehyde and gingerol, were the main compounds incorporated in the synthesis of cinnamon and ginger AgNPs, respectively. The purity and crystalline nature of the AgNPs have been confirmed by energy dispersive X-ray and X-ray Diffraction analysis. The results of antimicrobial activity showed that MIC of ginger, cinnamon, and chemical AgNPs were 37.64, 725.7, and 61.08 μg/ml, respectively. On studying the antibiofilm activity of AgNPs at sub-MIC values (1/2, 1/4, and 1/8 MIC), the results revealed that it was concentration dependent. Therefore, further studies were carried out to evaluate the antibiofilm activity of AgNPs at a concentration of 18 μg/ml. The results showed that ginger and chemical AgNPs reduced the formed biofilm to 39.14% and 65.32% and the number of adherent cells on the urinary catheter surface to 42.73% and 69.84%, respectively, as compared to that of the control, while cinnamon AgNPs showed no significant activity. Accordingly, ginger AgNPs had the most potent antibacterial and antiadherent activity against biofilm-associated enterococcal isolates.

Among the enterococci, environmental factors may interfere in the expression of virulence factors. Horizontal gene transfer plays an important role in the spread of resistance and virulence genes.

Enterococci are a common cause of urinary tract infections. The severity of enterococcal infections is associated with their ability to form biofilms. Morus leaves are known as a natural antibacterial, however, their antibiofilm activity against Enterococcus remains unveiled. This study aimed to evaluate the ability of four polyphenol-rich Morus leaves extracts (Morus nigra, M. rubra, M. macroura, and M. alba) to inhibit biofilm formed by enterococcal clinical isolates in relation to their metabolic profiling. Results revealed that 48% of the isolates formed strong biofilm, 28% formed moderate biofilm, 20% formed weak biofilm, and only 4% did not form a biofilm. The strong biofilm-forming isolates were E. faecalis, and hence were chosen for this study. The antibiofilm activity of the four polyphenol-rich Morus leaves extracts revealed that the M. nigra extract exhibited the highest percentage of biofilm inhibition followed by M. rubra then M. macroura and the least inhibition was detected in M. alba, and these results were in accordance with the phenolic and flavonoid contents of each extract. UPLC-ESI-MS/MS identified 61 polyphenolic compounds in the four extracts. Further, multivariate analysis confirmed clear segregation of M. nigra from the other species suggesting disparity in its metabolome, with accumulation of flavonoids, anthocyanidins, phenolic acids and coumarin derivatives. Quercetin and kaempferol glycosides were found to be positively and significantly correlated to the antibiofilm activity. In conclusion, M. nigra ethanolic extracts showed the highest phenolic content and antibiofilm activity and they could be developed as a complementary treatment for the development of antimicrobial agents.