Biofilm formation in enterococci: genotype-phenotype correlations and inhibition by vancomycin

Abstract

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.

Figure 1

ERIC-PCR analysis of the isolates. An nweighted pair group method using arithmetic overage algorithm (UPGMA) dendrogram in a radial format representing the ERIC-PCR pattern relatedness of all tested isolates. Isolate names are color coded according to their biofilm strength (Purple, strong; Red, medium; Green, weak; Black, non-biofilm-forming).

Figure 2

Comparative genomics screen for the 17 biofilm-associated protein-coding genes. Comparative genomics in a tabular format, representing patterns of gene presence and absence, analyzed by blastX against the 17 biofilm-related proteins and confirmed by blastN against primer pairs, in five representative E. faecalis strains. +: orthologous gene present; ±: paralogous gene present; −: gene absent; X: pseudogene. For Agg, more than one paralogous gene have been identified in some genomes. We differentiate the chromosomal and plasmid (p) copy of these genes.

Figure 3

Results of PCR analysis of the distribution of each of the genes involved in biofilm formation.

Figure 4

UPGMA analysis to cluster the isolates according to the pattern of their PCR results. A UPGMA cladogram representing the clustering relatedness of 31 isolates according to the pattern of their PCR results for biofilm-associated genes. Isolate names are color coded according to their biofilm strength (Purple, strong; Red, medium; Green, weak; Black, non-biofilm-forming).

Figure 5

Sensitivity of Enterococcus isolates to different antibiotics as analyzed by MIC. Tyg: tigecycline; Van: vancomycin; Cip: ciprofloxacin; Gen: gentamycin; Azi: azithromycin.

Figure 6

Effect of sub-MIC of some antibiotics on biofilm formation in a representative strong biofilm-forming isolate (E01).