Comparison of <i>Enterococcus faecalis</i> biofilm formation using a polystyrene dish assay and an ex vivo heart valve assay.

<p>A: The laboratory strain OG1RF and clinical isolates with high-level biofilm formation, as indicated by a biofilm index >1.0 in the standard polystyrene dish assay (strains 12, 1172, 1234, 1278, and 1288), were compared to OG1RF <i>ΔahrC</i> (a mutant with attenuated biofilm formation) and clinical isolates with low-level or no biofilm formation, as indicated by a biofilm index <0.1 (isolates 99w, 103, 1037, 1150, and 1211). Isolates were compared using the polystyrene dish assay with tryptic soy broth (TSB) (0.25% dextrose) and TSB without dextrose, and using an ex vivo porcine heart valve explant assay (with medium that contains 0.1% dextrose). Polystyrene dish assays were incubated for 24 hours; heart valve assays were incubated for 4 hours. *indicates p<0.01 for less biofilm formation compared to OG1RF (for the same conditions). Note that in the heart valve assay the only strain with a significant reduction in biofilm formation compared to OG1RF is OG1RF <i>ΔahrC.</i> B: For a selection of strains representing a variety of phenotypes, biofilm formation relative to OG1RF was similar whether TSB without dextrose or endothelial cell basal medium (the medium used for the heart valve assay) was used. Error bars represent standard deviation. r² = 0.98, p = 0.018.</p

Histogram of biofilm forming ability of clinical <i>Enterococcus faecalis</i> isolates.

<p>158 clinical <i>E. faecalis</i> isolates, including isolates from the clinical microbiology laboratory and fecal samples from healthy volunteers, were tested in the 96-well polystyrene dish biofilm assay using TSB without glucose and 24-hour incubation. To control for strain variation in planktonic growth, results are shown as each isolate’s biofilm index (Methods).</p

Characteristics of clinical <i>Enterococcus faecalis</i> study isolates.

a<p><i>E. faecalis</i> isolates were collected through the Minneapolis Veteran’s Affairs Medical Center from 1994–1999.</p>b<p>Aggregation substance gene.</p>c<p>Enterococcal surface protein gene.</p><p>Characteristics of clinical <i>Enterococcus faecalis</i> study isolates.</p

Comparison of safranin staining and quantitative colony-forming unit (CFU) counts using a polystyrene dish assay.

<p>Clinical <i>Enterococcus faecalis</i> isolates were examined for biofilm forming ability by staining for adherent biofilm biomass in a 96-well polystyrene dish biofilm assay using TSB without glucose after a 24-hour incubation. The microtiter wells were then scraped to measure CFUs present in the biofilms using quantitative culture. Error bars represent standard deviation. r² = 0.95, p<0.001.</p

Expression of <i>esp</i> in tryptic soy broth (TSB) with and without 0.75% dextrose as assessed by enzyme linked immunosorbent assay (ELISA).

<p><i>Enterococcus faecalis</i> isolates that were <i>esp</i>-negative (OG1RF and 108) or <i>esp</i> positive isolates (12, 90, 1288, and 1211) were assessed by ELISA for <i>esp</i> expression after growth in TSB without dextrose and TSB supplemented with dextrose (0.75% dextrose). The ELISA was repeated in duplicate with reproducible results. Some background signal, as seen in OG1RF and 108, is expected due to non-specific binding. Error bars represent standard deviation. *indicates p<0.05 for comparison of <i>esp</i> expression with and without dextrose.</p

Biofilm index of clinical <i>Enterococcus faecalis</i> isolates grown in tryptic soy broth (TSB) with and without dextrose.

<p>Six isolates that showed high-level biofilm formation (biofilm index >1) (OG1RF, 9, 1464, 1138, 263, 12, and 1288), one with moderate biofilm formation (biofilm index 0.4) (180), and five that showed low-level biofilm formation (biofilm index <0.1) (OG1RF <i>ΔahrC</i>, 1232, 90, 1130, 1028, and 1211) in the standard assay (TSB without dextrose) were evaluated using TSB supplemented with dextrose (0.75% dextrose). Error bars represent standard deviation. *indicates p<0.01 for comparison of biofilm formation with and without dextrose.</p

Modified Lactic Acid Bacteria Detect and Inhibit Multiresistant Enterococci

We designed <i>Lactococcus lactis</i> to detect <i>Enterococcus faecalis</i>. Upon detection, <i>L. lactis</i> produce and secrete antienterococcal peptides. The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of <i>L. lactis</i>. The enterococcal sex pheromone cCF10 serves as the signal for detection. Expression vectors derived from pCF10, a cCF10-responsive <i>E. faecalis</i> sex-pheromone conjugative plasmid, were engineered in <i>L. lactis</i> for the detection system. Recombinant host strains were engineered to express genes for three bacteriocins, enterocin A, hiracin JM79 and enterocin P, each with potent antimicrobial activity against <i>E. faecalis.</i> Sensitive detection and specific inhibition occur both in agar and liquid media. The engineered <i>L. lactis</i> also inhibited growth of multidrug-resistant <i>E. faecium</i> strains, when induced by cCF10. The presented vectors and strains can be components of a toolbox for the development of alternative antibiotic technologies targeting enterococci at the site of infection

Active immunization against AS⁺<i>E. faecalis</i> worsens infectious endocarditis after challenge with AS⁺ enterococci.

<p>^a−Determined with use of Fishers Exact Test.</p><p>^b−1+ vegetations were small in size and number (approximately 10 mg); 2+ vegetations were moderate in size and number (approximately 50 mg); and 3+ vegetations were large in size and numbers, often obstructing the aorta (>75 mg).</p

IgG Fabs against aggregation substance reduce the severity of enterococcal endocarditis after challenge with AS⁺<i>E. faecalis</i>.

<p>IgG Fabs against aggregation substance reduce the severity of enterococcal endocarditis after challenge with AS⁺<i>E. faecalis</i>.</p

Functional categories of genes found by microarray to be differentially expressed in <i>E. faecalis</i> OG1RF <i>in</i><i>vivo</i> during subdermal abscess infection.

<p>The numbers of up- (right) and down-regulated (left) genes at two (top) or eight (bottom) hours post-inoculation are indicated on the horizontal axis.</p