Effect of Dalbavancin on Staphylococcal Biofilms When Administered Alone or in Combination With Biofilm-Detaching Compounds

Abstract

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N-acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose-response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E-test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption.

Keywords: E-test; N-acetylcysteine; Staphylococcus; biofilm; dalbavancin; ficin; xCELLigence.

FIGURE 1

Dalbavancin effect on bacterial biofilm formation in Staphylococcus aureus 240 (A), S. aureus MRSA 4 (B), and Staphylococcus epidermidis 43040 (C) strains. The cell index (CI) values are measured by impedance in an xCELLigence equipment and correlate with total biofilm mass. Red asterisks indicate dalbavancin-free controls. Dalbavancin was added from the beginning of the experiment at concentrations from 0.0625 to 32 mg/L as indicated in the legend. Data are the means of two replicates.

FIGURE 2

Concentration-dependent effect of linezolid, vancomycin, cloxacillin, rifampicin, and dalbavancin on biofilms in Staphylococcus aureus 240, S. aureus MRSA4, and Staphylococcus epidermidis 43040 strains. (A–C) The antibiotics were added at the beginning of the experiment together with the bacterial inoculum. (D–F) The antibiotics were added when biofilms were already formed, at their exponential growth phase. All charts indicate biofilm formation at 20 h of growth expressed as the percentage of cell index (CI) compared with the control without antibiotic. Values below 100% indicate biofilm inhibition, whereas values over 100% indicate biofilm induction, in comparison with the biofilm mass achieved in the absence of each antibiotic.

FIGURE 3

Dalbavancin effect on established staphylococcal biofilms in Staphylococcus aureus 240 (A), S. aureus MRSA4 (B), and Staphylococcus epidermidis 43040 (C) strains. Dalbavancin was added at the exponential growth phase of the biofilm (marked by black arrows) at 6 h (S. aureus 240), 7 h (S. aureus MRSA4), or 9 h (S. epidermidis 43040) at the concentrations shown in the legend. Black asterisks indicate the antibiotic-free cell control. Data are the means of two replicates.

FIGURE 4

Effect on the biofilm growth of Staphylococcus aureus 240 (A,D), S. aureus MRSA4 (B,E), and Staphylococcus epidermidis strains (C,F), respectively, of the biofilm-detaching compounds ficin (F) (1,000, 100, 10 mg/L) and N-acetyl-L-cysteine (NAC) (8, 2, 0.5 g/L), and the antibiotic dalbavancin (D) (32, 4, 0.5 mg/L) alone or in combination. (A), (B) and (C) graphs correspond to the induction or inhibition values observed at 10 h of biofilm formation shown as percentage relative to the control, while (D), (E) and (F) to 20 h of biofilm growth, respectively. On the X axis, zero corresponds to biofilm mass of antimicrobial-free controls at 10 and 20 h of biofilm growth on an xCELLigence 96-well plate.

FIGURE 5

Biofilm disruption by ficin alone and in combination with dalbavancin. (A) Biofilm treatment by ficin [marked as F (1,000, 100, and 10 mg/L)] and dalbavancin [marked as D (32 mg/L)], either alone or in combination (F1000 D32), in methicillin-resistant Staphylococcus aureus strain MRSA4. Ficin, dalbavancin, or both was added on established biofilms after 7 h of growth in an xCELLigence equipment. (B) Effect of ficin on planktonic bacterial growth, measured as optical density in a 96-well plate. Bacteria were grown on TSB supplemented with 0.25% glucose at 37°C. Data are the means of three replicates.

FIGURE 6

Effect of 1,000 mg/L ficin (marked as F1000) and 32 mg/L dalbavancin (marked as D32) on preformed MRSA4 biofilms as measured by CV staining and viable CFU counting. (A) MRSA4 biofilm quantification by CV in flat-bottom 96-well Ibidi plates, performed by duplicate. (B) Biofilm formation capacity after different treatments and CV staining (measured as optical density). Data show average values from three biological replicates. (C) Bacterial viability of biofilm and planktonic MRSA4 cells after treatment by ficin, dalbavancin, and their combination for 24 h. Data show the average of log CFUs from three replicates. Statistical significance was assessed by t-test; asterisks indicate *p ≤ 0.05 and ***p ≤ 0.001.