Effects of antibiotics on biofilm and unattached cells of a clinical Staphylococcus aureus isolate from bone and joint infection

Abstract

Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51 200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected.Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections.

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms

“…The S. aureus LYO-S2 mCherry-tagged strain was constructed after insertion of pAH9 plasmid [22] into the LYO-S2 clinical strain [23] by electroporation, as described previously [24]. The S. aureus LYO-S2 mCherrytagged fluorescent strain, named S. aureus mCherry-LYO-S2, was selected onto Luria-Bertani (LB) agar containing erythromycin (10 μg/mL).…”

Fosfomycin and Staphylococcus aureus: transcriptomic approach to assess effect on biofilm, and fate of unattached cells

“…The patient underwent three antibiotic treatments over 147 days including fosfomycin combined with oxacillin for 55 days in the middle of the treatment period. The outcome was favorable after 3-year clinical follow-up.We previously demonstrated this strain's ability to form biofilm in vitro, and the in vitro anti-biofilm activity of fosfomycin at sub-MICs(4). We also showed that the effective concentrations of fosfomycin required for 24h-old biofilm eradication were unattainable by conventional antibiotic administration(4).…”

“…The outcome was favorable after 3-year clinical follow-up.We previously demonstrated this strain's ability to form biofilm in vitro, and the in vitro anti-biofilm activity of fosfomycin at sub-MICs(4). We also showed that the effective concentrations of fosfomycin required for 24h-old biofilm eradication were unattainable by conventional antibiotic administration(4). We therefore used S. aureus LYO-S2 as the model strain to study the molecular effects of fosfomycin on sessile cells using RNA-seq technology with both biofilm cells formed for 4h in the presence of fosfomycin at sub-MIC and pre-formed 24h-old biofilm cells later treated with sub-MBEC of fosfomycin for 24 hours.…”

“…The biofilm matrix can also act as a barrier to diffusion and thus reduces penetration of antibiotics into biofilms. The effectiveness of this barrier varies between antibiotics; gentamicin, tigecycline and daptomycin penetrate better than clindamycin, linezolid and vancomycin (3,4). The effects of antibiotics can also be affected by the particular microenvironment of biofilm, such as acidic pH and low levels of oxygen encountered in the deep layers of the aggregates (5).…”

“…MICs are determined in vitro with planktonic cells growing exponentially under conditions that are optimal for the action of the drug but do not provide a true estimation of the concentration of antibiotics required to treat a bacterial biofilm. In vitro and in vivo experiments demonstrated that minimum bactericidal concentrations for biofilm bacterial cells are usually much higher (about 10-10,000 times) than their counterpart planktonic cells (4,10) and therefore impossible to reach by conventional antibiotic administration. A combination of antibiotics with different and synergic 4/31 killing mechanisms is the best current solution for the treatment of biofilm infections.…”