Aims: To examine the response of Staphylococcus epidermidis isolates from prosthetic orthopaedic infections to vancomycin concentrations below the minimum inhibitory concentration.
Methods: Staphylococcal biofilms were grown in 96-well flat-bottomed cell culture plates under a variety of culture conditions and stained using an ammonium crystal violet solution. Optical density (450 nm wavelength) was recorded to estimate the biofilm density for each strain. Population analysis and time-kill studies were also performed on selected isolates.
Results: A range of responses were observed, including increased biofilm density at drug concentrations approaching the minimum inhibitory concentration. This increased density was associated with the presence of a more resistant population identified on population analysis but without an apparent effect on the time-kill curves.
Conclusions: The ability of some strains to show increased biofilm density could be a factor in the failure of vancomycin therapy reported in some cases. The demonstration that low concentrations of vancomycin may increase the density of newly forming S epidermidis biofilms may indicate an area of potential concern in the use of vancomycin in orthopaedic implants and intravascular catheter locks, and may partially account for some cases of treatment failure.
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Biofilms are a common mode of colonisation leading to catheter-related bloodstream infections, prosthetic valve endocarditis, and other complications.1 Staphylococcus epidermidis is one of many organisms capable of forming clinically significant biofilms2 and is frequently identified from intravascular catheter infections,3 orthopaedic infections4 and endocarditis.5
Antibiotic resistance within a biofilm can be up to 1000 times greater than for planktonic cells.6 Concentrations of vancomycin below the minimum inhibitory concentration (MIC) have been shown to have a variable effect on the adherence of coagulase-negative staphylococci (CoNS) to silicone rubber and polystyrene surfaces,7 and to reduce the production of a biofilm by cells attached to Teflon surfaces.8 Vancomycin is a common component of antimicrobial therapy in prosthetic joint infection,9 catheter infection3 10 11 and endocarditis,12 especially where meticillin resistance is detected. Vancomycin can attain levels within a biofilm that are above the minimum bactericidal concentration (MBC) of S epidermidis, but fail to eradicate the biofilm, although bacterial counts are reduced with rising antibiotic levels.13 Previous studies have shown that subinhibitory concentrations of vancomycin have no14 or small8 15 effects on biofilm density.
The series of experiments described here were designed to examine the density of biofilms formed by clinical S epidermidis isolates following exposure to vancomycin concentrations up to the MIC.
Eight clinical isolates of S epidermidis, identified by 16S rRNA sequencing, and known to be biofilm-forming strains and possessing the ica operon, were stored at 4°C on blood agar (Oxoid, Basingstoke, UK). The strains were originally recovered from prosthetic orthopaedic infections in a teaching hospital.
Working cultures were prepared in 10 ml tryptone soya broth (TSB; prepared in accordance with the manufacturer’s instructions with the addition of 0.25% w/v glucose; Oxoid) and incubated overnight at 36°C without agitation. The biofilm-forming S epidermidis reference strain RP62A was used as a control organism.
Determination of MICs and MBCs
The MICs and MBCs were estimated for each strain using doubling dilutions of vancomycin (Sigma-Aldrich, Gillingham, UK) in nutrient broth, to which was added one drop of overnight culture. These were incubated at 36°C for 24 h. The MBC was estimated by culturing non-turbid tubes on blood agar. Heterogeneous resistance screening was performed using brain-heart infusion agar (Oxoid) with 6 and 8 ug/ml additional vancomycin.16
Estimation of biofilm density
Staphylococcal biofilms were studied using a modification of a published method,17 using sterile polystyrene 96-well flat-bottomed cell culture plates (Nunc; Scientific Laboratory Supplies, Nottingham, UK). Different culture conditions are noted separately below. Plates were incubated at 36°C for 20 h and the well contents removed. Wells were individually washed with 200 μl phosphate-buffered saline three times, before they were dried for 2 h. No additional fixative was used. After drying, wells were stained with 150 μl 0.4% ammonium crystal violet solution for 10 min and washed under gently running water. Stained plates were then air-dried overnight and read on a Titertek Multiscan (Biological Instrumentation Services, Kirkham, UK) optical plate reader for optical density (OD) at 450 nm wavelength. In all experiments described below, the biofilm density for any isolate was calculated as the mean of eight replicates. In each case, the first column on microtitre plates was used as a blank as a zero control for OD measurement.
Effect of inoculum dilution on biofilm density
Briefly, 200 μl doubling dilutions (using fresh TSB) of overnight culture were made in the columns of a 96-well plate, from neat to 1:256. This procedure was performed three times for the clinical isolates and twice for RP62A. Biofilm density was estimated as described above.
Effect of vancomycin on biofilm density
Briefly, 200 μl of a 1:100 dilution of overnight culture was incubated with 0, 0.5, 1, 2 and 4 μg/ml vancomycin in microtitre plates. This was performed twice for all strains.
Effect of vancomycin exposure on pre-formed biofilms
Briefly, 200 μl of a 1:100 dilution of overnight culture was incubated overnight in microtitre plates. A 100 μl volume was then removed from each well and replaced with fresh TSB containing vancomycin at 0, 2.5, 5 and 30 μg/ml, and plates were incubated overnight.
Time-kill studies were performed as described previously.18 Briefly, for each strain, two conical flasks containing 20 ml TSB were inoculated with 100 μl overnight broth culture, with an initial sample collected to check that the initial concentration was ∼5×106 cfu/ml. These flasks were then incubated and agitated at 100 rpm at 36°C for 90 min before 20 μg/ml vancomycin was added (time 0). Samples were collected from all flasks at times 0, 1, 2, 4 and 6 h. The samples were diluted, and plated in triplicate to estimate the colony count.
Four strains tested (including RP62A) showed a generally steady biofilm density at vancomycin concentrations up to one-half MIC, followed by a fall in biofilm density at concentrations approaching the MIC. Three further test strains showed a steady reduction in biofilm density with increasing vancomycin concentration. Only the clinical strain s2 showed an increase in biofilm density at vancomycin concentrations up to the MIC, and this effect was reproducible suggesting a genuine response (fig 1). OD readings without vancomycin ranged from 0.338±0.053 to 0.764±0.135 for the mecA+ strains, with an OD of 0.196±0.049 for the mecA- strain. RP62A gave the highest OD readings of all tested samples. Strain s2 showed had an OD that was 39.7% higher at vancomycin concentrations of 4 μg/ml from a baseline level of 0.379. Further results are presented comparing the growth characteristics of this clinical isolate with RP62A.
Isolates s2 and RP62A had MIC of 4 μg/ml and an MBC/MIC ratio of 4, suggesting that neither strain was tolerant to vancomycin.19 The clinical isolate showed some growth on brain-heart infusion with 6 μg/ml vancomycin, indicating an heterogeneous population displaying intermediate susceptibilities;16 neither strain showed any growth with 8 μg/ml vancomycin.
Figure 2 shows the effect of the different vancomycin concentrations on pre-formed biofilms. In contrast to fig 1 (newly forming biofilms), there was no significant difference between the biofilm densities at different antibiotic concentrations.
Figure 3 shows the time-kill curves for the two strains. Overlaying the curves suggests that RP62A and s2 show similar susceptibilities to vancomycin. The clinical strain s2 showed a greater than 1 log10 reduction in colony counts after 6 h, again indicating the absence of vancomycin tolerance in this strain; this reduction was not seen in RP62A, suggesting the presence of vancomycin tolerance not revealed by the MBC/MIC ratio.19
Figure 4 shows the responses of the two strains to different inoculum densities. RP62A showed higher overall densities. Strain s2 showed an apparent increased density at lower concentrations, but the overlapping SDs suggest that these differences were not significant, and so the two strains showed broadly the same response to the size of the initial inoculum.
A variety of responses to subinhibitory concentrations of vancomycin were seen across the different strains tested. These ranged from a gradual decrease in density with increasing antibiotic concentrations, through a steady-state response, to an increasing biofilm density in the presence of vancomycin. This increased biofilm density was reproducible, and occurred with newly forming biofilms only. Plasma concentration levels of vancomycin and teicoplanin have been previously shown to have greater effect on young biofilms than on old biofilms formed by S epidermidis.20 A similar response seen previously for the strain RP62A21 was not replicated here, suggesting that the effect may depend on environmental factors as well as the strain itself. This phenomenon does not appear to be related to the presence of glycopeptide tolerance as examined by time-kill analysis, but may represent the effect of the presence of a small subset of vancomycin-intermediate cells. It does not appear to be related to the inoculum density, as RP62A and s2 show similar responses to an increasing initial density. One possible mechanism for this effect is the thickening of the staphylococcal cell wall in response to antibiotics.22 23 An alternate explanation may be that exposure to low concentrations of vancomycin may affect the expression of genes involved in biofilm production in some strains; subinhibitory concentrations of tetracycline and quinupristin/dalfopristin have been shown to increase ica expression and biofilm formation.14 This suggestion may be supported by the recent demonstration that the accumulation associated protein (Aap), which has a role in biofilm formation, is carried on the surface of subpopulations of S epidermidis cells.24 The possibility that the numerous genetic systems responsible for biofilm formation in S epidermidis may have a variable expression levels within strain populations should be examined. Increased biofilm formation may also indicate an increased biofilm-forming potential of a small population of more resistance cells within an isolate.
Coagulase-negative staphylococci are frequently associated with infections of prosthetic materials, including intravascular catheters,3 10 11 orthopaedic infection4 and endocarditis.5 S epidermidis is the most frequently identified CoNS species.3 4 The ability of some strains to show increased biofilm density may also be a factor in the failure of vancomycin therapy reported in some cases, for example endocarditis.25 26 The demonstration that low concentrations of vancomycin may increase the density of newly forming S epidermidis biofilms, even at apparently low frequency, may be of concern in some cases where vancomycin is used either in orthopaedic cement27 or on a surface carrier,28 although the high concentration of vancomycin found in cement should ensure that these sub-MIC effects are not relevant. A similar concern may surround the use of intravascular catheter locks, although lock solutions are designed to dwell within the catheter lumen for a period of time at antibiotic concentration far in excess of those found using systemic antibiotics,29 and successful CoNS treatment has been shown to be more frequent with lock therapy than with systemic antibiotics alone.9 As the effect is noticeable for new biofilms, a further concern may be the embolic/metastatic seeding of infection from a single source,30 especially if vancomycin therapy is subtherapeutic. The difference in response to reduced vancomycin concentrations warrants further investigation of the mechanisms of biofilm formation in clinically relevant S epidermidis isolates.
Some strains of Staphylococcus epidermidis from clinical infections may show increased biofilm formation in the presence of low concentrations of vancomycin.
This effect may partially explain some aspects of treatment failures with vancomycin when treating coagulase-negative staphylococcal infection.
The presence of this effect requires further investigation to establish both the frequency and the potential clinical impact and the mechanisms of biofilm formation elicited by these isolates.
The authors thank Stephanie Sandiford for molecular typing of S epidermidis isolates.
Part of this work was presented at the 15th European Congress on Clinical Microbiology and Infectious Diseases, Copenhagen, 2005, and at the 156th Meeting of the Society for General Microbiology, Edinburgh, 2005
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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