Abstract
Biofilm-associated infections in wounds or on implants are difficult to treat. Eradication of the bacteria is nearly always impossible, despite the use of specific antibiotics. The bactericidal effects of high-energy extracorporeal shock waves on Staphylococcus aureus have been reported, but the effect of low-energy shock waves on staphylococci and staphylococcal biofilms has not been investigated. In this study, biofilms grown on stainless steel washers were examined by electron microscopy. We tested ten experimental groups with Staph. aureus-coated washers and eight groups with Staph. epidermidis.
The biofilm-cultured washers were exposed to low-energy shock waves at 0.16 mJ/mm2 for 500 impulses. The washers were then treated with cefuroxime, rifampicin and fosfomycin, both alone and in combination. All tests were carried out in triplicate. Viable cells were counted to determine the bactericidal effect.
The control groups of Staph. aureus and Staph. epidermidis revealed a cell count of 6 × 108 colony-forming units/ml. Complete eradication was achieved using the combination of antibiotic therapy (single antibiotic in Staph. aureus, a combination in Staph. epidermidis) and shock wave application (p < 0.01).
We conclude that shock waves combined with antibiotics could be tested in an in vitro model of infection.
Staphylococci form biofilms on medical implants, damaged tissues and in-dwelling vascular catheters, and the associated infections are extremely difficult to treat for a variety of reasons, including failure of antibiotics to penetrate the biofilm.1
The treatment of urolithiasis by extra-corporeal lithotripsy was the first medical use of high-energy shock waves in non-invasive procedures.2 Later, the positive effect of shock waves on bone healing and neovascularisation led to the use of extracorporeal shock wave technology (ESWT) to treat pseudarthrosis, avascular necrosis and tendinopathies.3 These treatments were generally well tolerated. The bactericidal effects of high-energy extracorporeal shock waves on Staphylococcus aureus have been reported, but the more relevant potential effects of shock waves on biofilms has not yet been demonstrated.4,5
The purpose of this study was to investigate the in vitro impact of low-energy shock waves on staphylococci, and also the potential of these shock waves to enhance the penetration of antimicrobial agents into test strains of Staph. aureus and Staph. epidermidis biofilms.
Materials and Methods
Staph. aureus and Staph. epidermidis, susceptible to antibiotics in classic tests,6,7 were used in this study. The presence of organisms, colonisation of stainless steel washers and biofilm formation, including glycocalyx formation, were determined by scanning electron microscopy (SEM) after incubation times of 12, 24, 48 and 72 hours. Stainless steel washers were chosen because, despite similar colony inoculation loads, both bacteria adhere almost 150% better to stainless steel than to titanium.8Staph. aureus (Staph. aureus 15981, provided by F. Goetz, University of Tübingen, Germany) and Staph. epidermidis (RP 62, provided by B. Amorena, Agricultural Research Service, Zaragoza, Spain) were grown on stainless steel washers (stainless steel washers, 13.0/6.6 mm; Dorner Med Tech Europe GmbH, Wessling, Germany) to create biofilms. Slime production was confirmed using Congo Red agar (Congo Red Acid Morpholine Propane Sulfonic Acid Pigmentation; Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany), and biofilm production was assessed by SEM (Zeiss, Jena, Germany) after 24 and 72 hours. Ribotypes of the isolates were identified at the beginning and end of the study using the EcoR1 (Promega, Madison, Wisconsin) enzyme for verification.9,10 The minimum inhibitory concentrations (MIC) of Staph. aureus 15981 and Staph. epidermidis RP 62 were determined by the E-Test (AB Biodisk, Solna, Sweden).
The biofilm-covered washers from each bacterial strain were separated into groups, including positive controls, antibiotic treatment alone, ESWT treatment alone, and ESWT in conjunction with antibiotic treatment (Table I). Stainless steel washers (Dorner MedTech Europe GmbH, Wessling, Germany) routinely used in orthopaedic surgery were used in these tests.
Table I.
Experimental treatment groups: minimum inhibitory concentrations of test strains against antibiotic substances
| Staphylococcus aureus washers | Staphylococcus epidermidis washers |
|---|---|
| * ESWT, extracorporeal shock wave therapy | |
| † N/A, not available | |
| Positive control | Positive control |
| Cefuroxime 1.0 μg/ml | Cefuroxime 4.0 μg/ml |
| Fosfomycin 0.5 μg/ml | Fosfomycin 0.5 μg/ml |
| Rifampicin 0.016 μg/ml | N/A† |
| Cefuroxime + fosfomycin | Cefuroxime + fosfomycin |
| ESWT* alone | ESWT alone |
| Cefuroxime 1.0 μg/ml + ESWT | Cefuroxime 4.0 μg/ml + ESWT |
| Fosfomycin 0.5 μg/ml + ESWT | Fosfomycin 0.5 μg/ml + ESWT |
| Rifampicin 0.016 μg/ml + ESWT | N/A |
| Cefuroxime + fosfomycin + ESWT | Cefuroxime + fosfomycin + ESWT |
Bacterial cell growth and colony counting.
For biofilm formation, one colony of each test strain was incubated at 37°C in 1 ml of Tryptic Soy Broth (TSB) medium (Merck, Darmstadt, Germany) for 72 hours. Bacterial cell counts were performed using a method of sonication and log dilution/plating used to assess biofilm bacteria adhering to the washers.8
Scanning electron microscopy method.
This method was carried out for illustrative purposes, to allow the magnification of images of 72-hour biofilms and visualisation of the effect of a 24-hour treatment with antibiotics and/or ESWT. Colonised washers were prepared for SEM by fixation for three hours at 4°C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer containing 0.15% ruthenium red.
Antibiotic preparation methods.
The antibiotics were chosen according to their bactericidal action and efficacy as assessed in previously published studies.11 In preparation for treatment, the antibiotics were resuspended, filter sterilised (pore diameter 0.22 μm; Millipore Ltd, Livingston, United Kingdom) and diluted with TSB medium to a fourfold MIC. Fosfomycin (Sigma-Aldrich) was prepared by combining a fourfold concentration of the MIC with additional aliquots of glucose-6-phosphate (Sigma-Aldrich) in TSB medium. Further procedures were the same for cefuroxime.
Cefuroxime (Sigma-Aldrich) combined with fosfomycin was prepared in TSB medium such that the concentration of each single antibiotic corresponded to the fourfold concentration of MIC in the single-antibiotic solutions.
Rifampicin (Sigma-Aldrich) was diluted to a final concentration of a fourfold MIC (0.064 μg/ml) and was used as a single substance.
Procedure.
Test series (and replications) for both strains were performed in the same way and contained the following experimental groups;
Positive controls.
Positive control groups of biofilm-covered washers from each strain were used to confirm slime production, detection of the biofilm by SEM and for cell counting.
ESWT only.
Washer groups from both biofilm-producing strains were exposed to ESWT alone at 0.16 mJ/mm2 for 500 impulses. During the ESWT the washers were embedded in a cell culture cluster plate containing Ringer’s lactate solution. The plates were then covered with Parafilm (Pechiney Plastic Packaging, Chicago, Illinois). The ESWT-only groups were sonicated at a frequency of 35 kHz (Bandelin Sonorex RK102; Bandelin Electronic GmbH & Co. KG, Berlin, Germany) for two minutes and subjected to cell counting and SEM to determine the effect on biofilms and cell viability.
Antibiotic incubation (without shock waves).
Biofilm-coated stainless steel washers were rinsed with Ringer’s lactate solution to remove free bacteria from their surface and then exposed to each antibiotic solution (4 × MIC in 1 ml of TSB medium, 24 h/37°C), after which they were removed from the broth, rinsed with Ringer’s lactate, put into 1 ml Ringer’s lactate solution and sonicated at a frequency of 35 kHZ for two minutes. Several dilutions were then applied to a solid agar plate (blood agar) using a spiral plater (Whitley Automatic Spiral Plater; Don Whitley Scientific Limited, Shipley, United Kingdom), incubated for 24 hours at 37°C and then counted for viable cells. The plates were then covered with Parafilm, and the washers were subjected to SEM.
ESWT and antibiotic incubation.
The washers were put into a cell culture cluster plate, and the plate was filled up with Ringer’s lactate solution and covered with Parafilm, immediately after which the washers received 500 impulses at 0.16 mJ/mm2. They were then removed from the plate, rinsed with Ringer’s lactate solution, put into 1 ml of TSB medium containing the fourfold MIC of each antibiotic solution, and incubated for 24 hours. They were then removed from the broth, rinsed again with Ringer’s lactate and sonicated for two minutes in 1 ml of Ringer’s lactate solution. This solution was put on to a solid agar plate by means of a spiral plater and incubated for 72 hours at 37°C for counting of viable bacteria. In addition, washers were subjected to SEM. All tests were carried out in triplicate.
Statistical analysis.
Comparisons between cell counts before and after treatment in the different groups was performed using Student’s t-test. A p-value < 0.05 was considered to be statistically significant, and a p-value < 0.01 as highly statistically significant.
Results
A control group (untreated, i.e., without shock waves and without antibiotic incubation) was included in all cases and yielded a cell count within a range of 6 × 108 CFU/ml.
Samples including Staph. aureus and Staph. epidermidis treated with ESWT alone did not show a significant decrease in bacterial cell count (1 × 106 CFU/ml; p = 0.08, Student’s t-test) (Fig. 1). Samples of Staph. aureus treated with ESWT alone did not show a significant decrease in bacterial cell count (1 × 108 CFU/ml; p = 0.06, Student’s t-test) (Table II).
Table II.
Number of viable cells after different kinds of treatment
| Test set-up | Staphylococcus aureus cell count (CFU * /ml) | Staphylococcus epidermidis cell count (CFU/ml) |
|---|---|---|
| * CFU, colony-forming units | ||
| † ESWT, extracorporeal shock wave therapy | ||
| ‡ N/A, not available | ||
| 72 h after incubation | 4 × 108 | 8 × 108 |
| ESWT† (0.16 J/mm2 × 500 Impulses) | 1 × 108 | 4 × 106 |
| Cefuroxime | 1 × 108 | 1 × 108 |
| Fosfomycin | 3 × 107 | 1 × 107 |
| Rifampicin | 4 × 108 | N/A‡ |
| ESWT + cefuroxime | Sterile | 5 × 104 |
| ESWT + fosfomycin | 5 × 106 | 1 × 107 |
| ESWT + rifampicin | Sterile | N/A |
| Cefuroxime + fosfomycin | 4 × 105 | 1 × 107 |
| ESWT + cerfuroxime + fosfomycin | Sterile | Sterile |
Fig. 1
Scanning electron microscope image showing a multilayered cluster of Staphylococcus epidermidis covered by biofilm (72 hours old) after exposure to 500 impulses at 0.16 mJ/mm2 in a Ringer’s lactate solution (×5000 magnification).
The number of viable cells after the different kinds of treatment are shown in Table II.
Incubation with antibiotics alone (cefurozime, fosfomycin, rifampicin and the combination of cefurozime and fosfomycin) resulted in a final count of 107–8 CFU/ml. Only with Staph. aureus did the combination of cefuroxime and fosfomycin result in a decrease of viable cells to 4 × 105 CFU/ml.
Staph. aureus biofilm treated by ESWT followed by exposure to cefuroxime or rifampicin alone resulted in a complete eradication of viable bacteria. ESWT plus fosfomycin led to a reduction of Staph. aureus from 4 × 108 to 5 × 106. Samples of Staph. epidermidis biofilm treated by ESWT followed by exposure to cefuroxime alone showed a significant decrease (5 × 104; p < 0.01, Student’s t-test), but not complete eradication. Only the combination of cefuroxime and fosfomycin following ESWT led to a complete eradication of viable cells of Staph. epidermidis (p < 0.01, Student’s t-test). This was demonstrated by the growth control after 72 hours of incubation, where no bacterial growth could be detected.
Discussion
This study is the first reported investigation of the in vitro eradication of staphylococci growing in biofilm by means of low-dose ESWT followed by antibiotic exposure. There are several in vivo and in vitro reports about the limited effect of antibiotics on staphylococci forming biofilms. Nevertheless, fosfomycin, and cefuroxime followed by rifampicin have been shown to significantly affect the viability of younger (six-hour) biofilm cells, and were therefore used in this study.11 Due to the limited effect of antibiotics alone in the treatment of osteomyelitis, Lew and Waldvogel1 recommended a multidisciplinary approach involving orthopaedic, plastic and vascular surgery.
In accordance with other recent studies, no significant reduction in bacterial cell count was observed in this study exposing older (72 hours) staphylococcal biofilms to these antibiotics either alone or in combination.12 Dunne, Mason and Kaplan13 observed no sterilisation of a staphylococcal biofilm following the administration of rifampicin and vancomycin, and Darouiche et al14 described the failure of vancomycin in the treatment of prosthesis-related infections caused by Staph. epidermidis. This failure to affect the older biofilms may be due to the slow growth of biofilm bacteria, which may render the micro-organisms less susceptible to antibiotics.13 Several studies have suggested that the inefficacy of antibiotics in this situation may also result from poor penetration of the antibiotics into the inner layers of the biofilm, because exopolysaccharide or glycocalyx act as a barrier.1,14,15
Koshiyama et al16 have described damage to the bacterial membrane by shock waves, due to structural change in the phospholipid layer, allowing the penetration of water molecules.
In this study the biofilm layers were damaged by ESWT in order to facilitate antibiotic penetration. The bactericidal effect of ESWT on Staph. aureus has already been demonstrated,17 but only in high dosages and on bacterial suspensions, not on slime-producing staphylococci.
The incidence of complications of ESWT, including microfractures and haematoma formation, depends on the dose which is administered.18 Therefore, we used low-energy ESWT, which is known to induce neovascularisation and tissue regeneration, promoting the healing of tendon and bone.19,20
The application of ESWT alone to staphylococcal biofilms or exposure to antimicrobials alone did not lead to a significant reduction in viable cells in our study. However, the results clearly demonstrate that exposing older (72 hour) staphylococcal biofilms to low-energy ESWT, followed by antibiotic incubation, can eradicate viable cells in vitro. The effect of ESWT could be due not only to damage of the biofilm layers, but also to dispersal of bacteria from the biofilm into the medium, leading to increased susceptibility to antimicrobials.21
The need for a combination of antibiotics such as cefuroxime with fosfomycin to achieve eradication of Staph. epidermidis, in contrast to the need for only a single agent (cefuroxime) to eradicate Staph. aureus, could be due to two factors. First, the larger multilayered cluster of Staph. epidermidis compared to Staph. aureus, as observed by SEM, and secondly, the lack of the coupling agent fibronectin and plasma proteins in these in vitro conditions may affect the adherence of Staph. aureus when compared with Staph. epidermidis.8
Even though the antibiotic susceptibility of Staph. aureus and Staph. epidermidis exposed to low-energy ESWT has not been verified in vivo, the fact that complete eradication of staphylococcal biofilm has been achieved is encouraging. Moreover, in acute inflammation vascular channels are compressed and obliterated, leading to ischaemic necrosis, and antibiotics and inflammatory cells cannot reach the avascular areas.1 Therefore, low-energy ESWT, which is known to induce neovascularisation and tissue regeneration, could have additional beneficial effects in vivo. Nevertheless, caution is needed in relating the in vitro findings to the in vivo situation, particularly when considering differences in bacterial growth and the pharmacokinetics of the therapeutic agents. However, these encouraging results suggest that ESWT combined with antibiotic therapy could be tested in an in vivo model of infection.
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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