Influence of prophylactic antibiotics on tissue integration versus bacterial colonization on...

© 2012 Wichtig Editore - ISSN 0391-3988

Int J Artif Organs (2012; :00) 000-00000

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Influence of prophylactic antibiotics on tissue integration versus bacterial colonization on poly(methyl methacrylate)

Guruprakash Subbiahdoss, Thomas Aleyt, Roel Kuijer, Henk J. Busscher, Henny C. van der Mei

Department of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The NetherlandsDepartment of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The NetherlandsDepartment of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The NetherlandsDepartment of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The NetherlandsDepartment of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The NetherlandsDepartment of Biomedical Engineering, University Medical Center Groningen, Groningen and University of Groningen, Groningen - The Netherlands

ABSTRACT Purpose: Biomaterial-associated infections (BAI) remain a major concern in modern health care. BAI is difficult to treat and often results in implant replacement or removal. Pathogens can be introduced on implant surfaces during surgery and compete with host cells attempting to integrate the implant. Here we studied the influence of prophylactically given cephatholin in the competition between highly viru-lent Staphylococcus aureus and human osteoblast-like cells (U-2 OS, ATCC HTB-94) for a poly(methyl methacrylate) surface in vitro using a peri-operative contamination model. Method: S. aureus was seeded on the acrylic surface in a parallel plate flow chamber prior to adhe-sion of U-2 OS cells. Next, S. aureus and U-2 OS cells were allowed to grow simultaneously under shear (0.14 1/s) in a modified culture medium containing cephatholin for 8 h, the time period this drug is supposed to be active in situ. Subsequently, the flow was continued with modified culture medium for another 64 h. Results: In the absence of cephatholin, highly virulent S. aureus caused U-2 OS cell death within 18 h. In contrast, the presence of cephatholin for 8 h resulted in survival of U-2 OS cell up to 72 h during simultaneous growth of U-2 OS cells and bacteria. Not all adhering bacteria were killed however, but they showed a delayed growth. Conclusions: These findings are in line with the recalcitrance of biofilms against antibiotic treatment observed clinically, and represent another support for the use of in vitro co-culture models in mimick-ing the clinical situation.

KEY WORDS: Biomaterial-associated infection, Race for the surface, S. aureus, Biomaterials, Antibiotics, Biofilm

Accepted: August 17, 2012

ORIGINAL ARTICLE

DOI: 10.5301/ijao.5000155

INTRODUCTION

Biomaterial-associated infections (BAI) are a major prob-lem in modern medicine. BAI is often difficult to treat, as the biofilm mode of growth protects infecting pathogens against both the host defense system and antibiotics (1). In most cases, the final remedy of a BAI is removal of the implant associated with the infection. Bacterial contamina-

tion of biomaterial implants during the surgical procedure (peri-operative contamination) is the most common route of contamination. Whether or not infection develops de-pends on the outcome of the so-called “race for the sur-face” between successful tissue integration of the implant surface and colonization of the surface by pathogens (2). If this race is won by tissue cells, then the implant sur-face is covered by a cellular layer and is less vulnerable


Cephatholin influence on S. aureus-osteoblast interactions

the influence of cephatholin on the competition between S. aureus and U-2 OS cells for a poly(methyl methacrylate) (PMMA) surface in an in vitro, co-culture, peri-operative contamination model.

MATERIALS AND METHODS

Biomaterial

Poly(methyl methacrylate) (PMMA) (Vink Kunststoffen, Didam, The Netherlands) was used as a substratum. Samples were rinsed thoroughly with ethanol (Merck, Darmstadt, Germany) and washed with sterile, ultrapure water before use to yield a water contact angle of 73 ± 3 degrees (8).

Osteoblast-like cell culturing and harvesting

U-2 OS osteoblast-like cells (ATCC number: HTB-96; obtained from LGC standards, Wesel, Germany), origi-nally isolated from a human osteosarcoma, were chosen for this study because of their osteoblast-like nature and ease of growth. Although cancer cell lines may not rep-resent all aspects of primary cell behavior and possibly differ in integrin expression, previous studies have shown a relevant osteoblast-like behavior of U-2 OS cells in co-culture studies (8). U-2 OS cells were routinely cultured in Dulbecco’s modified Eagles Medium (DMEM)-low glucose supplemented with 10% fetal calf serum, 0.2 mM ascor-bic acid-2-phosphate. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2, and were passaged at 70% to 90% confluency. The cells were harvested by using trypsin/ethylenediaminetetraacetic acid. The har-vested cells were counted using a Bürker-Türk hemocy-tometer and subsequently diluted to a concentration of 6 × 105 cells/ml.

Bacterial growth conditions and harvesting

S. aureus ATCC 12600 was originally isolated from pleu-ral fluid. First, bacteria from a frozen stock were streaked on a blood agar plate and grown overnight at 37°C. The plate was subsequently kept at 4°C. For each experi-ment, a colony was inoculated in 10 mL of tryptone soya broth (TSB; Oxoid, Basingstoke, UK) and cultured for 24 h. This culture was used to inoculate a second culture,

to bacterial colonization. Alternatively, if the race is won by bacteria, the implant surface will become colonized by bacteria and tissue cell functions are hampered by bac-terial virulence factors and excreted toxins (2, 3). In the concept of the race for the surface, full coverage of an im-plant surface in vivo by a viable layer of tissue cells and a functional host defense mechanism will protect the im-plant against bacterial colonization or biofilm formation (4). Especially in case of orthopedic implants, establishment of a robust interface with a good integration of the biomaterial surface by host tissue is essential, requiring adhesion, pro-liferation and differentiation of tissue cells for successful implantation.Staphylococcus epidermidis and Staphylococcus aureus are the most frequently isolated pathogens from infections associated with biomaterial implant surfaces (2, 5). Almost 50% of the infections associated with catheters, artificial joints and heart valves are caused by S. epidermidis (5), whereas S. aureus is detected in approximately 23% of infections associated with prosthetic joints (6). Recently, we demonstrated the influence of different pathogen spe-cies in the competition with U-2 OS osteosarcoma cells for a biomaterial surface using an in vitro, co-culture mod-el (7, 8). U-2 OS cells were hampered in their adhesion, spreading, and growth on PMMA in the presence of ad-hering bacteria. Coverage of biomaterial surfaces by U-2 OS cells in the presence of S. epidermidis however, was less hampered than in the presence of other more virulent pathogens, like S. aureus and Pseudomonas aeruginosa, causing U-2 OS cell death within 24 h (8).In orthopedics and other biomaterial-implant related sur-geries, peri-operative prophylactic antibiotics have long been advocated to prevent biomaterial implant- associated infections (9). Antibiotics are mainly systemically applied and few techniques are established for local application of antibiotics to reduce infections related to orthopedic im-plants (10). Cephalosporins, prophylactic broad spectrum antibiotics, are the first choice as they provide the best bal-ance between minimal impact on the normal flora of the host and killing of infecting organisms (11). Preventing in-fections not only depends on the efficacy of the applied antibiotic but also on the time frame in which the antibiotic is given, in order to be effective just prior to and during the procedure (12). Guidelines for surgeons recommend 1 dose of prophylactic antibiotics given just before the sur-gical incision to prevent infection (9). Therefore based on this clinical practice, the aim of this study was to evaluate

© 2012 Wichtig Editore - ISSN 0391-3988 3

Subbiahdoss et al

which was grown for 17 h prior to harvesting. Bacteria were harvested by centrifugation at 5000 x g for 5 min at 10°C and washed twice with sterile, ultrapure water. Subsequently, the harvested bacteria were sonicated on ice (3 × 10 s) in sterile phosphate buffer saline (PBS, 10 mM potassium phosphate, 0.15 M NaCl, pH 7.0) in order to break bacterial aggregates. This suspension was further diluted in sterile PBS to 3 × 106 bacteria/ml. Prior to the experiments, growth and biofilm formation of the bacte-ria in modified culture medium (MCM: 98% DMEM with 10% fetal calf serum and 2% TSB, supplemented with 2% HEPES) (8) was confirmed by culturing bacteria in MCM for 48 h.

Determination of the minimal inhibitory and minimal bactericidal concentrations

In order to determine the minimal inhibitory concentration (MIC) of cephatholin, from a stock solution of cephatholin in PBS, a series of 2-fold dilutions in 100 µL MCM were made in a 96-wells plate. Next, wells were inoculated with 100 µL bacterial suspension in PBS with a concentration of 5 × 105 bacteria/ml suspended in MCM and left to incubate at 37°C for 24 h. The concentration of cephatholin in the well containing the lowest concentration that completely inhibited visual bacterial growth was taken as the MIC. The minimal bactericidal concentration (MBC) was determined by adding a droplet of 5 µL from each well showing no vis-ible bacterial growth on a TSB agar plate. The MBC was taken as the lowest concentration of cephatholin that pre-vented growth of the bacteria on agar.

Competitive assay for U-2 OS cell growth and staphylococcal biofilm formation

A competitive assay between S. aureus and U-2 OS cells was performed on the PMMA bottom plate of a parallel plate flow chamber (175 × 17 × 0.75 mm3), as described previously in detail (8). The flow chamber was equipped with heating elements and kept at 37°C throughout the ex-periments. Bacterial and U-2 OS cellular deposition were observed with a CCD camera (Basler AG, Ahrensburg, Germany) mounted on a phase-contrast microscope Olympus BH-2 (Olympus, Hamburg, Germany) with a 40x objective for bacteria and 10x objective for U-2 OS cells.Prior to each experiment, all tubes and the flow cham-ber were filled with sterile PBS, taking care to remove all

air bubbles from the system. Once the system was filled, and before the addition of the bacterial suspension, PBS was allowed to flow through the system at a shear rate of 11 s-1. Then, a suspension of S. aureus in PBS was perfused through the chamber at the same shear rate and phase-contrast images were obtained. As soon as a density of 103 adhering bacteria/cm2, was reached, flow was switched to sterile PBS to remove the bac-terial suspension and all non-adhering bacteria from the tubes and chamber. Then, a U-2 OS cell suspen-sion (6 × 105 cells/ml) in MCM containing cephatholin at 1 × MBC was allowed to enter the flow chamber. Once the entire volume of buffer inside the chamber was replaced by the cell suspension, flow was stopped for 1.5 h in order to allow U-2 OS cells to adhere and spread on the surface of the substratum. Subsequently, phase contrast images (nine images, 900 × 700 µm each) were taken and number of adhering cells per unit area as well as the area per spread cell were determined using Scion image analysis software. Then the flow was continued with MCM including cephatholin (1 × MBC) for another 6.5 h at a flow rate of 0.14 s-1 after which the flow was continued with MCM without cephatholin up to a total of 72 h. Phase contrast images were taken every 30 min. This procedure was chosen to mimic the clinical proce-dure of advocating 1-dose peri-operative prophylactic antibiotics to prevent infections.

Analysis of U-2 OS cell behavior

After 72 h, the surfaces were prepared for fluorescence staining to assess cell number and surface coverage. Phase contrast images could not be used for quantifica-tion because the images were too crowded after 72 h of growth and the spread area per cell was difficult to deter-mine. PMMA slides with adhering U-2 OS cells were fixat-ed using 3.7% formaldehyde in cytoskeleton stabilization buffer (0.1 M Pipes, 1 mM EGTA, 4% (w/v) polyethylene glycol 8000, pH 6.9). After 5 min, the fixation solution was replaced with 30 mL fresh cytoskeleton stabilization buffer for another 5 min. Subsequently, the slides were incubated in 0.5% Triton X-100 for 3 min, rinsed with PBS and stained for 30 min with 5 ml PBS containing 49 µL DAPI and 2 µg/ml of TRITC-phalloidin. Then, slides were washed four times in PBS. DAPI stained the nuclei blue and was used to count the number of cells. TRITC-phalloidin stained the actin cytoskeleton and was used to determine the area oc-



cupied by U-2 OS cells, as well as the area per cell. Images (nine images on different locations, 375 × 375 µm each) were taken with a confocal laser scanning microscope (CLSM) (Leica DMRXE with confocal TCS SP2 unit). The images were analyzed using Scion image analysis software and the number of adhering U-2 OS cells per unit area and the average area per spread cell were determined. The total surface coverage of the substratum surface by U-2 OS cells was calculated from the number of cells and the spread area.

Statistics

Experiments were performed in triplicate. Data are rep-resented as a mean with standard deviation. Statistical ANOVA analysis was performed followed by a Tukey’s HSD post-hoc test and p<0.05 was considered significant.

RESULTS

The MIC and MBC for cephatholin of S. aureus ATCC 12600 in its planktonic state amounted 0.1 µg/ml and 25.6 µg/ml, respectively. Images of simultaneous growth

of S. aureus and U-2 OS cells at different time points in the absence of cephatholin are shown in Figure 1. In the absence of cephatholin, after 18 h of co-culturing with S. aureus, U-2 OS cells were rounded up and detached, which is indicative of cell death (Fig. 2). In contrast, in the presence of 1 × MBC cephatholin for the first 8 h of co-culturing, U-2 OS cells survived the presence of S. aureus up to at least 72 h and no differences in cell morphology compared to U-2 OS cells in monoculture were observed (Fig. 3). Quantitative data did not show any differences at 1.5 h in the average number of U-2 OS cells adhering on the PMMA surfaces (3 × 104 cells/cm2; see Fig. 4A) or in the average surface coverage by cells, which was around 10% (Fig. 4B), regardless of the absence or presence of adher-ing staphylococci and cephatholin. Differences appeared after 72 h of growth, and the number of U-2 OS cells in co-cultures in the presence of S. aureus was significant-ly reduced compared to U-2 OS monocultures (p<0.05; Fig. 4A). However, addition of cephatholin completely eliminated this negative effect of staphylococcal pres-ence on U-2 OS cell numbers, although cephatholin did not kill all S. aureus and slow growth of S. aureus was ob-served after 72 h of simultaneous growth with U-2 OS cells

Fig. 1 - Phase contrast images of simultaneous growth of S. aureus and U-2 OS cells under flow at different time points (4 h, 10 h and 22 h) in the absence of cepha-tholin. White arrows indicate U-2 OS cell death. The bar denotes 25 µm.

Fig. 2 - Fluorescence live-dead images of U-2 OS cells after 48 h of simultaneous growth with S. aureus ATCC 12600 in the ab-sence of cephatholin. Live and dead U-2 OS cells appear green and red fluorescent, respectively. The dead U-2 OS cells appeared red in color and the live S. aureus are green fluorescent.


Subbiahdoss et al

(Fig. 5B). In the absence of cephatholin a significant re-duction in surface coverage by U-2 OS cells in co-culture with S. aureus compared to control (p<0.01) was observed (Fig. 4B), indicating U-2 OS cell death and no U-2 OS cells could be detected on the PMMA surface after 72 h.

DISCUSSION

This study describes the influence of a single dose of pro-phylactic antibiotics on the outcome of the race between adhering bacteria and tissue cells for a biomaterial surface in an in vitro model. S. aureus was allowed to adhere prior to cell adhesion and spreading, which mimics the situation of peri-operative bacterial contamination of implant sur-faces. The number of bacteria adhering on the PMMA sur-face prior to initiating U-2 OS cell adhesion and spreading was set to 103 bacteria/cm2. In the past, it has been docu-mented that during a surgical procedure of 1 h, the total number of bacteria carrying particles falling on the wound is about 270 bacteria/cm2 (13). These numbers are gener-ally higher during periods of activity and when more peo-ple are present in the operation theater (13). More recently, through the use of modern, better ventilated operation the-aters (20 changes of air per hour) and impermeable patient and personnel clothing, there may be less peri-operative bacterial contamination (14). However, many surgical pro-cedures in which implants are introduced into the body last longer than 1 h. Therefore, the level of bacterial con-tamination chosen in our experiments is considered to be realistic.

Fig. 3 - CLSM images of U-2 OS cells after 72 h of growth in the absence (A) and presence (B) of S. aureus ATCC 12600 and with cephatholin for 8 h. U-2 OS cells were stained with 5 mL PBS con-taining 49 µL DAPI and 2 µg/ml of TRITC-phalloidin. Bar denotes 75 µm.

Fig. 4 - A) Number of adhering U-2 OS cells after 1.5 h and 72 h of simultaneous growth with S. aureus ATCC 12600 on PMMA in the absence and presence of cephatholin influence for 8 h compared with growth in absence of bacteria and antibiotic (control). Error bars represent the standard deviations over three replicates, with sepa-rately cultured bacteria and osteoblast-like cells. *significantly differ-ent (p<0.05) from the control; B) Surface coverage by adhering U-2 OS cells immediately after seeding at 1.5 h and after 72 h of simulta-neous growth of S. aureus ATCC 12600 and U-2 OS cells on PMMA in the absence and presence of cephatholin influence for 8 h com-pared with growth in absence of bacteria and antibiotic (control). Error bar represents the standard deviation over three replicates, with separately cultured bacteria and osteoblast-like cells. *signifi-cantly different (p<0.05) from the control.



The current study was conducted with S. aureus, a patho-gen causative for BAI, and highly virulent through its pro-duction of toxins and tissue damaging exo-enzymes (15). In previous co-culture experiments, U-2 OS osteoblast-like cells on PMMA could only survive the presence of S. epi-dermidis, but not in the presence of adhering S. aureus and P. aeruginosa, and U-2 OS cell death was observed within 24 h (8). The influence of cephatholin (1 × MBC) for 8 h allowed U-2 OS cells to survive for at least 72 h in the presence of adhering S. aureus. Cephalosporins prevent cell wall synthesis by binding to enzymes, called penicillin binding proteins. These enzymes are essential for synthe-sis of the bacterial cell wall (6). However, complete killing of S. aureus was not observed, because cephatholin kills only dividing bacteria and delayed bacterial growth and subsequent biofilm formation were observed once the antibiotic was removed from the flow chamber after 8 h. These observations are in line with the clinically observed recalcitrance of BAI and suggest that antibiotic treatment with cephalosporins may have to be continued post- surgery (9). Similarly, a different study on the simultaneous growth of S. epidermidis and osteoblast-like cells in a microfluidic device showed that the addition of culture medium con-taining 1 × MBC of rifampicin to an existing 2-day-old bio-film of S. epidermidis resulted in reduction from 108 to 104 colony forming units/ml by day 4, while no further killing was observed (16), showing slow growth and metaboli-cal activity 2 days after stopping exposure to rifampicin. However, when rifampicin (1 × MBC) was added immedi-ately after bacterial adhesion as in the current study, com-plete killing of all adhering S. epidermidis occurred (16). This is in contrast with the results of our study in which S. aureus remained metabolically active after 72 h of

growth when exposed to cephatholin (1 × MBC) imme-diately after adhesion for 8 h. Rifampicin can penetrate freely into living cells and bacteria and blocks RNA syn-thesis (17). Therefore rifampicin can also kill adhering bacteria in absence of cell growth and division in con-trast to cephatholin requiring cell division before it kills bacteria.In summary, this study shows that co-culture studies on the simultaneous growth of bacteria and tissue cells on an acrylate surface in absence and presence of initial antibi-otics demonstrate features corresponding with the clinical situation. Therewith this study provides another support for the use of in vitro, co-culture models in mimicking the clinical situation. In view of societal pressure to reduce the number of animal experiments or possibly to fully replace animal experiments by improved in vitro models, this type of support is indispensable in the development of new strategies to prevent BAI.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Mihaela Crismaru for her

support with the MIC and MBC determination.

Financial Support: This work was funded by the University Medical Center Groningen, Groningen, The Netherlands.

Conflict of Interest Statement: None

Address for correspondence:Roel KuijerDepartment of Biomedical EngineeringUniversity Medical Center Groningen and University of GroningenPO Box 1969700 AD Groningen, The Netherlandse-mail: [email protected]

Fig. 5 - Phase-contrast images of adhering U-2 OS cells to PMMA after 72 h of growth in the absence (A) and presence (B) of S. aureus ATCC 12600 under the influence of cephatholin for 8 h. The bar de-notes 25 µm.


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