Unexpected synergistic and antagonistic antibiotic activity against Staphylococcus biofilms

Unexpected antibiotic activity in biofilms

GF Dall1, 2, 3, STJ Tsang*2, 3, 4, PJ Gwynne2, SP MacKenzie4, AHRW Simpson3, SJ Breusch4, and MP Gallagher2.

1. Department of Orthopaedic surgeryBorders General HospitalHuntlyburnMelroseTD6 9BSUnited Kingdom

2. School of Biological sciences University of Edinburgh Darwin Building King's Buildings Mayfield Road Edinburgh EH9 3JR United Kingdom

3. Department of Orthopaedic surgery University of Edinburgh Chancellor’s building 49 Little France Crescent Old Dalkeith Road Edinburgh EH16 4SBUnited Kingdom

4. Department of Orthopaedic surgeryRoyal Infirmary of Edinburgh51 Little France CrescentOld Dalkeith RoadEdinburghEH16 4SAUnited Kingdom

Corresponding authorMr Shao-Ting Jerry Tsang0131 242 6649Jerry.Tsang@ed.ac.uk

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Abstract

Objectives

To evaluate putative anti-staphylococcal biofilm antibiotic combinations used in the

management of periprosthetic joint infections (PJI)

Methods

Using the dissolvable bead biofilm assay, the minimum biofilm eradication

concentration (MBEC) was determined for the most commonly used antimicrobial agents

and combination regimens against staphylococcal PJI. The established fractional inhibitory

concentration (FIC) index was modified to create the fractional biofilm eradication

concentration (FBEC) index to evaluate synergism or antagonism between antibiotics.

Results

Only gentamicin (MBEC 64 mg/L) and daptomycin (MBEC 64 mg/L) were observed to

be effective anti-staphylococcal agents at clinically achievable concentrations.

Supplementation of gentamicin with daptomycin, vancomycin or ciprofloxacin resulted in a

similar or lower MBEC than gentamicin alone (FBEC index 0.25-2). Conversely, when

rifampicin, clindamycin or linezolid was added to gentamicin, there was an increase in the

MBEC of gentamicin relative to its use as a monotherapy (FBEC index 8-32).

Conclusion

This study found that gentamicin and daptomycin were the only effective single-

agent antibiotics against established Staphylococcus biofilms. Interestingly the addition of a

bacteriostatic antibiotic was found to antagonise the ability of gentamicin to eradicate

Staphylococcus biofilms.

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Introduction

Antibiotics remain an important adjunct to surgical debridement in the management

of orthopaedic biofilm-related infections such as prosthetic joint infection (PJI)1 and infected

fracture non-union.2 Currently the central tenets of this approach are to: firstly identify the

relevant organisms causing the infection; secondly, determine how susceptible they are to a

wide spectrum of antibiotics; and thirdly, select an antibiotic that is able to be

pharmacokinetically delivered, at a concentration that is pharmacodynamically (PD)

effective whilst maintaining an acceptable side-effect profile. The most important

treatment of PJI is surgical debridement in an attempt to eradicate the bacterial biofilm,3

but the opportunity to administer antibiotics locally remains an important adjunct. The

rationale of local antibiotics delivery is to enhance local antibiotic soft tissue concentration

in order to eradicate the remaining biofilm and prevent recolonisation. This depot is not

dependent on the presence of vascularized tissues and achieves concentrations that are

orders of magnitude higher than systemic administration would be able to safely deliver.

Despite the availability of biodegradable systems, antibiotic loaded acrylic bone cement

(ALAC) remains the most commonly used antibiotic carrier in the management of PJI. There

is evidence to show that ALAC is effective in minimising the risk of PJI following primary hip

and knee replacement.4–6 The use of locally-delivered antibiotics in the management of PJI

has been shown to be associated with eradication of infection in 75-91% cases.7,8 There is

however a paucity of evidence on whether local delivery of a single agent or a combination

is superior. The antibiotics commonly used in the management of PJI9 are listed in Table 1. It

is recognised that exposure to sub-inhibitory levels of one antibiotic can select for high level

tolerance to multiple and dissimilar antibiotics.10 It is feasible that even very high

concentrations remain ineffective against sessile biofilm organisms and persister cells, which

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are tolerant to a variety of stresses.11 Persister cells are a dormant, non-dividing sub-

population of cells found within biofilms that remain viable irrespective of the concentration

of the antibiotic. When the concentration of antibiotic reduces, persisters are thought to

repopulate the biofilm, which will shed off new planktonic cells, producing the relapsing

biofilm infection seen clinically.12

Antibiotic combinations are thought to work by: broadening the spectrum of

antibiotic activity in empirical therapy; utilising synergistic effects of combinations;

preventing resistance mechanisms evolving; enhancing intracellular penetration; and

limiting the effects of bacterial toxins and expression of other virulence factors.13,14 Several

commercial bone cements have been designed for treating PJI, which contain gentamicin,

with either clindamycin (Refobacin Revision® or Copal® G+C) or vancomycin (Vancogenx®).15,16

Meta-analyses of clinical trials evaluating antibiotic combinations in periprosthetic

joint infection have concluded that the patient populations and treatment algorithms were

too heterogeneous to draw any clinically useful inferences about the optimal antibiotic

regimen from the available data.17,18 In lieu of good quality clinical evidence, inferences

about the optimal antibiotic dose or combination for therapy must be made with in vitro

experiments and with animal models before a properly controlled trial comparing the most

promising candidate is commissioned. This study aimed to investigate which common

clinically used antibiotic combinations, at clinically achievable concentrations, were effective

against Staphylococcus biofilms.

Materials and methods

Bacterial Strains and media

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The following bacterial strains were used: a MSSA reference strain (ATCC #29213,

MSSA-N), which has been extensively studied in its biofilm state;19–21 and a coagulase-

negative Staphylococcus (CNS-J) isolated from an infected hip replacement. Bacterial strains

were cultured aerobically in LB overnight at 37°C prior to use. LB used in this study was:

Bacto tryptone (Difco) (10 g), Bacto yeast extract (Difco) (5 g) and NaCl (10 g), dissolved up

to 1L of dH2O. LB agar was solidified by adding 15 g/L agar (Difco) prior to autoclaving.

Antibiotics

The following antibiotics were used in this study: Linezolid - Zyvox® 2000mg/L

(Pharmacia, Sandwich, Kent, UK), Gentamicin - Cidomycin® 4000mg/L (Sanofi Aventis,

Guildford, UK) , Rifampicin - Rifadin® 400mg/L (Sanofi Aventis, Guildford, UK), Ciprofloxacin -

2000mg/L (PLIVA Pharma Ltd, Petersfield, UK), Vancomycin – 4000mg/L (Wockhardt UK

Limited Wrexham, UK) , Clindamycin - Clindamycin -Cleocin® 4000mg/L (Sanofi Aventis,

Guildford, UK), and Daptomycin – Cubicin® 4000mg/L (Cubist Pharmaceuticals,

Lexington,USA). All were stored and prepared as per the manufacturer’s instructions prior

to use22. Daptomycin was supplemented with 50 mg/L Ca2+, as it has been shown to result

in a more amphiphilic and bactericidal compound,23,24 improving in vitro activity.25 The

antibiotics examined in this study reflected the most commonly used agents in current

clinical practice.4,26–28

Dissolvable bead biofilm assay

The dissolvable bead biofilm assay was performed, as previously described,29 in order

to determine the minimum biofilm eradication concentration (MBEC) of each antimicrobial

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and antimicrobial combination. The MBEC is defined as the lowest concentration of

antimicrobial that eradicates 99.9% of the bacteria in a biofilm state compared to growth

controls in the same conditions.30,31 The 24-hour bacterial biofilms grown on the surface of

the alginate beads were exposed to the test antimicrobials, dissolved in LB, for three hours

to obtain an eradication curve. Following treatment the beads were washed in sterile water

and then dissolved using a sterile 0.05M Na2CO3 and 0.02M Citric acid (pH 6.8) solution. The

liberated organisms then underwent serial dilution and plated onto LB-agar using the Miles-

Misra technique.32 Each test condition was performed using three biological replicates, with

each replicate undergoing three technical repeats.

Data handling, graphical illustration and statistical analysis

Data was analysed using GraphPad Prism 6 for Mac OS X software (GraphPad

Software, San Diego California USA, www.graphpad.com) for statistical analysis and

graphing.

Results

Antibiotic activity against Staphylococcus biofilms

In order to investigate whether a single antimicrobial would be effective at

eradicating Staphylococcus biofilms, their effects were examined at concentrations between

4 and 2048mg/L. The MBEC was established for each antibiotic tested (Fig. 1 and 2). Figures

1 and 2 demonstrate that only gentamicin and daptomycin were observed to be effective

bactericidal agents at clinically achievable concentrations. Rifampicin, clindamycin, linezolid,

vancomycin and ciprofloxacin did not reduce cell viability substantially over the 3-hour

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exposure despite being orders of magnitude above typical minimal bactericidal

concentrations for planktonic staphylococci.22

Antibiotic activity of antibiotic combinations delivered locally in PJI

To investigate which combinations were efficacious in treating Staphylococcus

biofilms, five antibiotics suitable for local delivery were tested in combinations of two or

three. The concentration at which they are eluted in vivo from ALAC varies significantly and

is dependent on; cement porosity,33 hydrophilia of the polymethylmethacrylate structure,

volume of distribution,34,35 the surface area of the cement, the assay used to measure

elution, and the physical state and chemical structure of the antibiotic.36 Following a review

of relevant in vitro15,27,35,37–45 and in vivo studies23,38,39,41,46–52 a concentration of 128 mg/L was

chosen for the antibiotics commonly used in combination with gentamicin in ALACs to

reflect the midrange between the high initial interfacial gap levels and the human in vivo

levels recorded from sites more distant from the cement such as seroma. From the data

shown in Figures 3 and 4, combinations of gentamicin and either ciprofloxacin or

vancomycin, each at 128 mg/L, eradicated the biofilms of both isolates but combining

ciprofloxacin and vancomycin together did not. Interestingly, despite both strains being

susceptible to 128mg/L of gentamicin when tested alone (Fig 1 and 2), the combination of

clindamycin or linezolid with gentamicin appeared to reduce its bactericidal effect markedly

(Figure 3).

Antibiotic activity of antibiotic combinations in PJI delivered locally and systemically

As some antibiotics cannot be delivered locally because of practical or safety issues,

they are administered systemically in addition to the antibiotics delivered locally. Further

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experiments were undertaken to model the effect of systemic doses of rifampicin or

daptomycin administered as part of a therapeutic combination. The dissolving bead assay

was repeated in triplicate with rifampicin at 4mg/L and daptomycin between 4 and 16mg/L

to reflect the concentrations achieved by systemic delivery.49,51,52 The other five antibiotics

had a final concentration of 128mg/L. From Figures 5 and 6 it can be seen that a similar

trend was observed in both strains, with a three-log reduction seen in

gentamicin/daptomycin combinations. Whilst the addition of ciprofloxacin or

vancomycin to gentamicin/daptomycin had little effect, clindamycin appeared to be

antagonistic towards gentamicin/daptomycin. Similarly, despite both strains being

susceptible to 128mg/L of gentamicin when tested alone (Fig 1 and 2), the addition of

clindamycin or rifampicin to the combination reduced its bactericidal effect markedly.

Gentamicin activity against Staphylococcus biofilms when used in combination

The results in Figures 3-6 were unexpected and potentially clinically relevant, as any

antagonism may raise the MBEC above levels that are clinically achievable, leading to

treatment failure. Another series of experiments was therefore designed to investigate what

effect the addition of a second antibiotic had on the MBEC of gentamicin for CNS-J and

MSSA-N. Six of the antibiotics were tested at fixed concentrations felt to represent likely in

vivo levels, combined with a range of gentamicin concentrations that varied from 8-2048

mg/L. Figures 7 and 8 revealed the same trends in both organisms. The addition of

daptomycin, vancomycin and ciprofloxacin to gentamicin resulted in a similar or lower

MBEC than when gentamicin was used as a single agent. Conversely, when rifampicin,

clindamycin or linezolid was added to gentamicin, there was an increase in the MBEC

compared to when gentamicin was used as a single agent.

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The fractional biofilm eradication concentration index

In order to quantify the apparent interactions seen and enable comparisons with

previous and future work the fractional biofilm eradication concentration (FBEC) index was

calculated for gentamicin when used in each antibiotic combination. The FBEC is a

modification of the fractional inhibitory concentration (FIC) index,53 which is a method used

to detect synergism or antagonism between two antibiotics. The data displayed in Figures 7

and 8 was analysed together with that in Figures 2 and 3 was used to calculate the FBEC

index (Table 2). Below is shown how the FBEC for gentamicin was calculated:

FBEC index = MBEC of antibiotic in combination / MBEC of antibiotic on its own

FBEC index = FBEC (x) + FBEC (g)

Where (x) is an antibiotic tested in combination with gentamicin (g).

An FBEC index of ≤ 0.5 indicates a synergistic effect, > 0.5 to ≤ 4.0 no interaction, and > 4.0

an antagonistic effect.54

Discussion

This study found that gentamicin and daptomycin were the only effective single-

agent antibiotics, when used at clinically achievable doses, against coagulase-positive and

negative Staphylococcus biofilms. Other antibiotics were ineffective either alone or in

combination (Fig 3, 4, 5, and 6). Combinations of bactericidal antibiotics (except

daptomycin) with gentamicin conferred a synergistic effect, whilst the bacteriostatic

antibiotics examined were found to antagonise gentamicin action.

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The differences seen between the seven different antibiotics examined

cannot be explained simply by the differences in their ability to penetrate the glycocalyx of

the biofilm. Indeed gentamicin, ciprofloxacin,55 daptomycin,56 rifampicin,57 linezolid,58 and

clindamycin,59 have all demonstrated good penetration into Staphylococcus biofilms whilst

only vancomycin has been shown to be significantly hampered by it.55,58 While gentamicin

and daptomycin have different mechanisms of action, both impact membrane integrity,56,60–

62 suggesting this may be an important aspect of the anti-staphylococcal effect, including

MRSA. Although MRSA has been reported to have different mechanisms of biofilm

formation from MSSA;63 tolerance to non-penicillin antimicrobials S. aureus biofilms has

been reported to be independent to the resistance mechanisms associated with MRSA

strains.64–67

The lack of an agonistic effect seen with rifampicin combined with either vancomycin

or ciprofloxacin is intriguing, as these combinations are commonly used in surgical practice

with apparent success.7 Olson et al68 who performed an in vitro study of CNS biofilms using

steady state antibiotics, also found that the addition of 12mg/L rifampicin to 40mg/L

vancomycin did not result in additional killing. However, when repeating the experiment in a

small animal model they reported the presence of a synergistic effect. In addition Zimmerli

et al found the combination of rifampicin (trough 1mg/L to peak 8mg/L) and ciprofloxacin

(trough 0.11mg/L to peak 1mg/L) or vancomycin (trough 3mg/L to peak 9mg/L) much more

effective than monotherapy. A possible explanation for this discrepancy may be in the

dosing regimens, which are constant in vitro but vary in vivo. Lewis12 postulated that cyclic

dosing that allowed persisters to start repopulating the biofilm just as they are exposed to

another bactericidal wave may eventually result in their eradication. The effect of the host’s

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immune system might also serve to enhance discrepancies between laboratory and clinical

studies.

A synergistic effect against a S. aureus biofilm seen when combining bactericidal

antibiotics with gentamicin has been previously reported by Tsuji et al69 who also found

gentamicin and daptomycin to be the most efficacious as single antibiotics whilst

vancomycin was ineffective. However, the strong antagonism seen between gentamicin and

linezolid, rifampicin, and clindamycin has, to the authors’ knowledge, not been observed

before.

Previous studies have reported that in the presence of a sub-inhibitory concentration

of DNA synthesis-inhibiting antibiotic, the addition of a protein synthesis inhibitor increases

the steady state growth rate of planktonic Escherichia coli and S. aureus cultures.70,71

Bollenbach et al reported that there is an optimal ratio between protein synthesis and DNA

replication. They reported that when only one of these processes is hampered the

subsequent inhibition of planktonic bacteria growth is greater than when both protein

synthesis and DNA replication are impaired.72 Therefore, many protein synthesis inhibitors

and DNA synthesis inhibitors in combination could produce higher growth than just a single

inhibitor by itself, leading to an antagonistic interaction.73

There are two postulates for the observed antagonism. Either exposure to the

additional antibiotics increased tolerance to gentamicin, or a chemical interaction between

the two antibiotics rendered both less active. Given the effect was observed with narrow

inter-quartile ranges in three antibiotics for whom interactions have not been published

before, the development of tolerance seems more likely. Staphylococci have a diverse

network of regulators that modify gene expression and enable them to tolerate a wide

range of environmental stresses which include antibiotics and cationic antimicrobial

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peptides (CAMPs). They are able to alter the proportion of the negatively-charged

polysaccharide intercellular adhesin and positively-charged teichoic acids in their

extracellular polymeric matrix and cell membrane via the GraRS system.74 These

modifications can confer significant tolerance to positively-charged CAMPs and

antimicrobials such as gentamicin , vancomycin, and daptomycin.75,76 Although the envelope

stress response has been previously found not to be induced by gentamicin, vancomycin, or

daptomycin77 the effect of bacteriostatic antibiotics is unknown.

Recent efforts to augment the cellular uptake of gentamicin, either by degrading the

integrity of the cell membrane or increasing the proton motive force have been described.78

CAMPs are normally produced as part of the host’s response but can be isolated from non-

pathogenic bacteria and have been shown to be synergistic with gentamicin against S.

aureus at clinically achievable doses.78 CAMPs form complexes with the lipids in the cell

membrane resulting in poration, allowing gentamicin to enter the cell.79 Both

aminoglycoside uptake80 and hydroxyl radical formation by bactericidal agents62 are energy

dependent via the proton motive force. It may be that bacteriostatic antibiotics lead to a

reduction in metabolic activity, leading to reduced gentamicin uptake and subsequent

oxidative damage. Kohanski et al81 postulated that the common final pathway for all

bactericidal drugs is overwhelming oxidative damage from hydroxyl radical formation. While

bacteriostatic drugs do not cause oxidative stresses, their effects may deplete the pool of

redox-active metabolic intermediates such as NAD(H).

It has been previously reported that combinations of gentamicin and clindamycin are

synergistic in the inhibition of staphylococcal biofilm formation. Neut et al82 reported that

the presence of clindamycin (or fusidic acid) in gentamicin ALAC had no influence on the

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elution dynamics of gentamicin, but the authors also concluded that the combinations of

gentamicin and clindamycin or fusidic acid are more effective in preventing in vitro biofilm

formation than bone cements with gentamicin as a single drug.82 A further study conducted

by Ensing et al reported similar results with greater growth inhibition associated with

gentamicin/clindamycin ALAC compared with gentamicin ALAC alone.83 However the

gentamicin/clindamycin ALAC contained double the starting amount of gentamicin found in

the mono-antimicrobial ALAC. In addition the combination ALAC not only released a greater

amount of gentamicin overall but did so at a faster rate throughout the treatment period. It

would seem reasonable that this higher dose of gentamicin released from the combination

ALAC would explain these findings.

While this study focused on short antibiotic exposures, mimicking the first few hours

of treatment, longer time courses of exposure may reveal different effects.66 However, there

is consensus that the initial 48h postoperatively are the most important to prevent

recolonization and biofilm formation. It has been shown that the very high concentrations

that are seen initially in vivo, tail off rapidly, and unless dosing is consistent with in vivo

pharmacokinetically, then this short exposure time is a reasonable estimate of in vivo

effects. A further limitation was that only as a partial checkerboard analysis could be

performed due to limited resources.

This study is the first to describe the antagonistic effect of bacteriostatic antibiotics

on gentamicin action against both coagulase-positive and -negative Staphylococcus biofilms.

There remains an urgent clinical need to optimise the management of staphylococcal PJI by

establishing the most efficacious antimicrobial regime to complement surgical debridement.

Gentamicin and daptomycin were found to be the only effective single-agent antibiotics,

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when used at clinically achievable locally-delivered concentrations, against Staphylococcus

biofilms. Interestingly the addition of a bacteriostatic antibiotic, at commonly used

concentrations, was found to antagonise the ability of gentamicin to eradicate

Staphylococcus biofilms. Further investigation of these findings may be useful in evaluating

optimal use of antibiotic combinations in the management of PJI.

Funding. This work was supported by the British Hip Society (McMinn Bursary to G.D),

London, United Kingdom; and the Royal College of Surgeons Edinburgh (Cutner Fellowship

to ST), Edinburgh, United Kingdom.

Transparency declarations. None to declare

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References

1. Hansen E, Belden K, Silibovsky R et al. Perioperative Antibiotics. J Orthop Res 2014; 32:

S31–59.

2. Wang X, Luo F, Huang K, Xie Z. Induced membrane technique for the treatment of bone

defects due to post-traumatic osteomyelitis. Bone Joint Res 2016; 5: 101–5.

3. Khan W, Morgan-Jones R. Debridement: Defining something we all do. J Trauma Orthop

2016; 4.

4. Espehaug B, Engesaeter LB, Vollset SE et al. Antibiotic prophylaxis in total hip

arthroplasty. Review of 10905 primary total hip replacements reported to the Norwegian

arthroplasty register, 1987 to 1995. J Bone Jt Surg [Br] 1997; 79: 590–5.

5. Thierse L. [Experiences with Refobacin-Palacos with regard to deep late infections

following hip-joint endoprosthesis surgery. A 4-years’ study (author’s transl)]. Zeitschrift fur

Orthopadie und ihre Grenzgebiete 1978; 116: 847–52.

6. Chiu F-Y, Chen C-M, Lin C-FJ et al. Cefuroxime-impregnated cement in primary total knee

arthroplasty: a prospective, randomized study of three hundred and forty knees. J Bone

Joint Surg Am 2002; 84–A: 759–62.

7. Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med 2004; 351:

1645–54.

8. Stockley I, Mockford BJ, Hoad-Reddick A et al. The use of two-stage exchange arthroplasty

with depot antibiotics in the absence of long- term antibiotic therapy in infected total hip

replacement. J Bone Jt Surg [Br] 2008; 90: 145–8.

9. Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks.

Nat Publ Gr 2010; 8: 423–35.

10. Johnson PJT, Levin BR. Pharmacodynamics, Population Dynamics, and the Evolution of

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

16

Persistence in Staphylococcus aureus. PLoS Genet 2013; 9: 1–13.

11. Edwards AM. Phenotype switching is a natural consequence of Staphylococcus aureus

replication. J Bacteriol 2012; 194: 5404–12.

12. Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol 2007; 5: 48–

56.

13. Greimel F, Scheuerer C, Gessner A et al. Efficacy of antibiotic treatment of implant-

associated Staphylococcus aureus infections with moxifloxacin, flucloxacillin, rifampin, and

combination therapy: an animal study. Drug Des Devel Ther 2017; Volume 11: 1729–36.

14. Anagnostakos K, Fürst O, Kelm J. Antibiotic-impregnated PMMA hip spacers: Current

status. Acta Orthop 2006; 77: 628–37.

15. Gallo J, Bogdanová K, Šiller M et al. [Microbial and pharmacological characteristics of

VancogenX]. Acta Chir Orthop Traumatol Cech 2013; 80: 69–76.

16. Bertazzoni Minelli E, Della Bora T et al. Different microbial biofilm formation on

polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin.

Anaerobe 2011; 17: 380–3.

17. Stengel D, Bauwens K, Sehouli J et al. Systematic review and meta-analysis of antibiotic

therapy for bone and joint infections. Lancet Infect Dis 2001; 1: 175–88.

18. Iarikov D, Demian H, Rubin D et al. Choice and doses of antibacterial agents for cement

spacers in treatment of prosthetic joint infections: Review of published studies. Clin Infect

Dis 2012; 55: 1474–80.

19. Ceri H, Olson ME, Stremick C et al. The Calgary Biofilm Device: New Technology for Rapid

Determination of Antibiotic Susceptibilities of Bacterial Biofilms. J Clin Microbiol 1999; 37:

1771–6.

20. Pettit RK, Weber CA, Pettit GR. Application of a high throughput Alamar blue biofilm

362

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374

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379

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susceptibility assay to Staphylococcus aureus biofilms. Ann Clin Microbiol Antimicrob 2009;

8: 1–7.

21. Zimmerli W, Frei R, Widmer AF et al. Microbiological tests to predict treatment outcome

in experimental device-related infections due to Staphylococcus aureus. J Antimicrob

Chemother 1994; 33: 959–67.

22. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob

Chemother 2001; 48: 5–16.

23. Hawkey PM. Pre-clinical experience with daptomycin. J Antimicrob Chemother 2008; 62:

7–14.

24. Barry AL, Fuchs PC, Brown SD. In Vitro Activities of Daptomycin against 2,789 Clinical

Isolates from 11 North American Medical Centers. Antimicrob Agents Chemother 2001; 45:

1919–22.

25. Fuchs PC, Barry AL, Brown SD. Daptomycin susceptibility tests: interpretive criteria,

quality control, and effect of calcium on in vitro tests. Diagn Microbiol Infect Dis 2000; 38:

51–8.

26. Nandi SK, Mukherjee P, Roy S et al. Local antibiotic delivery systems for the treatment of

osteomyelitis - A review. Mater Sci Eng C 2009; 29: 2478–85.

27. Adams K, Couch L, Cierny G et al. In Vitro and In Vivo Evaluation of Antibiotic Diffusion

From Antibiotic-Impregnated Polymethylmethacrylate Beads. Clin Orthop Relat Res 1992;

278: 244–52.

28. Taggart T, Kerry RM, Norman P et al. The use of vancomycin-impregnated cement beads

in the management of infection of prosthetic joints. J Bone Joint Surg Br 2002; 84: 70–2.

29. Dall GF, Tsang STJ, Gwynne PJ et al. The dissolvable bead: A novel in vitro biofilm model

for evaluating antimicrobial resistance. J Microbiol Methods 2017; 142: 46–51.

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18

30. Clinical and Laboratories Standards Institute. Methods for Determining Bactericidal

Activity of Antimicrobial Agents; Approved Guidelines M26-A. CLSI, Wayne, PA, USA 1999.

31. Girard LP, Ceri H, Gibb AP et al. MIC Versus MBEC to Determine the Antibiotic Sensitivity

of Staphylococcus aureus in Peritoneal Dialysis Peritonitis. Perit Dial Int 2010; 30: 652–6.

32. Miles A, Misra S, Irwin J. The Estimation of the Bactericidal Power of the Blood. J Hyg

(Lond) 1938; 6: 732–49.

33. Meyer J, Piller G, Spiegel CA et al. Vacuum-mixing significantly changes antibiotic elution

characteristics of commercially available antibiotic-impregnated bone cements. J Bone Joint

Surg Am 2011; 93: 2049–56.

34. Hendriks JGE, Neut DL, Van Horn JR et al. The Release of Gentamicin from Acrylic Bone

Cements in a Simulated Prosthesis-Related Interfacial Gap. J Biomed Mater Res Part B Appl

Biomater 2003; 64B: 1–5.

35. Hendriks JGE, Neut D, Van Horn JR et al. Bacterial survival in the interfacial gap in

gentamicin-loaded acrylic bone cements. J Bone Jt Surg [Br] 2005; 87: 272–6.

36. Lewis G. Properties of antibiotic-loaded acrylic bone cements for use in cemented

arthroplasties: A state-of-the-art review. J Biomed Mater Res Part B Appl Biomater 2009;

89B: 558–74.

37. Dall GF, Simpson PMS, Breusch SJ. In vitro comparison of Refobacin-Palacos R with

Refobacin Bone Cement and Palacos R + G. Acta Orthop 2007; 78: 404–11.

38. Hoff SF, Fitzgerald RH, Kelly PJ. The depot administration of penicillin G and gentamicin

in acrylic bone cement. J Bone Joint Surg Am 1981; 63: 798–804.

39. Weiss BD, Weiss EC, Haggard WO et al. Optimized elution of daptomycin from

polymethylmethacrylate beads. Antimicrob Agents Chemother 2009; 52: 264–6.

40. Nelson CL, Griffin FM, Harrison BH et al. In Vitro Elution Characteristics of Commercially

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

19

and Noncommercially Prepared Antibiotic PMMA Beads. Clincial Orthop Relat Res 1992;

284: 303–9.

41. Anguita-Alonso P, Rouse MS, Piper KE et al. Comparative study of antimicrobial release

kinetics from polymethylmethacrylate. Clin Orthop Relat Res 2006; 445: 239–44.

42. Mader JT, Calhoun J, Cobos J. In Vitro Evaluation of Antibiotic Diffusion from Antibiotic-

Impregnated Biodegradable Beads and Polymethylmethacrylate Beads. Antimicrob Agents

Chemother 1997; 41: 415–8.

43. Wang J-S, Franzen H, Lidgren L. Interface gap after implantation of a cemented femoral

stem in pigs. Acta Orthop Scand 1999; 70: 234–9.

44. Stravinskas M, Horstmann P, Ferguson J et al. Pharmacokinetics of gentamicin eluted

from a regenerating bone graft substitute: In vitro and clinical release studies. Bone Joint

Res 2016; 5: 427–35.

45. Wendling A, Mar D, Wischmeier N et al. Combination of modified mixing technique and

low frequency ultrasound to control the elution profile of vancomycin-loaded acrylic bone

cement. Bone Joint Res 2016; 5: 26–32.

46. Chohfi M, Langlais F, Fourastier J et al. Pharmacokinetics, uses, and limitations of

vancomycin-loaded bone cement. Int Orthop 1998; 22: 171–7.

47. Anagnostakos K, Kelm J. Enhancement of antibiotic elution from acrylic bone cement. J

Biomed Mater Res - Part B Appl Biomater 2009; 90: 467–75.

48. Fink B, Vogt S, Reinsch M et al. Sufficient release of antibiotic by a spacer 6 weeks after

implantation in two-stage revision of infected hip prostheses. Clin Orthop Relat Res 2011;

469: 3141–7.

49. Acocella G. Clinical pharmacokinetics of rifampicin. Clin Pharmacokinet 1978; 3: 108–27.

50. Cortes NJ, Lloyd JM, Koziol L et al. Successful clinical use of daptomycin-impregnated

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

20

bone cement in two-stage revision hip surgery for prosthetic joint infection. Ann

Pharmacother 2013; 47.

51. Mader JT, Adams K. Comparative Evaluation of Daptomycin (LY146032) and Vancomycin

in the Treatment of Experimental Methicillin-Resistant Staphylococcus aureus Osteomyelitis

in Rabbits. Antimicrob Agents Chemother 1989; 33: 689–92.

52. Traunmuller F, Schintler M V., Metzler J et al. Soft tissue and bone penetration abilities

of daptomycin in diabetic patients with bacterial foot infections. J Antimicrob Chemother

2010; 65: 1252–7.

53. Greco WR, Bravo G, Parsons JC. The Search for Synergy: A Critical Review from a

Response Surface Perspective*. Pharmacol Rev 1995; 47: 332–85.

54. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J

Antimicrob Chemother 2003; 52: 1–1.

55. Singh R, Ray P, Das A et al. Penetration of antibiotics through Staphylococcus aureus and

Staphylococcus epidermidis biofilms. J Antimicrob Chemother 2010; 65: 1955–8.

56. Stewart PS, Davison WM, Steenbergen JN. Daptomycin Rapidly Penetrates a

Staphylococcus epidermidis Biofilm. Antimicrob Agents Chemother 2009; 53: 3505–7.

57. Zheng Z, Stewart PS. Penetration of Rifampin through Staphylococcus epidermidis

Biofilms. Antimicrob Agents Chemother 2002; 46: 900–3.

58. Mathur T, Singhal S, Khan S et al. Adverse effect of staphylococci slime on in vitro activity

of glycopeptides. Jpn J Infect Dis 2005; 58: 353–7.

59. Farber BF, Kaplan MH, Clogston AG. Staphylococcus epidermidis Extracted Slime Inhibits

the Antimicrobial Action of Staphylococcus epidennidis Extracted Slime Inhibits the

Antimicrobial Action of Glycopeptide Antibiotics. Source J Infect Dis 1990; 16121517: 37–40.

60. Silverman JA, Perlmutter NG, Shapiro HM. Correlation of Daptomycin Bactericidal

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

21

Activity and Membrane Depolarization in Staphylococcus aureus. Antimicrob Agents

Chemother 2003; 47: 2538–44.

61. Hobbs JK, Miller K, O’Neill AJ et al. Consequences of daptomycin-mediated membrane

damage in Staphylococcus aureus. J Antimicrob Chemother 2008; 62: 1003–8.

62. Kohanski MA, Dwyer DJ, Wierzbowski J et al. Mistranslation of Membrane Proteins and

Two-Component System Activation Trigger Antibiotic-Mediated Cell Death. Cell 2008; 135:

679–90.

63. Fitzpatrick F, Humphreys H, O’Gara JP. Environmental regulation of biofilm development

in methicillin-resistant and methicillin-susceptible Staphylococcus aureus clinical isolates. J

Hosp Infect 2006; 62: 120–2.

64. Kostenko V, Salek MM, Sattari P et al. Staphylococcus aureus biofilm formation and

tolerance to antibiotics in response to oscillatory shear stresses of physiological levels. FEMS

Immunol Med Microbiol 2010; 59: 421–31.

65. Kostenko V, Ceri H, Martinuzzi RJ. Increased tolerance of Staphylococcus aureus to

vancomycin in viscous media. FEMS Immunol Med Microbiol 2007.

66. Rose WE, Poppens PT. Impact of biofilm on the in vitro activity of vancomycin alone and

in combination with tigecycline and rifampicin against Staphylococcus aureus. J Antimicrob

Chemother 2009; 63: 485–8.

67. Fernandez-Hidalgo N, Gavalda J, Almirante B et al. Evaluation of linezolid, vancomycin,

gentamicin and ciprofloxacin in a rabbit model of antibiotic-lock technique for

Staphylococcus aureus catheter-related infection. J Antimicrob Chemother 2010; 65: 525–

30.

68. Olson ME, Slater SR, Rupp ME et al. Rifampicin enhances activity of daptomycin and

vancomycin against both a polysaccharide intercellular adhesin (PIA)-dependent and -

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

22

independent Staphylococcus epidermidis biofilm. J Antimicrob Chemother 2010; 65: 2164–

71.

69. Tsuji BT, Rybak MJ. Short-Course Gentamicin in Combination with Daptomycin or

Vancomycin against Staphylococcus aureus in an In Vitro Pharmacodynamic Model with

Simulated Endocardial Vegetations. Antimicrob Agents Chemother 2005; 49: 2735–45.

70. Chait R, Craney A, Kishony R. Antibiotic interactions that select against resistance.

Nature 2007; 446: 668–71.

71. Yeh P, Tschumi AI, Kishony R. Functional classification of drugs by properties of their

pairwise interactions. Nat Genet 2006; 38: 489–94.

72. Bollenbach T, Quan S, Chait R et al. Nonoptimal microbial response to antibiotics

underlies suppressive drug interactions. Cell 2009; 139: 707–18.

73. Beppler C, Tekin E, White C et al. When more is less: Emergent suppressive interactions

in three-drug combinations. BMC Microbiol 2017; 17: 1–9.

74. Weidenmaier C, Peschel A, Kempf VAJ et al. DltABCD- and MprF-Mediated Cell Envelope

Modifications of Staphylococcus aureus Confer Resistance to Platelet Microbicidal Proteins

and Contribute to Virulence in a Rabbit Endocarditis Model. Infect Immun 2005; 73: 8033–8.

75. Kelley WL, Lew DP, Renzoni A. Antimicrobial Peptide Exposure and Reduced

Susceptibility to Daptomycin: Insights Into a Complex Genetic Puzzle. J Infect Dis 2012; 206:

1153–6.

76. Cui L, Lian J-Q, Neoh H-M et al. DNA microarray-based identification of genes associated

with glycopeptide resistance in Staphylococcus aureus. Antimicrob Agents Chemother 2005;

49: 3404–13.

77. Yang S-J, Bayer AS, Mishra NN et al. The Staphylococcus aureus Two-Component

Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

23

Peptides. Infect Immun 2012; 80: 74–81.

78. Mataraci E, Dosler S. In vitro activities of antibiotics and antimicrobial cationic peptides

alone and in combination against methicillin-resistant Staphylococcus aureus biofilms.

Antimicrob Agents Chemother 2012; 56: 6366–71.

79. Zendo T, Yoneyama F, Sonomoto K. Lactococcal membrane-permeabilizing antimicrobial

peptides. Appl Microbiol Biotechnol 2010; 88: 1–9.

80. Taber HW, Mueller JP, Miller PF et al. Bacterial uptake of aminoglycoside antibiotics.

Microbiol Rev 1987; 51: 439–57.

81. Kohanski MA, Dwyer DJ, Hayete B et al. A Common Mechanism of Cellular Death

Induced by Bactericidal Antibiotics. Cell 2007; 130: 797–810.

82. Neut DD, de Groot EP, Kowalski RSZ et al. Gentamicin-loaded bone cement with

clindamycin or fusidic acid added: Biofilm formation and antibiotic release. J Biomed Mater

Res Part A 2005; 73A: 165–70.

83. Ensing GT, Horn JR van, Mei HC van der et al. Copal Bone Cement Is More Effective in

Preventing Biofilm Formation than Palacos R-G. Clin Orthop Relat Res 2008; 466: 1492.

530

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532

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539

540

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542

543

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24

Table 1. Antibiotics commonly used to treat Staphylococcal periprosthetic joint infections

and

their modes of action. Adapted from Kohanski et al.6

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Class Examples Cellular Target Effect

Fluoroquinolones Ciprofloxacin and levofloxacin

Synthetic

DNA gyraseBactericidal by interfering with DNA replication and cell division

RifamycinsRifampin

Semisynthetic derivative of naturally occurring Rifamycin SV produced by Streptomyces mediterranei

DNA-dependent RNApolymerase

Both bacteriostatic by blocking RNA transcription and bactericidal damaging DNA if oxidised

β-lactams

Penicillin - naturally derived from Penicillium fungi

Semisynthetic - Ampicillin, Flucoxacillin, Cephalosporins and Carbapenems

Penicillin- binding proteins in cell wall

Bactericidal by preventing peptidoglycan cross linkage in cell wall, cell division and autolysin activation

Glycopeptides

Vancomycin and teicoplanin

Semisynthetic derived from Actinobacteria Spp.

Terminal dipeptide of Peptidoglycan in cell wall

Bactericidal by preventing transglycosylation, transpeptidation of cell wall and autolysin activation. Less rapid than penicillin as larger molecule cannott penetrate entire wall.

LipopeptidesDaptomycin and Polymixin B

Derived from Streptomyces Spp.

Cell membraneBactericidal. Rapid depolarisation of the membrane potential.

Aminoglycosides

Gentamicin, Tobramycin, Streptomycin Kanamycin Natural and semi-synthetic amino sugars derived from Streptomyces and Micromonospora Spp.

30S ribosome Bactericidal. Protein translation (mistranslation by tRNA mismatching)

TetracyclinesTetracycline and Doxycycline.

Semi-synthetic -derived from Streptomyces Spp

30S ribosomeBacteriostatic by blocking aminoacyl tRNA binding to ribosome and preventing translation of proteins

MacrolidesErythromycin Clindamycin and Azythromycin.

Semi-synthetic derived from Streptomyces Spp.

50S ribosomeBacteriostatic by inhibition of elongation and translocation steps during protein translation and free tRNA depletion.

Oxazolidinone Synthetic - Linezolid 50S ribosomeBacteriostatic. Inhibits protein translation through inhibition of initiation step.

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574 575

26

Table 2: Fractional biofilm eradication concentration index (FBEC) for MSSA-N and CNS-J.

Efficacy of gentamicin as a monotherapy in relation to commonly used antibiotic combinations

(data from Figures 2, 7, and 8).

Antibiotic Vancomycin Daptomycin Clindamycin Ciprofloxacin Rifampicin Linezolid

MSSA-N

MBEC in combination 128 4 128 128 4 128

MBEC in isolation 2048 16 2048 2048 2048 2048

FBEC 0.0625 0.2500 0.0625 0.0625 0.0020 0.0625Gentamicin

MBEC in combination

32 128 2048 64 2048 2048

Gentamicin MBEC in isolation

256 256 256 256 256 256

FBEC 0.1250 0.5000 8.0000 0.25 8 8

FBEC index0.1875

Synergistic

0.7500

No interaction

8.0625

Antagonistic

0.3125

Synergistic

8.0020

Antagonistic

8.0625

AntagonisticCNS-J

MBEC in combination 128 8 128 128 4 128

MBEC in isolation 2048 32 2048 2048 2048 2048

FBEC 0.0625 0.2500 0.0625 0.0625 0.0020 0.0625Gentamicin

MBEC in combination

32 16 1024 64 512 256

Gentamicin MBEC in isolation

32 32 32 32 32 32

FBEC 1.0000 0.500 32.0000 2.0000 16.0000 8.0000

FBEC index1.0625

No interaction

0.7500

No interaction

32.0625

Antagonistic

2.0625

No interaction

16.0020

Antagonistic

8.0625

Antagonistic

576

577

578

579

580581

27

Figure 1. Median log density (fitted circles) & inter-quartile ranges (bars) of CNS-J after exposure to single

antibiotics. MBEC represents a log reduction of 3 from growth controls.

Figure 2. Median log density (fitted circles) & inter-quartile ranges (bars) of MSSA-N after exposure to single

antibiotics. MBEC represents a log reduction of 3 from growth controls.

582

583

584

585

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588

589

590

28

Figure 3. Median & inter-quartile ranges (bars) of CNS-J recovery after 3- hour exposure to a combination of

2 or 3 antibiotics at 128mg/L concentrations. (G–Gentamicin, V-Vancomycin, CL- Clindamycin, CP-

Ciprofloxacin, LIN-Linezolid)

Figure 4. Median & inter-quartile ranges (bars) of MSSA-N recovery after 3- hour exposure to a combination

of 2 or 3 antibiotics at 128mg/L concentrations. (G–Gentamicin, V-Vancomycin, CL- Clindamycin, CP-

Ciprofloxacin, LIN-Linezolid)

591

592

593

594

595

596

597

598

599

600

29

Figure 5. Median & inter-quartile range of CNS-J recovery after 3-hour exposure to a combination of 2 or

3 antibiotics at 128, 16 or 4mg/L concentrations. (G128 – 128mg/L Gentamicin, R4 – 4mg/L Rifampicin,

D16 – Daptomycin 16mg/L, V- Vancomycin, CL- Clindamycin, CP-Ciprofloxacin, LIN-Linezolid)

601602

603

604

605606

607

608

30

Figure 6. Median & inter-quartile range of MSSA-N recovery after 3-hour exposure to a combination of 2

or 3 antibiotics at 128, 16 or 4mg/L concentrations. (G128 – 128mg/L Gentamicin, R4 – 4mg/L

Rifampicin, D16 – Daptomycin 16mg/L, V-Vancomycin, CL- Clindamycin, CP-Ciprofloxacin, LIN-Linezolid)

Figure 7a. Median log density (fitted circles) & inter-quartile ranges (bars) of MSSA-N recovery after 3-hour

exposure to gentamicin alone or in combination with daptomycin, ciprofloxacin or vancomycin.

Figure 7b. Median log density (fitted circles) & inter-quartile ranges (bars) of CNS-J recovery after 3-hour

exposure to gentamicin alone or in combination with daptomycin, ciprofloxacin or vancomycin.

609

610

611

612

613

614

615

616

617

618

619

620

31

Figure 8a. Median log density (fitted circles) & inter-quartile ranges (bars) of MSSA-N recovery after 3-hour

exposure to gentamicin alone or in combination with rifampicin, clindamycin or linezolid.

Figure 8b. Median log density (fitted circles) & inter-quartile ranges (bars) of CNS-J recovery after 3-hour

exposure to gentamicin alone or in combination with rifampicin, clindamycin or linezolid.

621

622

623

624625

626

627

628

629

32