Anti-staphylococcal activity and mode of action of clofazimine

Brunello Oliva1, Alexander John O’Neill2, Keith Miller2, William Stubbings2 and Ian Chopra2,*

1 Department of Experimental Medicine, University of L’Aquila, Coppito-67100, L’Aquila, Italy; 2 Antimicrobial Research Centre and Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

Received 27 August 2003; returned 8 December 2003; revised 15 December 2003; accepted 16 December 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Infections caused by Staphylococcus aureus might be treated with agents whose primary indications are for other infections. Clofazimine, an established anti-mycobacterial drug, could be such a candidate. However, the anti-staphylococcal properties of clofazimine have not been fully described and its mode of action, possibly involving inhibition of both RNA polymerase and a membrane-located target, has not been explored in detail. We have now conducted experiments to address these issues.

Methods: Using established procedures, we examined the activity of clofazimine against a range of clinical isolates of S. aureus and determined whether it was bactericidal, exhibited a post-antibiotic effect (PAE), or interacted synergically with other agents. The potential for emergence of clofazimine-resistant mutants was also examined. Mode of action studies involved macromolecular synthesis assays, cross-screening against rifampicin-resistant mutants, susceptibility of RNA polymerase to clofazimine in vitro and several methods to detect drug-induced membrane damage.

Results: Clofazimine demonstrated good anti-staphylococcal activity encompassing MSSA, MRSA and GISA. It was bactericidal and resistant mutants could not be isolated. Clofazimine did not exhibit a PAE and failed to act synergically with other drugs. No evidence for specific inhibition of RNA polymerase was obtained. Clofazimine caused non-specific inhibition of DNA, RNA and protein synthesis, consistent with membrane-damaging activity that was detected in three independent assays for membrane disrupting agents.

Conclusions: Clofazimine is a potent anti-staphylococcal agent. It appears to be a membrane-disrupting agent and does not inhibit RNA polymerase.

Keywords: Staphylococcus aureus, mechanisms of antibiotic action, membrane-disrupting agent


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance to antibiotics is becoming an increasingly difficult problem in the management of bacterial infections.1 The situation is particularly critical for Staphylococcus aureus where methicillin-resistant (MRSA) and vancomycin intermediate resistant (VISA) strains have emerged, that are also frequently resistant to multiple classes of antibiotics.2 Recent reports of high-level vancomycin resistance in MRSA as a result of acquisition of the vanA determinant from enterococci3,4 and the emergence of MRSA resistant to linezolid5 are further disturbing trends in the evolution of antimicrobial resistance in S. aureus.

One approach to the problem of antibiotic resistance in bacteria, including S. aureus, involves the discovery and development of new antimicrobials.1,2 However, possibilities for introducing new drugs are diminishing since the pharmaceutical industry is now placing less emphasis on research and development of antibacterial agents.1,6 It may therefore be necessary to consider other strategies to control bacterial infections, including those caused by S. aureus.

One possibility could be extension of the therapeutic application of agents used for other infections to indications involving S. aureus. A similar strategy was recently suggested for Mycobacterium tuberculosis after it was noted that a number of commonly used anti-fungal and anti-helmintic drugs displayed significant anti-mycobacterial activity in vitro.7 During a search for drug candidates that might have application for the therapy of staphylococcal infections, we noted that the synthetic phenazine drug clofazimine, which is effective in the treatment of leprosy,8 also possesses anti-staphylococcal activity.911 In addition to reports that clofazimine is active against S. aureus there are other interesting aspects of this drug that could make it attractive for application as an anti-staphylococcal agent. In S. aureus (and other organisms), clofazimine is reported to stimulate phospholipase A2 activity leading to generation of lysophospholipids that specifically disrupt potassium transport.11,12 However, the possibility that RNA polymerase might also be a target for clofazimine is suggested by reports of cross-resistance between clofazimine and rifampicin and claims for direct inhibition of the enzyme by this phenazine drug.13,14 A dual mode of action for clofazimine could be advantageous in minimizing emergence of resistance in staphylococci since mutations might be required in both targets to confer resistance.15

Although clofazimine might have potential against S. aureus, the accepted pre-clinical studies16 required for a comprehensive evaluation of this drug as a possible anti-staphylococcal agent have not been carried out. Furthermore, whether clofazimine interferes with potassium transport and RNA polymerase in S. aureus is unknown. In this paper we present the results of experiments that have examined both the in vitro antimicrobial activity of clofazimine against S. aureus and its mode of action. In some cases, the anti-staphylococcal activity of clofazimine was also directly compared with other anti-staphylococcal drugs that are in clinical use.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. aureus strains

S. aureus 8325-4 was used as a standard laboratory strain. Rifampicin-resistant mutants of 8325-4 have been described elsewhere.17 Clinical strains used for comparative susceptibility testing were isolates maintained in a culture collection belonging to the Division of Microbiology, University of Leeds.

Growth media and antimicrobial agents

Mueller–Hinton broth (MHB) and agar (MHA), and Iso-Sensitest broth (ISB) and agar (ISA) were purchased from Oxoid (Basingstoke, UK).

Ciprofloxacin, mupirocin, linezolid and protegrin IB-367 were gifts, respectively, from Bayer AG (Leverkusen, Germany), GlaxoSmithKline Beecham Pharmaceuticals (Harlow, Essex, UK), Pharmacia & Upjohn (Kalamazoo, MI, USA) and IntraBiotics Pharmaceuticals (Mountain View, CA, USA). Other antimicrobial agents, including clofazimine, were purchased from Sigma–Aldrich (Poole, UK).

Biochemical reagents and radiochemicals

The BacLight kit to assess membrane damage was from Molecular Probes, Inc. (Eugene, OR, USA). Sigma factor-saturated Escherichia coli RNA polymerase was purchased from Amersham Biosciences (Amersham, UK) and the plasmid pGEM ß-gal from Promega (Southampton, UK). All other reagents were obtained from standard commercial sources.

The following radiolabelled chemicals were purchased from Amersham Biosciences: [methyl-3H]thymidine (70–85 Ci/mmol), [5-3H]uridine (25–30 Ci/mmol), L-[3,4-3H]glutamine (20–50 Ci/mmol) and [5-3H]UTP (14 mCi/mmol).

Determination of susceptibility to antimicrobial agents

Minimum inhibitory concentrations (MICs) were determined by agar dilution on MHA or ISA using an inoculum in MHB, or ISB, of 106 cfu/spot.18 The MIC was defined as the lowest concentration of antibiotic completely inhibiting visible growth after 18–24 h of incubation at 37°C. Nisin was activated as previously described.19 The in vitro anti-staphylococcal activity of clofazimine has only been reported for a limited number of strains.9,11 Furthermore, comparative data with other anti-staphylococcal agents are not available. Therefore the activity of clofazimine against a number of clinical isolates of S. aureus (MSSA, MRSA and VISA) was determined by dilution in MHA and compared with cefotaxime, ceftriaxone, cefepime, fusidic acid, linezolid, oxacillin, quinupristin–dalfopristin and vancomycin.

Effects of clofazimine on culture turbidity and bacterial viability

The effects of clofazimine on the growth and survival of strain 8325-4 over a 6 h period were determined for liquid cultures grown in MHB.20

Determination of mutation frequencies for resistance to antimicrobial agents

This was carried out as described by O’Neill et al.21 using ISA. Both standard and concentrated cell techniques were used, whereby mutation frequencies as low as 1 in 10–11 can be detected. Plates were usually incubated at 37°C for 24 h, but were incubated for up to 96 h if no resistant colonies were detectable at 24 h.

Measurement of post-antibiotic effect (PAE)

PAEs were determined with strain 8325-4 using a standard viable count method as previously described.20 Bacteria were exposed to x5 the MIC of the antimicrobial agent for 60 min in MHB before removal of the drug by a 10–3 dilution.

Chequerboard testing for synergic interactions between clofazimine and other antimicrobial agents

This was carried out with S. aureus 8325-4 cultured in MHB using the broth culture procedure described by Eliopoulos & Moellering.22

Macromolecular synthesis

DNA, RNA and protein synthesis were monitored in mid-exponential phase cultures of S. aureus 8325-4 (108 cfu/mL in MHB) by the incorporation of the radiolabelled precursors [methyl-3H]thymidine, [5-3H]uridine and L-[3,4-3H]glutamine acid into the macromolecular (trichloroacetic acid-precipitable) fraction as previously described.20 Final concentrations were 1 µCi/mL for each of the precursors which were added to cultures 3 min before the addition of antibiotics (x4 MIC). After a further 10 min incubation at 37°C in the presence of antibiotics, samples were taken for determination of radioactive incorporation into DNA, RNA or protein and the data expressed as a percentage of incorporation into a drug-free control as described by Hilliard et al.23

Effect of clofazimine on RNA polymerase in vitro

RNA polymerase was assayed in vitro as described previously.24 This involved determining the incorporation of [5-3H]UTP into RNA catalysed by sigma-saturated E. coli RNA polymerase using the plasmid template pGEM ß-gal.

Assays for bacterial membrane damage

Three assays were used to detect bacterial membrane damage induced by clofazimine. These were: (a) the BacLight method of Molecular Probes Inc. as previously described,20 (b) leakage of ATP,25 and (c) loss of 260 nm absorbing material.26 In each assay, S. aureus 8325-4 was exposed to clofazimine and other antimicrobial agents used as controls at x4 MIC for 10 min and the results related to untreated, drug-free, bacterial suspensions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro anti-staphylococcal activity of clofazimine

Clofazimine was active against all organisms in the panel of 67 strains tested, displaying MICs in the range 0.25–4 mg/L. This included activity against strains resistant to established agents (Table 1).


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Table 1. Activity of clofazimine and other antimicrobial agents against clinical isolates of S. aureus in MHA
 
Susceptibility of S. aureus 8325-4 to various antimicrobial agents including clofazimine

A number of antimicrobial agents, including clofazimine, were used in subsequent, more detailed studies with strain S. aureus 8325-4. The MIC values of these agents against 8325-4 are recorded in Table 2.


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Table 2. Activity of clofazimine and other antimicrobial agents against S. aureus 8325-4
 
Effect of clofazimine on growth and survival of S. aureus 8325-4

Clofazimine was bactericidal, since addition of the agent at x4 MIC resulted in a 90% decline in viability over 6 h (Figure 1). However, loss of viability was not accompanied by lysis of bacteria, since culture turbidity did not decline during the 6 h period of exposure to clofazimine (Figure 2).



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Figure 1. Effect of clofazimine on survival of S. aureus 8325-4. Clofazimine (CLM) was added at x4 MIC to an early logarithmic phase culture in MHB at time zero and samples taken at the times indicated for determination of viable bacteria. The remainder of the culture served as a drug-free control.

 


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Figure 2. Effect of clofazimine on growth of S. aureus 8325-4. Clofazimine (CLM) was added at x4 MIC to an early logarithmic phase culture in MHB at time zero and samples taken at the times indicated for determination of culture absorbance at 600 nm. The remainder of the culture served as a drug-free control.

 
PAE of clofazimine and synergy with other antimicrobial agents

Cultures of S. aureus 8325-4 were exposed to x5 the clofazimine MIC for 60 min before washout, and the PAE determined as described in the Materials and methods section. Clofazimine demonstrated no PAE (data not shown). In contrast, rifampicin, which was used as a control agent, demonstrated a PAE of 180 min. This value agrees with data from the literature.20

Possible synergy between clofazimine and a number of other anti-staphylococcal agents (mupirocin, fusidic acid, rifampicin and vancomycin) was examined using a chequerboard technique. In each case, indifferent responses were observed. Thus the results obtained when clofazimine was combined with each of the other drugs did not differ from the inhibition observed with the most effective drug alone (data not shown).

Mutation frequency for resistance to clofazimine and comparison with other agents

Spontaneous mutants of S. aureus 8325-4 resistant to clofazimine could not be recovered (frequency < 10–11) by direct plating of organisms onto ISA containing the antimicrobial agent at x4 MIC. In contrast, mutants resistant to fusidic acid, rifampicin, norfloxacin, mupirocin and ciprofloxacin were recovered at frequencies between 7 x 10–6 and 1 x 10–8.

Effect of clofazimine on macromolecular synthesis in S. aureus 8325-4

Clofazimine was tested at x4 MIC in three macromolecular synthesis assays to evaluate its effect on the incorporation of radioactive precursors into DNA, RNA and protein during a period of short exposure to the drug. The effects observed with clofazimine were compared to those exhibited by known, specific, inhibitors of DNA, RNA and protein synthesis (respectively ciprofloxacin, rifampicin and tetracycline) each at x4 MIC. The control antibiotics behaved as expected in this assay23 with ciprofloxacin preferentially inhibiting DNA synthesis, rifampicin preferentially inhibiting RNA synthesis and tetracycline preferentially inhibiting protein synthesis (Figure 3). In contrast, clofazimine caused moderate inhibition of incorporation of precursors into all three macromolecular classes and did not display preferential inhibition (Figure 3).



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Figure 3. Effect of clofazimine and control agents at x4 MIC on DNA, RNA and protein synthesis in S. aureus 8325-4 measured by incorporation of radiolabelled precursors. Incorporation is shown as a percentage of that in drug-free controls.

 
Activity of clofazimine on mutants of S. aureus 8325-4 with defined rifampicin resistance mutations in rpoB

Cross-resistance between clofazimine and rifampicin has been reported in mycobacteria.13,14 However, using a set of rifampicin-resistant mutants of S. aureus 8325-4 with defined mutations in rpoB17 we were unable to demonstrate cross-resistance with clofazimine (data not shown).

Effect of clofazimine on RNA polymerase activity

It has been claimed that the antimicrobial activity of clofazimine may reflect inhibition of prokaryotic RNA polymerase.13,14 Although we have not developed RNA polymerase assays with the enzyme from S. aureus, we have established a related assay with purified E. coli RNA polymerase.24 Accordingly, we examined the activity of clofazimine in the E. coli-based in vitro enzyme assay. At concentrations up to 256 mg clofazimine/L (i.e. x128 the MIC for S. aureus 8325-4), no inhibition of RNA polymerase activity was observed (data not shown). Rifampicin, included as a positive control, exhibited a 50% inhibitory concentration of 0.02 mg/L in this assay.

Membrane-damaging activity of clofazimine

The antimicrobial activity of clofazimine against S. aureus and other bacteria has been attributed to specific effects on membrane-located potassium transport systems.11,12 However, for reasons presented in the discussion, we sought evidence that clofazimine has more general membrane-damaging effects in S. aureus.

Bacterial membrane damage was initially examined using the BacLight assay (Table 3). In this assay, agents which produce permeability values <=40% of the control are considered to be membrane-damaging agents.23 As expected, CTAB, nisin and protegrin severely compromised the staphylococcal membrane with BacLight assay values <20% of control, whereas ciprofloxacin and tetracycline had no effect on membrane integrity. Clofazimine exhibited a mean BacLight value of 41.7% (Table 3), indicating moderate membrane-damaging activity in this assay. However, clofazimine displayed strong membrane-damaging activity, comparable to the known membrane disrupting agents CTAB, nisin and protegrin when assays for leakage of 260 nm absorbing material and ATP were employed (Table 3). As expected, tetracycline and ciprofloxacin, which do not exhibit short-term membrane damaging effects, displayed negative responses in these assays.


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Table 3. Effects of clofazimine and other antimicrobial agents at x4 MIC on membrane integrity in S. aureus 8325-4 measured by the BacLight, 260 nm leakage and ATP release assays
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently there has been considerable interest in using older, relatively unexploited antimicrobial agents, for the treatment of current bacterial infections, especially as resistance to established agents continues to emerge.2729 In this context clofazimine, which is an established anti-mycobacterial drug,8 has already been suggested as a possible agent for treatment of infections caused by Enterococcus faecalis and Streptococcus pneumoniae.30,31 Detailed studies on its potential application as an anti-staphylococcal agent have not been reported. However, the results reported here indicate that clofazimine exhibits some interesting anti-staphylococcal properties. Thus clofazimine demonstrated good anti-staphylococcal activity encompassing MSSA, MRSA and GISA. Furthermore, clofazimine is bactericidal and spontaneous mutants resistant to the drug do not readily arise, at least in strain 8325-4. Nevertheless, although resistance did not develop in this laboratory strain there is a possibility that it could emerge in other strains of S. aureus in vitro, or during clinical application of the drug. Although clofazimine is well tolerated and free of toxicity in man in doses up to 100 mg/day, it does cause reversible skin discolouration. Therefore, although some of the in vitro anti-staphylococcal properties of clofazimine described in this paper are quite promising, the compound can probably only be regarded as a potential lead molecule for future medicinal chemistry approaches to improve antimicrobial potency and minimize adverse side effects. Indeed, synthesis of clofazimine analogues that produce less skin pigmentation, yet retain anti-mycobacterial activity has already been achieved.32

The mode of action of clofazimine is intriguing. Despite earlier claims that the agent displays cross-resistance with rifampicin-resistant mycobacteria, and inhibits RNA polymerase,13,14 we found no evidence that this enzyme is a target for clofazimine. Thus no cross-resistance was exhibited with rifampicin-resistant (rpoB) S. aureus mutants and clofazimine failed to inhibit E. coli RNA polymerase in vitro. However, it should be noted with respect to the RNA polymerase assay that the enzyme is derived from an organism (E. coli) that is naturally resistant to clofazimine.10 Furthermore, in whole cell macromolecular synthesis assays with S. aureus, clofazimine did not preferentially inhibit RNA synthesis, whereas the known RNA polymerase inhibitor rifampicin was a highly selective inhibitor of this process.

In S. aureus (and other organisms), clofazimine apparently stimulates phospholipase A2 activity, leading to generation of lysophospholipids that specifically interfere with potassium transport.1012 Nevertheless, we doubt that clofazimine has such a specific mode of action. Protein synthesis is particularly susceptible to depletion of potassium.33 However, in our whole cell macromolecular synthesis assays with S. aureus, clofazimine failed to preferentially inhibit protein synthesis and indeed caused similar levels of inhibition for DNA, RNA and protein synthesis. These effects on macromolecular synthesis are consistent with non-specific membrane damage.20,23 Indeed, further evidence that clofazimine caused generalized membrane disruption was obtained in the BacLight assay and the ATP and 260 nm leakage experiments. We therefore conclude that the bactericidal activity and low potential for resistance exhibited by clofazimine in S. aureus are associated with generalized membrane-disrupting effects. Such properties are shared with other membrane-damaging agents.34


    Footnotes
 
* Corresponding author. Tel: +44-113-233-5604; Fax: +44-113-233-5638; E-mail: i.chopra{at}leeds.ac.uk Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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