In vitro activities of cefotaxime, vancomycin, quinupristin/dalfopristin, linezolid and other antibiotics alone and in combination against Propionibacterium acnes isolates from central nervous system infections

Francine Mory1, Sébastien Fougnot1, Christian Rabaud2, Hélène Schuhmacher2 and Alain Lozniewski1,*

1 Laboratoire de Bactériologie-Virologie, UMR-CNRS 7565, Faculté de Médecine, 54500 Vandoeuvre-les-Nancy; 2 Service des Maladies Infectieuses et Tropicales, Hôpital de Brabois, 54500 Vandoeuvre-les-Nancy, France


* Corresponding author. Tel: +33-3-83851814; Fax: +33-3-83852673; Email: a.lozniewski{at}chu-nancy.fr

Received 5 August 2004; returned 27 September 2004; revised 23 October 2004; accepted 1 November 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To evaluate the antibiotic susceptibilities of Propionibacterium acnes isolates from central nervous system (CNS) infections to agents used in current treatment regimens.

Methods: MICs of 16 reference antibiotics were determined by an agar dilution method for 24 consecutive strains of P. acnes isolated from individual patients with intracranial empyema or brain abscess. Bactericidal activities of antibiotics against P. acnes PAN14 were studied at 0.25–2 x MIC using a time–kill method.

Results: All of the isolates were resistant to fosfomycin, intermediate or resistant to metronidazole and susceptible to all the other antibiotics tested, except for nine strains, which were intermediate to ofloxacin. Among antibiotics tested alone in time–kill experiments, vancomycin was the most effective drug and exhibited bactericidal activity after 24 h at 1x and 2 x MIC, whereas cefotaxime and ciprofloxacin were bactericidal after 48 h at 2 x MIC. No significant bactericidal activity could be demonstrated with the other antibiotics tested alone. The addition of cefotaxime to vancomycin resulted in bactericidal activity at lower concentrations (0.5 x MIC), whereas synergy was observed between quinupristin/dalfopristin and cefotaxime at 2 x MIC. In contrast, antagonism was observed between cefotaxime and linezolid, and ciprofloxacin and clindamycin.

Conclusion: Our data suggest that P. acnes isolates causing CNS infections remain highly susceptible to most antibiotics used for the treatment of such infections. Moreover, we showed that cefotaxime, vancomycin and ciprofloxacin possess good bactericidal activities against P. acnes, and that these activities may be enhanced when vancomycin is combined with cefotaxime or when cefotaxime is combined with quinupristin/dalfopristin.

Keywords: P. acnes , susceptibility , time–kill assay


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Propionibacterium acnes is a Gram-positive, non-spore-forming, anaerobic to aerotolerant bacillus that may normally inhabit human skin and mucosa. It is regarded as a major aetiological agent of inflammatory acne, but also as a cause of bacterial infections of endogenous origin, including central nervous system (CNS) infections such as empyemas or even brain abscesses.1,2 Antimicrobial therapy for intracranial bacterial infections should include agents that penetrate into the infection site and have bactericidal activity. To treat such infections adequately, empirical therapy usually relies on the use of ß-lactams (penicillins, third-generation cephalosporins) combined with metronidazole and/or vancomycin, but other antibiotics such as clindamycin or fluoroquinolones may also be used depending on the circumstances.3 P. acnes is usually susceptible to these antibiotics, except for metronidazole. In the present study, we evaluated the antibiotic susceptibilities of 24 clinical P. acnes isolates obtained from intracranial pus and investigated the in vitro bactericidal activities of cefotaxime, vancomycin, clindamycin, quinupristin/dalfopristin, linezolid and ciprofloxacin, alone or in combination, against P. acnes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

Twenty-four consecutive strains of P. acnes (PAN1-24), isolated from individual patients with subdural empyema (n=13) or brain abscess (n=11) at the University Hospital Centre of Nancy, were studied. All strains were kept frozen in Brucella broth containing 15% (w/v) glycerol at –80°C until use. Prior to testing, strains were subcultured three times onto Brucella agar (Becton Dickinson, Le Pont de Claix, France) supplemented with 5% defibrinated sheep blood, 5 mg/L haemin (Sigma, Saint Quentin Fallavier, France) and 1 mg/L vitamin K1 (Sigma).

Antibiotics

The following antibiotics were used and obtained from the indicated sources: penicillin G, amoxicillin, cefotaxime, chloramphenicol, clindamycin, vancomycin, metronidazole and rifampicin (Sigma); quinupristin/dalfopristin, teicoplanin and ofloxacin (Aventis, Vitry-sur-Seine, France); linezolid (Pharmacia-Upjohn, Guyancourt, France); piperacillin (Wyeth Lederlé, Paris, France); imipenem (Merck Sharp, Paris, France); ciprofloxacin (Bayer Pharma, Wuppertal, Germany); fosfomycin (Sanofi Winthrop, Gentilly, France).

MIC assays

MICs of each antibiotic were determined for all isolates by agar dilution, as recommended by the NCCLS,4 using supplemented Brucella agar. For fosfomycin, 25 mg/L of glucose-6-phosphate (Merck, Darmstadt, Germany) was added. After plates were inoculated (105 cfu/spot) with a multipoint inoculator (Denley, Billingshurst, UK), they were incubated in an anaerobic chamber (Don Whitley Scientific Ltd, Shipley, UK) in an atmosphere of 85% N2, 10% CO2 and 5% H2 at 35 °C for 48 h. MICs were interpreted as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie.5

Time–kill assays

In vitro bactericidal activities of antibiotics alone (cefotaxime, vancomycin, clindamycin, quinupristin/dalfopristin, linezolid and ciprofloxacin) or in combination (cefotaxime plus vancomycin or quinupristin/dalfopristin or linezolid, vancomycin plus quinupristin/dalfopristin, ciprofloxacin plus clindamycin) were studied against one representative strain (P. acnes PAN14) using a standard time–kill method at concentrations of 0.25–2 x MIC. P. acnes PAN14 was isolated from a patient with post-operative subdural empyema and had an antibiotic susceptibility profile (resistant to metronidazole and fosfomycin, intermediate to ofloxacin and susceptible to all other antibiotics tested) similar to that observed for the other isolates. For P. acnes PAN14, MICs of all the antibiotics were also determined by microdilution, according to the recommendations of the NCCLS.4 These MICs were not significantly different (≤ one two-fold dilution) from those found by agar dilution (data not shown).

Time–kill assays were performed in pre-reduced Brucella broth supplemented with haemin (5 mg/L), vitamin K1 (1 mg/L) and 5% defibrinated sheep blood. Inocula were prepared inside the anaerobic chamber. Colonies from Brucella agar plates were harvested, and a cell suspension equal to a 1.5 McFarland standard was prepared in supplemented Brucella broth without blood. A 0.5 mL aliquot of this suspension was delivered by syringe into 23 x 75 mm borosilicate screw-capped tubes with screw-thread open top screw caps and butyl-faced rubber septa. Each tube contained 10.5 mL of pre-reduced supplemented Brucella broth and 4 mL of antibiotic dilution prepared in the same medium without blood. The final inoculum was ~5 x 106 cfu/mL. Controls without antibiotics were included in each run. Tubes were then removed from the chamber and incubated in a shaking water bath at 35°C. Aliquots (0.1 mL) of bacterial culture were taken at 0, 6, 12, 24 and 48 h from each tube, serially diluted and plated onto supplemented Brucella agar plates. Colonies were counted after incubation inside the chamber for 48 h. Drug carryover was prevented by dilution (>10–1). Tests were performed in duplicate; results are expressed as mean log10 cfu/mL. Bactericidal activity was defined as a reduction in viable bacteria of ≥3 log10 cfu/mL. Synergy and antagonism were defined, respectively, as a ≥2 log10 decrease and a ≥2 log10 increase in the viable count for bacteria treated with the combination compared with the viable count in the presence of the more active of the two antibiotics used alone. Indifference was defined as a <1-log10-unit change in cfu/mL, with the combination compared with the most active single agent.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of MIC studies are presented in Table 1. All of the 24 clinical isolates studied were resistant to fosfomycin, intermediate or resistant to metronidazole and susceptible to all the other antibiotics tested, except for nine strains intermediate to ofloxacin.


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Table 1. Antimicrobial activity of 16 antibiotics against 24 strains of P. acnes

 
Among antibiotics tested alone in time–kill experiments, vancomycin was the most effective drug and exhibited bactericidal activity after 24 h at 1–2 x MIC, whereas cefotaxime and ciprofloxacin were only bactericidal after 48 h at 2 x MIC (Table 2). A slight decrease in viable count was observed with clindamycin or linezolid only at 2 x MIC after 24–48 h of incubation, whereas no killing effect of quinupristin/dalfopristin was found at any time point or any concentration studied.


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Table 2. Killing activities of antibiotics alone or in combination against P. acnes PAN14

 
The addition of cefotaxime to vancomycin resulted in an overall improvement in killing effect and in significant bactericidal activity at lower concentrations (0.5xMIC) from 24–48 h. The combination of cefotaxime with quinupristin/dalfopristin at 2 x MIC was synergistic from 6–24 h, and resulted in a bactericidal effect at 12 h instead of 48 h. The addition of quinupristin/dalfopristin to vancomycin resulted in a slight increase of the killing effect at 2 x MIC between 6–24 h. The addition of linezolid to cefotaxime resulted in a slight reduction of the killing effect after 24–48 h at 1 x MIC and after 24 h at 2 x MIC, whereas antagonism was observed at 2 x MIC after 48 h. Such antagonism was also seen with the combination of ciprofloxacin and clindamycin at 2xMIC after 48 h.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our study, all of the P. acnes clinical isolates demonstrated high-level susceptibility to all of the antibiotics tested, except for metronidazole, fosfomycin and, to a lesser extent, ofloxacin. These results are similar to those reported by Behra-Miellet et al.6 However, in contrast to other authors,7 we did not detect any ciprofloxacin- or clindamycin-resistant strain. This suggests that the prevalence of antibiotic-resistant propionibacteria may be lower in patients with CNS infections than in acne patients treated for several months with topical antibiotics.7 However, further studies are necessary to confirm this observation.

We showed that cefotaxime, vancomycin and ciprofloxacin were bactericidal at concentrations achievable in intracranial abscess formations.3 Our results are in agreement with those reported for ciprofloxacin by Spangler et al.8 Additivity between levofloxacin and clindamycin against P. acnes has been demonstrated by Credito et al.9 In our study, we observed no such effect between another fluoroquinolone, ciprofloxacin, and clindamycin, and even observed antagonism at 2 x MIC. However, we showed that the addition of cefotaxime to vancomycin resulted in a bactericidal effect at lower concentrations than those required with vancomycin alone. These results suggest that the combination of cefotaxime with vancomycin, which is recommended for the empirical treatment of post-traumatic and post-neurosurgical brain abscesses, may be effective in the treatment of CNS infections caused by P. acnes. Multidrug-resistant Gram-positive cocci may be involved in post-operative CNS infections. It has been suggested that the treatment of such infections may include clindamycin, quinupristin/dalfopristin and linezolid.10,11 These antibiotics may therefore also be used in polymicrobial infections in which P. acnes could be involved. However, we detected no significant bactericidal activity when these drugs were used alone against P. acnes, and when they were used in combination with other antibiotics no significant enhancement of the killing rate could be demonstrated, except when quinupristin/dalfopristin was combined with cefotaxime at a concentration of 2 x MIC, which may be achieved in vivo after intrathecal administration.11

In conclusion, our data suggest that P. acnes isolates from CNS infections remain highly susceptible to most antibiotics used for the treatment of such infections. Moreover, we showed that cefotaxime, vancomycin and ciprofloxacin possess good bactericidal activities against P. acnes and that these activities may be enhanced when vancomycin is combined with cefotaxime, or when cefotaxime is combined with quinupristin/dalfopristin. In vivo studies are needed to validate these in vitro findings.


    Acknowledgements
 
We are grateful to Marie-Renée Balland for her technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ghalayini, S. R. A., Likhith, A. M. & Golash, A. (2004). Propionibacterium acnes causing delayed subdural empyema — a case report and review of literature. Journal of Clinical Neuroscience 11, 677–9.[CrossRef][ISI][Medline]

2 . Barazi, S. A., Gnanalingham, K. K., Chopra, I. et al. (2003). Delayed postoperative intracerebral abscess caused by Propionibacterium acnes: case report and review of the literature. British Journal of Neurosurgery 17, 336–9.[CrossRef][ISI][Medline]

3 . Tunkel, A. R., Wispelwey, B. & Scheld, W. M. (2000). Brain abscess. In Principles and Practice of Infectious Diseases, 5th edn (Bennett, J. E. & Dollin, R., Eds), pp. 1016–28. Churchill Livingstone, Philadelphia, PA, USA.

4 . National Committee for Clinical Laboratory Standards. (2003). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Sixth Edition: Approved Standard M11-A6. NCCLS, Wayne, PA, USA.

5 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (2004). Communiqué 2004. SFM, Paris, France.

6 . Behra-Miellet, J., Dubreuil, L. & Jumas-Bilak, E. (2002). Antianaerobic activity of moxifloxacin compared with that of ofloxacin, ciprofloxacin, clindamycin, metronidazole and ß-lactams. International Journal of Antimicrobial Agents 20, 366–74.[CrossRef][ISI][Medline]

7 . Vogt, K., Hermann, J., Blume, U. et al. (1992). Comparative activity of the topical quinolone OPC-7251 against bacteria associated with acne vulgaris. European Journal of Clinical Microbiology and Infectious Diseases 11, 943–5.[ISI][Medline]

8 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Bactericidal activity of DU-6859a compared to activities of three quinolones, three ß-lactams, clindamycin, and metronidazole against anaerobes as determined by time-kill methodology. Antimicrobial Agents and Chemotherapy 41, 847–9.[Abstract]

9 . Credito, K. L., Jacobs, M. R. & Appelbaum, P. C. (2000). Anti-anaerobic activity of levofloxacin alone and combined with clindamycin and metronidazole. Diagnostic Microbiology and Infectious Disease 38, 181–3.[CrossRef][ISI][Medline]

10 . Roche, M., Humphreys, H., Smyth, E. et al. (2003). A twelve-year review of central nervous system bacterial abscesses; presentation and aetiology. Clinical Microbiology and Infection 9, 803–9.[CrossRef][ISI][Medline]

11 . Garey, K. W., Tesoro, E., Muggia, V. et al. (2001). Cerebrospinal fluid concentrations of quinupristin/dalfopristin in a patient with vancomycin-resistant Enterococcus faecalis ventriculitis. Pharmacotherapy 21, 748–50.[CrossRef][ISI][Medline]





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