Antistaphylococcal activity of the novel cephalosporin ...

Advance Access published February 18, 2005

JAC

Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dki003

Antistaphylococcal activity of the novel cephalosporin CB-181963 (CAB-175) Keith Miller, Christopher Storey†, William J. Stubbings, Anthony M. Hoyle, Joanne K. Hobbs and Ian Chopra* Antimicrobial Research Centre and School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT UK Received 14 October 2004; returned 2 December 2004; revised 6 December 2004; accepted 6 December 2004

Methods: Using established procedures, we examined the activity of CB-181963 against methicillinsusceptible (MSSA) and methicillin-resistant (MRSA) strains of Staphylococcus aureus in both planktonic and biofilm culture. We also determined whether CB-181963 exhibited a post-antibiotic effect (PAE). A radioactive competition assay with 3H-labelled benzylpenicillin was used to determine penicillin-binding protein (PBP) affinities of CB-181963, including binding to PBP2a from MRSA. The potential for emergence of CB-181963-resistant mutants in MSSA and MRSA strains was examined using plating procedures. Results: CB-181963 showed excellent activity against MRSA strains resistant to other cephalosporins in both planktonic and biofilm cultures. However, in common with other cephalosporins it was unable to eradicate biofilms. CB-181963 had a short PAE compared with other b-lactam antibiotics. CB-181963 retained activity against a strain expressing type A b-lactamase and demonstrated affinity for PBP2a of MRSA. Mutants resistant to CB-181963 were not recovered in either MSSA or MRSA. Conclusions: CB-181963 is a potent antistaphylococcal agent with better activity against MRSA than other cephalosporins. The anti-MRSA activity is correlated with elevated binding to PBP2a. CB-181963 may have a role in the treatment of staphylococcal infections, including those caused by MRSA and in the prophylaxis of biofilm-associated MSSA and MRSA infections. However, because of its short PAE, CB-181963 may have to be administered more frequently than other b-lactam antibiotics, or given via prolonged infusion. Keywords: methicillin-resistant Staphylococcus aureus, penicillin-binding proteins

Introduction During the last 40 years, a range of semi-synthetic cephalosporins has been developed for the treatment of bacterial infections. Interest in these antibiotics continues to be maintained, with particular emphasis on the discovery and development of novel cephalosporins possessing activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus

(MRSA), Enterococcus faecalis and penicillin-resistant Streptococcus pneumoniae.1,2 CB-181963 (formerly CAB-175) (Figure 1) is a novel cephalosporin, possessing an azomethine substituent at position 3 of the cephem nucleus.3 The activity of CB-181963 against MRSA is one of the most interesting properties of this cephalosporin.3 Other cephalosporins active against MRSA possess enhanced binding to penicillin-binding protein (PBP) 2a, which is uniquely

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*Corresponding author. Tel: +44-113-343-5604; Fax: +44-113-343-3167; E-mail: [email protected] †Present address. Jackson’s Mill, University of Manchester, Sackville Street, Manchester M60 1QD, UK. ..........................................................................................................................................................................................................................................................................................................................................................................................................................

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Objectives: We examined the antistaphylococcal activity of the novel cephalosporin CB-181963 (formerly known as CAB-175), with emphasis on its microbiological activity and penicillin-binding protein specificities.

K. Miller et al. the test antibiotic. The system was then incubated for 24 h at 378C on a rocking platform. The MIC was defined as the lowest concentration of antibiotic completely inhibiting visible growth after this incubation. After the MIC was recorded, the lid assembly was again washed twice in PBS to remove planktonic cells and remaining antibiotic, and then placed into fresh drug-free media. The system was incubated for a further 24 h and the MBEC was defined as the lowest concentration of antibiotic completely inhibiting the re-establishment of planktonic growth.

Figure 1. Structure of CB-181963.

Materials and methods Bacterial strains S. aureus strains 8325– 4, SH1000 and PC1 (all MSSA), and EMRSA-15 and EMRSA-16 are maintained in a culture collection belonging to the Division of Microbiology, University of Leeds.

Antibiotics, chemicals and growth media

Measurement of post-antibiotic effect (PAE) PAEs were determined with strain 8325– 4 using a standard viable count method in triplicate, as previously described.7 Bacteria were exposed to 5 the MIC of the antimicrobial agent for 60 min in MHB before removal of the drug by a 10 3 dilution.

Determination of mutation frequencies for resistance to antibiotics This was performed as described by O’Neill et al.8 using MH growth media. In each case, cultures were started from single colonies of the test strain that were shown to be sensitive to the antibiotic under investigation. Antibiotic-resistant mutants were identified on medium containing the selective antibiotic at a concentration that was four-fold higher than the respective MIC value for an individual strain. Mutation frequencies were expressed as the number of resistant mutants recovered as a fraction of total viable bacteria.8

PBP binding assays

Chemicals were from Sigma-Aldrich (Poole, UK) unless stated otherwise. Antibiotics were also from Sigma-Aldrich (Poole, UK), with the exception of CB-181963, samples of which were gifts from Sandoz GmbH (Vienna, Austria) and Cubist Pharmaceuticals (Lexington, MA, USA). Mueller – Hinton broth (MHB) and agar (MHA) were from Fisher (Loughborough, UK).

Determination of susceptibility to antimicrobial agents MICs were determined by agar dilution according to BSAC guidelines5 on MHA. The MIC was defined as the lowest concentration of antibiotic completely inhibiting visible growth after 18– 24 h of incubation at 378C.

Determination of susceptibility to antimicrobial agents in biofilms Biofilm MICs and minimum biofilm eradication concentrations (MBEC) were determined in the Calgary Biofilm Device (Nunc Inc., Roskilde, Denmark) as described by Ceri et al.6 This involves the following steps. Aliquots (200 mL) of exponential phase culture were added to each well of a 96-well microtitre tray. The lid assembly, which has 96 polystyrene pegs corresponding to each well, was then replaced and the system incubated for 24 h at 378C on a rocking platform. Following this, a biofilm of 107 cfu matured on each peg. The lid was then washed twice in phosphate-buffered saline (PBS) to remove residual planktonic growth and then placed into a microtitre tray with fresh media containing doubling dilutions of

PBP assays were performed using membranes prepared from S. aureus 8325– 4 (MSSA) according to Komatsuzawa et al.,9 or purified soluble PBP2a kindly provided as a gift by Dr W. P. Lu10 (Proctor and Gamble Pharmaceuticals, Mason, USA). The use of soluble PBP2a was required because of difficulty in separating PBP2a from other PBPs during electrophoresis of membranes prepared from MRSA. Membranes from S. aureus 8325– 4 (MSSA) were used as a source of PBPs 1 – 4 for binding studies with CB-181963 and the comparator agent cefotaxime. Membranes prepared according to Komatsuzawa et al.9 were resuspended in PBS containing DNAse1 (25 mg/L) and stored at 80 8C. For PBP binding assays, 10 mL of membrane preparation (50 mg of protein) was added to 10 mL of b-lactam solution (various concentrations) in PBS. Samples were incubated at 35 8C for 20 min, then 2 mL of [3H]benzylpenicillin (Amersham Pharmacia Biotech, Amersham, UK) (37 mBq/mL; final concentration 0.5 mg/L) was added. After a further 20 min at 358C, the reaction was stopped by the addition of 20 mL of SDS sample buffer [0.06 M Tris-HCl, 1.4 M 2-mercaptoethanol, 4% (w/v) sodium dodecyl sulphate, 20% (v/v) glycerol, 0.1% (w/v) Bromophenol Blue]. Samples were boiled for 5 min and then centrifuged (30000 g, 10 min, 4 8C). Proteins were separated by SDS – PAGE and the gels analysed, essentially as described9 after autoradiography for 28 days, using Amplify (Amersham Pharmacia, UK) as the fluor. Binding assays with purified soluble PBP2a from MRSA were performed as described for membranes, and 1 mg aliquots applied to gels for electrophoresis.

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expressed in MRSA.1,2 However, there is no published information on the affinity of CB-181963 for the PBPs of S. aureus. In this paper, we report binding affinities of CB-181963 for staphylococcal PBPs and conclude that the anti-MRSA activity of CB-181963 is indeed related to potent binding activity for PBP2a. S. aureus is a major cause of intravascular device-related infections involving the formation of biofilms. Antimicrobial prophylaxis is frequently used to prevent the establishment of biofilms, with cephalosporins used regularly in vascular surgery.4 Consequently, we have also examined the ability of CB-181963 to prevent the formation of biofilms by methicillinsusceptible S. aureus (MSSA) and MRSA in vitro.

Cephalosporin CB-181963 Table 1. Activity of CB-181963 and a range of cephalosporins against planktonic and biofilm cultures of S. aureus

Table 2. PAEs induced by various antibiotics in S. aureus Mean PAE (h)a

Antibiotic Strain

Planktonic MIC (mg/L)

Biofilm MIC (mg/L)

MBEC (mg/L)

CB-181963

SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16 SH1000 EMRSA-15 EMRSA-16

0.5 4 2 0.5 64 128 0.064 16 64 2 128 256 4 256 256 2 256 256 16 128 256

0.5 8 8 0.5 64 128 0.064 32 64 4 128 512 16 256 256 2 256 512 32 128 256

> 256 > 256 > 256 > 1024 > 1024 > 1024 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024 > 1024

Cefalothin Cefaloridine Cefadroxil Cefoxitin Cefotaxime Ceftazidime

CB-181963 Cefotaxime Ceftriaxone Ampicillin Amoxicillin Imipenem Meropenem Tetracycline Fusidic acid Erythromycin Gentamicin Linezolid a

± S.D .

Table 3. Binding affinities of CB-181963 and cefotaxime for PBPs from S. aureus 8325– 4 (MSSA) and soluble PBP2a from MRSA IC50a (mg/L)

CB-181963 Cefotaxime

Results

a

In vitro anti-Gram-positive activity

0.50 ± 0.42 1.93 ± 0.39 2.29 ± 0.09 2.50 ± 0.28 2.16 ± 0.19 1.99 ± 0.03 1.84 ± 0.11 0.27 ± 0.29 0.22 ± 0.38 1.02 ± 0.25 2.26 ± 0.65 1.04 ± 0.09

PBP1

PBP2

PBP2a

PBP3

PBP4

0.75 0.2

0.2 0.2

1.0 > 250

0.25 0.15

16.0 32.0

Concentration that inhibits 50% of binding by penicillin.

Selection of CB-181963 resistant mutants

CB-181963 was active against planktonic cultures of MSSA and EMRSA (MIC range 0.5– 4 mg/L), whereas other cephalosporins had poor activity against EMRSA (MIC range 16–256 mg/L) (Table 1). In addition, CB-181963 appears to be stable against degradation by staphylococcal b-lactamases, since its MIC against S. aureus PC1, which expresses type A b-lactamase constitutively,2 was 0.5 mg/L. Furthermore, the MIC was also lower than for other cephalosporins against EMRSA-15 and EMRSA16, which also exhibit b-lactamase activity.

We were unable to recover mutants in MSSA or MRSA host backgrounds by direct selection, indicating a mutation frequency of < 10 9 (data not shown). We were also unable to record mutation frequencies in MSSA with other b-lactam antibiotics. In contrast, with similar selections in the MSSA strain 8325–4, mutants resistant to fusidic acid arose with a frequency of 7.6 ± 1.3 10 7 and those resistant to mupirocin with a frequency of 7.2 ± 0.93 10 8.

Penicillin-binding protein affinities Anti-biofilm activity The activity of CB-181963 against S. aureus biofilms was compared with a range of other cephalosporins (Table 1). CB181963 prevented biofilm formation by MSSA and EMRSA, whereas other cephalosporins were generally less active, especially for preventing biofilms formed by the EMSRA strains (Table 1). However, all cephalosporins, including CB-181963, were ineffective at eradicating the biofilm since MBEC values exceeded 256 mg/L (Table 1).

PAE of CB-181963 and comparator antibiotics Cultures of S. aureus 8325 –4 were exposed to 5 the MIC of the drugs for 60 min before washout, and the PAE determined, as described in the Materials and methods section. CB-181963 demonstrated a PAE of 0.5 h compared with PAEs of between 0.22 and 2.5 h for other antibiotics (Table 2).

Data for competition of CB-181963 and cefotaxime with [3H]benzylpenicillin for the various PBPs are presented in Table 3. As the PBP assay is based on competitive binding, these data are expressed as IC50 values, i.e. the concentration of cephalosporin required to inhibit subsequent binding of [3H]benzylpenicillin by 50%. CB-181963 demonstrated strong binding to PBP1, 2, 2a and 3 of S. aureus (Table 3). These binding affinities are consistent with the potency of CB-181963 against MRSA. In contrast, although cefotaxime bound to PBPs 1–4 from the MSSA strain, it failed to bind PBP2a (Table 3) consistent with its poor activity against MRSA (Table 1).

Discussion Antimicrobial options for the treatment of MRSA are currently limited. CB-181963, in common with a number of other cephalosporins under investigation, possesses stability to staphylococcal b-lactamase and potent binding to PBP2a from MRSA.1,2

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Antibiotic

K. Miller et al. Furthermore, CB-181963 prevents the formation of biofilms by MRSA. In addition, spontaneous mutants resistant to CB-181963 did not arise either in MSSA or MRSA hosts under conditions where mutants resistant to other antistaphylococcal antibiotics could be recovered. Human pharmacokinetic or pharmacodynamic data for CB-181963 are currently not available. Nevertheless, this novel cephalosporin may have a role in the treatment of planktonic infections and the prophylaxis of biofilm infections caused by MRSA. However, because of its short PAE, CB181963 may have to be administered more frequently than other b-lactam antibiotics, or given via prolonged infusion. Regulatory approval of CB-181963 will ultimately depend upon acceptable clinical efficacy and safety profiles.

Acknowledgements

References 1. Fujimura, T., Yamano, Y., Yoshida, I. et al. (2003). In vitro activity of S-3578, a new broad-spectrum cephalosporin active against methicillin-resistant staphylococci. Antimicrobial Agents and Chemotherapy 47, 923– 31. 2. Malouin, F., Blais, J., Chamberland, S. et al. (2003). RWJ54428 (MC-02,479), a new cephalosporin with high affinity for penicillin-binding proteins, including PBP 2a, and stability to

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This work was supported by a research grant to Professor Ian Chopra from Sandoz GmbH, Vienna, Austria. Joanne Hobbs was supported by a vacation studentship from the British Society for Antimicrobial Chemotherapy.

staphylococcal b-lactamases. Antimicrobial Agents and Chemotherapy 47, 658– 64. 3. Huang, V., Brown, W. J. & Rybak, M. J. (2004). In vitro activities of a novel cephalosporin, CB-181963 (CAB-175), against methicillinsusceptible or -resistant Staphylococcus aureus and glycopeptideintermediate susceptible staphylococci. Antimicrobial Agents and Chemotherapy 48, 2719–23. 4. Haas, D. W. & Kaiser, A. B. (1994). Antimicrobial prophylaxis of infections associated with foreign bodies. In Infections Associated with Indwelling Medical Devices, 2nd edn (Bisno, A. L. & Waldvogel, F. A. Eds), pp. 375 –88. ASM Press, Washington DC, USA. 5. Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1– 50. 6. Ceri, H., Olson, M. E., Stremick, C. et al. (1999). The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. Journal of Clinical Microbiology 37, 1771– 6. 7. McDonald, P. J., Craig, W. A. & Kunin, C. M. (1977). Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. Journal of Infectious Diseases 135, 217–23. 8. O’Neill, A. J., Cove, J. H. & Chopra, I. (2001). Mutation frequencies for resistance to fusidic acid and rifampicin in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 47, 647 –50. 9. Komatsuzawa, H., Choi, G. H., Ohta, K. et al. (1999). Cloning and characterization of a gene, pbpF, encoding a new penicillin-binding protein, PBP2B, in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 1578–83. 10. Lu, W. P., Sun, Y. P., Bauer, M. D. et al. (1999). Penicillinbinding protein 2a from methicillin-resistant Staphylococcus aureus: kinetic characterization of its interactions with b-lactams using electrospray mass spectrometry. Biochemistry 38, 6537– 46.