In vitro activity of a new cephalosporin, RWJ-54428, against streptococci, enterococci and staphylococci, including glycopeptide-intermediate Staphylococcus aureus

Jana M. Swenson* and Fred C. Tenover

Anti-infectives Investigation Section, Epidemiology and Laboratory Branch, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Mailstop G08, 1600 Clifton Road N.E., Atlanta, GA 30333, USA

Received 23 October 2001; returned 6 December 2001; revised 24 January 2002; accepted 5 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The need for new antimicrobial agents with activity against Gram-positive organisms has become increasingly important because of emerging resistance. We compared the activity of a new b-lactam antimicrobial agent, RWJ-54428 (MC-02 479), with representatives of other classes of antimicrobial agents against 76 Staphylococcus aureus (including four glycopeptide- intermediate strains), 50 coagulase-negative staphylococci, 20 Enterococcus faecalis, 20 Enterococcus faecium, 10 Enterococcus gallinarum/Enterococcus casseliflavus, 54 Streptococcus pneumoniae and 22 viridans streptococcal isolates. The MIC90 of RWJ-54428 was <=2 mg/L for all groups of bacteria tested except E. faecium. The activity against four strains of glycopeptide-intermediate S. aureus was similar to that for other methicillin-resistant S. aureus isolates (range 0.5–2.0 mg/L).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the last decade, increasing resistance among Gram-positive bacteria,13 including oxacillin-resistant staphylococci, vancomycin-resistant enterococci and penicillin-resistant streptococci, has emphasized the need for new, more potent, compounds effective against these organisms. Recent reports of staphylococci with increasing resistance to vancomycin have caused even more concern.4

RWJ-54428 (MC-02 479) is a new parenteral cephalosporin (Microcide Pharmaceuticals, Inc., Mountain View, CA, USA), originally developed by the R. W. Johnson Pharmaceutical Research Institute (Raritan, NJ, USA), that has been shown to be active against resistant strains of staphylococci, enterococci and pneumococci.5 In the present study, we investigated the activity of RWJ-54428 against recent isolates of Gram-positive bacteria and included strains of staphylococci with decreased susceptibility to vancomycin. The study focused primarily on more resistant organisms in each group and compared the in vitro activity of the new agent with the activity of other agents, such as penicillin and vancomycin, and also antibacterial agents with enhanced activity against this group of organisms, such as quinupristin/dalfopristin and linezolid.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria tested

Strains of staphylococci, streptococci and enterococci were selected from the culture collection of the Centers for Disease Control and Prevention (CDC) to comprise a large number of resistant organisms. These included staphylococci resistant to oxacillin (confirmed by the presence of the mecA gene), streptococci resistant to penicillin and enterococci resistant to ampicillin. The total number of each species and the number of strains that were resistant to the selected antimicrobial agent noted above (indicated in brackets) were: 76 (40) Staphylococcus aureus, 50 (29) coagulase-negative staphylococci, 54 (44) Streptococcus pneumoniae, 22 (9) viridans group streptococci, 20 (14) Enterococcus faecium, 20 (0) Enterococcus faecalis and 10 (0) Enterococcus gallinarum/Enterococcus casseliflavus. Among the oxacillin-resistant S. aureus tested, four were characterized as glycopeptide-intermediate (GISA, vancomycin MIC 8 mg/L). Included among the 36 oxacillin-susceptible S. aureus were four ‘hyper b-lactamase-producing’ strains (i.e. strains showing resistant oxacillin MICs that were reduced to a susceptible level in the presence of clavulanic acid) and three strains with the modified penicillin binding protein (MOD) phenotype (mecA-negative strains with borderline resistant oxacillin MICs that are not reduced in the presence of clavulanic acid). The number and species of coagulase-negative staphylococci tested included 27 Staphylococcus epidermidis and 23 iso-lates of other coagulase-negative species. Of enterococcal isolates tested, 35 were vancomycin resistant (MICs > 4 mg/L); nine expressed the VanA phenotype and were positive for vanA, and 20 expressed the VanB phenotype and were positive for vanB. All 10 E. gallinarum/E. casseliflavus isolates were positive for the vanC gene, but only four demonstrated a non-susceptible MIC of vancomycin (8 mg/L). The number and species of viridans group streptococci tested included two Streptococcus milleri, seven Streptococcus mitis, three Streptococcus mutans, four Streptococcus salivarius and six Streptococcus sanguis isolates.

Antimicrobial agents

Broth microdilution plates were prepared for testing streptococci using amoxicillin (Sigma, St Louis, MO, USA), cefepime (Bristol-Myers Squibb, Princeton, NJ, USA), ceftriaxone (Sigma), clarithromycin (Abbott Laboratories, Abbott Park, IL, USA), erythromycin (Eli Lilly & Co., Indianapolis, IN, USA), linezolid (Pharmacia & Upjohn, Kalamazoo, MI, USA), meropenem (Astra-Zeneca, Wilmington, DE, USA), penicillin (Sigma), trovafloxacin (Pfizer, Groton, CT, USA) and RWJ-54428. For testing staphylococci and enterococci, plates contained ampicillin (Sigma), erythromycin, gentamicin (Sigma), linezolid, minocycline (Wyeth-Ayerst, Pearl River, NY, USA), oxacillin (Sigma), penicillin, quinupristin/dalfopristin (Rhone-Poulenc Rorer, Collegeville, PA, USA), trovafloxacin, vancomycin (Eli Lilly & Co.) and RWJ-54428. (Use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service or the US Department of Health and Human Services.)

MIC method

Testing was performed by the NCCLS broth microdilution method6 using cation-adjusted Mueller–Hinton broth (Difco brand; BD Biosciences, Sparks, MD, USA; cations adjusted in house). For testing streptococcal and pneumococcal isolates, the plates were supplemented with 5% lysed defibrinated horse blood. Plates were prepared with all the agents except RWJ-54428 and frozen at –70°C. RWJ-54428 was prepared on each day of testing, diluted and then dispensed into wells just before inoculation. Size of inoculum (c. 5 x 105 cfu/mL) and length and conditions of incubation of all plates were as specified by the NCCLS.6


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MICs of the antimicrobial agents for staphylococci, enterococci and streptococci are shown in Table 1. Activity is expressed as MIC50, MIC90 and MIC range. Comparison of activity is based on the MIC90 values.


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Table 1. Activity of RWJ-54428 and other antimicrobial agents against Gram-positive bacteria
 
Staphylococci

RWJ-54428 was active against all staphylococci tested, including mecA-positive strains and strains with decreased susceptibility to vancomycin (data not shown), with MIC90s <= 2 mg/L. The only antimicrobial agent with greater activity than RWJ-54428 against mecA-positive S. aureus strains was quinupristin/dalfopristin; both quinupristin/dalfopristin and minocycline demonstrated comparable activity against mecA-positive coagulase-negative staphylococci. For the seven S. aureus strains for which the oxacillin MICs were borderline (both ß-lactamase mediated and MOD strains), the activity of RWJ-54428 was similar to that of other mecA-negative strains (data not depicted separately).

Enterococci

Of the 50 enterococci tested, 18 (all E. faecium) were penicillin resistant (MIC >= 16 mg/L). Of those 18 strains, 14 were also resistant to ampicillin and the MICs of RWJ-54428 for those 14 strains were 4–8 mg/L. For the four strains that demonstrated resistance to penicillin (MICs 16–32 mg/L) but susceptibility to ampicillin (MICs 4–8 mg/L), the MICs of RWJ-54428 were 1 mg/L. Therefore, there is better correlation of RWJ-54428 activity with ampicillin than with penicillin. Against the ampicillin-susceptible enterococci tested, RWJ-54428 demonstrated the best overall activity.

For the 29 strains possessing the vanA or vanB gene, the MIC range of RWJ-54428 was <=0.06–8 mg/L, which was similar to the range for strains that did not possess a vancomycin resistance gene (0.25–8 mg/L). For the 10 E. gallinarum/E. casseliflavus strains that possessed the vanC gene, the MIC range of RWJ-54428 was 0.25–0.5 mg/L.

Streptococci

The MIC90s of RWJ-54428 for penicillin-susceptible, -intermediate and -resistant pneumococci were, respectively, 0.03, 0.5 and 1 mg/L. Likewise, for penicillin-intermediate or -resistant strains of viridans group streptococci, the MIC90 was 1 mg/L. Based on MIC90s, trovafloxacin demonstrated greater activity against penicillin-resistant S. pneumoniae than RWJ-54428; however, RWJ-54428 was the most active agent against viridans group streptococci with decreased susceptibility to penicillin.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The novel cephalosporin, RWJ-54428, was shown in this study to be active against a wide range of multiply resistant Gram-positive pathogens, including oxacillin-resistant S. aureus (MRSA), vancomycin-resistant E. faecalis, ampicillin-susceptible E. faecalis and E. faecium, and penicillin-resistant pneumococci and streptococci. Ampicillin-resistant E. faecium appeared to be the one group of bacteria for which the MICs of RWJ-54428 were typically >=4 mg/L.

Most resistance to oxacillin in staphylococci is due to the presence of the mecA gene, which regulates production of a new penicillin-binding protein (PBP) with decreased affinity for b-lactam agents. The failure of most current b-lactam agents to adequately treat infections caused by mecA-positive strains is due to the failure of the agents to bind to this alternative PBP, called PBP2' (or PBP2a).7 RWJ-54428 is a parenteral cephalosporin that binds PBP2' in staphylococci5 and shows in vitro activity against other Gram-positive bacteria, as well as Haemophilus influenzae and Moraxella catarrhalis.5

Conventional wisdom has held that MRSA should be considered resistant to all b-lactams and reported as such,810 even though these organisms may appear to be susceptible when tested in vitro. Should RWJ-54428 or similar agents with increased affinity for PBP2' eventually become available for treatment of resistant staphylococci, microbiologists will need to be re-educated regarding the testing and reporting of such compounds, to which MRSA would typically be reported as resistant based on oxacillin test results.

In summary, RWJ-54428 is a novel cephalosporin with broad in vitro activity against a variety of multiply resistant Gram-positive pathogens, including staphylococci, enterococci and pneumococci. Of the bacteria tested, the only group of organisms for which the MIC90 was >2 mg/L was ampicillin-resistant E. faecium.


    Acknowledgements
 
This study was presented in part at the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 26–29 September 1999, Abstract 394.


    Footnotes
 
* Corresponding author. Tel: +1-404-639-0196; Fax: +1-404-639-1381; E-mail: jms1{at}cdc.gov Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Doern, G. V., Brueggemann, A. B., Huynh, H., Wingert, E. & Rhomberg, P. (1999). Antimicrobial resistance with Streptococcus pneumoniae in the United States, 1997–98. Emerging Infectious Diseases 5, 757–65.[ISI][Medline]

2 . Gerberding, J. L., McGowan, J. E. & Tenover, F. C. (1999). Emerging nosocomial infections and antimicrobial resistance. Current Clinical Topics in Infectious Diseases 19, 83–98.[Medline]

3 . Cormican, M. G. & Jones, R. N. (1996). Emerging resistance to antimicrobial agents in gram-positive bacteria. Enterococci, staphylococci, and nonpneumococcal streptococci. Drugs 51, Suppl. 1, 6–12.[ISI][Medline]

4 . Tenover, F. C., Lancaster, M. V., Hill, B. C., Steward, C. D., Stocker, S. A., Hancock, G. A. et al. (1998). Characterization of staphylococci with reduced susceptibility to vancomycin and other glycopeptides. Journal of Clinical Microbiology 36, 1020–7.[Abstract/Free Full Text]

5 . Chamberland, S., Blais, J., Hoban, D. J., Dinh, C., Cotter, D., Bond, E. et al. (2001). In vitro activities of RWJ-54428 (MC-02 479) against multiresistant gram-positive bacteria. Antimicrobial Agents and Chemotherapy 45, 1422–30.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

7 . Chambers, H. F. & Sachdeva, M. (1990). Binding of ß-lactam antibiotics to penicillin-binding proteins in methicillin-resistant Staphylococcus aureus. Journal of Infectious Diseases 161, 1170–6.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

9 . Acar, J. F., Courvalin, P. & Chabbert, Y. A. (1970). Methicillin-resistant staphylococcemia: bacteriological failure of treatment with cephalosporins. Antimicrobial Agents and Chemotherapy 10, 280–5.

10 . Richmond, A. S., Simberkoff, M. S., Schaefler, S. & Rahal, J. J. (1977). Resistance of Staphylococcus aureus to semisynthetic penicillins and cephalothin. Journal of Infectious Diseases 135, 108–12.[ISI][Medline]