and against two methicillin-susceptible Staphylococcus aureus strains in 105-CFU/ml inocula. Synergy between. GV and gentamicin was observed against an E.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1996, p. 2142–2146 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 40, No. 9
In Vitro Activity of the Trinem Sanfetrinem (GV104326) against Gram-Positive Organisms KAVINDRA V. SINGH,1 TERESA M. COQUE,1
AND
BARBARA E. MURRAY1,2*
The Division of Infectious Diseases, Department of Internal Medicine,1 and Department of Microbiology and Molecular Genetics,2 The University of Texas Medical School, Houston, Texas 77030 Received 20 February 1996/Returned for modification 2 May 1996/Accepted 9 July 1996
The in vitro activity of the trinem sanfetrinem (formerly GV104326) (GV) was compared with that of vancomycin, ampicillin, and/or nafcillin against 287 gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and multiresistant enterococci, by the agar and microbroth dilution methods. GV demonstrated 2 to 16 times more activity than ampicillin and nafcillin against the majority of these organisms. The MIC range of GV was 16 to 64 mg/ml for 19 Enterococcus faecium strains that were highly resistant to ampicillin (ampicillin MIC range, 64 to 512 mg/ml) and vancomycin resistant and 0.25 to 32 mg/ml for resistant Rhodococcus spp. Similar activities (61 dilution) were observed by either the agar or the broth microdilution method. GV demonstrated bactericidal activity against a b-lactamase-producing Enterococcus faecalis strain and against two methicillin-susceptible Staphylococcus aureus strains in 105-CFU/ml inocula. Synergy between GV and gentamicin was observed against an E. faecalis strain that lacked high-level gentamicin resistance. The activity of GV suggests this compound warrants further study. In recent years, there has been an increase in the number of gram-positive clinical isolates resistant to the beta-lactam antibiotics currently available (1, 2, 8, 23, 24). Overproduction and the low affinities of penicillin-binding proteins, in addition to penicillinase, have been shown to be the key mechanisms responsible for these resistances in enterococci, staphylococci, and pneumococci (7, 9, 12, 14, 16, 17, 27, 35, 36). Sanfetrinem (GV104326) (GV) is the first member of a new class of tricyclic beta-lactam (trinem) antibiotics (6). Previous reports have demonstrated that GV is resistant to hydrolysis by many b-lactamases and by renal dehydropeptidases (6, 30) and has broadspectrum antibacterial activity against many organisms, including gram-negative, gram-positive, and anaerobic bacteria, and moderate activity against enterococci (6, 13). However, the number of enterococci studied previously was small. Recent attention has been focused on enterococci because of their increasing resistance to vancomycin and high-level resistance (HLR) to aminoglycosides and penicillin, thus limiting the effective therapeutic alternatives for enterococcal infections (10, 11, 19–24, 27, 32, 33, 37). In this study, we determined the activity of sanfetrinem by agar dilution and microbroth dilution and compared this to the activities of vancomycin, ampicillin, and/or nafcillin against 287 gram-positive organisms (including 145 enterococci) that display various degrees of resistance to antimicrobial agents that is caused by different mechanisms. The bactericidal activity of GV against five multiresistant strains and its ability to produce synergism with gentamicin against an Enterococcus faecalis strain were also determined.
Enterococcus faecium strains were mainly isolated in Texas, New York, Wisconsin, and Belgium. Some E. faecium strains with HLR to ampicillin and vancomycin and E. faecalis strains which were highly resistant to gentamicin and vancomycin and/or which produced b-lactamase were obtained from California, Delaware, Florida, Massachusetts, Wisconsin, Virginia, Connecticut, Argentina, and the Centers for Disease Control and Prevention over the last 7 to 8 years. Such organisms as Pediococcus spp., Lactobacillus spp., Corynebacterium spp., Rhodococcus spp., Leuconostoc spp., and Streptococcus pneumoniae were obtained from M. B. Coyle, Seattle, Wash.; G. Wauters, Brussels, Belgium; J. A. Washington, Cleveland, Ohio; A. L. Barry, Tualatin, Oreg.; R. Jones, Iowa City, Iowa; and Ken Rolston and A. Wanger, Houston, Tex. The strains used in the study were identified by defined biochemical tests or with commercially available Analytab Products strips (bio Merieux SA, Plainview, N.Y.). The identification of rhodococci was based on the phenotypic and biochemical characteristics (29) of these organisms, with the biochemical tests present in Coryne Analytab Products strips (bio Merieux SA) being used. MIC determination. The following agents were used: sanfetrinem (formerly GV104326) (Glaxo, Verona, Italy) and ampicillin, vancomycin, and nafcillin (Sigma Chemicals, St. Louis, Mo.). The MIC of each antimicrobial agent was determined by following the National Committee for Clinical Laboratory Standards guidelines for antimicrobial susceptibility testing by the agar dilution and broth microdilution methods (26). Mueller-Hinton medium (Difco Laboratories, Detroit, Mich.) with serial twofold dilutions of antibiotics was used for nonfastidious bacteria. This medium was supplemented with 5% lysed sheep blood (Bethyl Laboratories, Montgomery, Tex.) for the testing of streptococci, Pediococcus spp., Rhodococcus spp., Leuconostoc spp., and Corynebacterium spp. Incubation was carried out at 35 6 18C for 24 h for enterococci, staphylococci, and Rhodococcus spp., while other organisms were incubated at 35 6 18C in a 5% CO2 atmosphere. The standard reference strains used were E. faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, Escherichia coli ATCC 29212, S. pneumoniae ATCC 49619, and Pseudomonas aeruginosa ATCC 27853. The susceptibilities to ampicillin, nafcillin, and vancomycin were interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (26). Bactericidal activity. Time-kill studies were performed to determine the bactericidal activity (20, 25) of GV in comparison with that of ampicillin or nafcillin against one E. faecalis (Bla1), one E. faecium (highly resistant to ampicillin and vancomycin), one methicillin-resistant S. aureus (MRSA) (Bla2), and two methicillin-susceptible S. aureus (MSSA) (Bla1) strains. Organisms were used at final inoculum concentrations of 105 and 107 CFU/ml in brain heart infusion (BHI) broth (Difco) with or without the test drugs. The drug concentrations selected for time-kill studies were twofold higher than the microdilution MIC for that particular organism and fourfold higher for MRSA. Tenfold serial dilutions for colony counts were performed at 0, 4, and 24 h of incubation, and a 25-ml aliquot was plated in duplicate on BHI agar plates for the recovery of viable cells. For the MRSA and MSSA strains, 1-ml aliquots of cells were removed from the experimental flasks at 24 h, washed once with 0.9% saline, and then serially diluted and plated on BHI agar plates for colony counts to eliminate a possible carryover effect. Experimental cultures in flasks or the colony count plates were
MATERIALS AND METHODS Bacterial isolates. Most of the enterococcal isolates were obtained from different locations between 1980 and 1995. Staphylococci (b-lactamase positive [Bla1] and negative [Bla2]) and streptococci (groups A and B and alpha-hemolytic) were collected in Texas between 1989 and 1995. Vancomycin-resistant
* Corresponding author. Mailing address: The Division of Infectious Diseases, University of Texas Medical School at Houston, 6431 Fannin St., 1.728 JFB, Houston, TX 77030. Phone: (713) 745-0131. Fax: (713) 745-0130. 2142
TABLE 1. In vitro activities of Glaxo’s sanfetrinem compared with those of vancomycin, ampicillin, and/or nafcillin MIC (mg/ml) Organism(s) (no. of isolates)
E. faecalis (94) Bla1 strains with or without HLRG (14)a Bla2 strains with HLRG (33)b Other strains (47)c E. faecium (51) Vanr strains with HLR to ampicillin (19)d Vans strains with HLR to ampicillin (11)d Amps-Ampi Vanr (8)e Amps-Ampi Vans (13)e S. aureus (36) MSSA (20) MRSA (16) Streptococci (39) Group A streptococci (9)
Group B streptococci (10)
S. pneumoniae (10)
Alpha-hemolytic streptococci (10)
Other gram-positive spp. (67) Rhodococcus spp. (14)
Leuconostoc spp. (12)
Pediococcus spp. (17)
Corynebacterium spp. (13)
Lactobacillus spp. (11)
Drug
Range
50%
90%
Sanfetrinem Ampicillin Vancomycin Sanfetrinem Ampicillin Vancomycin Sanfetrinem Ampicillin Vancomycin
0.5–8 1–4 1–2 0.125–8 0.125–4 1–.256 1–8 1–16 0.5–.256
2 2 2 2 1 1 1 1 1
2 4 2 2 2 2 2 2 2
Sanfetrinem Ampicillin Vancomycin Sanfetrinem Ampicillin Vancomycin Sanfetrinem Ampicillin Vancomycin Sanfetrinem Ampicillin Vancomycin
16–64 64–512 32–.256 16–32 64–256 0.5–1 4 2–8 .256 1–8 1–16 0.5–2
16 256 .256 16 64 1
32 512 .256 32 256 1
2 2 1
4 16 2
Sanfetrinem Nafcillin Vancomycin Sanfetrinem Nafcillin Vancomycin
0.062–0.25 0.25–1 0.5–1 0.25–.64 8–.256 0.5–2
0.062 0.5 0.5 8 64 1
0.062 0.5 1 .64 256 1
Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin
0.003–0.007 0.003–0.031 0.003–0.015 0.25 0.031–0.062 0.25 0.25 0.5 0.007–0.25 0.031–16 0.062–16 0.25 0.007–2 0.031–32 0.062–64 0.25–1
0.062 0.25 0.25 0.5 0.031 0.062 0.125 0.25 0.062 0.25 4 0.5
0.062 0.25 0.25 0.5 0.25 2 8 0.25 2 32 64 0.5
Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin Sanfetrinem Ampicillin Nafcillin Vancomycin
0.25–32 0.25–.512 8–.256 0.25–.256 0.5–16 0.125–2 0.5–32 .256 1–8 1–8 16–128 .256 0.007–.64 0.062–256 1–.256 0.25–4 0.125–8 0.5–4 4–16 0.5–.256
a
HLRG, high-level resistance to gentamicin. Includes three endocarditis and three Vanr strains. Includes 12 endocarditis and 1 Vanr strains. d HLR is defined here as an MIC of ampicillin of $64 mg/ml. e Amps-Ampi, susceptible or intermediate to ampicillin, defined here as an MIC of ampicillin of #16 mg/ml. b c
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4 512 .256 .256 8 1 8 .256 1 2 32 .256 0.25 32 64 0.5 0.5 0.5 4 0.5
32 .512 .256 .256 16 2 16 .256 2 4 32 .256 .64 256 .256 1 4 1 16 256
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TABLE 2. Comparative killing of enterococcal and staphylococcal isolates by sanfetrinem in time-kill studies
Strain
MIC (mg/ml) in broth (concn [mg/ml] used for time-kill experiment) Sanfetrinem
Ampicillin
2 (4)
16 (32)
E. faecium A358
32 (64)
64 (128)
MRSA 26 (Bla2)
4 (16)
MSSA JT 9 (Bla1)
0.062 (0.125)
16
MSSA JT 16 (Bla1)
0.062 (0.125)
8
E. faecalis HH22 (Bla1)
8
Initial inoculum for sanfetrinem (log10)
Initial inoculum for ampicillin or nafcillin (log10)
5.3 7.2 5.1 7.0 4.7 6.8 5.0 6.8 5.1 6.7
5.2 7.2 5.0 7.2 4.7 6.7 4.9 6.7 5.1 6.6
Nafcillin
8 .256 32 (128) 0.25 (0.5) 0.5 (1)
incubated at 358C. A reduction of $3 log10 CFU/ml from the concentration of the starting inoculum was defined as a bactericidal effect (25). Synergy experiment. The synergy study was performed in BHI broth with E. faecalis JH2-7 at an inoculum concentration of ;107 CFU/ml and GV at 2 mg/ml and gentamicin at 5 mg/ml, separately and together, being used. Colony counts at 0, 4, and 24 h were performed as described above. The carryover effect at the end of a 24-h incubation was also determined.
RESULTS The results of susceptibility testing of 287 organisms with GV and other antibiotics are summarized in Table 1. For E. faecalis strains with various resistance phenotypes and origins (e.g., b-lactamase producing, gentamicin resistant, and vancomycin resistant; from endocarditis patients; from Bangkok, Thailand; from Argentina; and from the United States), the MICs of GV and those of ampicillin as determined by agar dilution were the same or within 1 dilution. For E. faecium, GV was generally 4 to 16 times more active than ampicillin. For the nine strains of E. faecium for which the MICs of ampicillin were lowest (MIC, 1 to 4 mg/ml), GV varied from being two times less active to two times more active than ampicillin, i.e., 61 dilution. For the 12 strains of E. faecium for which the MICs of ampicillin were 8 to 16 mg/ml, GV was two to four times more active than ampicillin (MIC of GV, 2 to 8 mg/ml). For 25 strains of E. faecium with HLR to ampicillin (MIC, 64 to 256 mg/ml), GV was four to eight times more active than ampicillin. For five strains of E. faecium for which the MICs of ampicillin were 512 mg/ml, the MICs of GV were 32 mg/ml for four isolates and 64 mg/ml for one isolate; in other words, GV was 8 to 16 times more active against the most highly ampicillin-resistant strains. Many of our ampicillin-resistant strains were also vancomycin resistant. However, some of the vancomycin-resistant strains were susceptible to ampicillin. GV at the concentration at which 90% of the isolates are inhibited (MIC90) was eight times more active than nafcillin against MSSA strains. For 6 of 16 MRSA strains studied, the MICs of GV ranged from 0.25 to 2 mg/ml whereas nafcillin had MICs ranging from 8 to 64 mg/ml; for 5 other strains, GV was four to eight times more active (MIC, 8 to 32 mg/ml) than nafcillin (MIC, 32 to 128 mg/ml). For the remaining five MRSA strains, the MICs of nafcillin were $256 mg/ml and the MICs of GV were $64 mg/ml. GV was two to four times more active than the other betalactams against all group B streptococci and against most group A streptococci. The MIC90 of GV was 0.25 mg/ml for S. pneumoniae strains, while ampicillin and nafcillin were 8 and 32 times less active than GV (the MIC90s of ampicillin and
Change from baseline (log10) Sanfetrinem
Ampicillin or nafcillin
4h
24 h
4h
24 h
22.1 21.3 20.6 20.4 21.5 20.1 22.8 22.0 21.8 21.5
23.7 23.7 21.5 22.5 22.2 0.8 25 24.9 23.8 21.2
21.7 21.6 20.1 20.1 20.6 20.6 22.3 21.9 21.3 21.3
23.5 1.4 21.8 21.2 22.2 0.7 22.2 22.5 22.5 21.6
nafcillin were 2 and 8 mg/ml, respectively). Against alphahemolytic streptococci, GV was 16 times more active at the MIC90 than ampicillin and showed 32 times more activity than nafcillin. The MIC90s of GV were 2 to .64 mg/ml for Leuconostoc spp., Pediococcus spp., Corynebacterium spp., and Lactobacillus spp., while the MIC90s of ampicillin and nafcillin were 1 to 256 and 16 to .256 mg/ml, respectively. MICs of vancomycin for these organisms were 1 to .256 mg/ml. Against rhodococci, GV at the MIC90 demonstrated at least 8 to 16 times more activity than nafcillin, ampicillin, and vancomycin at the MIC90. Broth microdilution MIC results for GV, ampicillin, nafcillin, and vancomycin with all the test organisms were either the same as or within one dilution of the agar dilution MIC values (data not shown). The results of the time-kill activity tests are summarized in Table 2. Bactericidal activity of GV against one b-lactamaseproducing E. faecalis strain and one of the MSSA strains (JT 9) at inoculum concentrations of 105 and 107 CFU/ml was observed at 24 h. Ampicillin showed bactericidal activity against the b-lactamase-producing E. faecalis strain at an inoculum concentration of 105 CFU/ml but not at 107 CFU/ml. GV was also bactericidal for another MSSA strain (JT 16) at an inoculum concentration of 105 CFU/ml but was not bactericidal for this organism at 107 CFU/ml. Nafcillin was not bactericidal for either the MRSA or MSSA strains at either inoculum concentration. Neither GV nor ampicillin was bactericidal for the E. faecium Ampr or the MRSA strain. Against an E. faecalis strain, GV at 2 mg/ml demonstrated a synergistic effect with gentamicin at 5 mg/ml (Fig. 1). Gentamicin by itself at 5 mg/ml did not have any inhibitory effect, while GV by itself at 2 mg/ml caused an ;2-log10 reduction in the starting inoculum. No significant carryover effect was observed with GV alone or with GV combined with gentamicin. DISCUSSION Sanfetrinem, a new trinem developed by Glaxo, has been shown to have broad-spectrum activity against many gramnegative bacteria, MRSA, and MSSA and against the limited number of enterococci tested (6). This compound has also been shown to be quite stable in the presence of clinically relevant b-lactamases and was not degraded by rodent or human renal dehydropeptidases (6, 30). The present study is the first study of this compound in which a large number of multiresistant E. faecalis and E. faecium strains as well as MRSA,
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gentamicin against an E. faecalis strain, GV appears to be a promising drug for use against multiresistant enterococci and other gram-positive organisms. ACKNOWLEDGMENTS This work was supported by a grant from Glaxo, Verona, Italy. We thank M. B. Coyle, Seattle, Wash.; G. Wauters, Brussels, Belgium; J. A. Washington, Cleveland, Ohio; A. L. Barry, Tualatin, Oreg.; R. Jones, Iowa City, Iowa; and Ken Rolston and A. Wanger, Houston, Tex., for providing some of the strains. REFERENCES
FIG. 1. Time-kill curve for GV at 2 mg/ml (GV2), gentamicin at 5 mg/ml (Gen5), and the two drugs together against E. faecalis JH2-7.
MSSA, rhodococci, streptococci, Corynebacterium spp., Lactobacillus spp., Leuconostoc spp., and Pediococcus spp. were evaluated. In this study, the MIC range of GV for all the E. faecalis strains studied was consistent with that previously reported for 10 E. faecalis strains (6). The activities of GV at MIC50 and MIC90 against our ampicillin- and vancomycin-susceptible E. faecium strains were also consistent with those reported previously (6), but the activities of GV against highly ampicillinand vancomycin-resistant E. faecium strains were superior to those of ampicillin. The overproduction or low affinity of PBP5 has been demonstrated (7, 16) to be one of the mechanisms in E. faecium resistance to beta-lactam compounds. The superior activity of GV in the present study against the highly ampicillin-resistance E. faecium strains could be due to the better affinity of the penicillin-binding proteins of these organisms for this compound. The MICs of GV against MSSA and 11 of the MRSA strains were similar to those reported earlier (6), but 5 of our MRSA strains showed GV MICs of $64 mg/ml, which were higher than those in the previously published report. In the present study, the six MRSA strains for which the MICs of GV were lowest (0.25 to 2 mg/ml) showed nafcillin MICs of 8 to 64 mg/ml. Other gram-positive organisms, such as Streptococcus spp., Corynebacterium spp., Rhodococcus spp., Lactobacillus spp., Pediococcus spp., and Leuconostoc spp., have also been attracting the attention of clinicians and microbiologists because of the vancomycin resistance of some of these microbes, the multiresistance of others, and the occasional occurrence of severe infections caused by these organisms (3–5, 15, 18, 28, 31, 34). In the present study, GV was equally active as or more active than ampicillin against most isolates of Pediococcus spp. and Corynebacterium spp. and 8 to 16 times more active than vancomycin and ampicillin against Rhodococcus spp. It was also more active against a-streptococci, especially ampicillin-resistant strains. On the basis of the MICs of GV for highly ampicillin- and/or vancomycin-resistant enterococci and other resistant grampositive organisms, its bactericidal activity against a b-lactamase-producing E. faecalis strain, and its synergistic effect with
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