1 Anti-Infective Research Laboratory, Department of Pharmacy Services, Detroit Receiving Hospital/University Health Center, 2 Wayne State UniversityCollege of Pharmacy and Allied Health Professions and 4 Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, Detroit, MI 48201; 3 The University of New MexicoCollege of Pharmacy, Albuquerque, NM 87131, USA
Received 16 July 2001; returned 9 November 2001; revised 18 December 2001; accepted 14 March 2002
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Abstract |
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Introduction |
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Oritavancin has antimicrobial activity unique among glycopeptides currently available; rapid, concentration-dependent killing has been demonstrated in several in vitro analyses.11,15 Oritavancin is bactericidal against some strains of VRE, supporting the possibility of monotherapy for VRE.11,12 In vitro investigations have evaluated the activity of oritavancin under varying conditions, such as in the presence of albumin, serum and large inoculum; however, the effects of pH and growth phase on oritavancin are currently unknown.11 These conditions become increasingly important in deep-seated infections such as endocarditis, where vegetations may consist of inocula as high as 10910 cfu/gram.17 In such environments, bacteria exist in a stationary growth phase within acidic conditions and may become less susceptible to antibiotics such as cell wall-active agents.17,18 Previous studies with an investigational agent, daptomycin, reported that increased bacterial inoculum, decreased pH and stationary growth phase resulted in decreased antimicrobial activity.19 The effect of pH and growth phase on antimicrobial activity has been reported for quinolones against S. aureus, as well as aminoglycosides and quinolones against Pseudomonas aeruginosa.2022
The objectives of this study were to compare the effects of pH and growth phase on the activity of oritavancin and vancomycin against S. aureus and E. faecium.
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Materials and methods |
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The following clinical isolates were tested: MRSA-494, vancomycin-susceptible E. faecium (VSEF-679) and vancomycin-resistant (vanA) E. faecium (VREF-7303). The S. aureus bloodstream isolate was obtained from the University of Michigan Medical Center (Ann Arbor, MI, USA) and the E. faecium isolates were obtained from William Beaumont Hospital (Royal Oak, MI, USA).
Antibiotics
Vancomycin analytical powder was obtained from Sigma (St Louis, MO, USA) and oritavancin (LY333328) powder was supplied by Eli Lilly & Co. (Indianapolis, IN, USA). Stock solutions of each antibiotic were prepared in distilled and deionized sterile water to achieve a concentration of 1 mg/mL for each antibiotic. Further dilutions were made to achieve the desired target concentrations for each specific experiment.
Media
MuellerHinton broth (Difco, Detroit, MI, USA) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) (SMHB) was used for susceptibility testing and kill curve experiments. Tryptic soy agar (TSA) (Difco) was used for carrying out colony counts.
In vitro antibiotic susceptibility tests
MICs and minimum bactericidal concentrations (MBCs) were determined for each drug (oritavancin and vancomycin) by broth microdilution following the technique described by the National Committee for Clinical Laboratory Standards.23 MICs were determined at a standard inoculum of c. 5 x 105 cfu/mL for all three isolates.
Timekill studies
Oritavancin and vancomycin were both tested against the MRSA and VSE strains, whereas only oritavancin was tested against the VRE isolate. For all test tube experiments, antibiotics were added at 4 x MIC for the strain tested. Appropriate dilutions were made to minimize the effect of antibiotic carryover. Each test tube experiment was carried out in duplicate. Bacterial colonies from an overnight growth on TSA were used to prepare the starting inoculum. An initial inoculum of log10 106 cfu/mL was obtained by diluting 1 mL of a 0.5 McFarland suspension in 9 mL of SMHB or saline and then adding 0.8 mL of the diluted 0.5 McFarland suspension to 7.2 mL of SMHB containing antibiotics. Further dilutions were made as appropriate for the exponential growth phase experiments as described below.
Stationary growth phase. Sampling immediately after transferring the organism from saline to SMHB simulated the stationary growth phase. All experiments were carried out in duplicate and samples (100 µL) were taken at 0, 0.5, 1, 2, 4, 8 and 24 h. Serial dilutions (10-fold) were carried out and 20 µL aliquots plated in triplicate on TSA. The plates were incubated at 37°C for 1824 h, organisms were quantified and the resultant log10 cfu/mL was plotted versus time to produce killing curves.
Exponential growth phase. Exponential growth phase experiments were assessed by incubating each isolate in SMHB for 3 h before the addition of antibiotics. Preliminary growth curve data confirmed that both the S. aureus and E. faecium strains reached exponential growth phase at 2 h and demonstrated an increase in inoculum of 1 log10 cfu by 3 h. Initial bacterial inocula of 104 cfu/mL were prepared for all isolates tested in SMHB and incubated at 37°C. Sampling was carried out every hour for 3 h before antibiotic inoculation, to document exponential growth.
pH variation. The effects of pH on the activity of oritavancin and vancomycin were assessed using pH-adjusted SMHB. Studied pHs of 6.4, 7.4 and 8.0 were obtained by titration with either 1 M hydrochloric acid or 0.1 M NaOH. The pH was monitored throughout the experiment at each sampling time.
Statistical analysis
The number of log10 cfu/mL at 24 h was compared among treatment groups and growth controls using an ANOVA with Tukeys test for multiple comparisons of significance. A P value of 0.05 was considered significant in these analyses. The time required to achieve 99.9% killing was determined by linear regression or visual inspection of the killing curves and compared using a KaplanMeier survival test. An r2 value of
0.9 was considered significant in these analyses.
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Results |
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The MIC/MBCs for all isolates tested are summarized in Table 1.
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Killing curves for MRSA, VSE and VRE in stationary and exponential growth phase are depicted in Figures 13, respectively. There was no significant difference in the log10 cfu/mL at 24 h between stationary and exponential phase for vancomycin against either the MRSA or VSE isolate (Figures 1 and 2); however, there was a strong tendency for vancomycin to be more effective in killing VSE at 24 h (Figure 2) in exponential phase than in stationary phase (P = 0.052). Moreover, there was also a tendency for a more rapid killing rate when vancomycin was tested against the MRSA isolate (Figure 1) in an exponential growth phase versus a stationary one (P = 0.09). Oritavancin was equally effective in killing both the MRSA and VSE strains in the stationary and exponential growth phase at 24 h. Oritavancin appeared to have a greater rate of killing against the MRSA strain (Figure 1), with a 99.9% inoculum reduction in the exponential phase in 0.51 ± 0.02 h, compared with the stationary phase (2.1 ± 0.29 h; P = 0.02). Oritavancin was equally effective in the rate of killing of VSE (Figure 2) in both growth phases as measured by time to 99.9% reduction in log10 cfu/mL: 0.8 ± 0.13 h versus 0.5 ± 0.0 h, respectively. Although there were no significant differences in reduction of bacterial density for the MRSA strain (Figure 1) between vancomycin and oritavancin at 24 h, time to achieve 99.9% kill was significantly less for oritavancin (1.0 ± 0.01 h; P < 0.02). Against the VRE isolate (Figure 3), oritavancin had significantly greater activity versus vancomycin for the first 8 h for both growth phases (P = 0.02). These differences were also noted at the 24 h time point, except for vancomycin in exponential growth phase. No other significant differences were found between these antibiotics and this isolate of E. faecium. Time to achieve 99.9% kill occurred in 0.6 ± 0.01 h for oritavancin against the VRE strain in exponential phase, whereas in the stationary growth phase, oritavancin did not decrease the inoculum by 99.9% in <24 h (Figure 3).
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Discussion |
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The activity of oritavancin against MRSA and VRE has been tested under varying in vitro conditions. An inoculum effect was seen with MRSA exposed to oritavancin at 4 x MIC. Starting with an inoculum of 107108 cfu/mL, timekill studies demonstrated decreased activity, and regrowth occurred at 24 h.11 Although not examined in this study, protein binding is another factor affecting the activity of oritavancin. We have previously reported that the activity of oritavancin was decreased against MRSA and VRE when measured with SMHB plus albumin in comparison with SMHB alone. Increasing concentrations of oritavancin to 8 x MIC appeared to restore activity.11
Decreased binding to the penicillin-binding proteins (PBPs) and the loss of PBPs may be responsible for the decreased activity of ß-lactam antibiotics against stationary growth phase streptococcal infections.17 Other classes of antibiotics, such as the quinolones, aminoglycosides, carbapenems, lipopeptides and the glycopeptide antibiotic teicoplanin, have also been investigated and found to be affected by the growth phase of the organism.9,17,18,2022 Eng et al.9 have demonstrated that against slow-growing S. aureus strains, aminoglycosides are the least affected, followed by quinolones, which remain bactericidal. The carbapenems (meropenem and imipenem) and nafcillin are bactericidal against rapidly growing S. aureus, but only become static when the growth phase of the organism becomes minimal. It has already been shown that oritavancin is affected by the size of the inoculum and there is a slower growth of bacteria as the inoculum size is increased.11 However, the fact that growth phase did not seem to influence oritavancins activity against MRSA and VSE is important, since it has been demonstrated that sequestered infections such as bacterial endocarditis, abscesses and osteomyelitis may contain organisms in various stages of growth.23 Even though the rate of killing of oritavancin was optimal against an exponential phase S. aureus, such as that shown previously with daptomycin, the rate at which oritavancin decreased the stationary inoculum was still faster than that reported with vancomycin.9,11
Another important goal of our study was to investigate the effect of pH alteration on the activity of oritavancin. Previous studies have shown that the rate of killing of different antibiotics may vary depending upon the pH of the environment.18,19 Lamp et al.19 demonstrated that daptomycin, a lipopeptide with similar pharmacodynamic properties to oritavancin, was affected by changes in pH. As the pH increased, the activity of daptomycin was increased against S. aureus. However, these experiments were carried out over only 6 h as opposed to our 24 h killing curves; we do not know whether these differences would have remained significant after 24 h of exposure to daptomycin. In contrast, the change in pH did not affect oritavancins rate of killing or the colony counts during the first 6 h of the timekill studies or at 24 h. Vancomycin killed the S. aureus isolate more effectively when the broth pH was adjusted to 8.0 rather than 6.4. A similar study looking at the effect of pH changes reported a trend for vancomycin to kill S. aureus more rapidly at higher pH,18 although against the E. faecium isolate vancomycin killed more effectively at lower pH. These experiments were repeated several times obtaining the same results, and to our knowledge there are limited data looking at the effect of pH changes against E. faecium, which limits our ability to understand and elucidate this phenomenon.
In summary, oritavancin is a potent glycopeptide with activity against resistant Gram-positive organisms. Its favourable pharmacodynamic properties observed in this study may favour its use for treatment of a variety of infections. Future animal and human studies are needed to confirm our findings.
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Acknowledgements |
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Footnotes |
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Corresponding author. Tel: +1-313-745-4554; Fax: +1-313-993-2522; E-mail: m.rybak{at}wayne.edu
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References |
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