Effect of growth phase and pH on the in vitro activity of a new glycopeptide, oritavancin (LY333328), against Staphylococcus aureus and Enterococcus faecium

Renee-Claude Mercier1,2,3,*, Carmine Stumpo2 and Michael J. Rybak1,2,4,§

1 Anti-Infective Research Laboratory, Department of Pharmacy Services, Detroit Receiving Hospital/University Health Center, 2 Wayne State University—College 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 Mexico—College of Pharmacy, Albuquerque, NM 87131, USA

Received 16 July 2001; returned 9 November 2001; revised 18 December 2001; accepted 14 March 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oritavancin (LY333328) is a novel glycopeptide with activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. We compared the effects of pH and growth phase on the activity of oritavancin and vancomycin against methicillin-resistant S. aureus and vancomycin-susceptible and -resistant E. faecium. Killing curve methods were used to evaluate the effect of growth phase (stationary versus exponential) and pH (6.4, 7.4 and 8.0). An inoculum of 106 cfu/mL was used for all experiments. Growth phase of S. aureus and vancomycin-susceptible E. faecium did not influence the rate and killing activity of oritavancin. The rate of killing by oritavancin against the vancomycin-resistant E. faecium strain was significantly faster and the reduction in cfu/mL at 24 h was significantly greater when the organism was in exponential compared with stationary growth phase (P < 0.05). In exponential growth phase, time to 99.9% killing was achieved in 0.6 ± 0.01 h for the vancomycin-resistant strain, whereas in stationary growth phase, oritavancin did not decrease the inoculum by 99.9% within 24 h. Oritavancin’s activity against S. aureus and vancomycin-susceptible E. faecium was not influenced by the pH conditions tested. Oritivancin’s killing activity against the vancomycin-resistant E. faecium strain was significantly enhanced when tested at pH 7.4 and 8.0 (P < 0.05). Our study has demonstrated that oritavancin’s activity does not seem to be influenced by the growth phase of the organisms or the pH of the environment when tested against sensitive strains of S. aureus and E. faecium. However, oritavancin’s activity might be reduced against vancomycin-resistant E. faecium strains in stationary growth phase, as seen in infective endocarditis or when organisms are exposed to an acidic environment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
New antimicrobials have been developed in response to vancomycin-resistant enterococci and the potential emergence of vancomycin-resistant Staphylococcus aureus.13 Oritavancin (LY333328) is a novel glycopeptide that is similar in structure to vancomycin. The mechanism of antimicrobial activity appears to involve inhibition of the transglycosylation of the cell wall, via inhibition of the peptidoglycan intermediates at the D-alanyl-D-alanine terminus. Additionally, the drug might also impair RNA synthesis.4 Oritavancin is active against many Gram-positive organisms, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE), but has little activity against Gram-negative organisms.515 Oritavancin’s activity against VRE is not fully understood; however, it may be related to the formation of drug dimers at low concentrations and the ability to associate with bacterial membranes in a manner similar to teicoplanin.16

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 109–10 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.


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

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

Mueller–Hinton 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.

Time–kill 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 18–24 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 Tukey’s 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 Kaplan–Meier survival test. An r2 value of >=0.9 was considered significant in these analyses.


    Results
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility testing

The MIC/MBCs for all isolates tested are summarized in Table 1.


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Table 1.  Summary of MICs and MBCs (mg/L)
 
Time–kill studies

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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Figure 1. Methicillin-resistant S. aureus-494 growth phase. Stationary phase: black circles, oritavancin; black triangles, vancomycin; black squares, growth control. Exponential phase: white circles, oritavancin; white triangles, vancomycin; white squares, growth control.

 


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Figure 3. Vancomycin-resistant E. faecium-7303 growth phase. Stationary phase: black circles, oritavancin; black triangles, growth control. Exponential phase: white circles, oritavancin; white triangles, growth control.

 


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Figure 2. Vancomycin-susceptible E. faecium-679 growth phase. Stationary phase: black circles, oritavancin; black triangles, vancomycin; black squares, growth control. Exponential phase: white circles, oritavancin; white triangles, vancomycin; white squares, growth control.

 
The effects of pH on the activity of oritavancin and vancomycin against MRSA, VSE and VRE are represented in Figures 46, respectively. Overall, pH did not vary significantly from baseline during the various killing curve experiments. Vancomycin against the MRSA strain (Figure 4) tested in SMHB adjusted to pH 8.0 was significantly more effective in decreasing the inoculum than when studied at pH 6.4 (P = 0.01). However, when vancomycin was tested at pH 6.4 against the VSE isolate (Figure 5), the cfu/mL at 24 h was significantly lower than when analysed at pH 7.4 and 8.0 (P < 0.01). There was no difference in the activity of oritavancin detected for the MRSA or VSE isolate (Figures 4 and 5). However, oritavancin’s killing activity against the VRE strain (Figure 6) was significantly enhanced when tested at pH 7.4 or 8.0 (P < 0.05).



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Figure 4. Effect of changes of pH against methicillin-resistant S. aureus-494. Oritavancin: black circles, 6.4; white circles, 7.4; black downward triangles, 8.0. Vancomycin: white triangles, 6.4; black squares, 7.4; white squares, 8.0. Growth control: black diamonds, 6.4; white diamonds, 7.4; black upward triangles, 8.0.

 


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Figure 6. Effect of changes of pH against vancomycin-resistant E. faecium-7303. Oritavancin: black circles, 6.4; white circles, 7.4; black triangles, 8.0. Growth control: white triangles, 6.4; black squares, 7.4; white squares, 8.0.

 


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Figure 5. Effect of changes of pH against vancomycin-susceptible E. faecium-679. Oritavancin: black circles, 6.4; white circles, 7.4; black downward triangles, 8.0. Vancomycin: white triangles, 6.4; black squares, 7.4; white squares, 8.0. Growth control: black diamonds, 6.4; white diamonds, 7.4; black upward triangles, 8.0.

 
Against MRSA and VSE (Figures 4 and 5), the log10 cfu/mL at 24 h was significantly lower at all pH values studied with oritavancin versus vancomycin (P < 0.001). Vancomycin never achieved 99.9% kill against either isolate, whereas oritavancin decreased the inoculum by 99.9% after 3.1 ± 0.1 h (pH = 6.4), 3.6 ± 0.75 h (pH = 7.4) and 2.8 ± 1.9 h (pH = 8.0) against the MRSA isolate. At all pH values tested against the VSE isolate (Figure 5), oritavancin achieved 99.9% kill in <0.5 ± 0 h. Time to 99.9% kill for oritavancin was achieved after 8 h for the VRE isolate (Figure 6) at pH 7.4 and 8.0. However, this target was not achieved against the VRE isolate when tested at pH 6.4.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oritvancin’s activity against VRE was affected by an acidic pH. However, pH did not influence oritavancin’s activity against S. aureus or VSE. The rapid bactericidal effects of oritavancin against staphylococci and enterococci illustrated above are consistent with previous studies.6,10,14 An in vitro analysis by Schwalbe et al.14 studied the activity of oritavancin against a total of 219 enterococcal and staphylococcal strains. Oritavancin was active against all methicillin-resistant staphylococci strains tested, with MBCs equal to or a two-fold dilution higher than the MICs. The MBCs ranged from 1 to 16 mg/L for enterococci, with higher values associated with vancomycin resistance.14 Other in vitro analyses support our results, demonstrating favourable MIC:MBC ratios for oritavancin against staphylococcal isolates and VRE.6,10,13

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 107–108 cfu/mL, time–kill 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 oritavancin’s 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 oritavancin’s rate of killing or the colony counts during the first 6 h of the time–kill 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.


    Acknowledgements
 
We are very grateful to Dr Glenn Kaatz, Wayne State University—School of Medicine and Dr Marcus Zervos, William Beaumont Hospital for providing us with the MRSA-494 and E. faecium strains, respectively.


    Footnotes
 
* Present address. The University of New Mexico, College of Pharmacy, 2502 Marble NE, Albuquerque, NM 87131, USA. Back

§ Corresponding author. Tel: +1-313-745-4554; Fax: +1-313-993-2522; E-mail: m.rybak{at}wayne.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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23 . Johnson, C. C. (1996). In vitro testing: correlations between bactericidal susceptibility, body fluid levels, and effectiveness of antibacterial therapy. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., ed.), pp. 813–34. The Williams & Wilkins Co., Baltimore, MD.