The in vitro and in vivo antibacterial characterization of vancomycin and linezolid against vancomycin-susceptible and -resistant enterococci

Shuichi Miyazaki*, Toshihiko Fujikawa, Intetsu Kobayashi, Tetsuya Matsumoto, Kazuhiro Tateda and Keizo Yamaguchi

Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143, Japan

Received 29 July 2002; returned 10 September 2002; revised 12 September 2002; accepted 12 September 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study was designed to compare in vitro antibacterial activities of linezolid and vancomycin against vancomycin-susceptible Enterococcus faecalis (VSEF) and vancomycin-resistant enterococci (VRE) isolated in Japan with those of quinupristin–dalfopristin, teicoplanin and minocycline, and the in vitro short time bactericidal activity and the in vivo activities of linezolid and vancomycin against vancomycin-susceptible and -resistant E. faecalis. The MIC90s of linezolid, quinupristin–dalfopristin, vancomycin, teicoplanin and minocycline for VSEF and VRE were both 2 mg/L, both 2 mg/L, 2 and >128 mg/L, 0.25 and >128 mg/L, and both 32 mg/L, respectively. The efficacy of linezolid for mice with bacteraemia caused by VSEF was similar to that of vancomycin, but the elimination ratio of viable organisms from the blood of mice treated with vancomycin was significantly higher than in linezolid-treated and untreated mice at 2 h post-administration, and those of the two groups at 4 and 6 h were significantly higher than in untreated mice. Moreover, linezolid was highly active in mice with bacteraemia caused by vancomycin-resistant E. faecalis_because this drug had potent in vitro activity against the organisms. Our results indicate that linezolid is suitable for the treatment of VRE and VSEF bacteraemia, and vancomycin is suitable for VSEF bacteraemia.

Keywords: linezolid, vancomycin, VSEF, VRE


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vancomycin-resistant enterococci (VRE) bacteraemia is reported to be associated with extensive co-morbid pathology, prolonged hospitalization, heavy exposure to antimicrobial agents such as vancomycin, metronidazole and ß-lactams and high crude mortality rates.1 One important_strategy is often recommended to prevent colonization of VRE organisms: restrict vancomycin use in hospitals. Hence, there continues to be a need for new therapeutic agents for treatment of patients with VRE. At present, there are some reports on in vivo evaluation of efficient antimicrobial agents for VRE infection, using experimental models.2,3 However, in these reports, the drugs were evaluated using laboratory animals with intra-abdominal abscess and endocarditis. Clinically, the majority of VRE infections manifest as bacteraemia, and this type of infection is often associated with colonization of VRE organisms in the alimentary tract.1,46 Thus, the bacteraemia model in animals is insufficient to address this question.

Based on the above background, we recently established a model of bacteraemia caused by VRE in mice.7 Using this model in the present study, we assessed the in vitro and in vivo antibacterial activities of linezolid and other antimicrobial agents against vancomycin-susceptible Enterococcus faecalis (VSEF) or VRE.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

Vancomycin-resistant E. faecalis v583, which was isolated from blood culture, was a kind gift from Dr B. E. Murray (The University of Texas, Houston, TX, USA).8 VSEF TMS5 was isolated from a blood sample of a Japanese patient with bacteraemia. Thirty strains of VSEF and 17 strains of VRE (E. faecalis, three isolates; Enterococcus faecium, eight isolates; Enterococcus gallinarum, six isolates), including vancomycin-intermediate enterococci, were clinically isolated from in-patients in various Japanese hospitals.

In vitro susceptibility tests

The MICs of vancomycin, linezolid, quinupristin–dalfopristin, teicoplanin and minocycline for test isolates were determined by the broth microdilution method in 0.1 mL volumes of cation-adjusted Mueller–Hinton broth.9 Incubation was for 20 h at 35°C. Microdilution plates were inoculated with an automatic pin inoculator (MIC-2000; Dynatech Laboratories, Inc., Alexandria, VA, USA) to a final inoculum of ~5 x 105 cfu/mL.

Animals and treatment

C57BL/6 strain mice, 5 weeks old, were a generous gift from Charles River Japan (Atsugi, Kanagawa, Japan). Bacteraemia was induced in mice using the method described previously.7 Briefly, leucopenia was induced in mice by pre-treatment with 250 mg/kg cyclophosphamide (Shionogi Co., Osaka, Japan) intraperitoneally, 3 days before oral inoculation of VSEF or vancomycin-resistant E. faecalis organisms. Furthermore, to reduce normal intestinal flora, a combination of metronidazole, kanamycin and vancomycin was administered orally once a day for 3 days at a dose of 100 mg/kg, commencing on the same day as administration of cyclophosphamide. One day after treatment with antimicrobial agents and cyclophosphamide, mice were orally inoculated with ~3–5 x 109 organisms prepared by centrifugation of overnight culture in brain–heart infusion broth. Six hours after oral inoculation of organisms, each mouse received subcutaneous injection of the test drug (vancomycin or linezolid) at a dose of 25 mg/kg and saline (control) twice a day for 3 days. The survival of mice infected with vancomycin-susceptible or vancomycin-resistant E. faecalis was monitored over a period of 14 days.

In another experiment performed in order to understand the decreasing level of viable organisms from blood in mice treated with linezolid and vancomycin, mice were infected intraperitoneally with 1–2 x 106 VSEF or vancomycin-resistant E. faecalis organisms (0.5 mL), then subcutaneously injected with 25 mg/kg vancomycin or linezolid dissolved in 0.2 mL of distilled water and saline (control) at 3 h post-infection. Blood samples were obtained from infected mice (n = 5) at 1, 2, 4 and 6 h post-administration of each drug, which were serially diluted and plated on blood agar plates to quantify the number of live microorganisms. In a preliminary experiment, antibiotic carry-over from blood did not affect the viable counts when 0.1 mL portions of blood samples or diluents were spread on plates containing 15 mL of blood agar. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Toho University School of Medicine.

Statistical analysis

All data were expressed as mean ± S.E.M. Differences in viable bacterial counts between groups were examined for statistical significance by the Mann–Whitney U-test. Differences in survival rates of mice were analysed by the Prism’s {chi}2 test. A P value <0.05 denoted the presence of a statistically significant difference.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro antibacterial activities of five antimicrobial agents

The MIC90s of linezolid, quinupristin–dalfopristin, vancomycin, teicoplanin and minocycline for clinical isolates of VSEF and VRE were both 2 mg/L, both 2 mg/L, 2 and >128, 0.25 and >128 mg/L, and both 32 mg/L, respectively (Table 1). The MICs of linezolid for VSEF TMS5 and vancomycin-resistant E. faecalis v583 were both 2 mg/L, and those of vancomycin for these strains were 4 and 128 mg/L, respectively.


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Table 1.  Antibacterial activities of five antimicrobial agents against VSEF and VRE
 
In vivo efficacy

In mice orally inoculated with VSEF TMS5, treatment with vancomycin or linezolid was associated with survival of all 30 mice, whereas the number of survivals among untreated mice was only 25 animals. The mortality rate was significantly different (P < 0.05) between treated and untreated groups. In mice orally inoculated with vancomycin-resistant E. faecalis v583, treatment with linezolid was associated with survival in 29 of 30 mice, whereas the numbers of survivals among vancomycin-treated and untreated mice were 25 and 24 mice, respectively. Thus, the mortality rate was significantly lower (P < 0.05) in the linezolid treatment group than the vancomycin treatment and untreated groups.

Elimination of viable organisms from blood

In mice injected intraperitoneally with VSEF TMS5, viable organisms in blood samples from both vancomycin- and linezolid-treated groups decreased gradually after subcutaneous administration of each drug (Figure 1). The viable count tended to be lower in blood samples of vancomycin-treated mice than in linezolid-treated mice, and that in the former, at 2 h post-administration of a drug, was significantly lower than in the latter and untreated mice. In addition, the viable counts in both vancomycin- and linezolid-treated mice at 4 and 6 h after administration of a drug were significantly lower than in untreated mice. In those injected intraperitoneally with vancomycin-resistant E. faecalis, viable organisms in blood samples from linezolid-treated mice decreased gradually after administration of the drug (Figure 2). The viable counts at 4 and 6 h after administration of linezolid were significantly lower than in vancomycin-treated and untreated mice.



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Figure 1. Elimination of E. faecalis from blood of mice treated with vancomycin (open circles), linezolid (closed squares) and untreated mice (closed circles) after intraperitoneal challenge with VSEF TMS5. Data are means ± S.D. of five mice. *P < 0.05, compared with linezolid-treated and untreated mice at the corresponding time. **P < 0.01, ***P < 0.5, compared with untreated mice at the corresponding time interval.

 


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Figure 2. Elimination of E. faecalis from blood of mice treated with vancomycin (open circles), linezolid (closed squares) and untreated mice (closed circles) after intraperitoneal challenge with vancomycin-resistant E. faecalis v583. Data are mean ± S.D. of five mice. *P < 0.01, compared with untreated mice at the corresponding time interval.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following the NCCLS breakpoints, the breakpoints of quinupristin–dalfopristin, vancomycin, teicoplanin and minocycline are <=1, <=4, <=8 and <=4 mg/L, respectively. The tentative breakpoint of linezolid was <=2 mg/L, following the recommendation of Wise et al.10 In the present study, the percentages of isolates susceptible to linezolid, quinupristin–dalfopristin, vancomycin, teicoplanin and minocycline against VSEF were 90%, 49%, 100%, 100%, 26%, respectively, and those of these drugs against VRE were 82%, 41%, 0%, 41% and 41%, respectively. This result indicates that linezolid is probably the most active drug for VRE infections, although vancomycin is the representative agent against vancomycin-susceptible enterococci. These results confirm that vancomycin is effective for treatment of mice with bacteraemia caused by vancomycin-susceptible enterococci, whereas linezolid is suitable for experimental VRE bacteraemia.

Recently, we established an experimental model of bacteraemia caused by VRE in mice.7 The reasons for the establishment of this model included the following: (i) the main mode of infection with VRE is through the oral route in most cases of VRE infection; (ii) bacteraemia is the most important clinical problem; and (iii) the incidence of this form of infection is the highest among VRE infections.1,46,11 To our knowledge, there are studies that have examined the in vivo activity of linezolid in experimental infection models such as endocarditis, subcutaneous abscess and otitis media caused by VRE, penicillin-resistant_Streptococcus pneumoniae (PRSP) or methicillin-resistant Staphylococcus aureus (MRSA).2,3,10,12 In the present study, the VRE bacteraemia model of intestinal translocation in mice was used because this was a clinically important infection. Since there is a report that described the concentration of linezolid in mouse plasma after administration of 25 mg/kg, the administration dose used was 25 mg/kg.13

In mice with bacteraemia caused by oral inoculation of E. faecalis, linezolid showed good efficacy in both types of experimental model used in our study, and vancomycin showed good efficacy in the VSEF infection model. Of course, one of the most important factors affecting the efficacy of those drugs is the in vitro activity of these agents against the infecting organisms. One other factor is the in vivo activity of the test drug. In the present study, VRE organisms were delivered intraperitoneally because the mortality rate was <20% by oral inoculation of VRE organisms. Thus this model was not fit to assay the viable counts in blood after challenge of VRE organisms. When we compared the ratio of elimination of viable organisms in blood samples obtained from mice infected with VSEF, the number of organisms eliminated by vancomycin tended to be higher than in the linezolid treatment group. Rybak et al.14 recently reported that MBC/MIC values of linezolid for Gram-positive cocci were generally four times higher than those of vancomycin, and the in vitro time–kill activities of vancomycin against MRSA, methicillin-resistant Staphylococcus epidermidis and vancomycin-intermediate S. aureus were higher than those of linezolid. These results indicate that the killing activity of vancomycin early in infection is higher than that of linezolid.

Recent studies have reported the isolation of linezolid-resistant VRE and linezolid-resistant S. aureus in the USA.15,16 This indicates the possible spread of linezolid-resistant organisms by increasing the usage frequency and dosage. Thus, while linezolid is an efficacious agent for chemotherapy of patients infected with vancomycin-resistant organisms, its use should be controlled (restricted) in order to prevent an increase in linezolid-resistant organisms.


    Footnotes
 
* Corresponding author. Tel: +81-3-3762-4151; Fax: +81-3-5493-5415; E-mail: shuichi{at}med.toho-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lucas, G. M., Lechtzin, N., Puryear, D. W., Yau, L. L., Flexner, C. W. & Moore, R. D. (1998). Vancomycin-resistant and vancomycin-susceptible enterococcal bacteraemia: comparison of clinical features and outcomes. Clinical Infectious Diseases 26, 1127–33.[ISI][Medline]

2 . Schulin, T., Thauvin-Eliopoulos, C., Moellering, R. C., Jr & Eliopoulos, G. M. (1999). Activities of the oxazolidinones linezolid and eperezolid in experimental intra-abdominal abscess due to Enterococcus faecalis or vancomycin-resistant Enterococcus faecium. Antimicrobial Agents and Chemotherapy 43, 2873–6.[Abstract/Free Full Text]

3 . Patel, R., Rouse, M. S., Piper, K. E. & Steckelberg, J. M. (2001). Linezolid therapy of vancomycin-resistant Enterococcus faecium experimental endocarditis. Antimicrobial Agents and Chemotherapy 45, 621–3.[Abstract/Free Full Text]

4 . Montecalvo, M. A., Horowitz, H., Gedris, C., Carbonaro, C., Tenover, F. C., Issah, A. et al. (1994). Outbreak of vancomycin-, ampicillin-, and aminoglycoside-resistant Enterococcus faecium bacteraemia in an adult oncology unit. Antimicrobial Agents and Chemotherapy 38, 1363–7.[Abstract]

5 . Edmond, M. B., Ober, J. F., Weinbaum, D. L., Pfaller, M. A., Hwang, T., Sanford, M. D. et al. (1995). Vancomycin-resistant Enterococcus faecium bacteraemia: risk factors for infection. Clinical Infectious Diseases 20, 1126–33.[ISI][Medline]

6 . Edmond, M. B., Wallace, S. E., McClish, D. K., Pfaller, M. A., Jones, P. R. & Wenzel, R. P. (1999). Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clinical Infectious Diseases 29, 239–44.[ISI][Medline]

7 . Miyazaki, S., Fujikawa, T., Kobayashi, I., Matsumoto, T., Tateda, K. & Yamaguchi, K. (2001). Development of systemic bacteraemia after oral inoculation of vancomycin-resistant enterococci in mice. Journal of Medical Microbiology 50, 697–701.

8 . Sahm, D. F., Kissinger, J., Gilmore, M. S., Murray, P. R., Mulder, R., Solliday, J. et al. (1989). In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 33, 1588–91.[ISI][Medline]

9 . 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, USA.

10 . Wise, R., Andrews, J. M., Boswell, F. J. & Ashby, J. P. (1998). The in-vitro activity of linezolid (U-100766) and tentative breakpoints. Journal of Antimicrobial Chemotherapy 43, 466–8.

11 . Garbutt, J. M., Ventrapragada, M., Littenberg, B. & Mundy, L. M. (2000). Association between resistance to vancomycin and death in cases of Enterococcus faecium bacteraemia. Clinical Infectious Diseases 30, 466–72.[ISI][Medline]

12 . Pelton, S. I., Figueira, M., Albut, R. & Stalker, D. (2000). Efficacy of linezolid in experimental otitis media. Antimicrobial Agents and Chemotherapy 44, 654–7.[Abstract/Free Full Text]

13 . Dailey, C. F., Dileto-Fang, C. L., Buchanan, L. V., Oramas-Shirey, M. P., Batts, D. H., Ford, C. W. et al. (2001). Efficacy of linezolid in treatment of experimental endocarditis caused by methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 45, 2304–8.[Abstract/Free Full Text]

14 . Rybak, M. J., Hershberger, E., Moldovan, T. & Grucz, R. G. (2000). In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and -resistant strains. Antimicrobial Agents and Chemotherapy 44, 1062–6.[Abstract/Free Full Text]

15 . Gonzales, R. D., Schreckenberger, P. C., Graham, M. B., Kelkar, S., DenBesten, K. & Quinn, J. P. (2001). Infections due to vancomycin-resistant Enterococcus faecium resistant to linezolid. Lancet 357, 1179.[ISI][Medline]

16 . Tsiodras, S., Gold, H. S., Sakoulas, G., Eliopoulos, G. M., Wennersten, C., Venkataraman, L. et al. (2001). Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358, 207–8.[ISI][Medline]





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