Effect of LY333328 against vancomycin-resistant Enterococcus faecium in a rat central venous catheter-associated infection model

Mark E. Ruppa,*, Paul D. Feya and G. Matthew Longob

a Department of Internal Medicine and b Department of Surgery, 984031 University of Nebraska Medical Center, Omaha, NE 68198-4031, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A rat central venous catheter (CVC) infection model was used to assess the activity of LY333328 against vancomycin-resistant Enterococcus faecium (VRE). Via the CVC, animals were challenged with 106 cfu of Enterococcus faecium with the VanA phenotype. Eight rats received a single dose of LY333328 and eight rats received saline. Seventy-five per cent of control animals had peripheral bacteraemia and 87.5% had VRE recovered from explanted CVCs at the time they were killed, as compared with 0 and 12.5%, respectively, of the LY333328-treated animals (P < 0.01). All animals in the control group had evidence of metastatic disease compared with none of the treated group (P < 0.01). LY333328 was effective against the strain of VRE tested in this model.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Enterococci are a common cause of nosocomial infection.1 In 1999, 25.2% of enterococci recovered from intensive care units in the United States were resistant to vancomycin.2 Unfortunately, treatment of these infections is problematic and emphasizes the need for antimicrobial agents with activity against multidrug-resistant enterococci. LY333328, a new semi-synthetic glycopeptide, is an N-alkyl derivative of LY264826, a naturally occurring compound similar to vancomycin.3 In vitro, LY333328 is highly active against Gram-positive cocci and exhibits bactericidal action against glycopeptide-resistant enterococci.3 In previous studies we demonstrated that LY333328 was effective against Staphylococcus aureus in a rat central venous catheter (CVC) infection model.4 The purpose of this study was to assess the activity of LY333328 against a glycopeptide-resistant enterococcus in this model.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacteria/antibiotics/in vitro susceptibility tests

VRE1227 is a bloodstream isolate of Enterococcus faecium exhibiting a VanA phenotype. LY333328 was obtained from Eli Lilly & Co. (Indianapolis, IN, USA). Susceptibility testing of LY333328, vancomycin and teicoplanin was by broth microdilution as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).5

Rat CVC infection model

Silastic catheters were placed in the jugular veins of 16 male 400 g Sprague–Dawley rats (Charles River, Wilmington, MA, USA) as described previously.6 Twenty-four hours after CVC placement, blood cultures were obtained from the catheters to ensure sterility, and 106 cfu of VRE1227 were injected into the catheters. The inoculum of VRE was allowed to dwell for 15 min and the catheters were then flushed with a heparin solution. Twenty-four hours after the inoculation of bacteria, eight rats were treated with a single 20 mg/kg injection of LY333328 administered via the CVC and eight rats received an equal volume of saline. The single dose of 20 mg/kg was based on earlier studies of LY333328 against staphylococci in this in vivo model.4 On day 8 the animals were killed. Peripheral blood was obtained and quantitatively cultured on sheep blood agar (SBA) (Remel, Lenexa, KS, USA). The location of the distal tip of the CVC in the superior vena cava was confirmed visually and the catheters and surrounding venous tissue were removed aseptically. The catheters/venous tissues were vortex washed in phosphate-buffered saline (PBS) and the wash fluid was quantitatively cultured. Previous studies, in which culture results were confirmed by electron microscopy, documented complete removal of adherent organisms through use of this procedure.7 To ascertain the extent of metastatic disease, the heart, lungs, liver and kidneys were aseptically harvested, weighed, homogenized in 1 mL of PBS and quantitatively cultured on SBA. Bacteria recovered from the catheters, blood or tissues were identified to species level.

To limit the number of experimental animals used to the minimum that would reveal a significant therapeutic effect, inoculum size experiments were performed. Before conducting the comparative studies described above, the inoculum of VRE1227 that reliably resulted in a CVC infection was determined in dose-ranging inoculum studies. CVCs were placed as described above. Three animals each were challenged with an inoculum of either 103, 104, 105 or 106 cfu of VRE1227. Animals were evaluated for the presence of infection as described above.

The {chi}2 test was used to assess whether there was a significant difference in infection rate between the two groups of animals. The Mann–Whitney test was used to analyse data regarding the number of organisms recovered from the CVC and levels of bacteraemia and metastatic disease. Statistical tests were performed using Graphpad Prism 2.0 (San Diego, CA, USA).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The inoculum studies showed that all of the animals inoculated with 106 VRE1227 developed CVC infection with metastatic disease (data not shown). None of the rats inoculated with 103 cfu had evidence of infection as compared with one of the three rats inoculated with 104 cfu and two of the three rats inoculated with 105 cfu. Therefore, the 106 cfu inoculum was used in the larger LY333328 therapeutic trial.

VRE1227 expressed the VanA phenotype and the presence of the vanA gene complex was demonstrated by PCR (data not shown). The broth microdilution MIC (mg/L) for the antimicrobials tested were as follows: vancomycin, 512; teicoplanin, 256; LY333328, 2. The MBC for LY333328 was 4 mg/L.

The overall CVC infection rate, defined as the recovery of VRE from the blood, CVC or organs at the time animals were killed was 100% (8/8) for the untreated rats as compared with 12.5% (1/8) for the rats treated with LY333328 (P < 0.01, {chi}2 test). As shown in the TableGo, 87.5% (7/8) of the untreated animals had VRE recovered from the CVC at the time when animals were killed (mean ± s.d., 3.4 x 104 cfu/catheter ± 3.3 x 104), compared with 12.5% (1/8) of the animals treated with LY333328 (625 cfu/catheter) (P < 0.01, {chi}2 test for rate of infection, P < 0.01, Mann–Whitney test for level of infection). The LY333328 MIC for the isolate recovered from the CVC of the treated rat was 2 mg/L. Seventy-five per cent (6/8) of the untreated animals had VRE recovered from the peripheral blood at the time animals were killed (1.1 x 103 cfu/mL ± 7.3 x 102), compared with 0% (0/8) of the rats treated with LY333328 (P < 0.01, {chi}2 test for rate of infection, P < 0.01, Mann– Whitney test for level of bacteraemia).


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Table. Effect of treatment with LY333328 on rats with VRE CVC-associated infection
 
The table summarizes results from studies defining the burden of metastatic disease in rats treated with LY333328 compared with controls. In all of the organ systems examined there were marked differences. None of the rats treated with LY333328 had evidence of metastatic disease, compared with 75–100% (depending on the organ system) of untreated animals. For all organ systems examined these differences were statistically significant (P < 0.01, for rate of infection, P < 0.01 Mann–Whitney test for burden of metastatic disease).

It is believed that most nosocomial primary bloodstream infections stem from intravascular devices. A recent paper detailing the use of quinupristin/dalfopristin for the treatment of 396 VRE infections reported that 36.6% were CVC infections or primary bacteraemia.8 Therefore, it is appropriate that we have chosen to study the efficacy of LY333328 against VRE in an in vivo CVC infection model. LY333328 exhibited good in vitro activity against VRE1227. The in vitro activity correlated with efficacy in the rat CVC infection model. Of eight rats receiving a single dose of LY333328, only one had VRE recovered from the CVC. None of the LY333328-treated animals were bacteraemic or had evidence of metastatic disease at the time animals were killed. These findings were dramatically different from those with untreated control animals, all of which had large numbers of VRE recovered from the bloodstream, CVC or peripheral organs.

A number of pharmacokinetic features of LY333328 are emphasized in this study. The half-life of LY333328 in rats is approximately 10 h, or almost 14 times greater than the half-life of vancomycin.9 LY333328 has a long post-antibiotic effect (18.7 h at 10 x MIC).10 Also, it appears that LY333328 is associated with uptake and persistence in tissues.10 Lastly, in previous studies with S. aureus in the rat CVC infection model, we found that dosing at 96 h intervals was more effective than adminstration of smaller doses more frequently.4 Therefore, evidence suggests that effective therapy with LY333328 may be achieved with infrequent dosing, which was supported by the current study.

In conclusion, LY333328, administered as a single dose, exhibited excellent activity against VRE1227 in a rat model of CVC infection. This compound has demonstrated activity against multi-resistant Gram-positive pathogens in in vitro and in vivo systems. LY333328 merits further clinical evaluation and may become a welcome addition to the antimicrobial armamentarium.


    Acknowledgments
 
This work was presented in part at the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 26–29 September, 1999, and was supported by a grant from Eli Lilly & Co.


    Notes
 
* Corresponding author. Tel: +1-402-559-5276; Fax: +1-402-559-8300; E-mail: merupp{at}unmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Centers for Disease Control and Prevention. (1997). National Nosocomial Infections Surveillance (NNIS) report, data summary from October 1986–April 1997, issued May 1997. American Journal of Infection Control 25, 477–87.[ISI][Medline]

2 . Centers for Disease Control and Prevention Hospital Infection Program. [On-line.] http://www.cdc.gov/ncidod/hip/nnis (14 November 2000, date last accessed).

3 . Nicas, T. I., Mullen, D. L., Flokowitsch, J. E., Preston, D. A., Snyder, J. J., Zweifel, M. J. et al. (1996). Semisynthetic glycopeptide antibiotics derived from LY264826 active against vancomycinresistant enterococci. Antimicrobial Agents and Chemotherapy 40, 2194–9.[Abstract]

4 . Rupp, M. E. & Ulphani, J. (1998). Efficacy of LY333328 in a rat model of Staphylococcus aureus central venous catheterassociated infection. In Programs and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract F111, p. 260. American Society for Microbiology, Washington, DC.

5 . 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, Villanova, PA.

6 . Ulphani, J. S. & Rupp, M. E. (1999). Model of Staphylococcus aureus central venous catheter-associated infection in rats. Laboratory Animal Science 49, 283–7.[Medline]

7 . Rupp, M. E., Ulphani, J., Fey, P. D., Bartsch, K. & Mack, D. (1999). Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infection and Immunity 67, 2627–32.[Abstract/Free Full Text]

8 . Moellering, R. C., Linden, P. C., Reinhardt, J., Blumberg, E. A., Bompart, F. & Talbot, G. H. (1999). The efficacy and safety of quinupristin/dalfopristin for the treatment of infections caused by vancomycin-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 44, 251–61.[Abstract/Free Full Text]

9 . Lin, Y., Stratford, R. E., Sornes, L. L., Confer, W. L., Vasudevan, V., Jones, T. W. et al. (1995). Non-clinical pharmacokinetics of LY333328, a semisynthetic glycopeptide antibiotic active against vancomycin-resistant enterococci. In Programs and Abstracts of the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Abstract F254, p. 157. American Society for Microbiology, Washington, DC.

10 . Baltch, A. L., Smith, R. P., Ritz, W. J. & Bopp, L. H. (1998). Comparison of inhibitory and bactericidal activities and postantibiotic effects of LY333328 and ampicillin used singly and in combination against vancomycin-resistant Enterococcus faecium. Antimicrobial Agents and Chemotherapy 42, 2564–8.[Abstract/Free Full Text]

Received 25 July 2000; returned 6 November 2000; revised 2 January 2001; accepted 25 January 2001