The activity of vancomycin against heterogeneous vancomycin-intermediate methicillin-resistant Staphylococcus aureus explored using an in vitro pharmacokinetic model

Jonathan Turnera,b, Robin A. Howea,b, Mandy Woottona,b, Karen E. Bowkera,b, H. Alan Holta,b, Vyvyan Salisburya,c, Peter M. Bennetta,d, Timothy R. Walsha,d and Alasdair P. MacGowana,b,*

a Bristol Centre for Antimicrobial Research and Evaluation; b Bristol Centre for Antimicrobial Research and Evaluation, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS1 5NB; c Faculty of Applied Sciences, Department of Biological and Biomedical Sciences, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY; d Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may account for treatment failure with vancomycin and act as a precursor of vancomycin-intermediate or -resistant S. aureus. The activity of vancomycin was assessed against vancomycinsusceptible, hVISA and VISA strains in a dilutional pharmacokinetic model. Over a 48 h period, total bacteria and cells with a vancomycin-intermediate phenotype were quantified. Total counts of hVISA were reduced by vancomycin in a similar way to a vancomycin-susceptible control. The vancomycin-intermediate sub-population was eradicated from the model within one dose interval. Exposure to low vancomycin concentrations did not result in an increase in the proportion of cells which were vancomycin intermediate. Short-term exposure of hVISA to vancomycin at gradient concentrations did not increase the proportion of cells with vancomycin-intermediate phenotype.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) has been defined as strains that are susceptible to vancomycin, (MIC <= 4mg/L) according to breakpoints recommended by the National Committee for Clinical Laboratory Standards1 but contain a sub-population of cells at a frequency of >=10-6 that exhibit intermediate susceptibility, MIC >4 mg/L but <32 mg/L. It has been suggested that hVISA is a precursor of S. aureus strains that have vancomycin MICs of >4 mg/L, hence it is important to study further the effects of vancomycin on these strains.2 The activity of vancomycin on vancomycin-intermediate S. aureus (VISA) as defined by an MIC of >4–<=32 mg/L has already been reported using in vitro models.3,4 Here we used a dilutional in vitro pharmacodynamic model to simulate the serum concentrations of vancomycin dosed as 1 g bd in adults with normal clearance and measured the antibacterial effect in terms of total bacterial count and vancomycin-intermediate (MIC > 4 mg/L) sub-population. One strain of vancomycin-susceptible methicillin-resistant S. aureus (MRSA), two strains of hVISA (Mu3 and SMH 26) and a VISA strain (Mu50) were studied.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Pharmacokinetic model

The serum concentrations of vancomycin were simulated using a New Brunswick Bioflo 1000 in vitro model (Hatfield, UK). The peak concentration (Cmax) was 30 mg/L and the 12 h trough (Cmin) was 7.5 mg/L. The apparatus consists of three connected chambers. The primary chamber is a reservoir of broth connected to the dosing chamber, which has an outflow connected to the third chamber, a waste vessel. Broth was pumped (37.6 mL/h) via red-to-red silicone tubing using a peristaltic pump. The dosing chamber was held at 37°C and stirred continuously by a magnetic stirrer.

Media

Tryptone soya broth (3%) (TSB; Beckton Dickinson, Cockeysville, MD, USA) was used for all experiments. Staphylococci were recovered on to brain–heart infusion (BHI) agar (Oxoid Ltd, Basingstoke, UK) for total viable counts and BHI containing 4 mg/L vancomycin to detect those cells with a vancomycin-intermediate phenotype.

Strains

S. aureus strains SMH26, Mu3, Mu50 and MRSA 15 were used. SMH26 is a hVISA, isolated from a patient who died following vancomycin therapy in Southmead Hospital, Bristol5 and MRSA 15 is a representative of epidemic clone 15 in the UK and is fully vancomycin susceptible.

Antibiotic

Stock solutions of vancomycin (Eli Lilly, Basingstoke, UK) were prepared according to BSAC guidelines6 and stored at –40°C. Vancomycin MICs were determined using BSAC methods.6

Bacterial killing curves

The central chamber of the model was inoculated with 1 mL of an overnight suspension of the test bacterium in TSB, then dosed with vancomycin once the population had reached equilibrium at a density of 5 x 107 cfu/mL, usually after c. 18 h incubation. Aliquots were removed from the central chamber at 0, 1, 2, 3, 4, 5, 6, 12, 24 and 48 h. The total bacterial count in each sample was determined using a spiral plater (Don Whitely, Spiral Systems, Shipley, UK). The bacterial counts of cells able to grow on BHI plus 4 mg/L vancomycin were determined by spiral plating of both neat and concentrated aliquots. Aliquots were concentrated 20-fold by centrifuging samples and removing 1330 µL then reconstituting the deposit and vortexing before plating. All aliquots were stored at 4°C and the vancomycin concentration assayed using Biostat Immunoassay.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The vancomycin MICs for each strain were as follows: MRSA 15 0.5 mg/L; SMH 26 1.5 mg/L; Mu3 3 mg/L and Mu50 8 mg/L; the mean (± s.d.) peak and trough concentrations simulated were 30.9 ± 2.4 and 7.1 ± 1.3 mg/L, respectively.

There was a 3–4 log reduction in viable count for MRSA 15 after exposure to vancomycin for 24 h; after 48 h the reduction was 5 logs. No cells able to grow in the presence of 4 mg/L vancomycin were recovered (Figure 1aGo).



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Figure 1. Bactericidal effect of vancomycin peak concentrations 30 mg/L every 12 h on S. aureus strains MRSA 15 (a), SMH 26 (b), Mu3 (c) and Mu50 (d). Symbols: {blacksquare}, total bacterial count; {square}, vancomycin-resistant bacteria. Error bars indicate standard deviations.

 
SMH 26 also exhibited a 3–4 log reduction in total viable count by 24 h and a 4–5 log reduction by 48 h. A vancomycin-intermediate sub-population at 10-3 was present before exposure to vancomycin but became undetectable by 6 h (Figure 1bGo).

Mu3 showed a 1–2 log reduction in total count after 24 h, and before exposure to vancomycin the vancomycin-intermediate population was present at a frequency of c. 10-5. No vancomycin-intermediate cells were detected after exposure to vancomycin (Figure 1cGo).

Mu50 total bacterial counts fell by 2–3 logs after 24 h and 4 logs after 48 h vancomycin exposure. Almost all the Mu50 cells were able to grow on 4 mg/L vancomycin plates before dosing the model with vancomycin. The frequency of the vancomycin-intermediate population was c. 10-5 cfu/mL by 6 h, then persisted at a low level of <10 cfu/mL until 48 h (Figure 1dGo).

In order to test whether sub-therapeutic vancomycin concentrations would result in an increase in the vancomycin-intermediate sub-population, three further simulations were performed with target peak vancomycin concentrations of 15, 3 and 0.75 mg/L using the Mu3 hVISA strain. The measured peak vancomycin concentrations were 15.2 ± 0.9, 3.1 ± 0.3 and <2 mg/L, respectively. The 15 mg/L peak vancomycin simulation produced similar results to the 30 mg/L simulation (data not shown). The results for the 3 and 0.75 mg/L simulations are shown in Figure 2Go. Vancomycin-intermediate cells were detected up to 6 h in the 3 mg/L simulation and up to 12 h in the 0.75 mg/L.



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Figure 2. Bactericidal effect of vancomycin peak concentrations of (a) 0.75 mg/L or (b) 3 mg/L against S. aureus Mu3. Symbols: {blacksquare}, total bacterial count; {square}, vancomycin-resistant bacteria.

 
The clinical significance of hVISA is unclear, both in terms of its potential impact on clinical outcomes with glycopeptide therapy, and whether hVISA is a precursor of VISA and, perhaps, VRSA.

So far, there have been no pharmacodynamic studies in animals or using in vitro pharmacokinetic models to provide information on vancomycin therapy for hVISA or the potential for the emergence of vancomycin resistance. These data indicate that clearance of the hVISA strains Mu3 and SMH26 from an in vitro model using conventional vancomycin dosing simulations is similar to a vancomycin-susceptible strain. In addition, there was no difference in clearance of the VISA Mu50 compared with the susceptible control. Although not fully established, it seems likely that the pharmacodynamic parameter T > MIC determines vancomycin therapeutic outcomes.7,8 In this series of experiments the T > MIC was 100% for all strains used (MRSA 15, Mu3, SMH26 and Mu50), hence it might be expected that clearance would be similar.

In contrast, the data demonstrate that there were clear differences between strains in the detection of a popula-tion demonstrating a vancomycin-intermediate phenotype. With hVISA strains Mu3 and SMH26 this population accounted for 10-4–10-6 of the total population before vancomycin exposure, but with both strains this popula-tion was undetectable after a single dose interval (12 h). With Mu50, almost all the population had a vancomycin-intermediate phenotype before vancomycin exposure, but this population also declined rapidly following addition of vancomycin, eventually reaching a low plateau after 12 h. Even low vancomycin concentrations (<3 mg/L) did not result in the proportion of cells with vancomycin- intermediate susceptibility increasing above pre-vancomycin exposure levels. However, the duration of exposure was only 48 h, which may have been too short to elicit any changes.

The data suggest that cells with the vancomycinintermediate phenotype are more likely to be cleared from the model than those that do not have this phenotype. These data are similar to some derived from VISA strains tested in animal and in vitro pharmacodynamic models, which indicated that the AUC/MIC ratio to produce bactericidal activity or 50% of Emax was five- to 10-fold lower for VISA than MRSA controls.3,9

In conclusion, these in vitro model data do not support the concept that exposure of hVISA to vancomycin results in an increase in the proportion of cells having the vancomycin-intermediate phenotype; however, we only tested two strains and exposed them to vancomycin for a short time. The limited number of strains tested here may be important as it has been shown that S. aureus strains vary in their ability to acquire the vancomycinintermediate phenotype on exposure to increasing vancomycin concentrations.10 Clearance from the model with vancomycin serum concentrations associated with conventional doses was the same despite MIC differences. However, these simulations do not include the potential impact of protein binding or tissue penetration on measurement of antibacterial effect. It remains to be seen whether these findings are confirmed in clinical practice where strains are exposed to vancomycin for much longer periods of time.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We wish to thank the Showering Fund for financial support of J.T.


    Notes
 
* Corresponding author. Tel: +44-117-959-5652; Fax: +44-117-959-3154; E-mail: macgowan_a{at}southmead.swest.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

2 . Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S. et al. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 1670–3.[ISI][Medline]

3 . Aeschlimann, J. R., Allen, G. P., Hershberger, E. & Rybak, M. J. (2000). Activities of LY333328 and vancomycin administered alone or in combination with gentamicin against three strains of vancomycin-intermediate Staphylococcus aureus in an in vitro pharmacodynamic infection model. Antimicrobial Agents and Chemotherapy 44, 2991–8.[Abstract/Free Full Text]

4 . Akins, R. L. & Rybak, M. J. (2000). In vitro activities of daptomycin, arbekacin, vancomycin and gentamicin alone and/or in combination against glycopeptide intermediate-resistant Staphylococcus aureus in an infection model. Antimicrobial Agents and Chemotherapy 44, 1925–9.[Abstract/Free Full Text]

5 . Howe, R. A., Bowker, K. E., Walsh, T. R., Feest, T. G. & MacGowan, A. P. (1998). Vancomycin resistant Stayphylococcus aureus. Lancet 351, 601–2.

6 . British Society for Antimicrobial Chemotherapy Working Party on Sensitivity Testing. (1999). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

7 . Dufful, S. B., Begg, E. J., Chambers, S. T. & Barclay, M. L. (1994). Efficacies of different vancomycin dosing regimens against Staphylococcus aureus determined with a dynamic in vitro model. Antimicrobial Agents and Chemotherapy 38, 2480–2.[Abstract]

8 . Knudsen, J. D., Fuursted, K., Espersen, F. & Frimodt-Moller, N. (1997). Activities of vancomycin and teicoplanin against penicillin-resistant pneumococci in vitro and in vivo and correlations to pharmacokinetic parameters in the mouse peritonitis model. Antimicrobial Agents and Chemotherapy 41, 1910–5.[Abstract]

9 . Dudley, M., Griffith, D., Corcoran, E., Liu, C., Sorensen, K., Tembe, V. et al. (1999). Pharmacokinetic–pharmacodynamic indices for vancomycin treatment of susceptible and intermediate Staphylococcus aureus in the neutropenic mouse thigh model. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2031, p. 49. American Society for Microbiology, Washington, DC.

10 . Pfeltz, R. F., Singh, V. K., Schmidt, J. L., Batten, M. A., Baranyk, C. S., Nadakavukaven, M. J. et al. (2000). Characterisation of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental background. Antimicrobial Agents and Chemotherapy 44, 294–303.[Abstract/Free Full Text]

Received 1 May 2001; returned 10 July 2001; revised 25 July 2001; accepted 8 August 2001





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