Comparison of agar dilution, microdilution, Etest and disc diffusion to test the activity of trovafloxacin against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae

Linda M. Kellya, Michael R. Jacobsb and Peter C. Appelbauma,*

a Departments of Pathology (Clinical Microbiology), Hershey Medical Center, 500 University Drive, Hershey, PA 17033; b Case Western Reserve University, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To investigate the ability of four in-vitro methods to test trovafloxacin activity, this study evaluated susceptibility of 101 isolates of each of Pseudomonas aeruginosa , methicillin- resistant Staphylococcus aureus and Streptococcus pneumoniae to trovafloxacin by agar dilution, microdilution, Etest and disc diffusion methodologies. MIC 50 and MIC 90 values were very similar for all three species with all four methods. For S. aureus and P. aeruginosa, good correlation was obtained between breakpoints of >= 17 mm, 14- 16 mm and <= 13 mm with agar and microdilution MICs. For both species, Etests yielded susceptibility rates lower than the other three methods. For pneumococci, excellent correlation was obtained with all four methods.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trovafloxacin is a new quinolone with activity against a wide variety of Gram-positive and -negative aerobes and anaerobes, including some bacterial pathogens resistant to currently available quinolones such as ciprofloxacin and ofloxacin. 1 ,2 ,3 ,4

Methods used in the routine laboratory to test activity of antimicrobials include agar dilution, microdilution, Etest and disc diffusion. The Etest has gained acceptance as a method for MIC determination, and is less labour-intensive than standardized agar or microdilution methodology. In this study, we tested activity of trovafloxacin by agar dilution, microdilution, Etest and disc diffusion against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae.


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

Strains comprised of a total of 101 recent clinical isolates each of P. aeruginosa, methicillin-resistant S. aureus and S. pneumoniae (31 penicillin susceptible, 35 intermediate, 35 resistant). Isolates were selected to cover as wide a range of trovafloxacin MICs as possible (allowing for investigation of error rates compared with standardized MIC methodology), chosen to comprise susceptible organisms as well as organisms with trovafloxacin MICs clustering around the breakpoint, except for pneumococci. All tests were done in duplicate and arithmetic means of nearly identical results taken.

Interpretive breakpoints

For pneumococci, NCCLS breakpoints of <=1.0 mg/L (susceptible), 2.0 mg/L (intermediate) and >=4.0 mg/L (resistant) were applied. 5 For P. aeruginosa and S. aureus, breakpoints of <=2.0 mg/L (susceptible), 4.0 mg/L (intermediate) and >=8.0 mg/L (resistant), recently approved by the FDA, were used.

Interpretive zone diameter breakpoints (mm) for P. aeruginosa and S. aureus of >=17, 14–16 and <=13, 6 recently approved by the FDA, were applied. Diameters used for S. pneumoniae were those recently approved by NCCLS 5 and FDA: >=19 mm (susceptible), 16–18 mm (intermediate) and <=15 mm (resistant).

Agar dilution MICs

These were performed according to standard methods 7 on Mueller- Hinton agar supplemented with 5% sheep blood for pneumococci. Inocula were prepared by suspending growth from overnight cultures in saline to a turbidity of a 0.5 McFarland standard. Final inocula contained 10 4 organisms/spot. Standard quality control organisms were included in each run.

Microdilution MICs

These were determined by the method recommended by NCCLS, 7 using cation-adjusted Mueller- Hinton broth supplemented with 5% lysed horse blood for pneumococci. Suspensions with a turbidity equivalent to that of a 0.5 McFarland standard were prepared by suspending growth from blood agar plates in 2 mL sterile saline, and further diluted 1:10 to obtain a final inoculum of 5 x 10 5 cfu/well. Trays were incubated overnight in air at 35°C. Standard quality control isolates (as above) were included in each run.

Etest MICs

Mueller- Hinton plates with 5% sheep blood for pneumococci were inoculated with a 0.5 McFarland standard of suspension harvested from plates; Etest strips (AB Biodisk, Solna, Sweden) were placed on each. 8,9 After overnight incubation at 37°C, the MIC was read as the intersect where the ellipse of growth inhibition intersects the strip. Where colonies occurred within the inhibition ellipse, the higher value was taken as the MIC. Etests for pneumococci were incubated under 5- 10% CO 2, whereas those for P. aeruginosa and S. aureus were incubated in ambient air.

Disc diffusion testing

This was performed by standard NCCLS methodology 10 using Mueller- Hinton plates with supplemented sheep blood for pneumococci, inoculated with a 0.5 McFarland standard. Trovafloxacin discs (10 µg) were applied. After overnight incubation at 35°C, zone diameters were read with calipers. Disc diffusions for pneumococci were incubated under 5- 10% CO 2, whereas P. aeruginosa and methicillin-resistant S. aureus were incubated in air.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For P. aeruginosa, MIC 50 and MIC 90 values with all three methods were 1.0–2.0 mg/L and 8.0–16.0 mg/L, respectively. The number of P. aeruginosa isolates susceptible, intermediate and resistant by disc diffusion were 60, 11 and 30, compared with 65, 15 and 21 with agar dilution, 73, 11 and 17 with microdilution, and 59, 20 and 22 with the Etest.

For methicillin-resistant S. aureus, MIC 50 and MIC 90 values with all three methods were 2.0 mg/L and 8.0–16.0 mg/L, respectively. The number of isolates susceptible, intermediate and resistant by disc diffusion were 74, 4 and 23, compared with 71, 7 and 23 with agar dilution, 72, 13 and 16 with microdilution, and 61, 24 and 26 with the Etest. All 101 pneumococci were susceptible with all four methods, with MIC 50 and MIC 90 values of 0.06–0.125 mg/L and 0.125–0.25 mg/L.

Error rates for disc diffusion compared with agar dilution MICs are presented in Table I. Three major, zero very major and 19 minor errors occurred with P. aeruginosa. Corresponding numbers with methicillin-resistant S. aureus were one, one and eight, respectively. No errors occurred with S. pneumoniae.


View this table:
[in this window]
[in a new window]
 
Table I. Disc diffusion error rates for trovafloxacin using FDA-approved criteriaa
 
Comparison of microdilution and Etest MICs with agar dilution MICs is presented in Table II. As can be seen, 91–101 isolates in each species gave essential agreement (± 1 log 2 dilution). Two isolates of P. aeruginosa gave very major errors with microdilution. Fourteen minor errors were found in P. aeruginosa and methicillin-resistant S. aureus with microdilution, and 20 and 23, respectively, with the Etest. No errors were found with S. pneumoniae.


View this table:
[in this window]
[in a new window]
 
Table II. Comparison of MIC methods using agar dilution as the reference method
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our study, results for P. aeruginosa and methicillin- resistant S. aureus showed that disc testing gave good correlation compared with agar and microdilution. Methicillin-resistant S. aureus gave better correlation, compared with P. aeruginosa. In both organisms, Etests yielded higher MICs compared with the other three methods. Correlation with pneumococci was excellent with all four methods, regardless of the fact that pneumococcal agar and microdilution tests were incubated in air, whereas disc diffusion and Etests were incubated in CO 2. Isolates of P. aeruginosa and methicillin-resistant S. aureus were specifically chosen to represent both low and high quinolone MICs for error rate analysis, and are not representative of the MIC distribution of routine isolates.

Results of MIC testing by agar dilution, which we consider to be the reference method, corresponded well with findings by others. 1 ,2 ,3 ,4 Similar to findings by Dembry et al., 8 microdilution MICs of some P. aeruginosa and S. aureus isolates were lower than those obtained by Etest (and also by agar dilution methodology) in our study. Correlation between the three MIC techniques for pneumococci was excellent. Jones et al. 9 have found the Etest to be a valid method for fluoroquinolone susceptibility testing of nearly 300 isolates belonging to 26 species (62.5% with documented ciprofloxacin resistance).

In summary, disc diffusion, agar and microdilution can be recommended for routine trovafloxacin susceptibility testing of P. aeruginosa and methicillin-resistant S. aureus. For these two organisms, and other species whose trovafloxacin MICs cluster around the breakpoint, the Etest cannot be recommended at this time because MICs are higher than by the other methods, above the susceptible breakpoint. In view of the current rarity of quinolone- resistant pneumococci, all four methods appear to be suitable for routine susceptibility testing of these isolates.


    Acknowledgments
 
This study was supported by a grant from Pfizer, Inc., New York, NY, USA.


    Notes
 
* Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psghs.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Eliopoulos, G. M., Klimm, K., Eliopoulos, C. T., Ferraro, M. J. & Moellering, R. C. (1993.) In vitro activity of CP-99,219, a new fluoroquinolone, against clinical isolates of Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 37, 366–70.

2 . Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (1995.) Activity of CP 99,219 compared with DU-6859a, ciprofloxacin, ofloxacin, levofloxacin, lomefloxacin, tosufloxacin, sparfloxacin and grepafloxacin against penicillin-susceptible and -resistant pneumococci. Journal of Antimicrobial Chemotherapy 35, 230–2.[ISI][Medline]

3 . Visalli, M. A., Bajaksouzian, S., Jacobs, M. R. & Appelbaum, P. C. (1997.) Comparative activities of trovafloxacin, alone and in combination with other agents, against Gram-negative nonfermentative rods. Antimicrobial Agents and Chemotherapy 41, 1475–81.[Abstract]

4 . Fass, R. J., Barnishan, J., Solomon, M. C. & Ayers, L. W. (1996.) In vitro activities of quinolones, ß -lactams, tobramycin, and trimethoprim-sulfamethoxazole against nonfermentative Gram-negative bacilli. Antimicrobial Agents and Chemotherapy 40, 1412–18.[Abstract]

5 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing— Eighth Informational Supplement: Approved Standard M100 S8. NCCLS, Wayne, PA.

6 . Jones, R. N. (1994.) Preliminary interpretive criteria for in vitro susceptibility testing of CP-99,219 by dilution and disc diffusion methods. Diagnostic Microbiology and Infectious Disease 20, 167–70.[ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (1996). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically— Fourth Edition: Approved Standard M7–A4. NCCLS, Wayne, PA.

8 . Dembry, L. M., Farrel, P. A., Orcutt, D. R., Gerrity, L. A. & Andriole, V. T. (1997.) In-vitro activity of trovafloxacin against sensitive and resistant aerobic bacteria using the standard microdilution broth method and Etest. Journal of Antimicrobial Chemotherapy 39, Suppl. B, 35–42.[Abstract]

9 . Jones, R. N., Erwin, M. E. & Croco, J. L. (1996.) Critical appraisal of Etest for the detection of fluoroquinolone resistance. Journal of Antimicrobial Chemotherapy 38, 21–5.[Abstract]

10 . National Committee for Clinical Laboratory Standards. (1996). Performance Standards for Antimicrobial Disk Susceptibility Tests— Sixth Edition: Approved Standard M2–A6. NCCLS, Wayne, PA.

Received 25 August 1998; returned 7 December 1998; revised 22 December 1998; accepted 11 January 1999





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Kelly, L. M.
Articles by Appelbaum, P. C.
PubMed
PubMed Citation
Articles by Kelly, L. M.
Articles by Appelbaum, P. C.