Influence of the decrease in ciprofloxacin susceptibility and the presence of human serum on the in vitro susceptibility of Streptococcus pneumoniae to five new quinolones

I. P. Balcabaoa, L. Aloua, L. Aguilarb, M. L. Gomez-Lusa, M. J. Giménezb and J. Prietoa,*

a Microbiology Department, School of Medicine, Universidad Complutense, 28040 Madrid; b Medical Department, GlaxoSmithKline, Tres Cantos, Madrid, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Sixty recent Streptococcus pneumoniae isolates with different susceptibilities to ciprofloxacin (14 with MIC 0.5 mg/L, 10 with MIC 1 mg/L, eight with MIC 2 mg/L, 11 with MIC 4 mg/L and 17 with MIC >=8 mg/L) were tested against five new quinolones using Todd–Hewitt broth with and without 80% serum. The final inoculum was 5 x 105 cfu/mL. Gemifloxacin and clinafloxacin exhibited the lowest MIC90 values and resistance rates (percentage above and defined breakpoint) with and without serum for strains with a ciprofloxacin MIC of >=4 mg/L. Other quinolones tested were less active against strains with reduced ciprofloxacin susceptibility. The presence of serum did not affect susceptibility to moxifloxacin, but increased the resistance rates to other new quinolones for strains with high ciprofloxacin MICs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Quinolones are a particularly attractive therapeutic option for pneumococcal respiratory tract infections in regions where there has been an increase in strains with high-level penicillin resistance associated with macrolide resistance.1 In a 1996–1997 Spanish nationwide surveillance study, 60% of Streptococcus pneumoniae were resistant to penicillin (36.5% high-level resistance, 23.6% intermediate strains).1 Fifty-three per cent of high-level penicillin-resistant strains were also macrolide resistant.1 In the adult population, in which quinolones are mainly used, 22.6% of strains had a ciprofloxacin MIC >= 2 mg/L, 6% an MIC >= 4 mg/L, 1.9% an MIC >= 8 mg/L and 0.6% an MIC >= 16 mg/L.2 Isolates with higher ciprofloxacin MICs have variable susceptibility to new fluoroquinolones, as suggested in a previous study where MICs of trovafloxacin were higher than those of gemifloxacin.3 While several studies have concluded that the in vitro susceptibility to new quinolones is not influenced by the presence of serum in the media,4,5 this has not been extensively studied for all new fluoroquinolones such as gemifloxacin.

This study explores the activity of five new quinolones against isolates with reduced susceptibility to ciprofloxacin, and the effect of the presence of human serum on the resistance rate.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
In a Spanish nationwide surveillance programme (1998– 1999), 60 S. pneumoniae strains, recently isolated from adult patients with community-acquired respiratory tract infections, were tested against ciprofloxacin, levofloxacin, trovafloxacin, moxifloxacin, clinafloxacin and gemifloxacin. Strains were chosen that had an MIC of ciprofloxacin >=0.5 mg/L (14 strains with MIC 0.5 mg/L, 10 with MIC 1 mg/L, eight with MIC 2 mg/L, 11 with MIC 4 mg/L and 17 with MIC >= 8 mg/L).

Susceptibility was determined using a broth microdilution method. MICs and MBCs were measured using Todd–Hewitt broth as the culture medium. To evaluate the effect of the presence of human serum, MICs and MBCs were also determined using 80% human serum (from a serum pool obtained from healthy donors who had not undergone drug treatment in the previous month) and 20% Todd–Hewitt broth. The final inoculum was 5 x 105 cfu/mL. Cultures were incubated for 24 h at 35°C in 5% CO2. Strains were tested in triplicate, and modal MIC and MIC90 values were calculated. S. pneumoniae ATCC 49619 was used as the control strain. Percentages of resistance were calculated using the susceptibility breakpoints for levofloxacin (<=2 mg/L), trovafloxacin (<=1 mg/L) and moxifloxacin (<=1 mg/L) established by the NCCLS,6 a susceptibility breakpoint of <=2 mg/L for ciprofloxacin (there being no NCCLS breakpoint)2 and a susceptibility breakpoint of <=1 mg/L (similar to the value for new quinolones) for clinafloxacin and gemifloxacin.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
MIC90s and percentages of resistance (with and without human serum) of all six quinolones for strains of S. pneumoniae with increasing ciprofloxacin MICs in broth are shown in the TableGo. For all five new quinolones, 100% susceptibility was obtained for all strains with a ciprofloxacin MIC <= 2 mg/L when tested with and without serum. The highest intrinsic activity was obtained for moxifloxacin, clinafloxacin and gemifloxacin with MIC90s in broth of <=0.06 mg/L; the lowest intrinsic activity of new quinolones was obtained for levofloxacin with an MIC90 in broth of 1 mg/L.


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Table. MIC90s for S. pneumoniae determined with broth (MIC90b) and serum (MIC90s), and percentages of resistance with broth (%Rb) and serum (%Rs) stratifying strains according to the MIC of ciprofloxacin in broth
 
The problem arose with a ciprofloxacin MIC of >=4 mg/L, where resistance rates of 9, 18 and 9% were obtained for levofloxacin, trovafloxacin and moxifloxacin, respectively. The resistance rates increased to >=65% for these three quinolones when isolates with a ciprofloxacin MIC of 16 mg/L were tested. All these highly ciprofloxacin-resistant strains were susceptible to clinafloxacin and gemifloxacin. This may be important in the clinical setting,7 where there are increasing rates of ciprofloxacin resistance.2

Although other studies have concluded that the presence of human serum does not influence the intrinsic activity of some new quinolones,4,5 the results of this study suggest that this conclusion cannot be extended to all fluoroquinolones. Other authors have shown that serum has no influence on the resistance rates for moxifloxacin.5 For the other quinolones the influence of serum is significant for isolates with a ciprofloxacin MIC of >=4 mg/L, since when the strains are tested in the presence of serum, the MIC measured exceeded the breakpoint. Differences from studies published previously4,5,8 could be the result of the fact that the percentage of serum used in this study (80%) is higher than that used before (up to 50% serum),4,8 and may be due to the MIC distribution of the strains tested. The effect of serum could not be attributed exclusively to protein binding since it is lower for ciprofloxacin (30%)9 than for moxifloxacin (50%),9 where the effect is not observed.

In conclusion, considering both factors (using strains with higher ciprofloxacin MICs and the presence of human serum), gemifloxacin and clinafloxacin exhibited the lowest MIC90 values (0.5 and 1 mg/L without and with serum, respectively) for the whole population of S. pneumoniae with ciprofloxacin MICs of >=0.5 mg/L, and the lowest resistance rates (0 and <=10% without and with serum, respectively). The other new quinolones (levofloxacin, trovafloxacin and moxifloxacin) exhibited resistance rates of >=22% for the population tested, with less activity against strains with higher ciprofloxacin MICs.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This study was supported by a grant from SmithKline Beecham, S.A., Madrid, Spain.


    Notes
 
* Corresponding author. Tel: +34-91-394-15-08, Fax: +34-91-394-15-11; E-mail: jprieto{at}med.ucm.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Baquero, F., García-Rodríguez, J. A., García de Lomas, J., Aguilar, L. & the Spanish Surveillance Group for Respiratory Pathogens. (1999). Antimicrobial resistance of 1113 Streptococcus pneumoniae isolates from patients with respiratory tract infections in Spain: results of a 1-year (1996–1997) multicenter surveillance study. Antimicrobial Agents and Chemotherapy 43, 357–9.[Abstract/Free Full Text]

2 . García-Rey, C., Aguilar, L. & Baquero, F. (2000). Influences of different factors on prevalence of ciprofloxacin resistance in Streptococcus pneumoniae in Spain. Antimicrobial Agents and Chemotherapy 44, 3381–2.[Abstract/Free Full Text]

3 . Fuentes, F., Giménez, M. J., Marco, F., Alou, L., Aguilar, L. & Prieto, J. (2000). In vitro susceptibility to gemifloxacin and trovafloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin. European Journal of Clinical Microbiology and Infectious Diseases 19, 137–9.[ISI][Medline]

4 . Eliopoulos, G. M. (1995). In vitro activity of fluoroquinolones against gram-positive bacteria. Drugs 49, Suppl. 2, 48–57.[Medline]

5 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In vitro activity of BAY 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 101–6.[Abstract]

6 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing—Eleventh Informational Supplement M100-S11. NCCLS, Wayne, PA.

7 . Ewing, S., Ruiz, M., Torres, A., Marco, F., Martínez, J. A., Sánchez. M. et al. (1999). Pneumonia acquired in the community through drug-resistant Streptococcus pneumoniae. American Journal of Respiratory and Critical Care Medicine 159, 1835–42.[Abstract/Free Full Text]

8 . Marchese, A., Debbia, E. A., Pesce, A. & Schito, G. C. (1996). Bactericidal activity, morphological alterations, and synergistic interactions of rufloxacin, a new fluoroquinolone, alone and in combination with its N-desmethylate d derivative (MF 922). Chemotherapy 42, 90–9.[ISI][Medline]

9 . Lister, P. D. & Sanders, C. C. (1999). Pharmacodynamics of levofloxacin and ciprofloxacin against Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 43, 79–86.[Abstract/Free Full Text]

Received 8 March 2001; returned 26 June 2001; revised 10 July 2001; accepted 17 September 2001