Journal of Antimicrobial Chemotherapy (2002) 49, 1015–1018 DOI: 10.1093/jac/dkf036
Characterization of clinical Streptococcus pneumoniae strains from Germany with decreased susceptibility to fluoroquinolones Ralf René Reinert1*, Adnan Al-Lahham1, Rudolf Lütticken1, Mechthild Boos2 and Franz-Josef Schmitz2 1Institute of Medical Microbiology, National Reference Center for Streptococci, University of Aachen, Pauwelsstrasse 30,
D-52074 Aachen; 2Institute of Medical Microbiology and Virology, University Hospital Düsseldorf, Germany Received 13 July 2001; returned 29 November 2001; revised 6 February 2002; accepted 19 February 2002
Fourteen pneumococcal strains isolated in three nationwide studies were characterized for amino acid changes in the enzymes GyrA, GyrB, ParC and ParE, and for the in vitro activity of eight fluoroquinolones and the new non-fluorinated quinolone BMS 284756. Gemifloxacin and BMS 284756 exhibited the best in vitro activity against all 14 isolates tested. In nine of the 14 isolates mainly classical alterations in ParC (D83N/Y, S79Y/F), as well as rarer alterations such as S80P and D78N, contributed to the decreased susceptibility to fluoroquinolones. In two of the 14 isolates the classical alteration in GyrA (S81F) was found. In only one isolate did alterations in ParC and GyrA exist in parallel.
Introduction Streptococcus pneumoniae is a leading cause of morbidity and mortality in the human population worldwide. Traditionally, treatment of pneumococcal infections has relied largely on the use of β-lactam compounds; however, in recent years the widespread emergence of β-lactam resistance and the associated multidrug resistance have presented a challenge to effective anti-pneumococcal therapies.1 Consequently, several fluoroquinolone compounds have been developed that show encouraging in vitro activity against Gram-positive organisms, including penicillin- and macrolide-resistant pneumococci. The aim of the present study was to characterize mutations in the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of pneumococcal strains isolated in Germany, with ciprofloxacin MICs of ≥4 mg/L. In addition, the study was aimed at determining the activity of a panel of quinolone agents against these strains and the detection of multiply resistant isolates.
Materials and methods Strains were collected in three multicentre studies. Ten strains were from a study on pneumococcal strain isolates from normally sterile body sites [PS collection (duplicates
excluded), n = 2733, range 150–504 isolates per year] between 1992 and 2000.2 Three strains (RESP collection, n = 961) were from a study on clinically significant pneumococcal strains from community-acquired infections prospectively collected by 19 different clinical microbiological laboratories between November 1998 and April 1999 from all regions in Germany,3 and one isolate was from a nationwide study on respiratory tract infections (MSR 86, n = 328) performed in 1999–2000 (R. R. Reinert, unpublished data). Details on each of the studies are presented in Table 1. Of the 14 strains investigated, six were recovered from blood and eight from the respiratory tract. Each isolate was confirmed as being S. pneumoniae by optochin sensitivity and bile solubility testing. MICs of penicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, moxifloxacin, sparfloxacin, grepafloxacin, gemifloxacin, BMS 284756, clinafloxacin, gatifloxacin and levofloxacin were determined by the microbroth dilution method using Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5% lysed horse blood (Oxoid, Wesel, Germany) as recommended by the NCCLS.4 Tests were repeated three times for all isolates. The antibiotics were kindly provided by the respective pharmaceutical companies. The MIC of ciprofloxacin was determined in the presence and absence of reserpine (20 mg/L; Sigma, Frankfurt, Germany), an inhibitor of efflux pumps, in order to determine the
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*Corresponding author. Tel: +49-241-80-89-787; Fax: +49-241-80-82-483; E-mail:
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1015 © 2002 The British Society for Antimicrobial Chemotherapy
R. R. Reinert et al. Table 1. Overview of fluoroquinolone resistance in three multicentre studies performed in Germany
Study period
Source of strains
Total number of strains
Number of fluoroquinolone-resistant strains (%)a
Number of strains included in present study
1992–2000 1998–1999 1999–2000
NSBS RTIs RTIs
2733 961 328
19 (0.7) 4 (0.4) 2 (0.6)
10 3 1
Reference Reinert et al.2 Reinert et al.3 R. R. Reinert (unpublished data)
NSBS, normally sterile body sites; RTIs, respiratory tract infections. aDefinition of fluoroquinolone resistance: ciprofloxacin MIC ≥ 4 mg/L.
presence of an efflux mechanism, as described previously.5 MIC determinations in the presence and absence of reserpine were conducted three times. Efflux activity was assumed if a difference of at least two doubling dilutions in ciprofloxacin MICs with and without reserpine was observed. As controls, isolates with no efflux activity or three doubling dilutions difference derived from selection experiments described by Boos et al.6 were used. Prepared chromosomal DNA was used as a template for PCR amplification of target QRDRs. The primers and PCR conditions were those previously defined.7–9 The PCR products were purified using a PCR purification kit (Qiagen, Ratingen, Germany) and sequenced using the dye terminator method in both forward and reverse directions. The products were then resolved and analysed automatically using an ABI PRISM 310 DNA sequencer. In all isolates with reduced susceptibility to fluoroquinolones and with decreased sensitivity to erythromycin, clindamycin or tetracycline, the underlying resistance mechanisms were characterized. For amplification of tetM the following primers were used: 5′TGG AAT TGA TTT ATC AAC GG-3′ (position 2496– 2515) and 5′-TTC CAA CCA TAC AAT CCT TG-3′ (position 3575–3556). For the detection of erm and mef the following primers were chosen: erm, 5′-CGA GTG AAA AAG TAC TCA ACC-3′ (position 362–382), 5′-GGC GTG TTT CAT TGC TTG ATG-3′ (position 978–958); mef, 5′-AGT ATC ATT AAT CAC TAG TGC-3′ (position 57–77), 5′-GTA ATA GAT GCA ATC ACA GC-3′ (position 551–532).3 Pneumococcal strains were serotyped by Neufeld’s Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark.
Results and discussion The prevalence of pneumococci with reduced susceptibility to fluoroquinolones remains low in Germany, as documented by the nationwide, multicentre study of invasive pneumococcal disease in Germany (Figure 1) and the studies on non-invasive pneumococcal infections (Table 1). Gemifloxacin and the new non-fluorinated quinolone BMS 284756 exhibited the best in vitro activities against all
Figure 1. Fluoroquionolone resistance (ciprofloxacin MIC ≥ 4 mg/L) in invasive pneumococcal strains (n = 2733) isolated in Germany, 1992–2000.
14 isolates tested, followed by clinafloxacin, moxifloxacin, gatifloxacin, grepafloxacin and sparfloxacin. Levofloxacin and ciprofloxacin showed the lowest in vitro activities (Table 2). Based on the NCCLS breakpoints for the 14 strains, one strain was moxifloxacin intermediate (breakpoint ≥2 mg/L), six were sparfloxacin resistant (≥2 mg/L), three were gatifloxacin resistant (≥4 mg/L) and one was levofloxacinresistant (≥8 mg/L). All strains were inhibited by ≤1 mg/L of clinafloxacin, gemifloxacin or BMS 284756. In nine of the 14 isolates, mainly classical alterations in ParC (D83N/Y, S79Y/F), as well as rarer alterations such as S80P and D78N, were observed. In only two of the 14 isolates was the classical alteration in GyrA (S81F) found. Furthermore, in only one isolate (RESP 766) did alterations in ParC and GyrA exist in parallel, together with a very rare alteration in GyrA (S114G). This strain showed a levofloxacin MIC of 16 mg/L. The role of the additional S114G mutation needs to be determined. In two isolates, no alterations were found in either ParC or GyrA. In strain MSR 86, which showed a ciprofloxacin MIC of 8 mg/L, only one alteration in GyrA was found, indicating the likelihood of other alterations that might lie outside the QRDR of the ParC subunit. The significance of parE and gyrB mutations in fluoroquinolone resistance remains unclear. Previous authors have assigned no role to alterations of ParE and GyrB in isolates with reduced susceptibility to fluoroquinolones.10 In our
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Characterization of German S. pneumoniae strains Table 2. Characteristics of 14 pneumococcal strains with reduced susceptibility to fluoroquinolones isolated in Germany, 1992– 2000 Alterations in target enzymesb
MIC (mg/L) Strain
ST Year
PS 97 PS 565 PS 625 PS 2378 PS 2505 RESP 754 RESP 974 PS 671 PS 551 PS 845 MSR 86 PS 2124 PS 710 RESP 766
15C 6A 7F 16F 9N 46 15B 22F 7F 23F 6B 14 23A 6B
1992 1992 1992 1996 1997 1999 1999 1993 1992 1993 1999 1996 1993 1999
Town Köln Stade Hannover Berlin Bremen Stuttgart Wülfrath Mannheim Stuttgart Trier Bad Hersfeld Köln Hannover Wülfrath
Source CIP CIP/Res MOX SPX GRE GEM GAT BMS CLX LVX GyrA
ParC
blood 4 blood 4 pleura 4 blood 4 pleura 4 nose 4 trachea 4 blood 8 blood 8 pleura 8 sputum 8 blood 16 BAL 16 sputum 32
D83N D83N S79Y n S80P n D83N S79F S79Y D78N n D83N D83Y S79F
1 0.5 4 0.125 0.5 0.5 0.5 0.5 4 2 1 4 2 16
0.125 0.5 0.125 0.06 0.125 0.06 0.125 0.125 0.125 0.06 0.5 0.5 0.125 2
1 2 1 2 1 1 1 1 2 0.125 4 4 1 8
1 1 1 0.25 0.5 0.5 0.5 1 0.5 0.25 1 2 1 4
0.03 0.03 0.03 0.03 0.03 0.03 0.015 0.06 0.06 0.03 0.03 0.125 0.06 0.25
1 4 1 0.5 1 1 1 1 1 0.5 4 2 1 8
0.03 0.03 0.03 0.03 0.06 0.03 0.015 0.03 0.03 0.03 0.03 0.125 0.06 0.5
0.06 1 0.06 1 0.06 2 0.06 2 0.06 1 0.06 1 0.06 1 0.06 2 0.125 2 0.06 2 0.125 4 0.25 4 0.125 2 0.25 16
n n n n n n n n n n S81F n n S81Fa
ST, pneumococcal serotype; BAL, bronchoalveolar lavage; n, no mutation; CIP, ciprofloxacin; CIP/Res, ciprofloxacin plus reserpine (efflux inhibition); MOX, moxifloxacin; SPX, sparfloxacin; GRE, grepafloxacin; GEM, gemifloxacin; GAT, gatifloxacin; BMS, BMS 284756; CLX, clinafloxacin; LVX, levofloxacin. aStrain also showed an S114G alteration. bSeven of 14 strains showed a ParE Ile-460→Val alteration; no alterations were observed in GyrB.
pneumococcal strains, no alterations were found in gyrB, whereas seven of 14 strains displayed the frequently identified Ile-460→Val change, which seemed to have no clear-cut effect on MICs. Eleven of the 14 S. pneumoniae strains displayed ciprofloxacin MICs two to five doubling dilutions lower in the presence of reserpine, indicating the important contribution of efflux to decreased susceptibility to quinolones. The greatest efflux activity was observed in a serotype 16F isolate (PS 2378), which exhibited no alterations in GyrA or ParC. The serotype distribution of strains with reduced susceptibility to fluoroquinolones differs widely from those found among penicillin non-susceptible (predominant serotypes 23F and 6B) and macrolide-resistant isolates (predominant serotypes 14 and 6B). Multiple resistance among strains with reduced susceptibility to fluoroquinolones is rare in Germany. Only four of 14 strains showed resistance equal to or more than three classes of antibiotic. Of the pneumococci showing decreased susceptibility to fluoroquinolones, one also showed reduced sensitivity to penicillin G, three strains were erythromycin resistant (one strain mef positive, one strain erm positive, one strain neither erm positive nor mef positive), and two strains were also tetracycline resistant (all tetM positive). Cross-resistance is more likely to occur among strains with resistance to β-lactams, macrolides or tetra-
cycline compared with those with reduced susceptibility to fluoroquinolones. In conclusion, the rate of strains showing decreased sensitivity to fluoroquinolones is very low in Germany, as documented by the present work and other studies from Germany. Nevertheless, the broad usage of new fluoroquinolones may lead to increases in the prevalence of fluoroquinolone resistance among pneumococci.
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