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. 2001 Mar;45(3):938–942. doi: 10.1128/AAC.45.3.938-942.2001

In Vitro Development of Resistance to Six Quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus

Mechthild Boos 1, Susanne Mayer 1, Ansgar Fischer 1, Karl Köhrer 1, Sibylle Scheuring 1, Peter Heisig 2, Jan Verhoef 3, Ad C Fluit 3, F-J Schmitz 1,3,*
PMCID: PMC90398  PMID: 11181385

Abstract

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

Fluoroquinolone resistance in gram-positive cocci is related to mutations in the DNA gyrase and topoisomerase IV genes (8–12, 16, 23) and the active efflux of agents (1, 3, 13, 21–25, 31, 44). Because fluoroquinolones differ in both their target affinity (8, 16, 33–36, 39) and their activation of efflux pumps (7, 13, 21, 22, 24, 25, 31, 42), one can speculate that the phenotypic expression of quinolone resistance will also differ. Studies have shown that fluoroquinolone resistance can be selected for in pneumococci and staphylococci (5, 6, 37).

In order to analyze the ability of newer fluoroquinolones to cause resistance development in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus, we repeatedly exposed six clinical strains of each species to ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin.

Approximately 5 × 107 CFU of each of the 18 strains was added to tubes containing 9.9 ml of appropriate broth containing antibiotic concentrations ranging from 3 doubling dilutions above to 3 doubling dilutions below the MIC of each of the six agents. The tubes were then incubated for 24 h at 37°C. Aliquots from the test tubes containing the highest drug concentration that permitted visible growth were used following a 1:100 dilution to inoculate a second set of serial drug dilutions. After overnight incubation, the bacteria were transferred again. Finally, after 10 serial transfers, the bacteria for which the MICs were the highest were collected, stored, and also subcultured on quinolone-free agar for 10 days to assess the stability of resistance.

MICs were determined by the microdilution methodology according to NCCLS guidelines (29, 30). Ciprofloxacin MIC determinations were conducted in the presence and absence of reserpine (20 μg/ml; tests were repeated three times) for all of the original isolates (n = 18) as well as for all of the selected mutants (n = 108) (7).

S. pneumoniae and S. aureus isolates were analyzed before and after transfers for mutations in the quinolone-resistance determining regions (QRDRs) of parC or grlA and gyrA, respectively (19, 40, 41).

The MIC results from subculturing as well as the mutations in the QRDRs of S. pneumoniae and S. aureus are summarized in the Tables 1 to 3. Subculturing with newer quinolones led to resistance development in all three species. This is in line with previous reports with regard to cephalosporins, macrolides, and older quinolones in pneumococci (5, 6, 37).

TABLE 1.

Resistance selection results for S. pneumoniaea

Strain Initial MIC (μg/ml)
Selected resistance
MIC (μg/ml) upon retesting
Alterationc in enzymes of:
CIP SPX GAT MOX CLI GEM Drug Fold increase in MICsb CIP SPX GAT MOX CLI GEM GyrA ParC
1 0.25 0.125 0.125 0.06 0.03 0.015 CIP 8 64 8 8 2 0.5 0.5 S81F S79Y
SPX 7 32 16 8 2 0.25 0.5 S81Y S79Y
GAT 8 32 32 32 16 8 1 S81F S79Y
MOX 10 64 32 64 64 8 8 S81F S79Y, D83N
CLI 5 64 16 8 4 1 2 S81F S79Y
GEM 9 32 32 128 32 8 8 S81F S79Y
2 0.25 0.06 0.06 0.03 0.03 0.015 CIP 5 8 0.125 0.5 0.06 0.25 0.125 S81F
SPX 5 4 2 1 0.5 0.125 0.125 S81F
GAT 4 4 0.5 1 0.5 0.25 0.125 S81F
MOX 5 16 4 4 1 0.5 0.25 S81F
CLI 4 4 2 1 0.5 0.5 0.125 S81Y
GEM 7 32 8 64 2 2 2 D83Y
3 1 0.125 0.25 0.125 0.125 0.015 CIP 6 64 8 8 2 1 0.5 S81F S79Y, D83G
SPX 6 32 8 4 2 0.5 0.25 S81F D83G
GAT 5 32 16 8 4 1 0.5 S81F D83G
MOX 5 64 32 16 4 2 2 S81F S79Y
CLI 3 32 8 4 2 1 0.5 S81F D83N
GEM 6 16 4 64 1 2 1 S81F
4 1 0.125 0.25 0.125 0.06 0.015 CIP 6 64 8 8 2 1 0.5 S81F S79Y, D83G
SPX 6 8 8 4 2 0.5 0.25 S81F
GAT 3 8 4 2 1 0.5 0.25 S81F
MOX 6 64 32 16 8 1 1 S81F S79F
CLI 3 4 2 2 1 0.5 0.25 S81F
GEM 8 16 4 64 2 1 4 S81F S79Y
5 1 0.125 0.25 0.125 0.06 0.015 CIP 1 2 0.25 0.5 0.25 0.125 0.06
SPX 7 8 16 4 4 0.5 0.5 S81F S79F
GAT 4 16 32 4 4 0.5 0.5 S81Y S79F
MOX 4 8 8 4 2 0.25 0.25 S81F
CLI 3 32 16 8 4 0.5 0.5 S81Y S79F
GEM 6 32 8 32 1 0.5 1 D83Y
6 0.25 0.03 0.06 0.03 0.03 0.015 CIP 8 64 4 4 2 0.5 0.5 S81F S79Y
SPX 5 2 1 0.5 0.5 0.125 0.06
GAT 10 8 8 64 16 4 2 S81F S79F
MOX 7 8 2 4 4 0.25 0.125 S81F S79A
CLI 4 2 1 1 0.5 0.5 0.03 S81Y
GEM 4 8 1 32 1 0.125 0.25 S81F
Open in a new tab
a

CIP, ciprofloxacin; SPX, sparfloxacin; GAT, gatifloxacin; MOX, moxifloxacin; CLI, clinafloxacin; GEM, gemifloxacin. 

b

Fold increase in MIC between the MICs for the quinolone-resistant strain selected in the quinolone-containing medium and the MIC for the parental strain. For example, the ciprofloxacin MIC for parent strain 1 was 0.25 μg/ml. After passages in ciprofloxacin-containing medium, the ciprofloxacin MIC had increased to 64 μg/ml, i.e., an eightfold increase. 

c

Alterations refer to changes between the parent strain and the derived strain. All parent strains had wild-type ParC and GyrA enzymes. 

TABLE 3.

Resistance selection for S. aureusa

Strain Initial MIC (μg/ml)
Selected resistance
MIC (μg/ml) upon retesting
Alterationsc in enzymes of:
CIP SPX GAT MOX CLI GEM Drug Fold increase in MICsb CIP SPX GAT MOX CLI GEM GyrA GrlA
1 0.25 0.06 0.06 0.06 0.015 0.015 CIP 7 32 8 4 2 0.5 2 S84L E84K
SPX 8 64 16 4 4 1 0.5 S84L S80Y
E84K
GAT 6 64 16 4 8 0.5 4 S84L S80Y
E84K
MOX 3 4 0.5 1 0.5 0.25 0.015 S84A S80F
CLI 7 128 8 16 2 2 4 S84L E84K
GEM 13 2,048 64 128 32 32 128 S84L S80F
E84K
2 0.25 0.06 0.06 0.06 0.03 0.015 CIP 6 16 8 4 1 0.5 0.06 S84L E84K
SPX 6 4 4 2 1 0.25 0.5 S84L S80F
E88G
GAT 4 8 0.5 1 0.5 0.125 0.25 S84L S80F
MOX 4 4 4 1 1 0.25 0.5 S84L E84K
CLI 4 16 8 4 1 0.5 0.5 E88K E84K
GEM 3 16 1 4 4 0.5 0.125 S84L S80F
3 0.25 0.06 0.06 0.06 0.03 0.015 CIP 5 8 0.125 0.5 0.125 0.125 0.25 S84L E84K
SPX 6 4 4 2 1 0.125 0.5 S84L S80Y
GAT 4 8 4 1 1 0.25 0.5 S84L S80Y
E84K
MOX 3 4 4 2 0.5 0.125 0.5 S84L S80F
CLI 4 16 8 4 2 0.5 2 S84L E84K
GEM 9 128 8 8 4 0.5 8 S84L S80F
4 0.125 0.06 0.06 0.06 0.03 0.03 CIP 11 256 1 8 1 2 2 S84L S80F
E84K
SPX 8 32 16 16 2 0.5 2 S84L S80Y
GAT 11 128 128 128 32 8 16 S84L S80Y
E88K
MOX 10 64 128 128 64 16 16 S84L S80F
E84K
CLI 5 128 64 4 1 1 1 S84L S80Y
GEM 6 16 1 1 8 0.25 1 S80F
5 0.25 0.06 0.06 0.06 0.03 0.015 CIP 8 64 8 8 4 1 2 S84L S80Y
SPX 8 32 16 4 4 1 2 S84L S80Y
GAT 7 32 16 8 4 1 2 S84L S80Y
MOX 6 64 16 4 4 1 2 S84L S80Y
CLI 6 64 16 4 4 2 2 S84L E84K
GEM 12 1,024 32 32 16 8 64 S84L S80Y
E84G
6 0.25 0.06 0.06 0.06 0.03 0.015 CIP 8 64 8 8 2 1 4 S84L S80Y
SPX 2 0.5 0.25 0.12 0.125 0.03 0.06
GAT 4 4 0.5 1 0.25 0.125 0.06 S80F
MOXI 0 0.25 0.06 0.06 0.06 0.03 0.015
CLI 0 0.25 0.06 0.06 0.06 0.03 0.015
GEM 5 16 1 1 2 0.25 0.5 S80F
Open in a new tab
a

CIP, ciprofloxacin; SPX, sparfloxacin; GAT, gatifloxacin; MOX, moxifloxacin; CLI, clinafloxacin; GEM, gemifloxacin. 

b

Fold increase in MICs between the MIC for the quinolone-resistant strain selected in the quinolone-containing medium and the MIC for the parental strain. For example, the ciprofloxacin MIC for parent strain 1 was 0.25 μg/ml. After passages in ciprofloxacin containing medium, the ciprofloxacin MIC had increased to 32 μg/ml, i.e., a sevenfold increase. 

c

Alterations refer to changes between the parent strain and the derived strain. All parent strains had wild-type GrlA and GyrA enzymes. 

Resistance was stable in all cases; i.e., the MICs for the 108 selected mutants remained within 1 doubling dilution after 10 transfers on quinolone-free agar. Similar results were reported by Davies et al. (5) for S. pneumoniae.

Gemifloxacin and clinafloxacin exhibited the best in vitro activities against all original isolates, followed by moxifloxacin and gatifloxacin. Ciprofloxacin showed the lowest in vitro activities (Tables 1 to 3). On the basis of a breakpoint of ≤1 μg/ml, 88 of the 108 selected mutants were inhibited by clinafloxacin, 65 were inhibited by gemifloxacin, 46 were inhibited by moxifloxacin, 32 were inhibited by gatifloxacin, 20 were inhibited by sparfloxacin, and 3 were inhibited by ciprofloxacin. This rank order of activity is in parallel with previous findings (2, 4, 7, 19, 20, 26).

On the basis of the results presented in Tables 1 to 3, none of the quinolones was associated with a clearly lower potential for resistance development, measured as an increase in MIC doubling dilutions, compared with the potential for resistance development of the other quinolones tested. On the basis of the limited available data, one must be cautious in drawing broad conclusions. Further studies will be needed to clarify whether resistance development is strain dependent or dichotomous, especially for the 8-methoxyfluoroquinolones (gatifloxacin, moxifloxacin) compared with the other compounds.

For five of the six original S. pneumoniae strains tested, ciprofloxacin MICs were 1 to 3 doubling dilutions lower in the presence of reserpine. After 10 serial passages in ciprofloxacin-containing medium, the ciprofloxacin MICs for the same five of the six S. pneumoniae mutants were again 1 to 3 doubling dilutions lower in the presence of reserpine than in its absence. The MICs for five of six of the mutants selected with moxifloxacin or clinafloxacin were 1 to 2 doubling dilutions lower, the MICs for five of six of the mutants selected with sparfloxacin or gatifloxacin were 1 to 3 doubling dilutions lower, and finally, the MICs for five of six of the mutants selected with gemifloxacin were 2 to 4 doubling dilutions lower.

Ciprofloxacin MICs for none of the six original S. pyogenes strains tested were lower in the presence of reserpine. After 10 serial passages, the ciprofloxacin MICs for only one mutant selected with gemifloxacin were decreased by 3 doubling dilutions.

Ciprofloxacin MICs for none of the six original S. aureus strains tested were lower in the presence of reserpine. After 10 serial passages, ciprofloxacin MICs for six of six mutants selected with ciprofloxacin were 2 to 3 doubling dilutions lower in the presence of reserpine, MICs for four of six mutants selected with sparfloxacin or gatifloxacin were 1 doubling dilution lower, MICs for three of six mutants selected with clinafloxacin were 2 doubling dilutions lower, and, finally, MICs for six of six mutants selected with gemifloxacin were 2 to 4 doubling dilutions lower.

These results illustrate the importance of efflux as an additional factor for the development of quinolone resistance in S. aureus, followed by S. pneumoniae, while efflux seems not to play a significant role in the process of quinolone resistance development in S. pyogenes. Furthermore, our data indicate that some of the newer fluoroquinolones, especially gemifloxacin, selected mutants which had reserpine-sensitive phenotypes for ciprofloxacin resistance. This phenomenon must be kept in mind during therapy with newer quinolones.

Mainly classical alterations in ParC (a Ser-79→Phe or Tyr or Asp-83→Asp) and GyrA (a Ser-81→Phe or Tyr) contributed to the resistance seen in most S. pneumoniae mutants (Table 1). These results confirm those from previous investigations (12, 15, 17, 19, 20, 27, 32, 38, 43).

In addition, mainly classical alterations in GrlA (a Ser-80→Phe or Tyr, a Glu-84→Lys) and GyrA (a Ser-84→Leu or Lys and a Glu-88→Lys or Val) contributed to the resistance in most S. aureus mutants (Table 3). These results are in line with previous observations (14, 18, 28, 40, 41).

In summary, sequential subculture in the presence of subinhibitory concentrations of newer fluoroquinolones led to resistance development in S. pneumoniae, S. pyogenes, and S. aureus. None of the quinolones tested was associated with a clearly lower potential of resistance development.

TABLE 2.

Resistance selection results for S. pyogenesa

Strain Initial MIC (μg/ml)
Selected resistance
MIC (μg/ml) upon retesting
CIP SPX GAT MOX CLI GEM Drug Fold increase in MICsb CIP SPX GAT MOX CLI GEM
1 0.5 0.25 0.25 0.125 0.03 0.03 CIP 2 2 1 1 0.5 0.125 0.06
SPX 5 4 8 1 0.5 0.125 0.25
GAT 3 2 4 2 1 0.25 0.125
MOX 3 2 4 1 1 0.25 0.125
CLI 3 1 1 0.5 0.5 0.25 0.06
GEM 3 2 4 1 2 0.25 0.25
2 0.125 0.06 0.06 0.06 0.03 0.06 CIP 7 16 16 4 2 0.5 0.25
SPX 9 16 32 16 8 2 2
GAT 6 16 16 4 4 0.5 0.5
MOX 8 16 32 32 16 4 4
CLI 4 8 8 4 2 0.5 0.125
GEM 3 8 4 2 2 0.25 0.5
3 2 0.5 0.25 0.25 0.06 0.06 CIP 2 8 4 2 2 0.5 0.125
SPX 4 8 8 4 2 0.5 0.5
GAT 4 16 8 4 2 1 0.25
MOX 6 32 64 16 16 2 2
CLI 6 32 64 32 16 4 1
GEM 5 16 32 4 8 0.5 2
4 0.5 0.25 0.25 0.125 0.06 0.03 CIP 6 32 32 4 4 0.5 0.5
SPX 7 16 32 4 4 0.5 0.5
GAT 2 2 2 1 1 0.25 0.25
MOX 3 2 2 1 1 0.125 0.125
CLI 1 2 1 0.5 0.5 0.125 0.06
GEM 6 16 16 8 8 2 2
5 0.5 0.25 0.25 0.125 0.03 0.03 CIP 2 2 2 1 1 0.25 0.06
SPX 4 4 4 4 1 0.25 0.25
GAT 2 2 4 1 1 0.125 0.25
MOX 4 4 16 2 2 0.25 1
CLI 2 1 1 0.5 0.25 0.125 0.125
GEM 3 4 2 1 1 0.25 0.25
6 0.5 0.5 0.5 0.25 0.03 0.03 CIP 3 4 1 1 0.5 0.25 0.06
SPX 4 8 8 1 1 0.25 0.25
GAT 2 4 2 2 1 0.25 0.125
MOX 3 4 2 2 2 0.25 0.125
CLI 3 4 4 2 1 0.25 0.25
GEM 6 16 32 8 8 2 2
Open in a new tab
a

CIP, ciprofloxacin; SPX, sparfloxacin; GAT, gatifloxacin; MOX, moxifloxacin; CLI, clinafloxacin; GEM, gemifloxacin. 

b

Fold increase in MICs between the MIC for the quinolone-resistant strain selected in the quinolone-containing medium and the MIC for the parental strain. For example, the ciprofloxacin MIC for parent strain 1 was 0.5 μg/ml. After passages in ciprofloxacin-containing medium, the ciprofloxacin MIC had increased to 2 μg/ml, i.e., a 2 twofold increase. 

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