Fleroxacin combined with rifampin

DIAGN MICROBIOLINFECTDIS 1991;14:23-27

23

Fleroxacin Combined with Rifampin Yong-Xin Zhang and Harold C. Neu

We determined the effect of the combination of rifampin and fleroxacin against Enterobacteriaceae and streptococcal species. None of the 65 isolates tested by checkerboard assay demonstrated synergy, 12% of isolates showed an additive effect; 86.7% were indifferent, and only 1 isolate showed antagonism. The mean FIC was 1.2. When using 2 and 8 i~g/ml of rifampin, fleroxacin MICs of 285 isolates of Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influen-

zae, staphylococci, streptococci, Bacteroides, and Clostridium were not increased, but synergy was not demonstrated. Time-kill studies against Escherichia coli, P. aeruginosa, Enterobacter cloacae, Staphylococcus aureus, and Enterococcus faecalis failed to show increased killing when the two agents were present at one-half the MBC. The fleroxacinrifampin interaction is one of indifference but provides coverage for species not adequately inhibited by fleroxacin.

Fleroxacin is a new trifluoroquinolone that has been shown to have excellent activity against most of the Enterobacteriaceae inhibiting 90% at concentrations <1 ~g/ml (Chin et al., 1986). It also has excellent activity against staphylococci, but many streptococci and Streptococcus pneumoniae would be considered resistant, as are anaerobic species. Fleroxacin has a long half-life of 9-10 hr (Wise et al., 1987). The purpose of this investigation was to determine whether the combination of fleroxacin and rifampin would provide broader in vitro activity or whether antagonism would occur, as the activity of older quinolones such as nalidixic acid is antagonized by rifampin.

lution method using Mueller-Hinton agar according to NCCLS guidelines (NCCLS, 1988). An inoculum of 104 CFU was applied to agar as a spot with a replicating device. Incubation was at 35°C for 18-20 hr. Broth dilution was performed by using a volume of I ml and an inoculum of 5 x 105 CFU. Bactericidal activity was determined from broth dilutions by plating 0.01 ml from tubes showing no growth onto antibiotic-free agar plates, indicating a 99.9% reduction in CFU according to the method of Pearson et al. (1980). Synergy was performed by using a checkerboard agar method with the same inoculum as used in the agar minimal inhibitory concentration (MIC) determinations. Synergy was defined as a fractional inhibitory concentration (FIC) ~0.5, partial synergy or addition >0.5 to <0.75, indifference >0.75 to <2, and antagonism >2 (Krogstad and MoeUering, 1986). Time-kill studies were performed with exponential phase-growing organisms. Flasks of MuellerHinton medium were inoculated to contain - 106 CFU/ml and placed on a rotary shaker. Antimicrobial agents were added to individual flasks at I x minimal bactericidal concentration (MBC), one-half MBC, and one-quarter MBC of both agents. If the MBC for rifampin was >8 ~g/ml, only 8 ~g/ml was used. Aliquots were removed and diluted in broth to prevent carryover of drug. Sample aliquots were plated on antibiotic-free medium, and CFU was determined at 2, 4, 6, 8, and 24 hr after addition of the antimicrobial agents.

The organisms used were obtained from patients hospitalized at the Columbia-Presbyterian Medical Center (New York, NY). All organisms were considered pathogens. Rifampin and fleroxacin were provided by Hoffmann-La Roche Inc. (Nufley, NJ). Fresh dilutions of the compounds were prepared daily. Susceptibility was determined by the agar diFrom the Departments of Medicine and Pharmacology,College of Physiciansand Surgeons, ColumbiaUniversity, New York, New York. Present address (Y.-X.Z.): Department of Medicine, Hua Shan Hospital, ShanghaiMedicalUniversity, Shanghai, People's Republicof China. Address reprint requests to: Harold C. Neu, M.D., Department of Medicine, 630 West 168 Street, New York, NY 10032. Received April 30, 1990;June 11, 1990. © 1991 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, New York 10010 0732-8893/91/$3.50

Table I shows the MIC range and 50% and 90% MICs

24

Y.-X. Z h a n g and H.C. N e u

for fleroxacin, rifampin, and the combination of 2 p~g/ml fleroxacin and 8 p,g/ml rifampin. At a dose of 600 rng/day of rifampin for an adult, a steady-state concentration w o u l d be achieved and levels w o u l d rarely fall below 2 txg/ml. The Enterobacteriaceae were generally inhibited b y -<1 txg/ml of fleroxacin, with the exception of Providencia. For n o n e of the organisms was the MIC of fleroxacin increased in the pres-

TABLE 1.

ence of either 2 or 8 ~xg/ml of rifampin. Conversely, fleroxacin MICs were significantly lower (reduction ->4-fold) only for Proteus vulgaris and Proteus mirabilis in the presence of 8 p,g/ml of rifampin. Addition of rifampin did not lower the MICs of fleroxacin against Pseudomonas aeruginosa, but 8 pog/rnl of rifampin lowered fleroxacin MICs against Acinetobacter species.

Antimocrobial Activity of Fleroxacin Alone and in Combination with Rifampin MIC (jxg/ml)

Organism (no. tested)

Agent"

Range

Escherichia coli (15)

Fleroxacin Rifampin F+R2 F+Rs

0.06-0.25 0.06->16 ~<0.008-0.5 ~<0.008-0.25

Klebsiella pneumoniae (15)

Fleroxacln Rifampin F+R2 F + Rs

Enterobacter cloacae (15)

50%

90%

0.13 6.65 0.09 0.02

0.125 8 0.125 ~0.008

0.25 >16 0.25 0.125

0.03-0.5 8-32 0.06-1 0.015-1

0.11 29.33 0.11 0.06

0.125 >16 0.06 0.06

0.25 >16 0.25 0.125

Fleroxacln Rifampin F+R2 F+R8

0.06-0.5 8->16 0.03-0.5 0.03-0.125

0.18 20.15 0.125 0.08

0.25 16 0.125 0.125

0.25 >16 0.25 0.125

Serratia marcescens (15)

Fleroxacin Rifampin F + R2 F+R8

0.125-1 16->16 0.125-2 0.125-1

0.35 30.54 0.35 0.27

Proteus vulgaris (15)

Fleroxacln 0.125-2 Rifampin 4-8 0.06-2 F + R2 ~0.008-0.03 F+Rs

0.26 5.53 0.19 0.01

0.125 4 0.125 ~0.008

1 8 0.5 0.03

Morganella morganii (15)

Fleroxacm 0.03-0.25 Rifampin 4->16 0.06-0.25 F+R2 ~0.008-0.25 F + R8

0.12 10.08 0,13 0.04

0.125 8 0.125 0.06

0.25 16 0.25 0.25

Proteus mirabilis (15)

Fleroxacin Rifampin F+R2 F+R8

0.26 3.82 0.18 0.01

0.25 4 0.25 ~0.008

0.5 4 0.25 ~0.008

Providencia stuartii (15)

Fleroxacln 0.06-4 Rifampin 4-16 0.06-4 F+R2 ~0.008-0.06 F+Rs

0.42 8 0.33 0.01

0.25 8 0.25 ~0.008

2 16 1 0.06

Pseudomonas aeruginosa (15)

Fleroxacin Rifampin F+R~ F + R8

0.54 16.75 1.15 0.4

0.5 8 1 0.5

Acinetobacter sp. (15)

Fleroxacin 0.125-2 Rifampin 0.06-8 ~0.008-2 F+R2 ~0.008 F+Rs

0.83 1.2 0.4 ~<0.008

1 2 0.15 ~0.008

0.125-0.5 2->16 0.03-0.25 ~0.008-0.125

0.5-4 8-32 0.5-8 0.03-4

Mean

0.25 >16 0.25 0.25

1 >16 1 0.5

2 >16 4 2 2 4 1 ~0.008

Note

25

TABLE 1.

Continued MIC (p,g/ml)

Organism (no. tested)

Agent~

Range

Mean

50%

90%

Haemophilus influenzae (18)

Fleroxacin 0.03-0.125 Rifampin 0.125-0.5 40.008 F+R2 40.008 F + Rs

0.07 0.31 40.008 40.008

0.06 0.25 40.008 40.008

0.125 0.5 40.008 40.008

Staphylococcus aureus (15)

Fleroxacm 1 4 Rifarnpin 40.008-0.015 40.008 F+R2 40.008 F + R8

1.14 0.05 40.008 40.008

1 0.015 40.008 40.008

8. 0.015 40.008 40.008

S. aureus, methicillin

Fleroxacrn 0.015-8 Rifarnpin ~<0.0084 F+Ra 40.008 40.008 F+R8

1.38 0.125 40.008 40.008

4 1 40.008 40.008

4 2 40.008 40.008

Fleroxacrn 0.015-2 Rifampin 40.008 F+R2 40.008 40.008 F+R8

0.29 40.008 40.008 40.008

0.5 40.008 40.008 40.008

2 40.008 40.008 40.008

Fleroxacin 4-16 Rifarnpin 40.008-8 40.008-8 F + R2 40.008 F+R8

7.19 0.125 0.01 ~<0.008

8 0.125 40.008 40.008

16 1 40.008 40.008

6.96 1.74 0.16 0.02

8 2 40.008 40.008

7.33 0.07 40.008 40.008

8 0.06 40.008 40.008

8 0.125 40.008 40.008

resistant (10)

S. epidermidis, methicillin resistant (10)

Streptococcus spp. group A, B, C, G (26)

Enterococcus faecalis (15)

Fleroxacin Rifarnpin F+R2 F + Rs

2-8 0.25-8 40.008-16 40.008-8

Streptococcus pneumoniae (18) Fleroxacin 4-16 Rifarnpin F+R2 F + Rs

0.03-0.25 40.008 40.008

8 8 8 8

Bacteriodes fragilis (13)

Fleroxacm 4-16 Rifarnpin <0.125-0.5 42 F+R2

9.9 0.22 ~<2

8 0.25 42

16 0.25 42

Clostridium (17)

Fleroxacin 1-4 Rifampin <0.125 42 F+R2

1.18 ~0.125 42

1 <0.125 42

4 <0.125 42

~F + R2 and F + R8 indicate combination of 2 ~g/ml fleroxacin and 8 p,g/ml rifampin, respectively.

Most of the staphylococci isolates were very susceptible to rifampin. There was a reduction in MICs of these isolates for the rifampin-fleroxacin combination with the methicillin-resistant isolates, a n u m b e r of which were resistant to fleroxacin (MIC />4 p,g/ml) and to rifampin (breakpoint considered 1 ~g/ml). All methicillin-resistant Staphylococcus epidermidis was highly susceptible to rifampin; therefore, the combination effect was not important. For the hemolytic streptococci g r o u p s A, B, C, and G, the fleroxacin MICs0 was 8 ~g/ml (range 4 16 ~g/ml). Rifampin was extremely active against

most streptococci, but some organisms had higher rifampin MICs and s y n e r g y was seen for the combination of rifampin a n d fleroxacin. The combination of fleroxacin-rifampin (2 ~g/ml) s h o w e d synergistic activity for half the isolates of Enterococcus faecalis. Rifampin and fleroxacin did not s h o w a synergistic effect against S. pneumoniae because all of the isolates were inhibited by <0.008 ~g/ml of rifampin. All Bacteroides and Clostridium were inhibited by < 2 p,g/ml rifampin. The effect of checkerboard combination was determined against a total of 65 isolates (Table 2). Synergy was not found, 12% showed addition, and 86.7%

26

Y.-X. Zhang and H.C. Neu

TABLE 2.

Checkerboard Activity of the Combination of Fleroxacin and Rifampina No. (%) Strains Showing

Organism b

Addition

Klebsiella pneumoniae Pseudomonas aeruginosa Enterobacter cloacae Providencia stuartii Escherichia coli Serratia marcescens Staphylococcus aureus Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Enterococcus faecalis Bacteroides fragilis Clostridium spp.

2 2

Total

1 2 1

1 9 (12)

Indifference

Antagonism

3 3 5 5 4 5 5 3 3 5 5 13 6 65 (86.7)

Mean FIC Index 0.90 0.98 1.1 1.4 1.15 1.4 1.4 0.95 1.45

1.3 1.16

1 (1.3)

1.25

aNo synergyobserved. bFive strains each.

showed indifference. One group B streptococcus showed antagonism. The mean FIC was 1.25 (range 0.9-1.45). Time-kill studies of the fleroxacin-rifampin combination were performed on single isolates of five species--Escherichia coli, Enterobacter cloacae, P. aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. Fleroxacin at its MBC caused a rapid fall in CFU of E. coli, E. cloacae, and S. aureus, with lesser decline in CFU of P. aeruginosa and E. faecalis. When rifampin was combined with fleroxacin, which was present at one-half or one-fourth the MBC, the reduction in CFU was always less than that for fleroxacin alone at the MBC. Thus, synergy could not be demonstrated by the killing curve technique. Combination of fleroxacin and rifampin at onehalf MBC combination was less effective than was fleroxacin at the MBC. Rifampin failed to increase the activity of fleroxacin against E. faecalis. These results are generally similar to those reported for combination of rifampin and ciprofloxacin against staphylococci and Enterobacteriaceae (Neu, 1989). In the case of ciprofloxacin, 1-2 ~g/ml will inhibit a major proportion of hemolytic streptococci and S. pneumoniae, so there is less of a requirement for a combination with an agent that inhibits these species. Fleroxacin has MICs generally out of the sus-

ceptible range (>2 p~g/ml) for many streptococci and pneumococci. This study shows that the serum concentrations of rifampin and fleroxacin that could easily be achieved with the usual oral doses of each agent taken once a day would inhibit Enterobacteriaceae, Haemophilus, hemolytic streptococci, S. pneumoniae, and S. aureus, but not E. faecalis. Furthermore, the study shows that with the exception of a single isolate, antagonism of the two agents was not encountered. What is not established from such a study is whether resistance to the rifampin would develop. We did not test the organisms that regrew in the case of E. faecalis and S. aureus. Whether a program of fleroxacin-rifampin would be useful in the United States is unknown. In other countries in which rifampin has been used for infections other than just tuberculosis, such a combination might find favor because fleroxacin enters tissues rapidly and could easily eradicate streptococci although resistance could develop. A clinical study of the combination rifampin-fleroxacin compared to fleroxacin and rifampin alone in respiratory infections might be of value if very close attention were given to the follow-up isolates to determine whether resistance to rifampin developed in the pneumococci or Haemophilus, as has occurred when rifampin has been used as a single agent.

Note

27

REFERENCES Chin NX, Brittain DC, Neu HC (1986) In vitro activity of Ro 23-6240, a new fluorinated 4-quinolone. Antimicrob Agents Chemother 29:675-680. Krogstad DJ, MoeUering RC Jr (1986) Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 2nd ed. Ed, V Lorian. Baltimore: Williams and Wilkins, pp 537595. National Committee for Clinical Laboratory Standards (NCCLS) (1988) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2nd ed. Standard M7-T2. Villanova, PA: NCCLS.

Neu HC (1989) Synergy of fluoroquinolones with other antimicrobial agents. Rev Infect Dis 11(Suppl 5):10251035. Pearson RD, Steigbigel RT, David HT, Chapman SW (1980) Method for reliable determination of minimal lethal concentrations. Antimicrob Agents Chemother 18:699708. Wise RB, Kirkpatrick B, Ashby J, Griggs DJ (1987) Pharmacokinetics and tissue penetration of Ro 23-6240, a new trifluoroquinolone. Antimicrob Agents Chemother 31:61-63.