New Antimicrobial Agents
In V i t r o A c t i v i t y o f T o s u f l o x a c i n ( A 60969) and Clarithromycin (A-56268, T E - 0 3 1 ) a g a i n s t R e s i s t a n t Haemophilus
influenzae, Streptococcus pneumoniae Branhamella catarrhalis I s o l a t e s
and
j. p. B r y a n 1, 2., C. W a t e r s 1, J. S h e f f i e l d 1, K. F. W a g n e r t,2 The activity of tosufloxacin (A-60969), a new oral quinolone, and clarithromycin (A-56268, TE-031), a new oral macrolide, was compared in vitro to that of other oral quinolones and betalactam antimicrobial agents against clinical isolates of ampicillin and/or cbloramphenicol resistant Haemophilus influenzae, penicillin resistant Streptococcus pneumoniae and betaiactamase producing Branhamella catarrhalis. Results were compared to those for sensitive isolates. Tosufloxacin was the most active compound against Haemophilus influenzaeand was more active than ciprofloxacin or ofloxacin against all strains of Streptococcus pneumoniae. Tosufioxacin was also more active than any of the beta-lactam drugs tested against penicillin resistant or relatively penicillin resistant isolates. Clarithromycin was the most active c o m p o u n d tested against both penicillin sensitive and penicillin resistant Streptococcus pneumoniae, and was as active as ciprofloxacin against Branhamella catarrhalls. In view of the favourable in vitro activity against common bacterial respiratory pathogens, tosufloxacin should be considered for clinical trials in adults with respiratory tract infections, while clarithromycin might be useful in treatment of infection with these organisms in all age groups.
Haernophilus influenzae type b, Streptococcus pneumoniae and Branharnella catarrhalis are important causes of upper and lower respiratory infections and otitis media (1-5), especially in children. Ampicillin and/or chloramphenicol resistance in Haemophilus influenzae (3, 4) and penicillin resistance in Streptococcus pneumoniae (5-7) have caused changes in the choice of therapy in some areas, especially Spain and South Africa. Therapeutic options in patients with suspected or proven infection with 1Division of Infectious Diseases, National Naval Medical Center, Bethesda,Maryland20814-5000,USA. 2Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland20814-4799, USA.
Vol, 9, No. 5
361
resistant organisms are limited. Tosufloxacin, (A-60969), a new quinolone, has a broad antimicrobial spectrum which includes anaerobic bacteria (8-10). Clarithromycin (A-56268, TE031), a macrolide, has improved in vitro activity against many bacterial genera compared to e r y t h r o m y c i n (11-14). In this report, we compare the in vitro activity of tosufloxacin and clarithromycin with that of selected quinolones, e r y t h r o m y c i n and beta-lactam antibiotics against resistant clinical isolates of Strepto-
coccus pneumoniae, Haeraophitus influenzae and BranhamelIa catarrhaIis. Materials and Methods. Bacterial strains were provided by the Centers for Disease Control (CDC), Atlanta, GA (Dr. C. Thornsberry); National Institutes of Health, Bethesda, MD (Dr. Wu); US Naval Hospital, Bethesda, MD; University of Virginia, Charlottesville, VA (Dr. W. Scheld); National Children's Hospital, Washington, DC (Dr. W. Rodriguez); and University of Washington, Seattle, WA (Dr. A. Smith). Six of the CDC isolates were from patients with ampicillin and chloramphenicol resistant Haemophilus influenzae infections in Barcelona, Spain (3). Streptococcus pneumoniae isolates were identified by standard techniques (15) and the identity confirmed serologically using the Phadebact Pneumococcal Test (Pharmacia Diagnostics, USA). Sources included the blood (n = 20), cerebrospinal fluid (n = 1), respiratory tract (n = 39) and site unknown (n = 6). Resistance or relative resistance to penicillin was determined using a disc containing 1 gg oxacillin. Resistance of a strain producing a zone of growth < 19 mm was confirmed by determination of the MIC. Isolates with an MIC in the range 0.12-1.0 gg/ml were considered relatively resistant. Isolates with an MIC of > 2.0 txg/ml were considered resistant (t5).
Haemophilus influenzae isolates were identified by standard techniques (16) and the identity confirmed using the Haemophilus Identification Quad Plate (Scott Laboratories, USA). The serotype was confirmed as type b by the Phadebact Haemophilus Test (Pharmacia Diagnostics). Sources included the cerebrospinal fluid (n = 44), conjunctiva (n = 5), blood (n = 3) and respiratory tract (n = 25). Production of beta-lactamase was detected by the nitrocefin disc test (Cefinase, BBL, USA) and benzyl penicillin saturated strip test (Beta Test, Micro Diagnostics, USA). Production of chloramphenicol acetyl transferase was detected using the C.A.T. Reagent Kit (Remel, USA). Isolates were considered resistant to ampicillin if the MIC was > 4 gg/mI and resistant to chloramphenicol if the MIC was > 8 Ixg/ml.
362
Branhamella catarrhalis isolates were identified by standard techniques (17), and the identity confirmed by the API Quadferm system (Analytab Products, USA). Beta-lactamase production was tested as above. All isolates were from the respiratory tract of patients thought to have invasive disease caused by those isolates. A-61827, the tosylated salt of tosufloxacin, and clarithromycin were provided by A b b o t t Pharmaceuticals, USA, cefixime by Lederle Laboratories, USA, and ofloxacin by Ortho Pharmaceutical, USA. The other antimicrobial agents were obtained from Sigma Chemical, USA. Microdilution trays of antimicrobial agents were prepared by two-fold dilution of freshly prepared antimicrobial stock solutions in Mueller-Hinton broth. Each isolate was subcultured twice on appropriate agar before testing. A suspension of organisms containing approximately 5 x 105 CFU/ml was prepared in broth. Mueller-Hinton broth was used for Branhamella catarrhalis, and was supplemented with Fildes supplement for Haemophilus influenzae or 2-3 % lysed horse blood for Streptococcus pneumoniae (18). Representative isolates of Haemophilus influenzae were tested in Haemophilus Test Medium (19) by an independent laboratory, as well as our own laboratory, with essentially identical results to those obtained using Fildes supplement. Fifty gl of the bacterial suspension were added to each well of a 96-well plate containing 50 ~tl of the test agents in Mueller-Hinton broth. Each isolate was tested against each agent in triplicate. ATCC quality control strains were tested simultaneously to clinical strains (19). After incubation for 18-24 h at 35 °C the MIC (the lowest concentration which inhibited visible growth) was determined for each antibiotic. Results and Discussion. A total of 77 Haemophilus influenzae strains (70 type b and 7 untyped) was tested. All isolates resistant to ampicillin produced beta-lactamase. Of 15 isolates resistant to chloramphenicol, seven produced detectable chloramphenicol acetyl transferase but eight did not, indicating a probable permeability barrier mechanisms of resistance (20, 21). The most active agent overall against Haernophilus influenzae was tosufloxacin (Table 1). The activity of the quinolones was not affected by ampicillin or chloramphenicol resistance. Clarithromycin was the more active of the two macrolides. A total of 69 Streptococcus pneumoniae strains was tested (Table 2). Tosuflo×acin inhibited 90 % of all isolates at a concentration of
Eur. J. Clin. Microbiol. Infect Dis.
0.5 gg/ml, which was considerably higher than the MIC of most beta-lactam antibiotics for penicillin sensitive organisms. H o w e v e r , tosufloxacin was more active than the betalactam antibiotics against penicillin resistant organisms and was four- to eight-fold more active than ciprofloxacin and ofloxacin. The quinolones were active against Streptococcus pneumoniae irrespective of the degree of sensitivity to penicillin. Clarithromycin was the most active compound tested against penicillin sensitive and penicillin resistant isolates. T o s u f l o x a c i n i n h i b i t e d all isolates of Branhamella catarrhalis at a concentration of 0.06 gg/ml, similarly to ciprofloxacin and ofloxacin (Table 3). Even although 12 of the 15 organisms produced beta-lactamase, ampicillin inhibited all isolates at a concentration of 0.5 gg/ml, as shown in multiple tests. Amoxicillin plus clavulanic acid inhibited all isolates at a concentration of 0.25 gg/ml. Clarithromycin was more active than the betalactam antibiotics and as active as the quinolones. Other investigators have found a disparity between production of beta-lactamase by Branhamella catarrhalis and susceptibility to ampicillin (22-24). On the basis of its in vitro activity, clarithromycin might be useful in all age groups for treatment of infections with Branhamella catarrhalis or Streptococcus pneumoniae. While most Haemophilus influenzae strains would also be sensitive to clarithromycin, synergism obtained by combination with a sulfonamide, as in the case of erythromycin plus sulfamethoxazole, might increase the clinical utility of this drug in use against these organisms. Other investigators have found clarithromycin to be active against Streptococcus pneumoniae (11-13) but activity against penicillin resistant pneumococci has to our knowledge not been reported. Erythromycin has been shown to be active against some isolates of penicillin resistant Streptococcus pneumoniae, and has been used with success to treat pneumonia due to such organisms (6). Other investigators have found clarithromycin to be active against Legionella spp., also common respiratory tract pathogens (14). Tosufloxacin was the most active of all the antimicrobial agents tested against Haemophilus influenzae, including beta-lactamase producing strains and strains demonstrating resistance to chloramphenicol by means of chloramphenicol acetyl transferase p r o d u c t i o n or o t h e r mechanisms. Ciprofloxacin has proved useful in the treatment of respiratory tract infections, including those caused by Streptococcus pneurnoniae, even although the MIC for
V0!,9,1990
363
Table 1: C o m p a r a t i v e in vitro activity of tosufloxacin and clarithromycin against 77 selected
HaemophiIus influenzae strains (ampicillin and chloramphenicol sensitive n = 41, ampicillin resistant only n = 21, chloramphenicol and ampicillin resistant n = 13, chloramphenicol resistant only n = 2). MIC (I.tg/ml)
Antimicrobial agent Range
MIC50
MIC90
Tosufloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0.001--0,016 < 0.001-0.016 < 0.001-0.016
0.002 0.004 0.008
0.008 0.008 0.016
Ciprofloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0,001--0,125 < 0.001-0.032 < 0.001--0.016
0.008 0.008 0.008
0.032 0.016 0.016
Ofloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.008--0.25 < 0.001-0.064 0.008-0.064
0.032 0.032 0.032
0.125 0.064 0.032
Clarithromycin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.254.0 0.25-8.0 0.5 --8.0
1.0 1.0 2.0
8.0 4.0 4.0
Erythromycin A m p s Chlor s A m p r Chlor s Chlor r A m p r or Amp s
1- > 8.0 0.5- > 8.0 2.0- > 8.0
8.0 4.0 > 8.0
> 8.0 > 8.0 > 8.0
Amoxicillin/clavulanate A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0.06--0.5 0.125-1.0 0.125-4.0
Cefuroxime axetyl A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p ~ Cefaclor A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.125 0.5 05
0.25 0+5 4.0
0.03-4.0 0.03-1.0 0.125-1.0
0.25 0.25 0+5
0.5 0.5 1.0
0.06-2.0 0.125-2.0 0.125-2.0
05 03 05
1.0 2.0 2.0
Streptococcus pneumoniae is c l o s e t o t h e a t tainable serum concentrations (25), Tosufloxacin was four to eight times more active than ciprofloxacin against penicillin sensitive and p e n i c i l l i n r e s i s t a n t Streptococcus pneumoniae. Tosufloxacin was also quite active against Branhamella catarrhalis. T o s u f l o x a c i n m i g h t thus be useful as an alternative agent in treatment of adults with respiratory tract infection caused by these pathogens.
biology and therapy. Reviews of Infectious Diseases 1987, 9: 16+26. 3. C a m p o s J, G a r c l a - T o r n e l S, Sanfellu !: Susceptibility studies of multiple resistant Haemophilus influenzae isoaltes from pediatric p a t i e n t s a n d contacts. Antimicrobial Agents and Chemotherapy 1984,25: 706-709.
4. Doern GV, .iorgensen JI-l, Thornsberry C, Preston DA, Tubert T, Redding.IS, Maher LA: National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of ttaemophilus influenzae. Antimicrobial Agents and Chemotherapy 1988, 32: 180-185.
5. Istre GR, Tarpay M, Anderson M, Pryor A, Welch D and the Pneumococcus Study Group: Invasive disease due to Streptococcus pneumoniae in an area with a high rate of relative penicillin resistance. Journal of Infectious Diseases 1987, 156: 732-735.
References 1. Hager H, Verghese A, Alvarez S, Berk SL: Bran-
hamella catarrhatis respiratory infections. Reviews of Infectious Diseases 1987, 9: 1140-1150.
2. van Hare GF, Shurin PA, Marehan! CD, Carlelli NA, Johnson CE, Fulton D, Carlin S, Kim CH: Acute otitis media caused by
Branhamelta catarrhatis:
6. Pallares R, Guidoi F, Linares J, Arlza ,i, Ruff G, Murquii L, Dorca J, Viladrich PF: Risk factors and r e s p o n s e to a n t i b i o t i c t h e r a p y in adults with bacteremic p n e u m o n i a caused by penicillin-resistant pncumococci. New E n g l a n d Journal of Medicine 1987, 317:18-22.
364
E u r . J. Clin. M i c r o b i o l . I n f e c t D i s .
Table 2: Comparative in vitro activity of tosufloxacin and clarithromycin against 69 Streptococcus pneumoniae strains (penicillin sensitive n = 55, relatively penicillin resistant n = 11, penicillin resistant n = 3). MIC (gg/ml)
Antimicrobial agent Range
MIC50
MIC90
Tosufloxacin Penicillin sensitive Penicillin resistant
< 0.015-0.5 0.03-0,5
0,03 0,125
0.125 0,25
Ciprofloxacin Penicillin sensitive Penicillin resistant
< 0.5-2.0 < 0.25-2.0
1,0 0.5
2.0 1.0.
Ofloxacin Penicillin sensitive Penicillin resistant
0.5-2.0 1.0-2.0
1.0 2.0
2.0 2.0
0.002 0.004
0.008 0.008 0,125 0.125
Clarithromycin Penicillin sensitive Penicillin resistant
< 0.002-0.008 0.02-0.008
Erythromycin Penicillin sensitive Penicillin resistant
0,002-0.025 0.0(0-4).5
0.06 0.06
Penicillin Penicillin sensitive Penicillin resistant
< 0,064),06 0.125- > 8.0
< 0,06 0.5
0.06 2.0
Ampicillin Penicillin sensitive Penicillin resistant
< 0.06-0.12 0.fir--4.0
< 0.06 025
0.06 2.0
Amoxicillin/clavulanate Penicillin sensitive Penicillin resistant
< 0.06--0.12 < 0.06-4.0
< 0.06 0.06
0.06 1.0
Cttloramphenicol Penicillin sensitive Penicillin resistant
1.0- > 8.0 0.5-8.0
2.0 1.0
2.0 4.0
Cefuroxime axetyl Penicillin sensitive Penicillin resistant
< 0.015-0.25 2.0 < 0,015-0.25 0.1X~--2.0 < 0.125-1.0 < 0.06-0.25
0.03 0.015 0.06 0.015 0.25 < 0.06 0.5 0.5 0.06 025 0.5 0.125
0.06 0.03 0.125 0.03 0.5 0.5 1.0 2.0 0.125 1.0 1.0 0.125
Vol. 9, 1990
7. Viladrich PF, Gudiol F, Linares J, Ruff G, Ariza J, Pillares R: Characteristics and antibiotic therapy of adult meningitis due to penicillin resistant pneumococci. American Journal of Medicine 1988, 84: 839846. 8. Espinoza AM, Chin N, Novelll A, Neu HC: Comparative in vitro activity of a new fluorinated 4quinolone, T-3262 (A-60969). Antimicrobial Agents and Chemotherapy 1988,32: 663-670. 9. F u j i m a k i K, Noumi T, Saikawa 1, Indue M, Mitsuhashi S: In vitro and in vivo antibacterial activities of T-3262, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 1988, 32: 827--833. 1(3. Fernandes PB, ChuDTW, Swanson RN, Ramcr NR, Hanson CW, Bower RR, Stamm JM, Hardy D J: A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 1988, 32: 27-32, 11. Barry AL, Thornsberry C, Jones RN: In vitro activity of a new macrolide, A-56268, compared with that of roxithromycin, erythromycin, and clindamycin. Antimicrobial Agents and Chemotherapy 1987, 31: 343-345. 12. Benson CA, Segreti J, Beaudette FE, Hines DW, Goodman L J, Kaplan RL, Trenhoime GM: In vitro activity of A-56268 (TE-031), a new macrolide, compared with that of erythromycin and ctindamycin against selected gram-positive and gram-negative organisms. Antimicrobial Agents and Chemotherapy 1987, 31: 328-330. 13. Floyd-Reising S, HindlerJA, Young LS: In vitro activity of A-56268 (TE-031), a new macrolide antibiotic, compared with that of erythromycin and other antimicrobial agents. Antimicrobial Agents and Chemotherapy 1987, 31: 640--642. 14. Fernandes PB, Bailer R, Swanson R, Hanson CW, McDonald E, Ramcr N, Hardy D, Shipkowitz N, Bower RR, Gade A: In vitro and in vivo evaluation of A-56268 (TE-031), a new macrolide. Antimicrobial Agents and Chemotherapy 1986, 30: 865-873. 15. Facldam RR, Carey BB: Streptococci and aerococci. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 154-175.
.....
365
16. Killian M: Haemophilus. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 387-393. 17. Moreiio JA, Jarda WM, Bohnhof[ M: Neisseria and Branhamella. In: Lennette EH, Balows A, Hausler WJ, S h a d o m y H J (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 176-192. 18. Thrupp LD: Susceptibility testing of antibiotics in liquid media. In: Lorian V (ed): Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore, MD, 1986, p. 93-150. 19. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: Tentative standard M7 - T2. NCCLS, Villanova, PA, i989. 20. Roberts MC, Swenson CD, Owens LM, Smith AL: Characterization of chloramphenicol-resistant tlaemophilus influenzae. Antimicrobial Agents and Chemotherapy 1980, 18: 610--615. 21. Burns JL, Mendelman PM, Levy J, Slull TL, Smith AL: A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 1985, 27: 46-54. 22. Doern GV , Tubcrt T: Disk diffusion susceptibility testing of Branhametla catarrhalis with ampicillin and seven other antimicrobial agents. Antimicrobial Agents and Chemotherapy 1987, 31: 1519-I523. 23. Sweeney KG, Verghese A, Needham CA: In vitro susceptibilities of isolates from patients with Branhamella catarrhalis pneumonia compared with those of colonizing strains. Antimicrobial Agents and Chemotherapy 1985, 27: 499-502, 24. Aivarez S, Jones M, Holtsclaw-Berk S, Guarderas J, Berk SL: In vitro susceptibilities and 13-1actamase production of 53 clinical isolates of Branhamella catarrhalis. Antimicrobial Agents and Chemotherapy 1985, 27: 646-647. 25. Raoof S, Wollsehlarger C, Khan F: Treatment of respiratory tract infections with ciprofloxacin. Journal of Antimicrobial Chemotherapy 1986, 18: Supplement D: 139-145.
In V i t r o A c t i v i t y o f T o s u f l o x a c i n ( A 60969) and Clarithromycin (A-56268, T E - 0 3 1 ) a g a i n s t R e s i s t a n t Haemophilus
influenzae, Streptococcus pneumoniae Branhamella catarrhalis I s o l a t e s
and
j. p. B r y a n 1, 2., C. W a t e r s 1, J. S h e f f i e l d 1, K. F. W a g n e r t,2 The activity of tosufloxacin (A-60969), a new oral quinolone, and clarithromycin (A-56268, TE-031), a new oral macrolide, was compared in vitro to that of other oral quinolones and betalactam antimicrobial agents against clinical isolates of ampicillin and/or cbloramphenicol resistant Haemophilus influenzae, penicillin resistant Streptococcus pneumoniae and betaiactamase producing Branhamella catarrhalis. Results were compared to those for sensitive isolates. Tosufloxacin was the most active compound against Haemophilus influenzaeand was more active than ciprofloxacin or ofloxacin against all strains of Streptococcus pneumoniae. Tosufioxacin was also more active than any of the beta-lactam drugs tested against penicillin resistant or relatively penicillin resistant isolates. Clarithromycin was the most active c o m p o u n d tested against both penicillin sensitive and penicillin resistant Streptococcus pneumoniae, and was as active as ciprofloxacin against Branhamella catarrhalls. In view of the favourable in vitro activity against common bacterial respiratory pathogens, tosufloxacin should be considered for clinical trials in adults with respiratory tract infections, while clarithromycin might be useful in treatment of infection with these organisms in all age groups.
Haernophilus influenzae type b, Streptococcus pneumoniae and Branharnella catarrhalis are important causes of upper and lower respiratory infections and otitis media (1-5), especially in children. Ampicillin and/or chloramphenicol resistance in Haemophilus influenzae (3, 4) and penicillin resistance in Streptococcus pneumoniae (5-7) have caused changes in the choice of therapy in some areas, especially Spain and South Africa. Therapeutic options in patients with suspected or proven infection with 1Division of Infectious Diseases, National Naval Medical Center, Bethesda,Maryland20814-5000,USA. 2Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland20814-4799, USA.
Vol, 9, No. 5
361
resistant organisms are limited. Tosufloxacin, (A-60969), a new quinolone, has a broad antimicrobial spectrum which includes anaerobic bacteria (8-10). Clarithromycin (A-56268, TE031), a macrolide, has improved in vitro activity against many bacterial genera compared to e r y t h r o m y c i n (11-14). In this report, we compare the in vitro activity of tosufloxacin and clarithromycin with that of selected quinolones, e r y t h r o m y c i n and beta-lactam antibiotics against resistant clinical isolates of Strepto-
coccus pneumoniae, Haeraophitus influenzae and BranhamelIa catarrhaIis. Materials and Methods. Bacterial strains were provided by the Centers for Disease Control (CDC), Atlanta, GA (Dr. C. Thornsberry); National Institutes of Health, Bethesda, MD (Dr. Wu); US Naval Hospital, Bethesda, MD; University of Virginia, Charlottesville, VA (Dr. W. Scheld); National Children's Hospital, Washington, DC (Dr. W. Rodriguez); and University of Washington, Seattle, WA (Dr. A. Smith). Six of the CDC isolates were from patients with ampicillin and chloramphenicol resistant Haemophilus influenzae infections in Barcelona, Spain (3). Streptococcus pneumoniae isolates were identified by standard techniques (15) and the identity confirmed serologically using the Phadebact Pneumococcal Test (Pharmacia Diagnostics, USA). Sources included the blood (n = 20), cerebrospinal fluid (n = 1), respiratory tract (n = 39) and site unknown (n = 6). Resistance or relative resistance to penicillin was determined using a disc containing 1 gg oxacillin. Resistance of a strain producing a zone of growth < 19 mm was confirmed by determination of the MIC. Isolates with an MIC in the range 0.12-1.0 gg/ml were considered relatively resistant. Isolates with an MIC of > 2.0 txg/ml were considered resistant (t5).
Haemophilus influenzae isolates were identified by standard techniques (16) and the identity confirmed using the Haemophilus Identification Quad Plate (Scott Laboratories, USA). The serotype was confirmed as type b by the Phadebact Haemophilus Test (Pharmacia Diagnostics). Sources included the cerebrospinal fluid (n = 44), conjunctiva (n = 5), blood (n = 3) and respiratory tract (n = 25). Production of beta-lactamase was detected by the nitrocefin disc test (Cefinase, BBL, USA) and benzyl penicillin saturated strip test (Beta Test, Micro Diagnostics, USA). Production of chloramphenicol acetyl transferase was detected using the C.A.T. Reagent Kit (Remel, USA). Isolates were considered resistant to ampicillin if the MIC was > 4 gg/mI and resistant to chloramphenicol if the MIC was > 8 Ixg/ml.
362
Branhamella catarrhalis isolates were identified by standard techniques (17), and the identity confirmed by the API Quadferm system (Analytab Products, USA). Beta-lactamase production was tested as above. All isolates were from the respiratory tract of patients thought to have invasive disease caused by those isolates. A-61827, the tosylated salt of tosufloxacin, and clarithromycin were provided by A b b o t t Pharmaceuticals, USA, cefixime by Lederle Laboratories, USA, and ofloxacin by Ortho Pharmaceutical, USA. The other antimicrobial agents were obtained from Sigma Chemical, USA. Microdilution trays of antimicrobial agents were prepared by two-fold dilution of freshly prepared antimicrobial stock solutions in Mueller-Hinton broth. Each isolate was subcultured twice on appropriate agar before testing. A suspension of organisms containing approximately 5 x 105 CFU/ml was prepared in broth. Mueller-Hinton broth was used for Branhamella catarrhalis, and was supplemented with Fildes supplement for Haemophilus influenzae or 2-3 % lysed horse blood for Streptococcus pneumoniae (18). Representative isolates of Haemophilus influenzae were tested in Haemophilus Test Medium (19) by an independent laboratory, as well as our own laboratory, with essentially identical results to those obtained using Fildes supplement. Fifty gl of the bacterial suspension were added to each well of a 96-well plate containing 50 ~tl of the test agents in Mueller-Hinton broth. Each isolate was tested against each agent in triplicate. ATCC quality control strains were tested simultaneously to clinical strains (19). After incubation for 18-24 h at 35 °C the MIC (the lowest concentration which inhibited visible growth) was determined for each antibiotic. Results and Discussion. A total of 77 Haemophilus influenzae strains (70 type b and 7 untyped) was tested. All isolates resistant to ampicillin produced beta-lactamase. Of 15 isolates resistant to chloramphenicol, seven produced detectable chloramphenicol acetyl transferase but eight did not, indicating a probable permeability barrier mechanisms of resistance (20, 21). The most active agent overall against Haernophilus influenzae was tosufloxacin (Table 1). The activity of the quinolones was not affected by ampicillin or chloramphenicol resistance. Clarithromycin was the more active of the two macrolides. A total of 69 Streptococcus pneumoniae strains was tested (Table 2). Tosuflo×acin inhibited 90 % of all isolates at a concentration of
Eur. J. Clin. Microbiol. Infect Dis.
0.5 gg/ml, which was considerably higher than the MIC of most beta-lactam antibiotics for penicillin sensitive organisms. H o w e v e r , tosufloxacin was more active than the betalactam antibiotics against penicillin resistant organisms and was four- to eight-fold more active than ciprofloxacin and ofloxacin. The quinolones were active against Streptococcus pneumoniae irrespective of the degree of sensitivity to penicillin. Clarithromycin was the most active compound tested against penicillin sensitive and penicillin resistant isolates. T o s u f l o x a c i n i n h i b i t e d all isolates of Branhamella catarrhalis at a concentration of 0.06 gg/ml, similarly to ciprofloxacin and ofloxacin (Table 3). Even although 12 of the 15 organisms produced beta-lactamase, ampicillin inhibited all isolates at a concentration of 0.5 gg/ml, as shown in multiple tests. Amoxicillin plus clavulanic acid inhibited all isolates at a concentration of 0.25 gg/ml. Clarithromycin was more active than the betalactam antibiotics and as active as the quinolones. Other investigators have found a disparity between production of beta-lactamase by Branhamella catarrhalis and susceptibility to ampicillin (22-24). On the basis of its in vitro activity, clarithromycin might be useful in all age groups for treatment of infections with Branhamella catarrhalis or Streptococcus pneumoniae. While most Haemophilus influenzae strains would also be sensitive to clarithromycin, synergism obtained by combination with a sulfonamide, as in the case of erythromycin plus sulfamethoxazole, might increase the clinical utility of this drug in use against these organisms. Other investigators have found clarithromycin to be active against Streptococcus pneumoniae (11-13) but activity against penicillin resistant pneumococci has to our knowledge not been reported. Erythromycin has been shown to be active against some isolates of penicillin resistant Streptococcus pneumoniae, and has been used with success to treat pneumonia due to such organisms (6). Other investigators have found clarithromycin to be active against Legionella spp., also common respiratory tract pathogens (14). Tosufloxacin was the most active of all the antimicrobial agents tested against Haemophilus influenzae, including beta-lactamase producing strains and strains demonstrating resistance to chloramphenicol by means of chloramphenicol acetyl transferase p r o d u c t i o n or o t h e r mechanisms. Ciprofloxacin has proved useful in the treatment of respiratory tract infections, including those caused by Streptococcus pneurnoniae, even although the MIC for
V0!,9,1990
363
Table 1: C o m p a r a t i v e in vitro activity of tosufloxacin and clarithromycin against 77 selected
HaemophiIus influenzae strains (ampicillin and chloramphenicol sensitive n = 41, ampicillin resistant only n = 21, chloramphenicol and ampicillin resistant n = 13, chloramphenicol resistant only n = 2). MIC (I.tg/ml)
Antimicrobial agent Range
MIC50
MIC90
Tosufloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0.001--0,016 < 0.001-0.016 < 0.001-0.016
0.002 0.004 0.008
0.008 0.008 0.016
Ciprofloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0,001--0,125 < 0.001-0.032 < 0.001--0.016
0.008 0.008 0.008
0.032 0.016 0.016
Ofloxacin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.008--0.25 < 0.001-0.064 0.008-0.064
0.032 0.032 0.032
0.125 0.064 0.032
Clarithromycin A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.254.0 0.25-8.0 0.5 --8.0
1.0 1.0 2.0
8.0 4.0 4.0
Erythromycin A m p s Chlor s A m p r Chlor s Chlor r A m p r or Amp s
1- > 8.0 0.5- > 8.0 2.0- > 8.0
8.0 4.0 > 8.0
> 8.0 > 8.0 > 8.0
Amoxicillin/clavulanate A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
< 0.06--0.5 0.125-1.0 0.125-4.0
Cefuroxime axetyl A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p ~ Cefaclor A m p s Chlor s A m p r Chlor s Chlor r A m p r or A m p s
0.125 0.5 05
0.25 0+5 4.0
0.03-4.0 0.03-1.0 0.125-1.0
0.25 0.25 0+5
0.5 0.5 1.0
0.06-2.0 0.125-2.0 0.125-2.0
05 03 05
1.0 2.0 2.0
Streptococcus pneumoniae is c l o s e t o t h e a t tainable serum concentrations (25), Tosufloxacin was four to eight times more active than ciprofloxacin against penicillin sensitive and p e n i c i l l i n r e s i s t a n t Streptococcus pneumoniae. Tosufloxacin was also quite active against Branhamella catarrhalis. T o s u f l o x a c i n m i g h t thus be useful as an alternative agent in treatment of adults with respiratory tract infection caused by these pathogens.
biology and therapy. Reviews of Infectious Diseases 1987, 9: 16+26. 3. C a m p o s J, G a r c l a - T o r n e l S, Sanfellu !: Susceptibility studies of multiple resistant Haemophilus influenzae isoaltes from pediatric p a t i e n t s a n d contacts. Antimicrobial Agents and Chemotherapy 1984,25: 706-709.
4. Doern GV, .iorgensen JI-l, Thornsberry C, Preston DA, Tubert T, Redding.IS, Maher LA: National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of ttaemophilus influenzae. Antimicrobial Agents and Chemotherapy 1988, 32: 180-185.
5. Istre GR, Tarpay M, Anderson M, Pryor A, Welch D and the Pneumococcus Study Group: Invasive disease due to Streptococcus pneumoniae in an area with a high rate of relative penicillin resistance. Journal of Infectious Diseases 1987, 156: 732-735.
References 1. Hager H, Verghese A, Alvarez S, Berk SL: Bran-
hamella catarrhatis respiratory infections. Reviews of Infectious Diseases 1987, 9: 1140-1150.
2. van Hare GF, Shurin PA, Marehan! CD, Carlelli NA, Johnson CE, Fulton D, Carlin S, Kim CH: Acute otitis media caused by
Branhamelta catarrhatis:
6. Pallares R, Guidoi F, Linares J, Arlza ,i, Ruff G, Murquii L, Dorca J, Viladrich PF: Risk factors and r e s p o n s e to a n t i b i o t i c t h e r a p y in adults with bacteremic p n e u m o n i a caused by penicillin-resistant pncumococci. New E n g l a n d Journal of Medicine 1987, 317:18-22.
364
E u r . J. Clin. M i c r o b i o l . I n f e c t D i s .
Table 2: Comparative in vitro activity of tosufloxacin and clarithromycin against 69 Streptococcus pneumoniae strains (penicillin sensitive n = 55, relatively penicillin resistant n = 11, penicillin resistant n = 3). MIC (gg/ml)
Antimicrobial agent Range
MIC50
MIC90
Tosufloxacin Penicillin sensitive Penicillin resistant
< 0.015-0.5 0.03-0,5
0,03 0,125
0.125 0,25
Ciprofloxacin Penicillin sensitive Penicillin resistant
< 0.5-2.0 < 0.25-2.0
1,0 0.5
2.0 1.0.
Ofloxacin Penicillin sensitive Penicillin resistant
0.5-2.0 1.0-2.0
1.0 2.0
2.0 2.0
0.002 0.004
0.008 0.008 0,125 0.125
Clarithromycin Penicillin sensitive Penicillin resistant
< 0.002-0.008 0.02-0.008
Erythromycin Penicillin sensitive Penicillin resistant
0,002-0.025 0.0(0-4).5
0.06 0.06
Penicillin Penicillin sensitive Penicillin resistant
< 0,064),06 0.125- > 8.0
< 0,06 0.5
0.06 2.0
Ampicillin Penicillin sensitive Penicillin resistant
< 0.06-0.12 0.fir--4.0
< 0.06 025
0.06 2.0
Amoxicillin/clavulanate Penicillin sensitive Penicillin resistant
< 0.06--0.12 < 0.06-4.0
< 0.06 0.06
0.06 1.0
Cttloramphenicol Penicillin sensitive Penicillin resistant
1.0- > 8.0 0.5-8.0
2.0 1.0
2.0 4.0
Cefuroxime axetyl Penicillin sensitive Penicillin resistant
< 0.015-0.25 2.0 < 0,015-0.25 0.1X~--2.0 < 0.125-1.0 < 0.06-0.25
0.03 0.015 0.06 0.015 0.25 < 0.06 0.5 0.5 0.06 025 0.5 0.125
0.06 0.03 0.125 0.03 0.5 0.5 1.0 2.0 0.125 1.0 1.0 0.125
Vol. 9, 1990
7. Viladrich PF, Gudiol F, Linares J, Ruff G, Ariza J, Pillares R: Characteristics and antibiotic therapy of adult meningitis due to penicillin resistant pneumococci. American Journal of Medicine 1988, 84: 839846. 8. Espinoza AM, Chin N, Novelll A, Neu HC: Comparative in vitro activity of a new fluorinated 4quinolone, T-3262 (A-60969). Antimicrobial Agents and Chemotherapy 1988,32: 663-670. 9. F u j i m a k i K, Noumi T, Saikawa 1, Indue M, Mitsuhashi S: In vitro and in vivo antibacterial activities of T-3262, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 1988, 32: 827--833. 1(3. Fernandes PB, ChuDTW, Swanson RN, Ramcr NR, Hanson CW, Bower RR, Stamm JM, Hardy D J: A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 1988, 32: 27-32, 11. Barry AL, Thornsberry C, Jones RN: In vitro activity of a new macrolide, A-56268, compared with that of roxithromycin, erythromycin, and clindamycin. Antimicrobial Agents and Chemotherapy 1987, 31: 343-345. 12. Benson CA, Segreti J, Beaudette FE, Hines DW, Goodman L J, Kaplan RL, Trenhoime GM: In vitro activity of A-56268 (TE-031), a new macrolide, compared with that of erythromycin and ctindamycin against selected gram-positive and gram-negative organisms. Antimicrobial Agents and Chemotherapy 1987, 31: 328-330. 13. Floyd-Reising S, HindlerJA, Young LS: In vitro activity of A-56268 (TE-031), a new macrolide antibiotic, compared with that of erythromycin and other antimicrobial agents. Antimicrobial Agents and Chemotherapy 1987, 31: 640--642. 14. Fernandes PB, Bailer R, Swanson R, Hanson CW, McDonald E, Ramcr N, Hardy D, Shipkowitz N, Bower RR, Gade A: In vitro and in vivo evaluation of A-56268 (TE-031), a new macrolide. Antimicrobial Agents and Chemotherapy 1986, 30: 865-873. 15. Facldam RR, Carey BB: Streptococci and aerococci. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 154-175.
.....
365
16. Killian M: Haemophilus. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 387-393. 17. Moreiio JA, Jarda WM, Bohnhof[ M: Neisseria and Branhamella. In: Lennette EH, Balows A, Hausler WJ, S h a d o m y H J (ed): Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985, p. 176-192. 18. Thrupp LD: Susceptibility testing of antibiotics in liquid media. In: Lorian V (ed): Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore, MD, 1986, p. 93-150. 19. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: Tentative standard M7 - T2. NCCLS, Villanova, PA, i989. 20. Roberts MC, Swenson CD, Owens LM, Smith AL: Characterization of chloramphenicol-resistant tlaemophilus influenzae. Antimicrobial Agents and Chemotherapy 1980, 18: 610--615. 21. Burns JL, Mendelman PM, Levy J, Slull TL, Smith AL: A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 1985, 27: 46-54. 22. Doern GV , Tubcrt T: Disk diffusion susceptibility testing of Branhametla catarrhalis with ampicillin and seven other antimicrobial agents. Antimicrobial Agents and Chemotherapy 1987, 31: 1519-I523. 23. Sweeney KG, Verghese A, Needham CA: In vitro susceptibilities of isolates from patients with Branhamella catarrhalis pneumonia compared with those of colonizing strains. Antimicrobial Agents and Chemotherapy 1985, 27: 499-502, 24. Aivarez S, Jones M, Holtsclaw-Berk S, Guarderas J, Berk SL: In vitro susceptibilities and 13-1actamase production of 53 clinical isolates of Branhamella catarrhalis. Antimicrobial Agents and Chemotherapy 1985, 27: 646-647. 25. Raoof S, Wollsehlarger C, Khan F: Treatment of respiratory tract infections with ciprofloxacin. Journal of Antimicrobial Chemotherapy 1986, 18: Supplement D: 139-145.
