1 Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada; 2 Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Canada; 3 Department of Pathology and Laboratory Medicine, The Ottawa Hospital, Ottawa, Canada; 4 Clinical Epidemiology Program, Ottawa Health Research Institute at The Ottawa Hospital, Ottawa, Canada
Received 4 April 2005; returned 12 May 2005; revised 27 June 2005; accepted 30 June 2005
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Abstract |
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Methods: We evaluated the in vitro susceptibility of three virulent strains of H. ducreyi to ceftriaxone, azithromycin, rifabutin and streptomycin, and each two-drug combination by the agar dilution method. We then tested each two-antibiotic combination for activity by the chequerboard method. Lastly, we chose the antibiotic combination with the lowest fractional inhibitory concentration index (FICI) and tested combined sub-therapeutic doses, the highest doses which had no effect alone on lesion healing compared with controls, for in vivo interaction in the temperature-dependent rabbit model of H. ducreyi infection.
Results: Each H. ducreyi strain was susceptible in vitro to each antibiotic and two-antibiotic combination, and combined ceftriaxone and streptomycin had the lowest FICI at 0.63. In five treated animals versus three untreated controls, combined sub-therapeutic doses of ceftriaxone (0.05 mg/kg) and streptomycin (10 mg/kg) reduced mean (SD) duration of culture positivity from 7.3 (1.1) to 2.6 (1.7) days (P < 0.001), time to 50% reduction in lesion size from 9.7 (1.5) to 5.8 (0.8) days (P < 0.005), and time to resolution of ulcer from 11.7 (2.3) to 6.6 (1.7) days (P < 0.05).
Conclusions: Ceftriaxone and streptomycin have in vivo synergic interaction against H. ducreyi lesions in the temperature-dependent rabbit model of infection. Antibiotic combinations may be evaluated clinically as single-dose therapy for chancroid.
Keywords: antimicrobial interactions , chancroid , H. ducreyi , synergy
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Introduction |
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In a 1988 survey of H. ducreyi strains isolated in South Africa, most were found to be resistant to penicillin, tetracycline, sulfamethoxazole, and trimethoprim.15 H. ducreyi, once susceptible to sulphonamides, penicillins, tetracyclines, and trimethoprim, has developed plasmid or chromosomally encoded resistance to each.1618 Discrete susceptibility profiles in different regions are also reported, often reflecting differential drug use.19 Several single-dose regimens have been shown to be effective for the treatment of chancroid, including ceftriaxone, ciprofloxacin, azithromycin, and sulfamethoxazole/trimethoprim.20 Despite documented H. ducreyi resistance to it,21 streptomycin is still an alternative treatment.22 With the growing prevalence of HIV co-infection, however, single-dose therapies of ceftriaxone, azithromycin, fleroxacin, ciprofloxacin, erythromycin and trimethoprim/sulfamethoxazole are associated with greater than 20% chancroid treatment failure rates.2326 Also, there is regional variation of chancroid response to azithromycin,27 and chancroid treatment failure attributable to herpes simplex virus (HSV) co-infection28 or drug resistance. As H. ducreyi is adept at acquiring new gene products through horizontal mechanisms, both in its genome, e.g. cytolethal distending toxin,29 and on stably transmitted plasmids,18,30 it may acquire resistance genes through horizontal transmission as well as through de novo mutation. Although clinical evidence of this complex problem is limited to simple observational study methods, theoretical and mathematical models show that effective combination therapy can limit the emergence of antibiotic resistance at the population level.31 Efficacious single-dose treatment regimens may improve compliance-related effectiveness of treatment programmes and reduce the emergence of antibiotic resistance,20 and are desirable as chancroid therapy. Combination antibiotic therapy has improved therapeutic efficacy in other bacterial infectious conditions and in the immunosuppressed patient.32
We aimed to demonstrate the principle, and propose that antibiotic combinations may be developed as single-dose, effective and sustained treatment of chancroid, suitable for treatment-based chancroid control programmes. We screened antibiotics of unrelated class and each of their two-antibiotic combinations for in vitro antimicrobial activity against susceptible H. ducreyi strains, and then evaluated the most promising antibiotic combination for in vivo treatment efficacy in the temperature-dependent rabbit model of chancroid.33
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Materials and methods |
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Type strain 35000 of H. ducreyi was originally isolated during an outbreak in Winnipeg, Manitoba, in 1975,34 while strains RO 40 and RO 34 are clinical isolates from Nairobi, Kenya, 1987. Strains 35000 and RO 34 are virulent in the temperature-dependent rabbit model of chancroid,33,35 and possess distinct outer membrane profiles.35 The strain RO 40 has a distinct outer membrane profile and is also virulent in the temperature-dependent rabbit model of chancroid (D. W. Cameron and J. E. Roy-Leon, unpublished data). Reference strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were obtained from the American Type Culture Collection, Rockville, MD, USA.
H. ducreyi strains were grown on chocolate agar (CA) plates consisting of 3.6% gonococcal (GC) agar base (B-D Microbiology Systems, Cockeysville, MD, USA) +1% bovine haemoglobin (B-D Microbiology Systems) supplemented with 1% IsoVitaleX (B-D Microbiology Systems) and 5% fetal bovine serum (FBS; Gibco BRL, Burlington, ON, Canada); and in broth consisting of a 1:1 (v/v) mixture of MuellerHinton broth (B-D Microbiology Systems) and -minimal essential medium (Gibco BRL) supplemented with 17% FBS. Plates were incubated at 33°C for 48 h with 5% CO2 and high humidity, while broth cultures were incubated for 12 h at 33°C in an environmental shaker at 175 rpm. Reference strains of E. coli and S. aureus were grown in CA or MuellerHinton broth supplemented with 1% IsoVitaleX. Plates were incubated for 24 h at 37°C in 5% CO2 and broth cultures were incubated for 2 h at 37°C in an environmental shaker (Lab-line, Melrose Park, IL, USA).
Animals
Animals were treated in accordance with the Animal Care and Veterinary Services procedures. The Animal Care Committee at the University of Ottawa approved all experiments. Male New Zealand White Rabbits (2.53 kg) (Charles River Laboratories, Montreal, PQ, Canada) were housed in an 11.7 m2 room at the Animal Care Facility at the University of Ottawa Health Science Centre. The temperature in the room was maintained at 14°C with a Thermo Air plus air conditioning unit. The backs of the rabbits were shaved prior to infection and when necessary. All rabbits were kept under identical conditions for the duration of the experiments.
Antibiotics
Ceftriaxone (Sigma Chemical Co., St Louis, MO, USA), streptomycin (Sigma), azithromycin (Pfizer, Pointe-Claire-Dorval, PQ, Canada), and rifabutin (Farmitaliz Carlo Erba, Nerviano, Italy) were used in the experiments.
In vitro antimicrobial susceptibility testing
MIC and fractional inhibitory concentration index (FICI) determinations were measured by agar dilution on CA plates supplemented with 1% gonococcal supplement B (Difco Laboratories, Detroit, MI, USA). Plates were incubated for 48 h at 33°C in 5% CO2 in a humid chamber. All strains were tested for their susceptibility to ceftriaxone, streptomycin, azithromycin and rifabutin. All stock solutions were prepared in double distilled water. Azithromycin and rifabutin were initially dissolved in methanol (<2% of the final volume) because of their lack of solubility in water.
MIC determination. MIC was determined using the agar dilution method of the NCCLS with modifications.36,37 Ceftriaxone, streptomycin, azithromycin and rifabutin were diluted in water and incorporated into CA at 50°C to yield the appropriate 2-fold dilution series. Final concentration ranges tested were 0.0018 mg/L for ceftriaxone, 0.063512 mg/L for streptomycin, 0.000054 mg/L for azithromycin, and 0.0022 mg/L for rifabutin. The agar was poured into 100 x 15 mm Petri dishes (Fisher Scientific, Nepean, ON, Canada), to a depth of 34 mm. Plates were used the same day or were stored at 4°C for use the following day. H. ducreyi strains were grown on CA plates for 48 h, and inoculated on new plates that were incubated for 24 h. The growth was suspended in MuellerHinton broth with 1% IsoVitaleX and allowed to sediment for at least 15 min at room temperature. The optical density of each supernatant was adjusted to that of a 0.5 McFarland barium sulphate standard. A few colonies of the reference strains were selected from plates grown for 24 h at 37°C and were inoculated into 4 mL of MuellerHinton broth supplemented with 1% IsoVitaleX. This suspension was incubated at 37°C for 2 h. All suspensions were diluted 1:10 and 2 µL (104 cfu) was inoculated onto the plates with a pipette. MIC was defined as the lowest concentration of an antibiotic that completely inhibits growth (disregarding a single colony) after 48 h at 33°C in 5% CO2 and high humidity. Assays were performed in duplicate.
FICI determination. FICI for H. ducreyi strains 35000, RO 40 and RO 34 exposed to varying concentrations of all two-drug combinations of ceftriaxone, azithromycin, rifabutin and streptomycin were determined using the chequerboard agar dilution method.38 Plates and inocula were prepared and incubated for MIC determination as described above. The assays were performed in duplicate in three separate experiments.
In vivo antimicrobial susceptibility testing
Experimental induction of infection. Experimental infections were induced and assayed as previously described.35,39 Briefly, broth-grown H. ducreyi strains 35000 and RO 40 were harvested at the late mid-log phase, defined by time of incubation, i.e. 46 h, by centrifugation at 3000 g for 10 min. Pellets were washed once with PBS (Gibco BRL), pH 7.2, and resuspended in MuellerHinton broth. Serial 10-fold dilutions of H. ducreyi broth, from 107 to 103 cfu/mL were prepared, and injected intraepithelially in triplicate in 100 µL doses, for a total of 15 injections, into the shaved backs of the animals, to give five final inocula doses of 106 to 102 cfu. The actual inoculum size was determined by colony count after diluting and plating 100 µL on CA plates and incubating at 33°C with 5% CO2 and high humidity.
Determination of lesion severity. Two of three lesions were measured and scored daily, for a period of 21 days for each inoculum size on each rabbit (0 = nil, 1 = erythema, 2 = induration, 3 = suppuration, 4 = ulceration). The third lesion was cultured for the presence of H. ducreyi by lateral injection of 0.1 mL of PBS at pH 7.2, manipulation, and aspiration. The aspirate was cultured on CA plates for 48 h at 33°C and examined for evidence of typical H. ducreyi colony morphology by the push test, Gram stain, and microscopic examination. Culture was discontinued after four consecutive days of negative culture results.
Determination of sub-therapeutic antibiotic dose. Preliminary experiments using single-dose, single-drug treatment with 0.055 mg/kg of ceftriaxone and 2.515 mg/kg of streptomycin established that 0.05 mg/kg of ceftriaxone and 10 mg/kg of streptomycin were the highest doses which had no effect on lesion healing relative to controls without antibiotic treatment in the temperature-dependent rabbit model of chancroid.
Efficacy of antimicrobial treatment. H. ducreyi 35000 was used to examine in vivo antibiotic efficacy. Inocula of 104 cfu were injected into the shaved backs of the rabbits in a 15-location grid pattern. Rabbits were treated with antibiotics upon ulceration of the lesions on day 4 post-infection. Single sub-therapeutic doses of 0.05 mg/kg of ceftriaxone (n = 4), 10 mg/kg of streptomycin (n = 4), and a combination of both antibiotics (n = 5) were administered intramuscularly. Control rabbits (n = 3) received no treatment. Odd numbered lesions (8/rabbit) were measured and scored daily for 17 days post-treatment. Even numbered lesions were cultured until negative for four consecutive days.
Statistical analysis
Analysis of variance (ANOVA) was used to compare the FICI between strains and the StudentNewmanKeuls method was used where ANOVA showed a significant difference. The KruskalWallis ANOVA on ranks was used in instances where normality failed. Comparative analysis of in vivo efficacy of treatment on ulcerative lesions was done on data from treatment day 0 (on the fourth day from infection) to day 17. Serial lesion sizes were expressed as a percentage of pre-treatment lesion size, so that all lesions were 100% on day 0. For each rabbit, for each of 17 treatment days, the median lesion size percentage and median lesion score were calculated from eight of the 15 inoculation sites. The mean time in days for a 50% decrease in median lesion size, and the mean time in days to reach a median score of 2 were compared between groups. For each rabbit, the median duration of lesion culture positivity was calculated. Mean time in days to culture positivity was compared between groups. For each rabbit, the median inoculum size was calculated, and the means of the groups were compared. Comparisons were carried out by ANOVA, followed by the Tukey post-hoc test where appropriate. Statistical analysis was performed using SigmaStat software (SPSS Science, Chicago, IL, USA).
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Results |
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MIC determination. MICs for H. ducreyi strains 35000, RO 40 and RO 34 show that they were susceptible to the four antibiotics tested, and the MICs for the reference strains E. coli ATCC 25922 and S. aureus ATCC 29213 were all within one dilution of the accepted ranges (Table 1).
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Experimental induction of infection. The lowest inoculum size of H. ducreyi strain 35000 to consistently produce ulcerative lesions in this model is 104 cfu.39,40 Rabbits were inoculated with 104 cfu of H. ducreyi strain 35000 (100 µL of 105 cfu/mL) in a pattern of 5 rows of 3 columns. The actual input inoculum size was measured by limiting dilution of residual inocula immediately after injection (Table 3). There was no statistically significant difference in the inoculum size for the different treatments (P = 0.4, ANOVA).
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Efficacy of antimicrobial treatment. To evaluate interaction, once the lesions had ulcerated on day 4 after infection, rabbits were either not treated (n = 3) or treated with intramuscular injections of 0.05 mg/kg of ceftriaxone (n = 4), 10 mg/kg of streptomycin (n = 4), or both (n = 5). Results are shown in Figure 1 and Table 3. Rabbits receiving a single 0.05 mg/kg dose of ceftriaxone or a single 10 mg/kg dose of streptomycin did not differ significantly from the untreated animals with respect to mean (SD) duration of culture positivity in days [7.0 (2.7) and 7.5 (2.1), versus 7.3 (1.1), P > 0.8]; mean (SD) time in days to a 50% decrease from pre-treatment lesion size [8.2 (1.7) and 8.5 (0.6) versus 9.7 (1.5), P > 0.2]; and mean (SD) time in days to reach a lesion score of 2 from a pre-treatment score of 4 [9.7 (2.7) and 9.4 (1.7) versus 11.7 (2.3), P > 0.2]. However, simultaneous treatment with the same concentrations of both antibiotics resulted in abatement of disease. Table 3 shows that the mean (SD) duration of culture positivity was 2.6 (1.7) days in the treated animals versus 7.3 (1.1) days in the untreated controls (P < 0.001), mean time to 50% reduction in lesion size was reduced from 9.7 (1.5) days to 5.8 (0.8) days (P < 0.005), and the mean time to reduction in lesion score from 4 to 2 was reduced from 11.7 (2.3) days in control animals to 6.6 (1.7) days (P < 0.05) with treatment.
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Discussion |
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This is the first time positive antibiotic interactions against H. ducreyi have been reported. This in vivo synergy is not unexpected, as there is evidence of broad synergic interactions with combination ß-lactam and aminoglycoside antibiotics, both in vitro43,44 and in vivo.45,46 The theoretical basis for this interaction is the increased uptake of the aminoglycoside in the presence of the cell-wall-active ß-lactam.47 Streptomycin and ceftriaxone have been used separately in the treatment of chancroid and are affordable. Ceftriaxone is well tolerated and safe, with occasional allergic reactions. Streptomycin is associated with ototoxicity and nephrotoxicity when used at high doses for extended periods of time.48,49
Although commonly used to determine in vitro antibiotic interactions, the chequerboard method has several limitations, such as reliance on a single time point, assumption of a linear doseresponse for all antimicrobials, and an all-or-nothing read out,38 and has been described as inherently unstable as a predictor of synergy because of its reliance on 2-fold dilutions.50 Timekill methods can address some of the shortcomings of the chequerboard method of susceptibility testing.51 It is recognized in the field of antibiotic testing that special measures are needed in dealing with fastidious organisms,52 but no standardized culture procedure exists for this fastidious organism, and there was no precedent for the evaluation of antimicrobial activity on H. ducreyi in liquid media. We opted for the chequerboard agar dilution technique, which has been used with H. ducreyi in MIC determination in the past.36,37
We chose streptomycin, an aminoglycoside, ceftriaxone, a cephalosporin, and azithromycin, an azalide, as they have all been used to treat chancroid. We chose rifabutin, a semi-synthetic ansamycin, as it may be appropriate for single-dose therapy because of high in vitro susceptibility of H. ducreyi, high tissue binding, and long elimination half-life. By evaluating sub-therapeutic doses of antibiotics, we were able to properly evaluate synergic, indifferent, or additive effects, and not antagonistic effects, of the selected antibiotic combinations. We did not test potentially additive or synergic rifabutin antibiotic combinations in vivo, as they lacked consistent in vitro activity. As with other organisms and antibiotics, however, in vitro activity may not always reflect in vivo efficacy, especially when the chequerboard agar dilution method is used to determine FICI,41 and other two-drug combinations including fluoroquinolones may well deserve consideration.
It may be reasonable to predict similar interactions of other aminoglycosides and cephalosporins against H. ducreyi, however, several authors have cautioned that interaction between antibiotics is dependent upon the antibiotics used, the organism being tested, and, to some extent, the medium or the host.53,54 This study demonstrates the in vivo therapeutic synergy of streptomycin and ceftriaxone against H. ducreyi in an animal model of infection and disease. Antibiotic combinations need clinical evaluation as chancroid treatment, especially where consistently efficacious treatment is lacking or improved single-dose observed therapy is desirable.
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Acknowledgements |
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References |
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2. Cameron DW, Heath-Chiozzi M, Danner S et al. Randomised placebo-controlled trial of ritonavir in advanced HIV-1 disease. The Advanced HIV Disease Ritonavir Study Group. Lancet 1998; 351: 5439.[CrossRef][ISI][Medline]
3. Plourde PJ, Plummer FA, Pepin J et al. Human immunodeficiency virus type 1 infection in women attending a sexually transmitted diseases clinic in Kenya. J Infect Dis 1992; 166: 8692.[ISI][Medline]
4.
Telzak EE, Chiasson MA, Bevier PJ et al. HIV-1 seroconversion in patients with and without genital ulcer disease. A prospective study. Ann Intern Med 1993; 119: 11816.
5. Blackmore CA, Limpakarnjanarat K, Rigau-Perez JG et al. An outbreak of chancroid in Orange County, California: descriptive epidemiology and disease-control measures. J Infect Dis 1985; 151: 8404.[ISI][Medline]
6. Jessamine PG, Brunham RC. Rapid control of a chancroid outbreak: implications for Canada. Can Med Assoc J 1990; 142: 10815.[Abstract]
7. Neumann RA, Knobler RM, Aberer E et al. Incidence of chancroid in Vienna from 1980 to 1988. Int J Dermatol 1989; 28: 3936.[ISI][Medline]
8. Ronald AR, Plummer FA. Chancroid and granuloma inguinale. Clin Lab Med 1989; 9: 53543.[ISI][Medline]
9. Limpakarnjanarat K, Mastro TD, Saisorn S et al. HIV-1 and other sexually transmitted infections in a cohort of female sex workers in Chiang Rai, Thailand. Sex Transm Infect 1999; 75: 305.[Abstract]
10. Wellington M, Ndowa F, Mbengeranwa L. Risk factors for sexually transmitted disease in Harare: a casecontrol study. Sex Transm Dis 1997; 24: 52832.[ISI][Medline]
11. Martin DH, DiCarlo RP. Recent changes in the epidemiology of genital ulcer disease in the United States. The crack cocaine connection. Sex Transm Dis 1994; 21: S7680.[ISI][Medline]
12. Marx R, Aral SO, Rolfs RT et al. Crack, sex, and STD. Sex Transm Dis 1991; 18: 92101.[ISI][Medline]
13. Grosskurth H, Mosha F, Todd J et al. Impact of improved treatment of sexually transmitted diseases on HIV infection in rural Tanzania: randomised controlled trial. Lancet 1995; 346: 5306.[CrossRef][ISI][Medline]
14. Wawer MJ, Sewankambo NK, Serwadda D et al. Control of sexually transmitted diseases for AIDS prevention in Uganda: a randomised community trial. Rakai Project Study Group. Lancet 1999; 353: 52535.[CrossRef][ISI][Medline]
15. Abeck D, Johnson AP, Dangor Y et al. Antibiotic susceptibilities and plasmid profiles of Haemophilus ducreyi isolates from southern Africa. J Antimicrob Chemother 1988; 22: 43744.[Abstract]
16. Knapp JS, Back AF, Babst AF et al. In vitro susceptibilities of isolates of Haemophilus ducreyi from Thailand and the United States to currently recommended and newer agents for treatment of chancroid. Antimicrob Agents Chemother 1993; 37: 15525.[Abstract]
17. McNicol PJ, Ronald AR. The plasmids of Haemophilus ducreyi. J Antimicrob Chemother 1984; 14: 5614.[ISI][Medline]
18. Willson PJ, Albritton WL, Slaney L et al. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi. Antimicrob Agents Chemother 1989; 33: 162730.[ISI][Medline]
19. Erbelding E, Quinn TC. The impact of antimicrobial resistance on the treatment of sexually transmitted diseases. Infect Dis Clin North Am 1997; 11: 889903.[CrossRef][ISI][Medline]
20. Pechere JC. Parameters important in short antibiotic courses. J Int Med Res 2000; 28: 3A12A.[ISI][Medline]
21. Morse SA. Chancroid and Haemophilus ducreyi. Clin Microbiol Rev 1989; 2: 13757.[ISI][Medline]
22. Prachayasittikul V, Lawung R, Bulow L. Episome profiles and mobilizable ß-lactamase plasmid in Haemophilus ducreyi. Southeast Asian J Trop Med Public Health 2000; 31: 804.[Medline]
23. Behets FM, Liomba G, Lule G et al. Sexually transmitted diseases and human immunodeficiency virus control in Malawi: a field study of genital ulcer disease. J Infect Dis 1995; 171: 4515.[ISI][Medline]
24. MacDonald KS, Cameron DW, D'Costa L et al. Evaluation of fleroxacin (RO 236240) as single-oral-dose therapy of culture-proven chancroid in Nairobi, Kenya. Antimicrob Agents Chemother 1989; 33: 6124.[ISI][Medline]
25. Tyndall MW, Agoki E, Plummer FA et al. Single dose azithromycin for the treatment of chancroid: a randomized comparison with erythromycin. Sex Transm Dis 1994; 21: 2314.[ISI][Medline]
26. Tyndall M, Malisa M, Plummer FA et al. Ceftriaxone no longer predictably cures chancroid in Kenya. J Infect Dis 1993; 167: 46971.[ISI][Medline]
27. Ballard RC, Ye H, Matta A et al. Treatment of chancroid with azithromycin. Int J STD AIDS 1996; 7: 912.
28. Malonza IM, Tyndall MW, Ndinya-Achola JO et al. A randomized, double-blind, placebo-controlled trial of single-dose ciprofloxacin versus erythromycin for the treatment of chancroid in Nairobi, Kenya. J Infect Dis 1999; 180: 188693.[CrossRef][ISI][Medline]
29.
Cope LD, Lumbley S, Latimer JL et al. A diffusible cytotoxin of Haemophilus ducreyi. Proc Natl Acad Sci USA 1997; 94: 405661.
30. Brunton JL, Clare D, Ehrman N et al. Evolution of antibiotic resistance plasmids in Neisseria gonorrhoeae and Haemophilus species. Clin Invest Med 1983; 6: 2218.[ISI][Medline]
31.
Bonhoeffer S, Lipsitch M, Levin BR. Evaluating treatment protocols to prevent antibiotic resistance. Proc Natl Acad Sci USA 1997; 94: 1210611.
32. Bouza E, Munoz, P. Monotherapy versus combination therapy for bacterial infections. Med Clin North Am 2000; 84: 13571389, v.[CrossRef][ISI][Medline]
33. Purcell BK, Richardson JA, Radolf JD et al. A temperature-dependent rabbit model for production of dermal lesions by Haemophilus ducreyi. J Infect Dis 1991; 164: 35967.[ISI][Medline]
34. Hammond GW, Lian CJ, Wilt JC et al. Comparison of specimen collection and laboratory techniques for isolation of Haemophilus ducreyi. J Clin Microbiol 1978; 7: 3943.[ISI][Medline]
35. Desjardins M, Filion LG, Robertson S et al. Inducible immunity with a pilus preparation booster vaccination in an animal model of Haemophilus ducreyi infection and disease. Infect Immun 1995; 63: 201220.[Abstract]
36. Hammond GW, Lian CJ, Wilt JC et al. Antimicrobial susceptibility of Haemophilus ducreyi. Antimicrob Agents Chemother 1978; 13: 60812.[ISI][Medline]
37. Slaney L, Chubb H, Ronald A et al. In-vitro activity of azithromycin, erythromycin, ciprofloxacin and norfloxacin against Neisseria gonorrhoeae, Haemophilus ducreyi, and Chlamydia trachomatis. J Antimicrob Chemother 1990; 25 Suppl A: 15.[ISI][Medline]
38. Eliopoulos GM, Moellering RC. Antimicrobial combinations. In: Lorian V, ed. Antibiotics in Laboratory Medicine, 4th edn. Baltimore, MD: Williams and Wilkins, 1996; 33096.
39. Desjardins M, Filion LG, Robertson S et al. Evaluation of humoral and cell-mediated inducible immunity to Haemophilus ducreyi in an animal model of chancroid. Infect Immun 1996; 64: 177888.[Abstract]
40.
Thomas KL, Leduc I, Olsen B et al. Cloning, overexpression, purification, and immunobiology of an 85-kilodalton outer membrane protein from Haemophilus ducreyi. Infect Immun 2001; 69: 443846.
41. Cleeland R, Squires E. Evaluation of new antimicrobials in vitro and in experimental animal infections. In: Lorian V, ed. Antibiotics in Laboratory Medicine, 3rd edn. Baltimore, MD: Williams and Wilkins, 1991; 73986.
42. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 2003; 52: 1.[CrossRef][ISI][Medline]
43. Joly-Guillou ML, Decre D, Herrman JL et al. Bactericidal in-vitro activity of ß-lactams and ß-lactamase inhibitors, alone or associated, against clinical strains of Acinetobacter baumannii: effect of combination with aminoglycosides. J Antimicrob Chemother 1995; 36: 61929.[Abstract]
44. Schlegel L, Sissia G, Fremaux A et al. In-vitro killing activity of combinations of ß-lactam agents with aminoglycosides against penicillin-resistant pneumococci. J Antimicrob Chemother 1997; 39: 958.[Abstract]
45. Clark RB, Pakiz CB, Hostetter MK. Synergistic activity of aminoglycosideß-lactam combinations against Pseudomonas aeruginosa with an unusual aminoglycoside antibiogram. Med Microbiol Immunol (Berl) 1990; 179: 7786.[ISI][Medline]
46. Dodge RA, Daly JS, Davaro R et al. High-dose ampicillin plus streptomycin for treatment of a patient with severe infection due to multiresistant enterococci. Clin Infect Dis 1997; 25: 126970.[ISI][Medline]
47. Moellering RC Jr, Weinberg AN. Studies on antibiotic synergism against enterococci. II. Effect of various antibiotics on the uptake of 14C-labeled streptomycin by enterococci. J Clin Invest 1971; 50: 25804.[ISI][Medline]
48. Bagger-Sjoback D. Effect of streptomycin and gentamicin on the inner ear. Ann NY Acad Sci 1997; 830: 1209.[ISI][Medline]
49. Wersall J. Ototoxic antibiotics: a review. Acta Otolaryngol Suppl 1995; 519: 269.[Medline]
50. Hsieh MH, Yu CM, Yu VL et al. Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infect Dis 1993; 16: 3439.[CrossRef][ISI][Medline]
51. Stratton C. In vitro testing: correlation between bacterial susceptibility, body fluid levels and effectiveness of antibacterial therapy. In: Lorian V, ed. Antibiotics in Laboratory Medicine, 3rd edn. Baltimore, MD: Williams and Wilkins, 1991; 84979.
52. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: special needs for fastidious organisms and difficult-to-detect resistance mechanisms. Clin Infect Dis 2000; 30: 799808.[CrossRef][ISI][Medline]
53. Acar JF. Antibiotic synergy and antagonism. Med Clin North Am 2000; 84: 1391406.[CrossRef][ISI][Medline]
54. Hessen MT, Kaye D. Principles of selection and use of antibacterial agents. In vitro activity and pharmacology. Infect Dis Clin North Am 2000; 14: 26579, vii.[CrossRef][ISI][Medline]
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