Infectious Diseases Research, Abbott Laboratories, Abbott Park, IL 60064-3537, USA
Received 10 September 2001; returned 13 November 2001; revised 16 January 2002; accepted 31 January 2002.
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Abstract |
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Introduction |
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In this study, the frequency at which ABT-773, erythromycin and rifampicin selected for mutants with reduced susceptibility in vitro was assessed using 12 respiratory tract pathogens. Susceptibilities of the mutants to ABT-773, erythromycin, clindamycin and ciprofloxacin were then evaluated.
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Materials and methods |
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ABT-773 was prepared at Abbott Laboratories (Abbott Park, IL, USA). Erythromycin, clindamycin, rifampicin and ciprofloxacin were purchased from the Sigma Chemical Company (St Louis, MO, USA). MuellerHinton agar (MHA) and cation-adjusted MuellerHinton broth (MHB) were pur-chased from Becton Dickinson and Co. (Cockeysville, MD, USA). Haemophilus Test agar was purchased from Accumedia Manufacturers, Inc. (Baltimore, MD, USA).
Bacterial strains
Strains were from the Abbott Laboratories collection or the American Type Culture Collection (Manassas, VA, USA). mef(A) efflux and erm methylase genes were identified by PCR.3
Selection of mutants
MICs for the parent strains were determined by agar dilution as described by the NCCLS4 in MHA for Staphylococcus aureus, MHA containing 5% sheep blood for streptococci or Haemophilus Test agar for H. influenzae. Plates were inoculated with 104 cfu per spot, and incubated at 36°C in ambient air for 1620 h for S. aureus or in an atmosphere containing 5% CO2 for 2024 h for streptococci and H. influenzae.
Parent strains of S. aureus were grown overnight in MHB at 36°C in ambient air. The cultures were concentrated by centrifugation at 600g for 15 min and the cell pellet was resuspended in broth at 1/10 the original volume. Parent strains were grown overnight at 36°C in an atmosphere containing 5% CO2 on three plates of MHA containing 5% sheep blood for the streptococci or Haemophilus Test agar for H. influenzae. Colonies were harvested and resuspended in MHB containing 3% lysed horse blood for the streptococci or MHB containing 15 µg/L haematin, 5 g/L yeast extract and 15 mg/L NAD for H. influenzae. The number of cfu per mL in the inocula was determined on drug-free agar media following overnight incubation. Concurrently, 10 plates of agar media containing ABT-773, erythromycin or rifampicin were inoculated with 0.1 mL of the concentrated suspension of staphylococci or H. influenzae, whereas 20 plates were used for selections from S. pneumoniae by ABT-773. Selection plates were incubated at 36°C for up to 72 h in ambient air for S. aureus or in an atmosphere containing 5% CO2 for streptococci and H. influenzae, and the numbers of cfu per mL were determined. The selection frequency was calcu- lated as the number of cfu per mL on antibiotic-containing medium divided by the number of cfu per mL in the original inoculum. Up to 10 colonies per selecting condition for experiments with ABT-773 and erythromycin or two colon-ies per selecting condition for experiments with rifampicin were subcultured on to the same drug selection agar and were then maintained on drug-free medium.
Determination of antibacterial activity
Susceptibilities for parent strains and mutants were determined by the broth microdilution method.4 Quality control met NCCLS standards.4
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Results and discussion |
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Mutants with reduced susceptibility to ABT-773 and erythromycin were selected on agar media containing drug concentrations 416 x MIC from 12 strains of respiratory tract pathogens using inocula ranging from 109 to 1012 cfu. Additionally, selection was carried out with ABT-773 and rifampicin at 2 x MIC (Table 1). Six strains were erythromycin susceptible and six were erythromycin resistant [inducible Erm(A) in S. aureus and Streptococcus pyogenes, Erm(B) in S. pneumoniae and S. pyogenes, and Mef(A) in S. pneumoniae and S. pyogenes]. All strains were rifampicin susceptible.
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S. pneumoniae mutants were selected at 72 h by ABT-773 from the erythromycin-susceptible, Mef(A) and Erm(B) strains at frequencies of 1010 to 1012 at 24 x MIC, and by erythromycin from the Mef(A) strain at a frequency of 108 at 416 x MIC.
S. pyogenes mutants were selected at 72 h by ABT-773 at a frequency of 1010 from the Mef(A) strain at 2 x MIC and from the Erm(B) strain at 2 x and 4 x MIC. Mutants were selected from the inducible Erm(A) strain at high frequency by erythromycin at 416 x MIC.
H. influenzae mutants were not selected by ABT-773 or erythromycin.
Mutants were selected more frequently by rifampicin than by ABT-773 or erythromycin for 12/12 or 8/12 strains, respectively. Moreover, rifampicin mutants were selected as frequently on 16 x as on 2 x MIC for 11/12 strains.
Phenotypes of mutants derived by selection
Most of the ABT-773 and erythromycin mutants demonstrated increased MICs of both compounds, and some also demonstrated changes in clindamycin MICs (Table 2). More than one profile, defined as diverging effects on ABT-773, erythromycin and clindamycin susceptibility, was found in mutants derived from seven strains. In contrast, parent strains and mutants had identical ciprofloxacin MICs (data not shown). Rifampicin MICs increased from 0.12 to >32 mg/L with unchanged ABT-773 and ciprofloxacin MICs in mutants selected by rifampicin, consistent with mutations in DNA-dependent RNA polymerase (data not shown).5
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Two mutants with ABT-773 MICs of 0.25 or 2 mg/L were selected from the erythromycin-susceptible strain of S. pneumoniae by ABT-773; they had increased erythromycin MICs and decreased clindamycin MICs. Four phenotypes selected from Mef(A) S. pneumoniae by ABT-773 or erythromycin were distinguished by susceptibility to ABT-773 and clindamycin, although erythromycin MICs were similar. ABT-773 MICs were elevated to either 0.03 or 0.5 mg/L for two mutants selected from Erm(B) S. pneumoniae by ABT-773.
Resistance was not stable after drug-free passage of mutants selected from the inducible Erm(A) S. pyogenes by erythromycin. Two phenotypes were selected from Mef(A) S. pyogenes by ABT-773 in which the ABT-773 and erythromycin MICs increased to similar levels although clindamycin MICs were different. One phenotype was selected from the Erm(B) strain of S. pyogenes by ABT-773, which increased the ABT-773 MIC two- to four-fold.
Erythromycin, telithromycin and clindamycin share overlapping binding sites near the peptidyl transferase centre of the ribosome.6,7 Mutations at A2058, A2059 and C2611 in domain V of 23S rRNA and in ribosomal protein L4 selected from S. pneumoniae with azithromycin in vitro,8 at U754 in domain II of 23S rRNA selected from a library of Escherichia coli strains by telithromycin in vitro,7 and at A2059 of the 23S rRNA and in ribosomal protein L4 in clinical isolates of S. pneumoniae9 result in cross-resistance to macrolides, clindamycin and telithromycin. The patterns of reduced susceptibility to ABT-773, erythromycin and clindamycin indicate that the mutations selected in vitro by ABT-773 and erythromycin may reside in genes associated with the macrolide-binding site. Moreover, ABT-773, erythromycin and clindamycin may interact differently with the ribosome since mutant profiles were identified that showed divergent effects on the relative loss of susceptibility to each compound. In addition, mutants derived from the Erm strains may have altered ribosome methylation since ketolide susceptibility in Erm(B) S. pneumoniae is proportional to the level of ribosome methylation,10 and non-inducing macrolides select for constitutive expression of inducible erm genes, resulting in high-level cross-resistance to macrolides and clindamycin.6
In conclusion, ABT-773 at concentrations above the MIC did not select for mutants with reduced susceptibility from four strains of respiratory tract pathogens, and selected mutants very infrequently from eight other strains. Moreover, the frequency of selection of mutants from erythromycin-resistant and -susceptible streptococci was similar. Except for mutants selected from inducible Erm(A) S. aureus, ABT-773 MICs for mutants ranged from 0.015 to 4 mg/L. Selection on ABT-773 or erythromycin resulted in mutants that had reduced susceptibility to ABT-773, erythro-mycin and, in some cases, clindamycin. Since mutants demonstrated more than one susceptibility profile, different mutations may be involved. Studies to identify the molecular mechanisms of reduced susceptibility in these mutants are under way.
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Footnotes |
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References |
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2
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