Antimicrobial Agents and Chemotherapy, November 2004, p . 4113-4119, Vol . 48, No . 11

The major resistance mechanism in H . influenzae in the United States and Europe is the production of ß-lactamases (TEM-1 and ROB-1) . A study performed in the United States in 1997 (10) documented the incidence of ß-lactamase production among 1,676 H . influenzae strains isolated throughout the United States to be 41.6% . The incidence of ß-lactamase-negative ampicillin-resistant (BLNAR) strains in the United States is <1% . Of the ß-lactams available for the treatment of infections caused by this organism, cefixime and cefpodoxime are the most active in terms of their MICs as well as their pharmacokinetics and pharmacodynamics, followed by amoxicillin-clavulanate and cefuroxime (4) . Other oral cephalosporins, such as cefprozil, cefaclor, and loracarbef, are less active against these organisms (5, 20) . Among the macrolides and azalides, azithromycin has the lowest MIC for H . influenzae, followed by erythromycin and clarithromycin (4, 7, 11, 14) . However, the pharmacokinetic and pharmacodynamic properties of these compounds cast doubt on their clinical efficacies against H . influenzae (8) .

GW 773546 and GW 708408 (Fig . 1) are two novel 14-membered macrolides from the clarithromycin scaffold . The study described here examined the activities of these new compounds against H . influenzae in vitro compared with those of telithromycin (the first commercially available ketolide), erythromycin, azithromycin, and clarithromycin by (i) microdilution testing of the MICs for 223 strains, (ii) time-kill testing of 9 strains, (iii) single-step and multistep resistance selection studies with 11 strains, and (iv) determination of the postantibiotic effects (PAEs) for 6 strains . Pharmacokinetic and pharmacodynamic studies were not performed in the present study .

 
Bacteria and antimicrobials. The MICs of the drugs tested for 223 strains were determined by the microdilution method . The strains were isolated from 1999 to 2002 and comprised 89 ß-lactamase-positive strains, 115 ß-lactamase-negative strains, and 19 BLNAR strains . An attempt was made to include as many genotypes related to ß-lactam susceptibility as possible . All strains were from clinical specimens, predominantly sputum, bronchial aspirates, blood, and cerebrospinal fluid . Isolates from the last two types of specimens were from countries in which the H . influenzae type b vaccine is not used . Testing for ß-lactamase production was performed by the nitrocefin disk method (Cefinase; BBL Microbiology Systems, Inc., Cockeysville, Md.) . Three ß-lactamase-positive strains, three ß-lactamase-negative strains, and three BLNAR strains were tested by the time-kill method . Five ß-lactamase-negative strains, five ß-lactamase-positive strains, and one BLNAR strain were tested in the resistance selection studies; and three ß-lactamase-negative strains, three ß-lactamase-positive strains, and one BLNAR organism were included in the PAE studies . The strains were stored at –70°C in double-strength skim milk (Difco Laboratories, Detroit, Mich.) before they were tested and were examined for purity throughout the study by culture and Gram staining . GW 773546, GW 708408, and telithromycin were obtained from GlaxoSmithKline Laboratories, Collegeville, Pa . Telithromycin was synthesized by GlaxoSmithKline Laboratories and was not obtained from Aventis Pharma . Other compounds were obtained from their respective manufacturers . The stock of cefixime powder used in the present study was obtained from Wyeth Lederle Laboratories, Pearl River, N.Y . Although the production of cefixime has recently been discontinued by Wyeth Lederle Laboratories, the Food and Drug Administration has recently approved its production and distribution through Lupine Laboratories .

MIC determinations. MICs were determined by the NCCLS microdilution method (12) with freshly prepared Haemophilus test medium in commercially prepared frozen trays (TREK, Inc., Westlake, Ohio) (3) . Inocula were prepared from chocolate agar plates incubated for 18 h by the direct colony suspension method . The final concentration in each well was approximately 5 x 10⁵ CFU/ml . The standard quality control strains H . influenzae ATCC 49766, H . influenzae ATCC 49247, and H . influenzae ATCC 10211 were used as controls . Inoculum checks were done, and only suspensions yielding 3 x 10⁵ to 7 x 10⁵ CFU/ml were used . The trays were covered and incubated at 35°C in air .

Time-kill studies. Time-kill studies were done as described previously (3) . Glass tubes containing 5 ml of freshly made Haemophilus test medium with doubling antibiotic concentrations were inoculated with approximately 5 x 10⁵ CFU of each organism/ml and were incubated at 35°C in a shaking water bath . Viability counts for the antibiotic-containing suspensions were performed at 0, 3, 6, 12, and 24 h by plating 10-fold dilutions of 0.1-ml aliquots of sterile Haemophilus test medium from each tube onto chocolate agar plates . The recovery plates were incubated for up to 48 h . Colony counts were performed for plates that yielded from 30 to 300 colonies . (3) . The lower limit of sensitivity of the colony counting method was 300 CFU/ml . The results were analyzed by determining the number of strains which yielded changes in the log₁₀ number of CFU per milliliter of –1, –2, and –3 at 3, 6, 12, and 24 h compared with the counts at 0 h . The lowest concentration of the antimicrobials that reduced the original inoculum by 3 log₁₀ CFU/ml (99.9%) at each of the time periods was considered bactericidal, and the lowest concentration that reduced the original inoculum by <3 log₁₀ CFU/ml was considered bacteriostatic .

Multistep selection studies. The multistep selection method was described previously (1) . Briefly, serial passages in freshly prepared Haemophilus test medium were performed daily with each strain in the presence of subinhibitory concentrations of each antimicrobial . For each subsequent daily passage, an inoculum was taken from the tube with the concentration nearest the MIC (usually 1 to 2 dilutions lower) which had the same turbidity as that of the antibiotic-free control . The latter inoculum was used to determine the next MIC . Daily passages were performed until a significant increase (more than fourfold) was obtained . A minimum of 14 passages were performed in each case unless MICs >64.0 µg/ml were detected, in which case subculturing in the presence of antibiotic ceased . The maximal number of passages was 50 . The stability of the acquired resistance was determined after 10 daily passages of the clone on chocolate agar (BBL) without antibiotics . The resistance mechanisms of the resistant clones and the parent strains were determined as described below .

Mechanism of macrolide resistance. The strains were examined for the presence of mutations in the L4 and L22 proteins and 23S rRNA by using the primers and conditions described previously (1) . After PCR amplification, the products were purified with a QIAquick PCR purification kit (Qiagen, Valencia, Calif.) . Nucleotide sequences were obtained by direct sequencing with a CEQ8000 genetic analysis system (Beckman Coulter, Fullerton, Calif.) .

Single-step selection studies. The frequency of spontaneous single-step mutations was determined by spreading approximately 10¹⁰ CFU/ml in 100-µl aliquots on Haemophilus test medium plates containing each compound at the MIC and at two, four, and eight times the MIC . After incubation at 35°C in 5% CO₂ for 48 to 72 h, the presence of resistant colonies was confirmed by replica plating on medium with antibiotics . The resistance frequency was calculated as the number of resistant colonies per inoculum . The resistance mechanisms of eight randomly chosen mutants from the single-step selection procedure were determined as described above .

MICs for resistant clones. The MICs of each drug for each resistant clone were determined by the standard microdilution method as described above .

Confirmation of strain identities. All strains were tested by pulsed-field gel electrophoresis before the initiation of resistance selection (1) . At the end of the study, all strains tested were again tested by pulsed-field gel electrophoresis analysis to see whether a stepwise change in the electrophoretic profile had taken place .

PAEs. The PAEs were determined by the viable plate count method (3) with freshly prepared Haemophilus test medium . The PAE was induced by exposure to each of the compounds at 10 times the MIC for 1 h . For PAE testing, tubes containing 5 ml of broth with antibiotic were inoculated with approximately 5 x 10⁶ CFU/ml . Inocula were prepared by suspending the growth from an overnight chocolate agar plate in broth . Growth controls with an inoculum but no antibiotic were included in each experiment . The inoculated test tubes were placed in a shaking water bath at 35°C for an exposure period of 1 h . At the end of the exposure period, the cultures were diluted 1:1,000 in prewarmed broth to remove the antibiotic by dilution . Antibiotic removal was confirmed by comparing the growth curve of a control culture containing no antibiotic with the growth curve of another culture containing the antibiotic at 0.01 the exposure concentration . Viability counts were determined before exposure, immediately after dilution (0 h), and then every 2 h until the turbidity of the tube reached that of a no . 1 McFarland standard . The PAE was defined as T – C, where T is the time required for the viability counts of an antibiotic-exposed culture to increase 1 log₁₀ above the counts immediately after dilution and C is the corresponding time for the growth control (2, 6) .

 
The MICs for the strains determined by the time-kill method are presented in Table 2, and the results of the time-kill analyses are presented in Table 3 . The MICs were unimodal and, as described above, were not influenced by the strains' ß-lactam susceptibilities . The killing kinetics of each drug were compatible with the MIC . All compounds except telithromycin were bactericidal (99.9% killing) against nine strains at two times the MIC after 24 h . Telithromycin was bactericidal against eight of the nine strains . In addition, at two times the MIC both novel macrolides and telithromycin showed 99% killing of all nine strains after 12 h and 90% killing of all strains after 6 h . After 24 h, clarithromycin at the MIC was bactericidal against four strains; all other compounds were bactericidal against six to seven strains .

 
Ten of 11 strains tested by multistep selection analysis yielded resistant clones after 14 to 43 passages in the presence of erythromycin . Resistant clones of all strains were obtained after 20 to 50 passages in the presence of azithromycin, and resistant clones of 9 of 11 strains were obtained after 14 to 41 passages in the presence of clarithromycin . By comparison, resistant clones of 9 of 11 strains were obtained after 14 to 44 passages in the presence of GW 708408, and resistant clones of 9 of 11 strains were obtained after 14 to 45 passages in the presence of GW 773546 . Resistant clones of 7 of 11 strains were obtained after 18 to 45 passages in the presence of telithromycin . All drugs selected for resistance in 7 of the 11 strains . Among the four strains for which not all drugs selected for resistance, in one strain resistance was selected for only with erythromycin and azithromycin, in another strain resistance was selected for with azithromycin and GW 773546, and in two strains resistance was selected for with all compounds except telithromycin . In most strains in which resistance was selected for by one compound, the MICs of the other compounds tested were increased for the clones selected . All drugs selected for a total of 56 resistant clones, and 30 strains had amino acid alterations in the L4 and L22 proteins or changes in 23S rRNA sequences . Among these strains, 14 had alterations in the L22 protein, 9 had changes in the L4 protein, 5 had altered L22 and L4 proteins, and 2 had substitutions in 23S rRNA . Moreover, most deletions and insertions were found in the L22 protein, while amino acid substitutions predominated in the L4 protein (Table 4) . Two clones selected with azithromycin possessed mutations in 23S rRNA (A2058G and A2059C) .

 
Single-step resistance selection studies at the MIC yielded spontaneous resistant mutants at frequencies of 1.5 x 10^–9 to 2.2 x 10^–6 for GW 773546, 1.5 x 10^–9 to 6.0 x 10^–4 for GW 708408, and 7.1 x 10^–9 to 3.8 x 10^–4 for telithromycin, whereas the frequencies were 1.3 x 10^–9 to 6.0 x 10^–4 for erythromycin and azithromycin and 2.0 x 10^–9 to 2.0 x 10^–3 for clarithromycin (Table 5) . Testing of eight mutants for their mechanisms of resistance showed that mutations in the L22 protein predominated . Six of eight strains tested had insertions or deletions within the region between amino acid positions 80 and 94 . By comparison, insertions 88AKA89 and 88AMPRAK89 were present in mutants selected by the multistep selection procedure . However, deletions 88AK, 80SM, and 94RIL were detected only in the single-step selection analysis . Changes in L4 protein amino acid sequences (E154D and S79A) were also present in mutants selected by the multistep procedure (Table 4) . No changes in the 23S rRNA sequence were detected in the eight strains analyzed .

 
The PAE results for seven strains are presented in Table 6 . GW 773546 and telithromycin produced the longest mean PAEs, followed by azithromycin, GW 708408, clarithromycin, and erythromycin . The PAEs of none of the drugs were influenced by ampicillin susceptibility or ß-lactamase production .

 
In our study, the MICs of both novel macrolides and telithromycin were between 0.03 and 4.0 µg/ml and similar to those of azithromycin reported previously (4, 11) . The killing kinetics of the drugs were also similar and were compatible with their MICs . The results are comparable to those previously reported for telithromycin, erythromycin, azithromycin, and clarithromycin (9, 16) . As in previous studies, in general, telithromycin was found to select for resistant mutants at rates lower than those for erythromycin, azithromycin, and clarithromycin (9, 16) .

Macrolide resistance mechanisms have been described for H . influenzae strains (1, 17), and mutations in 23S rRNA and ribosomal proteins L4 and L22 have been reported in multistep resistance selection studies: such mutations have been found in approximately 2% of clinical isolates from the Alexander Project (17) . Macrolide resistance as a result of alterations in 23S rRNA has been found in different gram-negative and -positive species (18) . For two resistant mutants that were selected for by exposure to azithromycin and that had mutations in 23S rRNA (A2058G and A2059C), the MICs of azithromycin, clarithromycin, and erythromycin were higher than normal, from 32 to 64 µg/ml . These substitutions were associated with increased MICs of the drugs mentioned above (1), although the MICs were higher (>128 µg/ml) . Resistance as a result of mutations in the L4 and L22 proteins has been reported in in vitro mutants of Escherichia coli (18) . Moreover, it has previously been shown (1) that mutations in the highly conserved region of the L4 and L22 proteins in H . influenzae (from amino acids 55 to 75 in the L4 protein sequence and amino acids 65 to 100 in the L22 protein sequence) are associated with increases in erythromycin, clarithromycin, and azithromycin MICs and contribute to increases in macrolide MICs for H . influenzae strain Rd, as observed in transformation studies (1) . These changes probably cause decreases in macrolide affinity by changing the ribosomal conformational structure (17) . All but one mutation in ribosomal proteins L4 and L22 in the mutants selected for in this study by exposure to clarithromycin and azithromycin have been described previously (1, 17) and were associated with high MICs of these antibiotics . Four mutants selected for by exposure to erythromycin, telithromycin, and GW 773546 had substitutions K90E, R88P, G91D, and A93E and deletion 96ILKR in the L22 protein sequence . These mutations have been described previously, but only among strains for which clarithromycin and azithromycin were MICs increased (1, 17) . New mutations were found in the conserved region of the L22 protein sequence among mutants selected for by exposure to erythromycin, GW 708408, and GW 773546 . Amino acid changes were associated with specific selection agents: insertions (104 HIT and 100 KRT) were found in two mutants selected for by exposure to erythromycin, but deletions (85VMPR, 89AKGRA, 89AK, 89AKGRA, 83KRVM, and 80PSM) and substitutions (K90N/E) characterized strains exposed to the new macrolides GW 708408 and GW 773546 . Two new alterations were observed in the conserved region of the L4-protein sequence: deletion 93GT and substitution S54T in mutants selected for by exposure to clarithromycin and azithromycin, respectively .

No mechanism of resistance was detected for 26 resistant mutants . The absence of modifications in the portion of 23S rRNA studied and in ribosomal proteins L4 and L22 shows that other ribosomal regions or proteins are likely to be involved in macrolide resistance . These as yet unknown mechanisms of resistance are being investigated in our laboratory .

Single-step mutation analysis showed that mutation frequencies were similar with all compounds tested for each organism, regardless of its ß-lactam susceptibility . The mechanisms of resistance in these clones were similar to those defined in multistep mutation selection studies .

Our results demonstrate that members of the ketolide-macrolide-azalide group have long PAEs against H . influenzae . The PAEs produced by the novel macrolides, telithromycin, and azithromycin were longer than those produced by erythromycin . These results are consistent with those from previous studies (3, 15) . The PAE would be important only for organisms for which the MICs are high and in cases in which the levels in serum would fall below the MIC . Additional pharmacokinetic studies will be necessary to determine the significance of these findings .

The clinical application of macrolides and ketolides with activities against H . influenzae in vitro is a complex problem (10) . Macrolides, azalides, and ketolides all have a unimodal MIC distribution for this species; and macrolide resistance mechanisms have recently been identified in clinical specimens (17) . Also, there is a question concerning the validity of the established breakpoints of this group of compounds for H . influenzae (19) . In light of the values for the pharmacokinetic and pharmacodynamic parameters and bacteriological outcomes in double-tap studies of otitis media (4, 5, 8, 19), it has been suggested that the breakpoints for azithromycin and clarithromycin for Haemophilus are considerably lower than those currently approved by NCCLS . Ketolides such as telithromycin, like azithromycin, exhibit pharmacodynamic properties which correlate best with the area under the concentration-time curve/MIC ratio for Staphylococcus aureus and Streptococcus pneumoniae (8) . More detailed pharmacokinetic and pharmacodynamic data for both novel macrolides in comparison with those for other agents are necessary to test the clinical validity of the in vitro data described above .

In summary, GW 773546 and GW 708408 had MICs, time-kill patterns, and resistance selection properties similar to those of azithromycin and telithromycin for H . influenzae . Pharmacokinetic and pharmacodynamic studies as well as studies with animals must be preformed before the clinical usefulness of these findings may be tested .

Strainantibiotic5.0 x 10^–7<1.0 x 10^–10<2.0 x 10^–10<2.0 x 10^–10GW 7085.8 x 10^–91.7 x 10^–10<1.7 x 10^–10<1.7 x 10^–10GW 7734.0 x 10^–92.0 x 10^–9<2.0 x 10^–10<2.0 x 10^–10 7ERY4.3 x 10^–84.3 x 10^–10<1.4 x 10^–10<1.4 x 10^–10AZI7.0 x 10^–93.0 x 10^–92.0 x 10^–93.0 x 10^–10CLARI4.0 x 10^–92.0 x 10^–92.0 x 10^–10<1.0 x 10^–10 TELI7.1 x 10^–94.3 x 10^–102.8 x 10^–102.8 x 10^–10GW 7081.5 x 10^–96.2 x 10^–10<1.5 x 10^–10<1.5 x 10^–10GW 7731.5 x 10^–95.0 x 10^–102.5 x 10^–10<2.5 x 10^–10 8ERY2.0 x 10^–91.2 x 10^–9<4.0 x 10^–10<4.0 x 10^–10AZI1.5 x 10^–73.5 x 10^–9<5.0 x 10^–10<5.0 x 10^–10CLARI3.0 x 10^–81.5 x 10^–91.5 x 10^–9<5.0 x 10^–10TELI5.0 x 10^–77.5 x 10^–95.0 x 10^–97.5 x 10^–10GW 7088.0 x 10^–88.0 x 10^–104.0 x 10^–10<2.0 x 10^–10GW 7733.3 x 10^–81.7 x 10^–95.0 x 10^–103.3 x 10^–10 9ERY1.2 x 10^–85.0 x 10^–9<1.2 x 10^–10<1.2 x 10^–10AZI1.0 x 10^–71.0 x 10^–9<3.3 x 10^–10<3.3 x 10^–10CLARI1.0 x 10^–76.7 x 10^–96.7 x 10^–10<3.3 x 10^–10TELI6.2 x 10^–81.2 x 10^–8<2.5 x 10^–10<2.5 x 10^–10GW 7084.4 x 10^–9<1.1 x 10^–10<1.1 x 10^–10<1.1 x 10^–10GW 7731.7 x 10^–91.3 x 10^–91.7 x 10^–10<1.7 x 10^–10 10ERY1.0 x 10^–81.7 x 10^–9<3.3 x 10^–10<3.3 x 10^–10AZI2.0 x 10^–8<4.0 x 10^–10<4.0 x 10^–10<4.0 x 10^–10CLARI2.5 x 10^–91.3 x 10^–96.3 x 10^–102.5 x 10^–10TELI9.2 x 10^–71.4 x 10^–72.1 x 10^–9<1.4 x 10^–10GW 7083.0 x 10^–81.0 x 10^–91.0 x 10^–10<1.0 x 10^–10GW 7731.0 x 10^–81.2 x 10^–91.0 x 10^–98.0 x 10^–10 11ERY5.0 x 10^–72.5 x 10^–7<2.5 x 10^–10<2.5 x 10^–10AZI4.0 x 10^–9<1.0 x 10^–9<1.0 x 10^–9<1.0 x 10^–9CLARI2.0 x 10^–87.0 x 10^–9<1.0 x 10^–9<1.0 x 10^–9TELI2.7 x 10^–72.7 x 10^–9<1.3 x 10^–10<1.3 x 10^–10GW 7085.0 x 10^–71.7 x 10^–9<1.7 x 10^–10<1.7 x 10^–10GW 7731.7 x 10^–91.3 x 10^–9<1.7 x 10^–10<1.7 x 10^–10

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