Department of Medical Microbiology, Royal Free and University College Medical School, London NW3 2PF, UK
Received 17 April 2002; returned 30 July 2002; revised 30 August 2002; accepted 9 September 2002
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Abstract |
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Methods: MICs and MBCs of ABT-773 were determined for 165 strains of pneumococci (113 resistant to erythromycin). Extended phenotypes for the erythromycin-resistant strains were described in terms of intrinsic susceptibility to, and induction of resistance by, the antibiotics listed above.
Results: Erythromycin-resistant strains could be divided into 10 extended phenotypes (designated IIXI), two of which (II and IX) predominated. ABT-773 at 0.12 mg/L inhibited 109 strains (median 0.03 mg/L). MICs for the other four strains (of phenotypes X and XI) were 0.251 mg/L. MICs were only slighter higher when measured on agar in CO2 than by the NCCLS method (in broth in air). MBCs were usually 2 x MIC, but for 10 strains (eight of phenotype X, one each of types IX and XI) MBCs were >1 mg/L, and three of the latter (all type X) were tolerant. Clones of reduced susceptibility (MICs 18 mg/L, increased by up to 32-fold) could be isolated from some strains of phenotypes VII, IX and X, but not from those of type II (efflux mechanism) or from erythromycin-sensitive strains.
Conclusions: ABT-773 was active against all 113 erythromycin-resistant pneumococci tested, which belonged to 10 phenotypes. Extended phenotyping of pneumococci revealed interesting and potentially useful subdivisions of the classical phenotypes.
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Introduction |
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We report here the activity of the ketolide ABT-773 against a collection of erythromycin-resistant pneumococci, extended phenotypes of which were determined by a simple disc method2 using various members of the macrolide-lincosamide-streptogramin (MLS) group.
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Materials and methods |
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Pure compounds were obtained as follows: erythromycin BP free base (Lilly Industries, Basingstoke, Hants, UK); dalfopristin and quinupristin (Rhone Poulenc Rorer, Collegeville, PA, USA); rokitamycin (ISF SpA, Milan, Italy); ABT-773 (Abbott Laboratories, Abbott Park, IL, USA).
Antibiotic-containing discs were obtained as follows: 15 µg erythromycin, 2 µg clindamycin, 15 µg quinupristin/dalfopristin (Synercid) from Oxoid (Basingstoke, Hants, UK); 15 µg ABT-773 from Abbott Laboratories; discs containing 15 µg quinupristin, dalfopristin or rokitamycin were made by imbibing 6 mm Whatman AA discs (A1 Lab Supplies, London, UK).
Media
MuellerHinton agar (MHA) and broth (MHB), brainheart infusion (BHI) broth and Columbia agar were from Oxoid. Blood agar (BA) was made by adding 5% whole horse blood to Columbia agar.
Bacterial strains
Streptococcus pneumoniae strains were identified by colonial appearance, Grams stain and sensitivity to optochin. The test group comprised 113 erythromycin-resistant strains, all isolated from clinical material. Seventy-eight were from the UK, 35 from Belgium; all were recent isolates, and comprised all the erythromycin-resistant strains in our laboratory collection. A control group of 52 erythromycin-sensitive strains was selected at random from the collection.
Sensitivity testing
MICs were determined according to the NCCLS guidelines,3 i.e. by microdilution in MHB + 5% lysed horse blood with an inoculum of 104 cfu, incubated in air, and also by agar dilution on MHA + 5% whole sheep blood with an inoculum of 104 cfu in 5% CO2. MBCs were determined by subculturing on to BA 0.01 mL from each well showing no growth in the microdilution test.
Susceptibility to other antibiotics was determined by the disc method, following NCCLS guidelines.4 Breakpoints were as given in the latter guidelines; for ABT-773 the suggested5 value of 1 mg/L was taken, and for rokitamycin the value for erythromycin was used.
Determination of extended phenotype
Conventional testing for macrolide resistance phenotype, involving disc testing with erythromycin and clindamycin, alone and in combination, allows a maximum of five classical phenotypes to be discerned, of which four (sensitive; M, associated with efflux; inducible MLSB; constitutive MLSB) are found in pneumococci.6 This analysis can be considerably augmented by including results of testing a ketolide, rokitamycin (a 16-membered macrolide) and the streptogramin A and B components dalfopristin and quinupristin, again alone and in combination, and thus determining the extended phenotype, as done previously with staphylococci.2
Bacterial growth was harvested from BA, suspended in water to McFarland 0.5 and spread with a swab on MHA + 5% horse blood. Each plate was set with discs (2 cm apart) arranged as shown in Figure 1. Sizes and shapes of zones were recorded after overnight incubation in air. If the nature of a particular interaction was unclear, the individual test was set up again with distances between the discs being varied as appropriate.
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The different phenotypes observed were described by writing their constitutive resistances followed by an oblique followed by resistances inducible by erythromycin (in almost all cases, only erythromycin acted as an inducing agent; in the rare instances where other agents acted as inducers, this is indicated separately). The antibiotics are abbreviated: M, erythromycin; L, clindamycin; K, ketolide; Mac, rokitamycin. Thus, for example, a strain resistant to erythromycin and clindamycin, and in which resistance to rokitamycin is induced by erythromycin would be designated ML/Mac. The different phenotypes determined are distinguished by roman numerals, I (fully sensitive; control group) to XI.
Timekill experiments
One hundred millilitre amounts of MHB + 5% lysed horse blood in 250 mL conical flasks were inoculated with 108 cfu (1 mL of a suspension of cells harvested from overnight cultures on BA, adjusted to McFarland 1), and various multiples (1 x, 2 x, 4 x) of the MIC of ABT-773 were added. Flasks were incubated with rotation (50 cycles/min) at 37°C, and samples were taken at intervals for viable count determination on BA.
Selection for resistance
Cultures (10 mL in BHI broth) were spun down and resuspended in 1 mL of water. A viable count was made, and 0.1 mL (109 cfu) spread on MHA + 5% sheep blood containing 10 x MIC of ABT-773. Colonies were counted after 48 h incubation, and the proportion of cells in the original inoculum able to grow was calculated. MIC of ABT-773 and extended resistance phenotype were determined for the outgrowers, as above.
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Results |
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Using results obtained only from susceptibility to, and interaction between, erythromycin and clindamycin, the 113 erythromycin-resistant strains could be divided between the three classical phenotypes: M (resistant only to erythromycin), 41 (36.3%); inducible MLSB (resistant to erythromycin, which induces resistance to clindamycin), seven (6.2%); and constitutive MLSB (resistant to erythromycin and clindamycin), 65 (57.5%).
However, using the extended phenotyping scheme described above, each of the classical resistance phenotypes could be broken down further, giving a total of 10 phenotypes (IIXI; I describes erythromycin-sensitive strains), as shown in Table 1. The three most commonly found phenotypes were II, IX and X, comprising together 87.6% of the strains.
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Antibiotics belonging to the MLSB group. All 52 of the strains in the control group (phenotype I) were by definition sensitive to erythromycin; they were also sensitive to ABT-773 (MICs 0.0080.03 mg/L, Table 3) and by the disc method, also to clindamycin and rokitamycin. In contrast, all 113 test strains were, by definition, resistant to erythromycin; all were sensitive to ABT-773 using the suggested breakpoint of 1 mg/L (Table 3), their susceptibility usually being slightly less (two- or three-fold) than that of the erythromycin-sensitive strains. However, four strains (A13, B20, T76 and T84), belonging to phenotypes IX and X, were clearly less susceptible than the others, MICs of ABT-773 being 0.251 mg/L. The 113 strains were also all sensitive to the quinupristin/dalfopristin combination (Synercid), despite the poor activity of the individual components (86% and 43%, respectively) were sensitive to rokitamycin (phenotypes IIIX) and clindamycin (phenotypes IIVII).
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Other antibiotics. There were different associations in the various phenotypes between resistances to unrelated antibiotics: thus, phenotype II was more likely to be resistant to ofloxacin (45%) and less to tetracycline (26%), while phenotype IX strains were less often resistant to ofloxacin (6%) but more frequently to chloramphenicol (48%). All the type X strains were sensitive to ofloxacin and resistant to tetracycline.
Mutant selection
Seventeen strains from various phenotypes were examined for the presence of individual cells able to grow at 10 x the respective MIC of ABT-773. No mutants were found from strains of phenotypes I (erythromycin sensitive) and II (M-type, efflux). Bacteria with decreased sensitivity were found among strains from other phenotypes (Table 5), but MICs usually did not exceed 2 mg/L, except in the case of strain T68, where the MIC was 8 mg/L. Phenotyping of the outgrowers showed that the inducible resistance to ABT-773 present in the parent strains had become constitutive, giving a phenotype of MLMacK, except for strain D12 (phenotype VII) where resistance was still inducible against a background of reduced intrinsic susceptibility.
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Three strains, of different phenotypes, were tested. For two (T18, type I, and T120, type IX) killing was slow (1 log in 6 h). However, the third strain (T34, type II) was killed more quickly, the viable count falling by 99.9% in
5 h.
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Discussion |
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There are also geographical differences with regard to resistance phenotypes. In the USA11 and Canada12 an efflux mechanism (the M phenotype) is at least as common as ribosomal modification (MLSB), whereas in many other countries the MLSB phenotype predominates, sometimes by 10-fold or more, as in France13 and Spain14, and in other locations in a less pronounced way, e.g. two- or four-fold in Italy15 and Germany.16
Our finding that all the erythromycin-resistant pneumococci tested, irrespective of phenotype (and thus of genotype), were sensitive to the ketolide ABT-773, taking a breakpoint of MIC 1 mg/L, is largely in agreement with results of others.1721 In two of these reports17,18 ermB strains requiring 2 and 4 mg/L for inhibition were found: these would be regarded as intermediate or resistant by the suggested breakpoints.5 It is noticeable that there are quite large differences between the MIC parameters reported in these studies, e.g. MIC90 for erythromycin-sensitive strains has ranged18,21 from 0.002 to 0.06 mg/L. Telithromycin appears somewhat less active than ABT-773 against sensitive pneumococci and those of mef(A) genotype, but the two ketolides exhibit similar activity against other strains.21
Pneumococci differ from staphylococci in that isolates of the former species showing the constitutive MLSB phenotype (VIIIXI) remain sensitive to ketolides, whereas in staphylococci this phenotype is accompanied by resistance.2,14 On the other hand, while ketolides do not themselves induce the enzyme responsible for MLSB resistance,22 resistance to ABT-773 could be induced by erythromycin or another related antibiotic in pneumococcal phenotypes III, V, VII, IX and X (61.1% of those tested). However, this does not present a significant practical problem, as it seems unlikely that a patient would be treated simultaneously with a ketolide and another macrolide.
We agree with Hyde et al.11 that strains with the M phenotype (e.g. our phenotype II organisms) were less likely to be resistant to chloramphenicol or tetracycline, while those of the MLSB phenotype (mostly phenotype IX) were more often resistant to those antibiotics.
Nilius et al.17 suggested that the difference between the activity of ABT-773 against constitutive ermB strains of Streptococcus pyogenes and pneumococci of similar genotype was due to methodological variation (broth versus agar, incubation in air versus in CO2), but they did not investigate this phenomenon further. However, we found (Table 4) only a minor difference in the activity of ABT-773 against pneumococci when tested in parallel in broth incubated in air (NCCLS recommendations) and on agar incubated in CO2. This suggests that the influence of methodology on apparent sensitivity may be species specific, as we have reported previously.23
In conclusion, ABT-773 is active against a wide range of resistant pneumococci, and the extended phenotyping scheme used here reveals interesting and possibly useful subdivisions of the classical phenotypes.
Strains of phenotype X (a sub-group of the classical phenotype constitutive MLSB) appear of particular interest, as they are less sensitive to ABT-773 than other types (Table 1), they may contain individuals of decreased susceptibility (Table 5), and they seem to be more difficult to kill than other phenotypes (the MBC for eight of the 13 was >1 mg/L); fortunately they are relatively rare (11.5% of all the resistant strains tested). They are readily distinguished from the more commonly found phenotype IX (which has the same classical phenotype) by being intrinsically resistant to the 16-membered macrolide rokitamycin. Extended phenotyping may be helpful in epidemiological studies, and may also act as a springboard to further investigation of the genetic basis of pneumococcal resistance to antibiotics of the macrolide-lincosamide-streptogramin-ketolide (MLSK) group.
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Acknowledgements |
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Footnotes |
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Present address. Department of Pharmaceutics, School of Pharmacy, Brunswick Square, London WC1N 1AX, UK.
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References |
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2
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Hamilton-Miller, J. M. T. & Shah, S. (2000). Patterns of phenotypic resistance to the macrolide-lincosamide-ketolide-streptogramin group of antibiotics in staphylococci. Journal of Antimicrobial Chemotherapy 46, 9419.
3 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
4 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard M2-A7. NCCLS, Wayne, PA, USA.
5 . Barry, A. L., Fuchs, P. C. & Brown, S. D. (1999). Comparative in vitro antimicrobial activity of ABT-773 and tentative disk interpretive criteria. In Abstracts of the Thirty-ninth International Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA. Abstract 2144, p. 348. American Society for Microbiology, Washington, DC, USA.
6 . Leclercq, R. (2002). Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clinical Infectious Diseases 34, 48292.[ISI][Medline]
7 . Naaber, P., Tamm, E., Putsepp, A., Koljalg, S. & Maimets, M. (2000). Nasopharyngeal carriage and antibacterial susceptibility of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in Estonian children. Clinical Microbiology and Infection 6, 67586.[ISI][Medline]
8
.
Hsueh, P.-R., Teng, L.-J., Lee, L.-N., Ho, S.-W., Yang, P.-C. & Luh, K.-T. (2001). High incidence of erythromycin resistance among clinical isolates of Streptococcus agalactiae in Taiwan. Antimicrobial Agents and Chemotherapy 45, 32058.
9 . Reinert, R. R., Hoban, D. J., Felmingham, D. & Pluim, J. (2001). Worldwide surveillance of antibiotic resistance among clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes during 1999/2000. In Abstracts of the Forty-first International Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA. Abstract C2-692, p. 130. American Society for Microbiology, Washington, DC, USA.
10 . Felmingham, D. & Reinert, R. R. (2001). Prevalence of antibiotic resistance among European respiratory tract pathogens (1999/2000). In Abstracts of the Forty-first International Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA. Abstract C2-685, p. 128. American Society for Microbiology, Washington, DC, USA.
11
.
Hyde, T. B., Gay, K., Stephens, D. S., Vugia, D. J., Pass, M., Johnson, S. et al. (2001). Macrolide resistance among invasive Streptococcus pneumoniae isolates. Journal of the American Medical Association 286, 185762.
12
.
Hoban, D. J., Wierzbowski, A. K., Nichol, K. & Zhanel, G. G. (2001). Macrolide-resistant Streptococcus pneumoniae in Canada during 19981999: prevalence of mef(A) and erm(B) and susceptibilities to ketolides. Antimicrobial Agents and Chemotherapy 45, 214750.
13 . Angot, P., Vergnaud, M., Auzou, M., Leclerq, R. & Observatoire de Normandie du pneumocoque. (2000). Macrolide resistance phenotypes and genotypes in French clinical isolates of Streptococcus pneumoniae. European Journal of Clinical Microbiology and Infectious Diseases 19, 7558.[ISI][Medline]
14
.
Oteo, J., Alos, J. I. & Gomez-Garces, J. L. (2001). Antimicrobial resistance of Streptococcus pneumoniae isolates in 1999 and 2000 in Madrid, Spain: a multicentre surveillance study. Journal of Antimicrobial Chemotherapy 47, 2158.
15 . Pantosti, A., DAmbrosio, F., Tarasi, A., Recchia, S., Orefici, G. & Mastrantonio, P. (2000). Antibiotic susceptibility and serotype distribution of Streptococcus pneumoniae causing meninigitis in Italy, 19971999. Clinical Infectious Diseases 31, 13739.[ISI][Medline]
16
.
Reinert, R. R., Simic, S., Al-Lahham, A., Reinert, S., Lemperle, M. & Lutticken, R. (2001). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients with respiratory tract infections in Germany from 1998 to 1999: results of a national surveillance study. Journal of Clinical Microbiology 39, 11879.
17
.
Nilius, A. M., Bui, M. H., Almer, L., Hensey-Rudloff, D., Beyer, J., Ma, Z. et al. (2001). Comparative in vitro activity of ABT-773, a novel antibacterial ketolide. Antimicrobial Agents and Chemotherapy 45, 21638.
18
.
Davies, T. A., Ednie, L. M., Hoellman, D. M., Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (2000). Antipneumococcal activity of ABT-773 compared to those of 10 other agents. Antimicrobial Agents and Chemotherapy 44, 18949.
19
.
Barry, A. L., Fuchs, P. C. & Brown, S. D. (2001). In vitro activity of the ketolide ABT-773. Antimicrobial Agents and Chemotherapy 45, 29224.
20
.
Singh, K. V., Malathum, K. & Murray, B. E. (2001). In vitro activities of a new ketolide, ABT-773, against multidrug-resistant Gram-positive cocci. Antimicrobial Agents and Chemotherapy 45, 36403.
21
.
Shortridge, V. D., Zhong, P., Cao, Z., Beyer, J. M., Almer, L. S., Ramer, N. C. et al. (2002). Comparison of in vitro activities of ABT-773 and telithromycin against macrolide-susceptible and -resistant streptococci and staphylococci. Antimicrobial Agents and Chemotherapy 46, 7836.
22 . Bonnefoy, A., Girard, A. M., Agouridas, C. & Chantot, J. F. (1997). Ketolides lack inducibility properties of MLSB resistance phenotype. Journal of Antimicrobial Chemotherapy 40, 8590.[Abstract]
23 . Hamilton-Miller, J. M. T. & Shah, S. (1999). Susceptibility testing of linezolid by two standard methods. European Journal of Clinical Microbiology and Infectious Diseases 18, 2257.[ISI][Medline]