MRL Pharmaceutical Services, a Herndon, VA; b Den Brielstraat 11, 3554 XD, Utrecht, The Netherlands; and c Brentwood, TN, USA
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Abstract |
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Introduction |
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Materials and methods |
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During the winter of 1997 to 1998, 1879 clinical isolates of S. pneumoniae, 2645 isolates of H. influenzae and 638 isolates of M. catarrhalis were collected from 48 sites located in Japan (six sites), China (four sites), France (seven sites), Germany (eight sites), Italy (seven sites), Spain (eight sites) and the UK (eight sites). Laboratories were asked to ship isolates regardless of specimen source and a laboratory coding system was used to prevent the acquisition of duplicate strains from the same patient. For the purpose of data analysis, we grouped isolates into four specimen source categories: blood (n = 211), respiratory (n = 4463), eye (n = 318) and unknown (n = 170). In the initial phase of the study, isolates were shipped via overnight courier to the central laboratory (MRL Pharmaceutical Services, Cypress, CA, USA) on chocolate agar slabs and later, to enhance the delivery of viable organisms, on Amies medium without charcoal (Technical Service Consultants, Haywood, UK). Confirmatory tests were performed at the central laboratory to verify the identification of isolate by site.
Antibiotics and susceptibility testing
Representative drugs from five antimicrobial classes were tested: ß-lactams (penicillin, ampicillin, amoxycillin/clavulanate, cefuroxime, cefriaxone), macrolides (azithromycin, clarithromycin), sulphonamides (co-trimoxazole), glycopeptides (vancomycin) and fluoroquinolones (levofloxacin). Susceptibility testing was performed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS).14,15 Overnight growth of the test organism was suspended in diluent to produce a turbidity equivalent to a 0.5 McFarland standard (approximately 1 x 108 cfu/mL) and this suspension was used to inoculate broth microdilution plates (Accumed International, Chicago, IL, USA) to obtain a final inoculum of approximately 5 x 105 cfu/mL. The media used were 25% lysed horse blood, cation-adjusted MuellerHinton broth for S. pneumoniae, Haemophilus test medium for H. influenzae and cation-adjusted MuellerHinton broth for M. catarrhalis. The inoculated MIC plates were incubated at 35°C for 20 to 24 h in ambient air prior to reading. The control strains used were S. pneumoniae ATCC 49619 and H. influenzae ATCC 49247. All results were within control limits. ß-Lactamase production in M. catarrhalis and H. influenzae isolates was detected with DrySlide Nitrocefin (Difco Laboratories, Detroit, MI, USA). NCCLS breakpoints were followed to determine the susceptibility categories of S. pneumoniae and H. influenzae. Because NCCLS has not established susceptibility breakpoints for M. catarrhalis, only MIC data were evaluated for these isolates.
Statistical analysis
For specimen sources and patient age groups, the categories were dichotomized and the prevalence of penicillin resistance was compared using the 2 test for each country.
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Results and discussion |
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The overall 19971998 prevalences of penicillin-resistant strains of S. pneumoniae and ß-lactamase-positive strains of H. influenzae and M. catarrhalis are summarized in Table I. Nearly 33% of S. pneumoniae isolates were not susceptible to penicillin, and 18% of H. influenzae strains and 89% of M. catarrhalis strains were ß-lactamase producers. Dramatic differences in the prevalence of these strains existed between countries.
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The susceptibility profiles of the S. pneumoniae isolates from each country are given in Table II. High-level penicillin resistance (MIC
2 mg/L) was encountered in each country and the prevalence ranged from 0.7% in Germany, to 24.7% in Spain and 33.9% in France. Previous studies have documented the differences that exist between countries in the S. pneumoniae penicillin resistance profile.6,10,12,1820 In a 19921993 study, Felmingham and coworkers reported no penicillin-resistant (MIC
1 mg/L) pneumococci in Germany, 15.7% of isolates resistant in France and 26% of isolates resistant in Spain.10
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In addition to indicating that the prevalence of penicillin-resistant pneumococci differs by country, our data suggest that the prevalence of isolates resistant to penicillin may be increasing. Yoshida and colleagues studied pneumococcal isolates in Japan between 1988 and 1992 and determined that the prevalence of penicillin resistance (MIC 2 mg/L) increased from 4.3 to 9.8%,21 and in our 19971998 study the prevalence of pneumococci with MICs
2 mg/L was 10.1%. A multicentre study in Italy of 1993 pneumococcal isolates showed 2.6% high-level resistance,22 which we found to be 4.9% in 19971998. In the UK, penicillinintermediate and -resistant pneumococci accounted for 3.4 and 3.7% of isolates, respectively, during the 19951996 respiratory season,20 and in our 19971998 study these percentages were 5.5 and 5.2%, respectively.
Although international comparisons of penicillin resistance in pneumococci are limited, country-specific data on the activity of other drug classes in pneumococci are even less common.912 Our results show, however, that penicillin should not be the sole comparator drug in surveillance, as the activity of different classes of therapeutically important drugs can vary independently by country (Table III). For example, the percentage of S. pneumoniae isolates susceptible to ceftriaxone was greater in Spain than France (73 versus 65%) but co-trimoxazole was more active in France than Spain (43 versus 32% of strains were susceptible). In China and Germany, penicillin-resistant isolates showed no co-trimoxazole resistance. A similar lack of correlation between the activity of different classes was seen in Asia: the percentage resistance to the macrolides was greater than 66% in both China and Japan, yet only 3% of pneumococci in China were cefuroxime resistant while 37% were resistant in Japan. This lack of association between the prevalence of resistance to the ß-lactams and the prevalence of resistance to the non-ß-lactams was also documented in a multicentre European study by Gruneberg et al.11
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Since penicillin resistance in S. pneumoniae has been correlated with multiple drug resistance,38,10 penicillin resistance should be monitored so that a change in susceptibility may be readily detected. Surveillance programmes that collect MIC distributions may be better equipped to discern a change in resistance patterns before interpretative categories are breached or MIC90s shift.26 MIC distributions allow subtle biological shifts to be tracked before the manifestation of clinical resistance, especially for pathogens such as S. pneumoniae, for which the interpretative breakpoints are controversial, or for M. catarrhalis, for which no interpretative breakpoints have been established. For S. pneumoniae, the 17% of strains from China one dilution below the 0.12 mg/L intermediate breakpoint and the 1819% of strains from Spain and France one dilution below the 2 mg/L resistant breakpoint are indications that the prevalence of resistance may rise in these countries. Although the impact of different interpretative breakpoint schemes on the reporting of antimicrobial resistance rates has not yet been established, MIC distribution data enable equivalent comparisons by any system.10,26
Few surveillance studies on international isolates of S. pneumoniae report susceptibility according to specimen source or patient age,17,23 but our findings emphasize the importance of tracking susceptibility by these parameters (Table V). Based on studies conducted on isolates from the USA, S. pneumoniae isolates from blood are usually more susceptible to penicillin than isolates from noninvasive sources (e.g. respiratory isolates).5,6 While the prevalences of penicillin-susceptible pneumococci were essentially the same for both specimen types in Germany, blood isolates from the UK, Spain, France and Italy appeared more susceptible to penicillin than non-invasive isolates, but the differences were not significant (P > 0.1 for all five European countries). Specimen source comparisons could not be conducted for isolates from Japan and China because an insufficient number of blood isolates was collected. Studies on pneumococcal isolates from the USA also show that penicillin-resistant S. pneumoniae are more likely to be isolated from children < 12 years of age than from adults.36 In our study, resistance was significantly higher amongst pneumococcal isolates from patients
12 years of age than from older patients (>12 years) for Japan (P < 0.05 ) and France (P < 0.001). These trends indicate the necessity of considering the specimen source of isolates and patient age group when comparing resistance profiles of different populations.
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Among H. influenzae the activity of ampicillin varied greatly from one country to another (range 6894%) and resistance to ampicillin was entirely mediated by ß-lactamase production, which ranged from 6% of isolates from Germany and China to 32% of isolates from Spain (Tables I and VI). Susceptibility to co-trimoxazole showed great variation (4883% of strains were susceptible). The lowest level of co-trimoxazole susceptibility occurred in isolates from Spain, which confirms results from a 19921994 study of the same five European countries.11 Other than cotrimoxazole, clarithromycin was the only antimicrobial with decreased activity in H. influenzae isolates all other antimicrobials were highly active, regardless of the isolate's country of origin. We found that 18% of 2645 H. influenzae isolated from Europe produced ß-lactamase (Table I
), which was similar to the prevalence found amongst 4155 European isolates in a 19921994 study by Gruneberg et al.11 (12.315.5%) but lower than the prevalence reported by Richard and colleagues12 in their 19941995 study of 593 strains from the same five European countries (24%). The percentage of H. influenzae isolates susceptible to ampicillin in Spain has remained relatively unchanged since 199227 and ß-lactamase production in H. influenzae conferred resistance to ampicillin in all countries.
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Based on MIC90s, all drugs except ampicillin seemed active against isolates of M. catarrhalis from all countries (Table VII). Other than variation in ß-lactamase production (7998%, Table I
), there were no notable differences in the antimicrobial activities of the isolates from the seven countries. Approximately 11% of M. catarrhalis isolates were ß-lactamase positive with ampicillin MICs < 0.25 mg/L (which is the NCCLS susceptibility breakpoint for Staphylococcus aureus14), suggesting that the level of ß-lactamase produced failed to affect the MIC noticeably. The low ampicillin MICs were confirmed by repeat testing. ß-Lactamase-positive isolates with ampicillin MICs < 0.25 mg/L were collected from all countries except China, where only one M. catarrhalis strain was isolated. Although the percentage of ß-lactamase-producing M. catarrhalis isolates detected in this study was quite high for all countries, it differed by country of origin (79% in Germany to 97.5% in Japan), a variation also noted by Gruneberg et al.11 amongst 19921994 isolates.
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Acknowledgments |
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Notes |
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References |
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Received 18 February 1999; returned 20 July 1999; accepted 23 September 1999