Resistance surveillance of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolated in Asia and Europe, 1997–1998

Daniel F. Sahma, Mark E. Jonesb,*, Mary L. Hickeya, David R. Diakuna, Satish V. Mani and Clyde Thornsberryc

MRL Pharmaceutical Services, a Herndon, VA; b Den Brielstraat 11, 3554 XD, Utrecht, The Netherlands; and c Brentwood, TN, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A multicentre, collaborative study was performed in Asia and Europe during the winter of 1997–1998 to determine the in vitro activity of selected antimicrobial agents against common respiratory pathogens. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolates were collected from 48 sites in China, France, Germany, Italy, Japan, Spain and the UK and tested in a central laboratory in the USA. Broth microdilution MICs were determined for ß-lactams (penicillin, amoxycillin/clavulanate, cefuroxime, ceftriaxone), macrolides (azithromycin, clarithromycin), sulphonamides (co-trimoxazole), glycopeptides (vancomycin) and fluoroquinolones (levofloxacin). The percentage of isolates susceptible to each antimicrobial class varied substantially by country. Penicillin susceptibility amongst pneumococci ranged from 34% in France and Spain to 92% in Germany, and macrolide susceptibility varied between 26% in China and 91% in the UK. In most countries ß-lactam, macrolide and cotrimoxazole resistance was more prevalent amongst penicillin-intermediate and -resistant S. pneumoniae isolates. However, little or no resistance was detected to levofloxacin (0.3% intermediate and resistant) or vancomycin (0% intermediate and resistant). For H. influenzae the prevalence of ß-lactamase production varied from 6% in China and Germany to 32% in Spain, and for M. catarrhalis, from 79% in Germany to 98% in Japan. With the exception of ampicillin, ß-lactamase production had a minimal effect on ß-lactam activity against H. influenzae or M. catarrhalis. Our findings demonstrate that antimicrobial resistance profiles of common respiratory isolates differ dramatically between countries in Asia and Europe.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Pneumococci with reduced susceptibility to penicillin were reported by Hansman & Bullen in 1967,1 and soon thereafter, such isolates were detected in other parts of the world, although the incidence was quite low. In a review of the literature detailing the development of worldwide pneumococcal resistance, Applebaum2 notes that penicillin-intermediate and -resistant strains were detected in Germany and Poland in the early 1970s and in Spain, Switzerland, South Africa, Japan and the UK in the mid- to late 1970s. In other parts of the word bacterial species recognized as respiratory tract pathogens are becoming increasingly resistant to antimicrobial agents. The rise in penicillin resistance in Streptococcus pneumoniae is of concern not only because it poses a treatment challenge, but pneumococci with decreased penicillin susceptibility also have an increased frequency of resistance to other drug classes.38 Since the prevalence of penicillin-resistant pneumococci varies significantly with geographical location,7 effective surveillance ideally should monitor resistance trends from several countries. Although recent surveillance reports have recognized the need for data from multiple countries,913 comprehensive reports on large numbers of recent respiratory pathogens with susceptibility profiles analysed on the basis of geographical area, site of infection and age of the patient are yet to be published. This report details such a comprehensive study of 1997– 1998 isolates of S. pneumoniae, Haemophilus influenzae and Moraxella catarrhalis collected from multiple sites in Japan, China, France, Germany, Italy, Spain and the UK.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

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 2–5% lysed horse blood, cation-adjusted Mueller–Hinton broth for S. pneumoniae, Haemophilus test medium for H. influenzae and cation-adjusted Mueller–Hinton 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 {chi}2 test for each country.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
This international surveillance study was conducted during the 1997–1998 respiratory disease season in order to provide an evaluation of the current resistance levels to common antimicrobial classes in several Asian and European countries. In addition to the standard MIC50/90, MIC range and susceptibility interpretative category parameters used to evaluate the results, country-specific data on antimicrobial resistance profiles were examined according to penicillin susceptibility categories, MIC distribution, specimen source and patient age. Although previous studies have recognized the need for surveillance across countries and focused on some of these evaluation parameters,913,16,17 we are unaware of country-specific surveillance studies with large numbers of recent clinical isolates that examined multiple drug classes using all of these parameters.

The overall 1997–1998 prevalences of penicillin-resistant strains of S. pneumoniae and ß-lactamase-positive strains of H. influenzae and M. catarrhalis are summarized in Table IGo. 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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Table I. Prevalence of penicillin-intermediate and -resistant strains of S. pneumoniae and ß-lactamase-producing strains of H. influenzae and M. catarrhalis
 
Susceptibility of S. pneumoniae isolates

The susceptibility profiles of the S. pneumoniae isolates from each country are given in Table IIGo. 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 1992–1993 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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Table II. Percentage of S. pneumoniae strains by susceptibility interpretative category in each country (determined by broth microdilution)a
 
Striking variations in susceptibility between countries can be seen in other drug classes as well. The geographical distribution of resistance to other ß-lactams followed that of penicillin resistance and the lowest levels of resistance were encountered with ceftriaxone. As observed with penicillin, ceftriaxone resistance was greatest in Spain and France (5%) but was not detected in China, the UK or Germany. Although resistance to any ß-lactam was found in <4% of isolates from China, resistance to macrolides (71.8–72.6%) and co-trimoxazole (27.4%) was highest in China. A 49% difference in macrolide susceptibility was detected between France and Germany (42 and 91%, respectively) and a 65% difference in co-trimoxazole susceptibility was detected between China and Japan (24 and 89%, respectively). Recent reports of susceptibility from individual countries support this variation: Richard et al.12 reported that macrolide susceptibility was 94% in Germany but only 65% in Spain for the 1994–1995 respiratory season; similarly, Wang et al.18 reported that only 24% of the 1997 nasopharyngeal pneumococcal isolates from China were susceptible to co-trimoxazole, yet Rikitomi and coworkers19 reported 100% co-trimoxazole susceptibility in 1991–1994 pneumococcal isolates from Japan. No vancomycin resistance was encountered and fluoroquinolone (levofloxacin) nonsusceptibility was found in only five isolates (one resistant isolate each from Germany and China, two resistant isolates from Japan and one intermediate isolate from Germany). Since our findings confirm that susceptibility differs both within and amongst countries,6,11,20 comprehensive surveillance studies should be designed to capture data across geographical regions.

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 1997–1998 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 1997–1998. In the UK, penicillinintermediate and -resistant pneumococci accounted for 3.4 and 3.7% of isolates, respectively, during the 1995–1996 respiratory season,20 and in our 1997–1998 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 IIIGo). 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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Table III. Percentage of antimicrobial-resistant S. pneumoniae isolates according to penicillin susceptibility category for each countrya
 
The data in Table IVGo also suggest that resistance to drugs other than vancomycin and levofloxacin was notably higher amongst penicillin-nonsusceptible strains than amongst penicillin-susceptible strains, thus supporting previous observations that penicillin resistance is associated with resistance to other antimicrobial classes.38,10 However, elevated macrolide and co-trimoxazole resistance has also been noted in the penicillin-susceptible population.6,23 Our study confirms the finding, especially among penicillinsusceptible strains from Japan (48% macrolide resistance), China (30% co-trimoxazole and 73–74% macrolide resistance) and Spain (11% co-trimoxazole resistance). In both the UK and Germany, resistance rates in the penicillinsusceptible populations were much lower (<3% for cotrimoxazole and <7% for the macrolides), which agree with the report by Goldsmith et al.24 of 4% macrolide resistance in penicillin-susceptible S. pneumoniae isolated in the UK.


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Table IV. Penicillin MIC distribution for S. pneumoniae isolates in each country
 
No glycopeptide resistance was detected among pneumococci in any country in this study and the occurrence of levofloxacin resistance was rare. As a result, the susceptibility differences between countries for these drug classes are negligible. Contrary to the findings of a recent study that linked increased fluoroquinolone resistance to penicillin resistance,25 the levofloxacin-resistant strains were isolated in countries with low levels of penicillin resistance (China and Germany) as well as in one with a high level (Japan). Levofloxacin resistance did not appear to be associated with decreased penicillin susceptibility, but in Japan, one of the 22 penicillin-resistant isolates was levofloxacin-resistant. The current paucity of international surveillance data for fluoroquinolone activity in pneumococci by country limits our ability to examine this association closely.12 Surveillance studies are needed to monitor susceptibility levels by geographic region since resistance to this class of agents could emerge anywhere, not just in countries with high levels of penicillin resistance.

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 18–19% 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 VGo). 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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Table V. Penicillin susceptibility of S. pneumoniae by specimen source and age in each country
 
Susceptibility of H. influenzae isolates

Among H. influenzae the activity of ampicillin varied greatly from one country to another (range 68–94%) 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 VIGoGo). Susceptibility to co-trimoxazole showed great variation (48–83% of strains were susceptible). The lowest level of co-trimoxazole susceptibility occurred in isolates from Spain, which confirms results from a 1992–1994 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 IGo), which was similar to the prevalence found amongst 4155 European isolates in a 1992–1994 study by Gruneberg et al.11 (12.3–15.5%) but lower than the prevalence reported by Richard and colleagues12 in their 1994–1995 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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Table VI. Susceptibility profile of H. influenzae according to country
 
Susceptibility of M. catarrhalis isolates

Based on MIC90s, all drugs except ampicillin seemed active against isolates of M. catarrhalis from all countries (Table VIIGo). Other than variation in ß-lactamase production (79–98%, Table IGo), 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 1992–1994 isolates.


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Table VII. Activity of antimicrobial agents against M. catarrhalis according to country
 
In conclusion, this study demonstrates that antimicrobial resistance profiles differ dramatically between countries, suggesting the importance of conducting surveillance studies on a global level with country-specific data. In addition, surveillance studies should include a substantial number of recent clinical isolates from geographically diverse locations so that common pathogens can be monitored for emerging resistance patterns as well as shifts in current resistance profiles. Our findings also indicate that penicillin resistance amongst pneumococci is correlated with resistance to other ß-lactams, macrolides and co-trimoxazole, but not to the glycopeptides (vancomycin) or the fluoroquinolones (levofloxacin). To assist in controlling the public health threat posed by infections caused by multi-resistant S. pneumoniae, comprehensive surveillance that monitors MIC distribution data, associated resistance patterns and susceptibility patterns by specimen source and patient age are necessary. In addition, data should be analysed on a timely basis so that any change in the susceptibility patterns of highly active agents, such as the glycopeptides and the fluoroquinolones, can be readily detected and reported.


    Acknowledgments
 
We would like to thank Geriann Piazza, MA, for editing and statistical analyses. This work, which was supported by Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), was presented as a poster at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, California, USA, September 24–27, 1998 and at the Sixth International Symposium on New Quinolones and Related Antibiotics, Denver, Colorado, USA, November 15–17, 1998. The authors gratefully acknowledge the following participating hospitals for their role in acquiring the strains necessary for this study: Juntendo University School of Medicine, Tokyo, Japan; Saga Medical School, Saga-city, Japan; Toho University School of Medicine Omori, Ota-ku, Yokyo, Japan; Nagasaki University School of Medicine, Nagasaki-city, Japan; Asahikawa City Hospital, Hokkaido, Japan; Iwate University School of Medicine, Iwate, Japan; Third Affiliated, SUN Yat-sen, University Medical Science, Guangzhou, China; Zhong Shan Hospital, Shanghai, China; Beijing Hospital, Dong Dan, Beijing, China; Peking Union Medical College Hospital, Beijing, China; Addenbrookes Hospital, Cambridge, UK; Aberdeen Royal Infirmary, Aberdeen, UK; University Hospital Birmingham, Birmingham, UK; City Hospital, Belfast, UK; Western General Hospital, Edinburgh, UK; Southmead Hospital, Bristol, UK; GR Micro Limited, London, UK; Southern General Hospital, Glasgow, UK; Max von Pettenkofer Institut, Munchen, Germany; Institut für Medizinische Mikrobiologie, Köln, Germany; University Regensburg, Regensburg, Germany; Institut für Medizinische Mikrobiologie, Aachen, Germany; Mikrobiologisches Labor, Gorlitz, Germany; Institut für Medizinische Mikrobiologie, Frankfurt, Germany; Institut für Med Mikrobiologie, Leipzig, Germany; Hygiene-Institut der Universität, Heidelberg, Germany; Hospital Gregorio Maranon, Madrid, Spain; Hospital La Fe, Valencia, Spain; Hospital San Pablo, Barcelona, Spain; Hospital Clinico/Salamanco, Salamanco, Spain; Hospital Ramon y Cajal, Madrid, Spain; Hospital Clinico/Madrid, Madrid, Spain; Hospital Virgen Macarena, Sevilla, Spain; Hospital Clinico/Zaragoza, Zaragoza, Spain; Bordeaux-Pelligrin Tripode, Bordeaux Cedex, France; St. Etienne-HP De Bellvue, St Etienne, France; Hospital St Vincent De Paul, Paris, France; Hopitaux Universitaires de Strasbourg, Strasbourg, France; Hopital Edouard Herriot, Lyon, France; Groupe Hospitalier Bichat-Claude Bernard, Paris, France; CHU Lyon Sud, Pierre Benite Cedex, France; Ospedale S. Maria Della Misericordia, Udine, Italy; Ospedale San Martino, Genova, Italy; Ospedale L. Sacco, Milan, Italy; Universita Degli Studi Di Milano, Milano, Italy; Universita Studi di Udine, Udine, Italy; University of Naples Federico II, Napoli, Italy and University of Ancona Medical School, Ancona, Italy.


    Notes
 
* Corresponding author. Tel: +31-30-265-1794; Fax: +31-30-265-1784; E-mail: mjones{at}thetsn.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received 18 February 1999; returned 20 July 1999; accepted 23 September 1999