GR Micro Ltd, 79 William Road, London NW1 3ER, UK
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Abstract |
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Introduction |
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Surveillance of susceptibilities of individual isolates locally, nationally and worldwide, is desirable to inform empirical therapeutic choice and to influence prescription habits so that the further development of resistance may be slowed.5,6 To be useful and comparable, such studies must be of high quality.
The Alexander Project was established in 1992 to monitor the susceptibility of the major lower respiratory tract bacterial pathogens to a variety of antimicrobials and to identify trends in the development of resistance over time.7 For the first 4 years of the study (19921995), 10 European and five USA centres were monitored. In 1996 and 1997, the study was expanded to include centres located in Central and South America, the Middle East, South Africa, Hong Kong and other European countries not included previously. The general characteristics of the project were not altered, with isolates tested centrally at the laboratory of GR Micro Ltd, London, UK, using the same standard procedures that had been in use since the project began in 1992.7 Detailed data from the examination of isolates collected during 1996 and 1997 are now presented and, where possible, related to trends seen in previous years.
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Materials and methods |
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During 19961997, the following centres and investigators took part in the study: UK, London (D. Felmingham); Republic of Ireland, Dublin (L. Fenelon, E. Smith); France, Toulouse (J. Lemozy); Belgium, Leuven (J. Verhaegen); The Netherlands (co-ordinator A. J. de Neeling); Spain, Barcelona (J. Liñares); Portugal (co-ordinator J. Melo Cristino); Italy, Genoa (G. C. Schito); Germany, Weingarten (H. Grimm); Austria, Vienna (U. Theuretzbacher); Czech Republic (co-ordinator P. Urbáková); the Slovak Republic (co-ordinator P. Urbá
ková); Hungary (M. Konkoly-Thege); Poland (K. Trzcinski); Switzerland (co-ordinator J. Bille); USA (New YorkU.-X. Chin, ClevelandJ. Washington, San Francisco (1997 only)M. York); Mexico, Mexico City (J. Sifuentes-Osornio); Brazil, São Paulo (C. Mendes); Saudi Arabia, Riyadh (A. M. Shibl); South Africa, Johannesburg (K. Klugman) and Hong Kong, Pokfulam (W. H. Seto).
Bacterial isolates
Inclusion criteria for bacterial isolates, and transportation, storage, re-identification and the microbroth dilution susceptibility testing methods have been described in detail previously.7,8 In brief, centres were requested to collect up to 400 isolates from patients with community-acquired lower respiratory tract infection. Centres were instructed to avoid sending duplicate isolates from the same patient. Originally, the project accepted isolates of S. pneumoniae, H. influenzae, Haemophilus parainfluenzae, M. catarrhalis, Staphylococcus aureus and Klebsiella pneumoniae. However, by 1995 the consensus view of the project group was that H. parainfluenzae and K. pneumoniae should no longer be included, in view of the low numbers submitted for testing and their lesser importance in community-acquired respiratory tract infections. Isolates of S. aureus were collected up to and including 1997, but were submitted by only a few centres. Analysis of the MIC data produced has not, therefore, been undertaken for this species and further isolates will not be collected.
Antimicrobial susceptibility testing
MICs were determined at the central laboratory in London, using a broth microdilution method (MuellerHinton) with an inoculum of c. 104 cfu in 50 µL of medium.7,8 Breakpoint concentrations used to interpret MIC data qualitatively were based upon those published by the National Committee for Clinical Laboratory Standards of the USA (NCCLS, 1998),9 and are indicated in the tables and text. During the period covered by the Alexander Project, re-appraisal of the breakpoints defining susceptibility to certain antimicrobials has been undertaken by the NCCLS, and these new breakpoints will be used in this analysis.10
The antimicrobials and concentrations tested in 1996 and 1997 were: penicillin (0.0048 mg/L), ampicillin (0.0048 mg/L), co-amoxiclav (0.0048 mg/L amoxycillin and 0.0024 mg/L clavulanic acid), cefaclor (0.034 mg/L), cefuroxime (0.01532 mg/L), cefixime (0.0364 mg/L), ceftriaxone (0.0044 mg/L), erythromycin (0.01532 mg/L), clarithromycin (0.01532 mg/L), azithromycin (0.01532 mg/L), doxycycline (0.01532 mg/L), chloramphenicol (0.0364 mg/L), ciprofloxacin (0.00816 mg/L), ofloxacin (0.00816 mg/L) and co-trimoxazole (trimethoprim/sulphamethoxazole, 0.03/0.5732/608 mg/L).
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Results |
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A total of 2160 isolates of S. pneumoniae were submitted in 1996 and 2036 in 1997. Numbers collected by individual centres, with the percentage of penicillin-susceptible, -intermediate (MIC 0.121 mg/L) and -resistant (MIC 2 mg/L) isolates are presented in Table I
.
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Of the western European countries that joined the project during 19961997, Portugal appears to be an established centre of penicillin resistance, though the rate is not yet as high as that seen in its geographical neighbour, Spain. Results of testing of isolates submitted from the Republic of Ireland also indicate the establishment of significant levels of penicillin resistance. Rates of resistance were low in Belgium, The Netherlands and Switzerland, and particularly low in Austria (0.9% in 1996). However, with only 2 years' data from these regions, general trends are more difficult to interpret.
Amongst eastern European countries, relatively high rates of penicillin resistance are present in the Slovak Republic, in comparison with much lower rates in its neighbour the Czech Republic. Data for Hungary also indicate a high rate of penicillin resistance (1996: 24.4% intermediate, 11.8% resistant). However, this is lower than reported in a study by Marton in 1995.11 In Poland, almost 10% of isolates are now resistant, with intermediate resistance falling in relative importance from 7.7% in 1996 to 5% in 1997. This may indicate a shift to a predominance of more highly resistant strains of S. pneumoniae in this country, and should be observed carefully.
During the lifetime of the Alexander Project, strains collected from a small number of centres in the USA have contained an increasing proportion of both intermediate and resistant isolates, with a switch from a predominance of intermediate to resistant isolates in 1994. Results presented here confirm a continued increase from 1996 to 1997, of intermediate isolates from 12.7 to 15.3%, and of resistant isolates from 16.6 to 18.6%.
In centres in Central and South America, high rates of penicillin resistance were seen in Mexico, with a change to a predominance of resistant isolates during the 2 years of monitoring. In São Paulo, Brazil, although the frequency of detection of intermediate isolates was high at 17.9% in 1996 and 14.1% in 1997, resistant isolates were seen infrequently.
Both Saudi Arabia (Riyadh) and South Africa (Johannesburg) were characterized by considerably higher rates of intermediate compared with fully resistant isolates. Conversely, in Hong Kong, low rates of intermediate resistance (5.6% in 1997), but the highest rate of resistant isolates in the study (55.5% in 1997) were observed.
Susceptibility of S. pneumoniae to ß-lactams
The comparative in vitro activities of the various ß-lactam antimicrobials for S. pneumoniae tested in the study are shown in Table II. No change in the overall activity of the compounds against penicillin-susceptible isolates was observed. However, a general two-fold increase in MIC90 for all compounds except penicillin was seen, indicating a small change in distribution of MIC for the aminopenicillins and cephalosporins. For both years, as in those preceding, the most active compounds were ceftriaxone and co-amoxiclav.
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Susceptibility of S. pneumoniae to non-ß-lactams
Overall rates of macrolide resistance for isolates of S. pneumoniae collected in 1996 and 1997 at 16.5 and 21.9%, respectively, now exceed those of penicillin resistance (10.4 and 14.1%, respectively), and are close to the combined rates of penicillin-resistant and penicillin-intermediate isolates (24.3% in 1997) (Table III). Similarly high resistance rates (21.8% in 1997) were found for doxycycline (Table IV
), with high rates amongst isolates from Hong Kong, Poland, Spain, France, Italy, Belgium, Hungary and Mexico and, in 1997, Saudi Arabia, South Africa and the USA.
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Resistance to co-trimoxazole was widespread amongst the isolates of S. pneumoniae tested (Table IV). Although closely associated with penicillin resistance, high rates of resistance amongst penicillin-susceptible isolates were found in Spain, Italy, Poland, Hungary, Brazil and Hong Kong.
During the period of the study, neither the MIC50, MIC90, mode MIC nor the proportion of isolates requiring 16 mg/L of ciprofloxacin or ofloxacin for inhibition have changed significantly, the vast majority of MICs following a unimodal distribution (Table V
). Of the 8081 S. pneumoniae isolates tested since the Alexander Project was initiated in 1992, 0.3% (27 isolates) required a MIC
16 mg/L of either ciprofloxacin or ofloxacin. In 1997, isolates with a MIC
16 mg/L came from France (n = 1), Spain (n = 1), Germany (n = 1), Poland (n = 1), USA (two isolates, both from New York) and Hong Kong (n = 5).
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Relationship between the susceptibilities of S. pneumoniae to different antimicrobials
Resistance to non-ß-lactam compounds was associated with penicillin resistance, with the exception of the fluoroquinolones (Tables III and IV). This association was most evident for co-trimoxazole, with more than 90% of penicillin-resistant isolates resistant to this agent. Approximately 50% of all penicillin-resistant isolates were also resistant to the macrolides, doxycycline and chloramphenicol.
Of particular interest are differences in the distribution of macrolide resistance amongst penicillin-susceptible and penicillin-resistant isolates from various locations. Isolates from France (Toulouse), Spain (Barcelona), Belgium (Leuven), Italy (Genoa) and Hong Kong exhibited high rates of macrolide resistance (Table III). However, within these five centres it is clear that different selective pressures are operating. For example, in Barcelona the majority of macrolide-resistant isolates are also penicillin resistant, whereas in Toulouse, Belgium and Hong Kong, macrolide resistance is also emerging rapidly in penicillin-susceptible isolates. A different pattern is apparent in Genoa, Italy, where macrolide resistance in excess of 20% is seen in penicillin-susceptible isolates. This is in an area in which the combined level of intermediate and resistant isolates is low (7.3% in 1997). The development of macrolide resistance independently of penicillin resistance is also seen in isolates from some, but not all, of those centres with more modest levels of macrolide resistance, including the UK, the Republic of Ireland, Switzerland, Hungary, Poland, the Slovak Republic, the USA, Mexico and Saudi Arabia. In Germany, Austria, the Czech Republic, The Netherlands, Portugal, Brazil and South Africa, macrolide resistance occurs at a relatively low level (<10%).
Antimicrobial resistance in H. influenzae
A total of 2820 isolates of H. influenzae were collected in 1996 and 2721 in 1997. The principal mechanism of resistance observed was the production of ß-lactamase, with an overall rate of 13.4% in both 1996 and 1997 (Table VI). Large numbers of ß-lactamase-producing strains were found in Hong Kong, Spain, France, Belgium, the Republic of Ireland, the USA, Mexico and Saudi Arabia. ß-Lactamase-negative, ampicillin-resistant strains (MIC
4 mg/L) were identified only rarely, with overall rates of 0.1% in 1996 and 0.3% in 1997. In 1996, four strains were found in Barcelona, Spain. In 1997, strains were isolated in Dublin, Republic of Ireland (n = 3), Portugal (n = 1) and the Slovak Republic (n = 2).
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Of the ß-lactam antimicrobials tested, co-amoxiclav, cefixime and ceftriaxone were the most active against H. influenzae, followed by cefuroxime and cefaclor (Table VII).
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Co-trimoxazole resistance was high in Barcelona, Spain, São Paulo, Brazil and Riyadh, Saudi Arabia and less than 10% in all other centres except those in Italy, Austria, the Slovak Republic, Hungary, Poland, USA, Mexico, South Africa and Hong Kong (Table VI).
Fluoroquinolone resistance was detected in four isolates in 1996 (one each in London, Toulouse, Barcelona and New York) and in one isolate from Portugal in 1997, making a total of 13 fluoroquinolone-resistant strains out of 11,539 isolates of H. influenzae examined in the Alexander Project since 1992an overall rate of 0.1%.
The susceptibility of H. influenzae isolates to the macrolides followed a unimodal distribution in the rank order, azithromycin (mode MIC 0.51 mg/L) > erythromycin (mode MIC 4 mg/L) > clarithromycin (mode MIC 48 mg/L) (Table IX). Only eight of 11,539 isolates required
8 mg/L azithromycin for inhibition.
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ß-Lactamase production was the only resistance mechanism of importance identified in the isolates of M. catarrhalis tested. Overall, 90.4% of the 655 isolates collected in 1996 and 91.6% of 685 in 1997, produced ß-lactamase (Table X).
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The rank order of activity of the three macrolides tested was azithromycin (MIC90 0.03 mg/L) > clarithromycin (MIC90 0.12 mg/L) > erythromycin (MIC90 0.25 mg/L). No macrolide resistance was detected in isolates from 1996 and 1997; all were inhibited by 1 mg/L erythromycin,
0.5 mg/L clarithromycin and 0.12 mg/L azithromycin (Table X
).
All isolates of M. catarrhalis tested were susceptible to the fluoroquinolones and chloramphenicol, at least 99.7% to doxycycline and 97% to co-trimoxazole.
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Discussion |
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S. pneumoniae resistance
New centres in western Europe and eastern Europe showed high rates of S. pneumoniae resistance, though further data are required in order to elucidate trends. In particular, data from Dublin, Republic of Ireland, indicate that the high rates of resistance observed in Belfast, UK (19921995), are also present in other areas of Ireland, compared with relatively low rates on the UK mainland. Others have seen a similar pattern.14
In the USA centres, the rate of combined (intermediate and resistant) penicillin resistance has increased from 5.6% in 1992 to 18.6% in 1997. Although the total number tested in each year has been small, this trend has also been observed in a larger study, where rates of up to 52.9% (overall 23.6%) have been reported.15
Expansion of the Alexander Project has highlighted Hong Kong (with a rate of penicillin resistance of at least 50%) and South-East Asia as areas in which antimicrobial resistance is a major concern.16
Fluoroquinolone-resistant pneumococci are still found only rarely, and rates have not increased during the study period (19921997). More fluoroquinolone compounds are to be included from 1998 onwards, in order to observe any effects of the introduction of new agents of this class for the treatment of respiratory tract infections.
H. influenzae resistance
Results from the Alexander Project continue to demonstrate considerable variability in the prevalence of ß- lactamase production amongst isolates of H. influenzae worldwide. The rising trend in the USA slowed in 1997. However, Jacobs et al.17 reported 41.6% of 1676 isolates of H. influenzae from the USA in 1997 to be ß-lactamase producers. The low levels observed in this study may reflect a change in reporting centre from Worcester, Massachusetts, which has high rates of resistance (30% in 1995) to San Francisco, California, where the levels reported are lower.
As with S. pneumoniae, Hong Kong had a particularly high rate of ß-lactam-resistant H. influenzae in comparison with other centres in the study. ß-Lactamase-negative, ampicillin-resistant strains were identified only rarely (0.3% in 1997). However, in the Republic of Ireland (1997 = 1.5%), Spain (1996 = 1.8%) and the Slovak Republic (1997 = 2.6%), levels of detection of these strains indicate the need for vigilance.
Throughout the duration of the Alexander Project, the susceptibility of H. influenzae to the three macrolides tested has not changed and the use of arbitrary breakpoints seems inappropriate. As indicated by others, considerably more pharmacodynamic and clinical trial data are necessary if useful breakpoints are to be established for those compounds.17 Only a small number of isolates (eight of 11,539) were detected for which the MIC of azithromycin was 1 mg/L. Further work is necessary to determine the mechanism(s) of resistance of these isolates.
M. catarrhalis resistance
Although strains of M. catarrhalis resistant to other classes of antimicrobials have been reported,18,19 results from the Alexander Project demonstrate clearly that the great majority of clinical isolates remain susceptible to co-amoxiclav, macrolides, doxycycline, chloramphenicol, the fluoroquinolones and co-trimoxazole, confirming the unique importance of ß-lactamase production as an antimicrobial resistance mechanism in this species. A large multi-centre USA study also found ß-lactamase production to be the most important mechanism of resistance in this organism.20
Future developments
It is anticipated that the complete, up-to-date Alexander Project data set will be made available in an electronic format in the near future. The results from 19921996 are available on the Alexander Network website (http://www.Alexander-Network.com). Bacterial isolates examined during the period of the study are stored, deep-frozen, in the central laboratory, representing an important resource for retrospective analysis.
The Alexander Project continues into 1998 and beyond. Additional testing centres have been established to enable processing of a greatly increased number of isolates from a wider geographical spread of collecting sites, underlining the global dimension of the study. The data collected thus far demonstrate increasing resistance rates in many countries and highlight the need for increased vigilance in monitoring and tracking changes in antimicrobial susceptibility in order to promote successful clinical therapy, maintain the utility of current agents and suggest targets for new therapeutic strategies. The results can be used to promote rational prescribing, by guiding clinicians in the choice of the most appropriate antimicrobial for the treatment of community-acquired respiratory tract infections in their area.
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Acknowledgments |
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Notes |
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References |
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2
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14
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17
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20 . Doern, G. V., Bruggemann, A. B., Pierce, G., Hogan, T., Holley, H. P. & Rauch, A. (1996). Prevalence of antimicrobial resistance among 723 outpatient clinical isolates of Moraxella catarrhalis in the United States in 1994 and 1995: results of a 30-center national surveillance study. Antimicrobial Agents and Chemotherapy 40, 28846.[Abstract]
Received 5 February 1999; returned 27 May 1999; revised 23 August 1999; accepted 11 October 1999