1 GR Micro Ltd, 79 William Road, London NW1 3ER; 2 Clinical Microbiology and Public Health Laboratory, Health Protection Agency, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, UK
Received 22 June 2004; returned 6 September 2004; revised 17 November 2004; accepted 17 November 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine the antimicrobial susceptibility of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae causing community-acquired lower respiratory tract infection in the UK during 2002/2003 and to compare susceptibilities determined locally by disc diffusion with agar dilution MICs determined at a central laboratory.
Methods:
H. influenzae, M. catarrhalis and S. pneumoniae were isolated in 30 laboratories and susceptibility determined locally by the BSAC standardized disc diffusion method. At a central laboratory, isolates were re-identified, tested for ß-lactamase production (H. influenzae and M. catarrhalis only) and MICs determined using the BSAC agar dilution method.
Results:
Five hundred and eighty-one H. influenzae, 269 M. catarrhalis and 519 S. pneumoniae were collected. Over 93% of M. catarrhalis and nearly 15% of H. influenzae were ß-lactamase positive rendering these sub-populations resistant to aminopenicillins. Overall, the antibacterial susceptibility rates for the isolates were high. However, macrolides showed poor activity against H. influenzae (0.861.38% susceptible by disc or MIC methods) and, compared with other antimicrobials, against S. pneumoniae (approximately 88% susceptible). Between 84% and 95% of H. influenzae, M. catarrhalis and S. pneumoniae were susceptible to cefuroxime but all isolates were susceptible to cefotaxime. Eighty-five percent of H. influenzae were susceptible to trimethoprim. The fluoroquinolones were very active against the isolates, with moxifloxacin showing lower MICs than levofloxacin against S. pneumoniae. Susceptibility determined locally by disc diffusion was in general agreement with that determined centrally by agar dilution MIC testing. However, there was one inconsistency with H. influenzae where disc diffusion indicated 22.9% and 46.8% resistance to clarithromycin and erythromycin, respectively but by MIC, only 0.9% and 6.9% were resistant, respectively.
Conclusions:
Rates of resistance within community-acquired respiratory tract isolates were relatively low in the UK, in agreement with other studies. Moxifloxacin was the only antibacterial with over 99% isolates susceptible for each of the three pathogens investigated where breakpoints are available. The comparison between disc susceptibility testing and MIC determination using BSAC methods indicated generally good correlation but has highlighted a methodological problem with macrolides against H. influenzae in particular.
Keywords: susceptibility testing , Streptococcus pneumoniae , Haemophilus influenzae , Moraxella catarrhalis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Resistance surveillance studies including different laboratories may be based on susceptibility testing carried out locally in the participating clinical laboratories, or the isolates may be collected centrally for testing by a reference method in one laboratory. The latter has the advantages of greater standardization of methodology and the use of quantitative methods, whereas the former is less likely to be restricted in the number of isolates tested.7 No study of RTI isolates has investigated comprehensively whether centralized testing with a quantitative reference method produces significantly different resistance rates from local testing by a standardized disc diffusion method (although a comparative study was carried out in 1990 before development of the BSAC standardized disc diffusion method8 ). Such a comparison was made in this study as all isolates were tested locally by the BSAC standardized disc diffusion method and centrally by the BSAC agar dilution method.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following antimicrobials were obtained from their respective manufacturers: moxifloxacin, ciprofloxacin (Bayer, Newbury, UK), levofloxacin, clarithromycin (Aventis Pharma, Romainville, France), linezolid (Pfizer, Sandwich, UK), co-amoxiclav (clavulanic acid from GlaxoSmithKline, Harlow, UK and amoxicillin from Sigma, Poole, UK). Erythromycin, ampicillin, penicillin G, cefuroxime, cefotaxime, tetracycline and trimethoprim were obtained from Sigma. These antimicrobials were used for susceptibility testing by the BSAC agar dilution method.9
Discs containing the same antimicrobials (except oxacillin replaced penicillin G) with contents as required for the BSAC disc diffusion method10 were purchased from Oxoid (Basingstoke, UK).
Ampicillin and co-amoxiclav were tested against H. influenzae and M. catarrhalis only. Trimethoprim and penicillin G (oxacillin) were tested against H. influenzae and S. pneumoniae only, respectively.
Collecting laboratories and isolates
Thirty collecting laboratories throughout the UK (24 in England, three in Scotland, two in Wales and one in Northern Ireland) were asked to collect up to 50 isolates from cases of community-acquired lower respiratory tract infection as follows: H. influenzae (20), S. pneumoniae (20) and M. catarrhalis (10) from between September 2002 and March 2003. Isolates from patients admitted to hospital were also included, but only if from specimens taken within 48 h of admission. Duplicate isolates from the same patient were excluded. The following demographic data were also collected: patient gender, patient age and specimen type.
Local laboratory disc diffusion susceptibility testing
All collecting laboratories determined the antibacterial susceptibility of isolates by the BSAC disc diffusion method.10 Before this, each centre was sent six organisms (three S. pneumoniae, two H. influenzae and one M. catarrhalis) to test as a quality assurance exercise to ensure that results were acceptable before commencement of testing of clinical isolates.
Central laboratory isolate identification and MIC determination
Collected isolates were sent to a central testing laboratory (GR Micro Ltd) where they were re-identified using standard procedures, as described previously.11 H. influenzae and M. catarrhalis were tested for the presence of ß-lactamase (nitrocefin, Oxoid, Basingstoke, UK). MICs of antibacterial agents were determined by the BSAC agar dilution method.9 MICs were interpreted into categories of susceptibility according to BSAC breakpoints (Table 1).9 The following reference strains were used for quality control: Escherichia coli ATCC 25922, Haemophilus influenzae ATCC 49247, Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus 29213.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolates from a total of 1369 patients were included in the study, 713 patients were male, 655 were female and gender information was not provided for one patient. Antibacterial activity was similar for isolates from patients of different genders (data not shown).
The age of patients was provided for 1364 isolates. Numbers of isolates from patients in the three age groups 1864 (48.1% total), 65 years (44.1% total) and < 18 years (7.8% total) were, respectively, H. influenzae 290, 264 and 26; M. catarrhalis 105, 134 and 28; S. pneumoniae 261, 204 and 52. The antibacterial MIC profiles for isolates from patients in the three age groups were indistinguishable (data not shown).
The vast majority of isolates submitted for inclusion in this study were from sputum (93.0%), the next most prevalent sources were nasopharyngeal aspirate (2.6%), bronchoalveolar lavage (1.8%) and blood (1.2%). Numbers of isolates from sources other than sputum were too small to analyse any possible relationship between source of isolate and antibacterial susceptibility.
Susceptibility of Haemophilus influenzae by MIC determination
All MIC results for the relevant reference strains were within one two-fold dilution of the target MIC.9
A total of 581 H. influenzae were collected by local laboratories and all were re-confirmed as H. influenzae at the central laboratory. The MIC distributions of the test antimicrobials against H. influenzae are shown in Table 2 and susceptibility rates in Table 3.
|
|
Susceptibility of Moraxella catarrhalis by MIC determination
All MIC results for the relevant reference strains were within one two-fold dilution of the target MIC.9
Overall, 269 isolates of M. catarrhalis were collected by local laboratories and all were re-confirmed as M. catarrhalis at the central laboratory. The MIC distributions and the susceptibility rates are included in Tables 4 and 5, respectively.
|
|
Susceptibility of Streptococcus pneumoniae by MIC determination
All MIC results for the relevant reference strains were within one two-fold dilution of the target MIC, except that the moxifloxacin MIC for S. pneumoniae ATCC 49619 was consistently 0.12 mg/L, rather than the documented target of 0.5 mg/L.9 Our results are in keeping with the expected moxifloxacin MIC for susceptible pneumococci.
Five hundred and nineteen isolates of S. pneumoniae were collected by local laboratories and all were re-confirmed as S. pneumoniae at the central laboratory. The MIC distributions and susceptibility rates are shown in Tables 6 and 7, respectively. The highest rate of resistance among the S. pneumoniae isolates was 11.8% to both macrolides (Table 7). Resistance to cefuroxime, ciprofloxacin and tetracycline was around 5%. Although only a small percentage of isolates were fully resistant to penicillin (0.8%), a larger proportion (6.6%) were intermediate (Table 7). No linezolid-resistant isolate was found (Table 7) and, as with H. influenzae, no resistance to cefotaxime was seen.
|
|
Susceptibility determined centrally by agar dilution MIC compared with susceptibility determined locally by disc diffusion
Correct susceptibility reports for all tests were returned by all participating laboratories for the six quality assessment strains distributed before testing clinical isolates. Tables 3, and 7 show a summary of susceptibility data determined centrally by an agar dilution method and locally by the 30 collecting laboratories using BSAC disc diffusion methodology for H. influenzae, M. catarrhalis and S. pneumoniae isolates, respectively. In total, 785 disagreements (i.e. where local disc susceptibility did not agree with central MIC determination susceptibility) occurred out of a total of 14 281 susceptibility determinations. This equates to an overall discrepancy rate of 5.5%. Of these, 352 (2.5%) were major disagreements (i.e. resistant by one method but susceptible by the other) and 433 (3.0%) were minor disagreements (i.e. resistant or susceptible versus intermediate) (Table 8). In general, there was good correlation between susceptibility results obtained at local laboratories and those obtained at the central laboratory. The biggest disagreement between the two testing methods occurred with macrolides tested against H. influenzae, which showed a 23.2% discrepancy with clarithromycin and 40.9% discrepancy with erythromycin (Table 8). Almost all of these discrepancies were minor, with isolates intermediate in susceptibility to erythromycin (236 isolates) and clarithromycin (128 isolates) by MIC being classified as fully resistant by disc diffusion. False erythromycin resistance with M. catarrhalis tested by the disc diffusion method was also observed (Table 8) but 15 out of the 19 isolates were borderline resistant (zone diameters 2627 mm).
|
Disagreements occurred with ampicillin against H. influenzae and M. catarrhalis (Table 8). If ß-lactamase results are used to predict ampicillin susceptibility there was only one MIC error (one M. catarrhalis isolate being ß-lactamase-negative but ampicillin-resistant). Some false resistance of H. influenzae to co-amoxiclav by disc diffusion testing was noted (Table 8); but all 12 isolates falsely reported susceptible by disc diffusion were borderline resistant by MIC (2 mg/L) and 18/26 isolates falsely reported resistant by disc diffusion were borderline resistant on zone diameter (1516 mm).
Screening for penicillin resistance in S. pneumoniae by the oxacillin disc diffusion method produced disagreements with MIC results for 49 isolates (9.5%), the majority of these being minor (Table 8). Thirty-two of the 34 minor disagreements were with isolates that were resistant by the oxacillin screen but borderline intermediate by penicillin MIC (1 mg/L). In addition, 9/15 of the major disagreements were with isolates that were resistant by oxacillin disc screening results but borderline susceptible by penicillin MIC (0.06 mg/L).
Results by the two susceptibility testing methods with linezolid, trimethoprim and fluoroquinolones were highly comparable except for 5.8% minor disagreements with ciprofloxacin against S. pneumoniae (Table 8), and all these discrepancies were between intermediate and resistant as the BSAC has no susceptible category for this combination.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, the macrolide class of antimicrobials showed poor potency against H. influenzae, and S. pneumoniae were less susceptible to macrolides than to the other antimicrobials tested. Cephalosporins showed varied activity, with cefotaxime being more active than cefuroxime. Penicillin G and tetracycline non-susceptibility in S. pneumoniae was high relative to other non-macrolides and ampicillin activity was compromised against M. catarrhalis and H. influenzae due to the presence of ß-lactamases. The presence of ß-lactamase did not affect the activity of co-amoxiclav, as would be expected. For ß-lactamase-negative H. influenzae, ampicillin MIC was generally one dilution lower than co-amoxiclav MIC. As both antibacterials have identical breakpoints, this may be the cause of minor anomalies with those isolates being ampicillin-susceptible but co-amoxiclav-resistant.
The fluoroquinolones levofloxacin and moxifloxacin were consistently active against respiratory pathogens with moxifloxacin being the more active of the two against S. pneumoniae. In accordance with this, the MIC results for these fluoroquinolones against the reference strain S. pneumoniae ATCC 49619 were 0.12 and 0.51 mg/L for moxifloxacin and levofloxacin, respectively. However, the BSAC reference document states targets of 0.5 mg/L for both fluoroquinolones. We therefore suggest that the target for moxifloxacin against S. pneumoniae ATCC 49619 should be re-evaluated. Both fluoroquinolones showed similar activity against H. influenzae or M. catarrhalis. Linezolid resistance was not found, but this agent is restricted to Gram-positive infections only.
Since 1999, the BSAC has coordinated a respiratory tract surveillance study within the UK and Ireland with 20 centres supplying isolates to a central laboratory for testing by the BSAC agar dilution MIC method.3 Nine out of the 30 centres enrolled in this study also participate in the BSAC study but the remaining 21 centres are not involved. Data from the BSAC surveillance study are available online as an interactive database.3 This enables direct comparisons to be made between this study and the BSAC surveillance study over the same period. The MIC distributions for the two studies are almost identical.
The second objective of this study was to compare susceptibilities determined locally at the collecting laboratories using BSAC disc diffusion methodology and those determined by BSAC agar dilution MIC testing at a central laboratory. Collecting laboratories were screened in a quality assessment exercise before enrolment to ensure that they were competent in carrying out the BSAC disc diffusion methodology. The comparison between results from the collecting laboratories and the central testing laboratory was encouraging because there was little difference in results using either BSAC method. Furthermore, the majority of the disagreements that did occur were caused by borderline susceptibility results from one or other testing method. This indicates that any hospital laboratory accurately using BSAC disc diffusion methods can compare their local results directly with larger surveillance studies based on the BSAC agar dilution method, such as this current study and the BSAC surveillance programme. It may also be possible to compare local results with surveillance studies using other testing methods, as has been demonstrated with the NCCLS microbroth dilution method.14
Nevertheless, care must be taken with some antimicrobials because breakpoints can differ significantly between methods. For example, NCCLS co-amoxiclav breakpoints for H. influenzae are susceptible 4 mg/L and resistant
8 mg/L.15
With these breakpoints, only 0.17% of isolates are resistant, compared with 5.85% with BSAC breakpoints.
Despite the good correlation between the BSAC agar dilution MIC and disc diffusion methods, one specific anomaly has been highlighted where a substantial proportion of H. influenzae isolates with intermediate susceptibility to erythromycin and clarithromycin by MIC were classified as resistant by disc diffusion. Erythromycin and clarithromycin MICs for many of the isolates were close to the intermediate-resistant breakpoint concentrations of 8 and
16 mg/L, respectively. The breakpoints for macrolides in the BSAC method are adjusted to report most H. influenzae as intermediate in susceptibility as most isolates form a unimodal distribution of intermediate susceptibility. Hence it would appear that the BSAC disc diffusion method is falsely reporting resistance in this study. Problems with testing macrolides against H. influenzae are well known and often relate to the use of CO2 during incubation.16,17
While the use of CO2 enhances bacterial growth it also antagonizes macrolide activity by reducing pH. Both BSAC methods require an atmosphere of 46% CO2 during incubation of H. influenzae9,10
so any effects of CO2 should be consistent for the two methods, although it is possible that the two susceptibility methods differ in their response to CO2.
Problems with correlation of BSAC agar dilution MIC and disc diffusion methods have been described with Pseudomonas aeruginosa, where poor correlation between the BSAC MIC and disc diffusion methods has been reported, especially with meropenem.18,19 In addition, a recent study of 3378 blood culture isolates, including Enterobacteriaceae, Acinetobacter spp., pseudomonads, staphylococci and enterococci, showed that susceptible results by the BSAC disc diffusion method were generally confirmed by full MIC determination.19 However, disc diffusion tended to over-report resistances, particularly with some strains having borderline susceptibility to some agents. It was notable that most of the discrepancies investigated were discounted when disc diffusion tests were repeated in the reference laboratory, indicating errors in testing in the local laboratories rather than problems with the method. Such a critical examination of correlation of MIC and disc diffusion has not been published for other methodologies and guidelines.
In conclusion, this study showed that rates of resistance among community-acquired respiratory tract isolates were relatively low in the UK, as seen in other studies. Moxifloxacin was the only antibacterial with over 99% isolates susceptible for each of the three pathogens investigated where breakpoints are available. The comparison between disc susceptibility testing and agar dilution MIC determination by BSAC methods generally showed good correlation but highlighted a methodological problem with the testing of macrolides against H. influenzae by disc diffusion which resulted in intermediate strains frequently being reported resistant.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Ball, P. (1995). Epidemiology and treatment of chronic bronchitis and its exacerbations. Chest 108, Suppl., 43S52S.[Medline]
3 . Anonymous. http://www.bsacsurv.org (20th August 2003, date last accessed).
4
.
Hoban, D. & Felmingham, D. (2002). The PROTEKT surveillance study: antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis from community-acquired respiratory tract infections. Journal of Antimicrobial Chemotherapy 50, Suppl., S1 4959.
5
.
Hoban, D. J., Bouchillon, S. K., Karlowsky, J. A. et al. (2000). A comparative in vitro surveillance study of gemifloxacin activities against 2,632 recent Streptococcus pneumoniae isolates from across Europe, North America, and South America. The Gemifloxacin Surveillance Study Research Group. Antimicrobial Agents and Chemotherapy 44, 300811.
6
.
Felmingham, D., Reinert, R. R., Hirakata, Y. et al. (2002). Increasing prevalence of antimicrobial resistance among isolates of Streptococcus pneumoniae from the PROTEKT surveillance study, and comparative in vitro activity of the ketolide, telithromycin. Journal of Antimicrobial Chemotherapy 50, Suppl., S1 2537.
7
.
Kahlmeter, G. & Brown, D. F. J. (2002). Resistance surveillance studiescomparability of results and quality assurance of methods. Journal of Antimicrobial Chemotherapy 50, 7757.
8 . Powell, M., McVey, D., Kassim, M. H. et al. (1991). Antimicrobial susceptibility of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella (Branhamella) catarrhalis isolated in the UK from sputa. Journal of Antimicrobial Chemotherapy 28, 24959.[Abstract]
9
.
Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl., 1 516.
10
.
Andrews, J. M. (2004). BSAC standardised disc susceptibility testing method (version 3). Journal of Antimicrobial Chemotherapy 53, 71328.
11
.
Felmingham, D. (2002). The need for antimicrobial resistance surveillance. Journal of Antimicrobial Chemotherapy 50, Suppl., S1, 17.
12 . Felmingham, D., Robbins, M. J., Dencer, C. et al. (1996). Antimicrobial susceptibility of community-acquired bacterial lower respiratory tract pathogens. Journal of Antimicrobial Chemotherapy 38, 74751.[ISI][Medline]
13 . Felmingham, D., Robbins, M. J., Tesfaslasie, Y. et al. (1998). Antimicrobial susceptibility of community-acquired lower respiratory tract bacterial pathogens isolated in the UK during the 19951996 cold season. Journal of Antimicrobial Chemotherapy 41, 4115.[Abstract]
14 . Reynolds, R., MacGowan, A., Felmingham, D., et al. (2001). Conversion between NCCLS microdilution and BSAC agar dilution methods for respiratory pathogens. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract D-173, p. 152. American Society for Microbiology, Washington, DC, USA.
15 . National Committee for Clinical Laboratory Standards (2003). Performance Standards for Antimicrobial Susceptibility Testing: Thirteenth Informational Supplement: Approved Standard M100-S13. NCCLS, Wayne, PA, USA.
16
.
Paton, R., Arnold, J., Cockburn, J. et al. (2000). Susceptibility testing of Haemophilus influenzae to clarithromycin. Journal of Antimicrobial Chemotherapy 45, 52931.
17
.
Johnson, M. M., Hill, S. L. & Piddock, L. J. (1999). Effect of carbon dioxide on testing of susceptibilities of respiratory tract pathogens to macrolide and azalide antimicrobial agents. Antimicrobial Agents and Chemotherapy 43, 18625.
18
.
Henwood, C. J., Livermore, D. M., James, D. et al. (2001). Antimicrobial susceptibility of Pseudomonas aeruginosa: results of a UK survey and evaluation of the British Society for Antimicrobial Chemotherapy disc susceptibility test. Journal of Antimicrobial Chemotherapy 47, 78999.
19
.
Potz, N. A. C., Mustaq, S., Johnson, A. P. et al. (2004). Reliability of routine disc susceptibility testing by the British Society for Antimicrobial Chemotherapy (BSAC) method. Journal of Antimicrobial Chemotherapy 53, 72938.
|