Journal of Antimicrobial Chemotherapy (2000) 45, 405-406
© 2000 The British Society for Antimicrobial Chemotherapy
Correspondence |
Effect of the incubation atmosphere on the susceptibilities of Haemophilus influenzae isolates to macrolides and amoxycillin as determined by the Etest
Departments of Microbiology, a Friarage Hospital, Northallerton, North Yorkshire, DL6 1JG, and b Harrogate District Hospital, Harrogate, North Yorkshire, UK
Sir,
Haemophilus influenzae is an important cause of lower respiratory tract infections (LRTIs), especially in patients with chronic obstructive pulmonary disease. In recent years, ß-lactamase-producing strains of this bacterium have become increasingly prevalent in many parts of the world, thereby reducing the efficacy of aminopenicillins and leading to a search for alternative therapeutic agents. Although the macrolides are candidates for this role, there are difficulties associated with assessing the in vitro activities of these antibiotics, which are known to be affected by the pH of the susceptibility test medium.1,2 In the present study we used the Etest to determine the susceptibilities of clinically significant strains of H. influenzae isolated from the sputa of patients with LRTIs to amoxycillin, azithromycin, clarithromycin and erythromycin. Duplicate cultures were incubated in an atmosphere containing 5% CO2 or in air in order to assess the effect of different atmospheres of incubation on the MICs.
During the 1 year period 1994–1995 a total of 316 non-replicate isolates of H. influenzae were collected. The strains were stored at –70°C until they were tested. MICs were determined with the Etest (AB Biodisk, Solna, Sweden)3 according to the manufacturer's instructions. The medium used was IsoSensitest agar supplemented with NAD at a concentration of 20 mg/L (Unipath, Basingstoke, UK) and 5% whole horse blood.4 Cultures of all 316 isolates were incubated at 37°C in an atmosphere containing 5% CO2 and those of 209 collected in the first 8 months of the study period were also incubated in air. Susceptibility categories were assigned according to MIC breakpoints recommended by the British Society for Antimicrobial Chemotherapy (BSAC).5 ß-Lactamase production was determined with penicillin-impregnated strips containing a pH indicator (Intralactam; Mast Diagnostics, Merseyside, UK). The activities of the antibiotics tested were compared by analysing each possible pairwise combination of agents with McNemar's test. McNemar's test was also used to compare the MICs of each antibiotic as determined in air with those determined in a CO2-containing atmosphere.
The MICs of the four antibiotics, as determined in air or an atmosphere containing 5% CO2, are shown in the Table
. Experience of using the Etest to assess the activities of novel antibiotics against fastidious organisms is limited,3,6 but the highly enriched medium used in the present study yielded consistently good growth and clear-cut, easy-to-read MICs, in contrast to the indistinct endpoints reported by others.6 Azithromycin was the most active of the four drugs tested (P = 0.001), irrespective of the atmosphere of incubation. Fifty-five (17.4%) isolates were resistant to amoxycillin (as determined in an atmosphere containing 5% CO2) and 80% of these were ß-lactamase producers. There was no apparent relationship between resistance to amoxycillin and that to macrolides.
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The activities of the macrolides, when susceptibility testing was performed in air, were significantly greater than those of the same drugs when susceptibility testing was performed in an atmosphere containing CO2 (P = 0.001). This discrepancy is presumably related to changes in drug activity associated with the altered pH of the medium.1,2 As the MICs of the macrolides for most of the strains tested were close to the recommended breakpoints, varying the atmosphere of incubation markedly influenced the susceptibility categories of the isolates.
The recommendation of the manufacturer of the Etest that "5% CO2 is used if necessary for growth" suggests that account should be taken of the effect of the atmosphere of incubation on the outcome of susceptibility testing, particularly in relation to antibiotics the MICs of which are affected by the presence of CO2. This might be facilitated by deriving breakpoints specific to each atmosphere. In contrast to the macrolides, the activity of amoxycillin was not affected by this variable.
Acknowledgments
This study was supported by a grant from Pfizer Limited.
Notes
J Antimicrob Chemother 2000; 45: 405–406
* Corresponding author. Tel: +44-1609-763033; Fax: +44-1609-764632; E-mail: n.weightman{at}onyxnet.co.uk 
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