Carbon dioxide requirements of Streptococcus pneumoniae and Haemophilus influenzae

Sir, It is common practice for sputum cultures to be incubated in an atmosphere containing 5% CO2 in order to maximize the isolation rate of Streptococcus pneumoniae and Haemophilus influenzae. Plates on which the susceptibilities of strains of S. pneumoniae are determined by the disc diffusion method are also routinely incubated in such an atmosphere. It has been shown, however, that CO2 can influence the diameter of the zone of inhibition when the Stokes or comparative method of susceptibility testing is u s e d . In our experience, concentrations of CO2 lower than 5% are adequate to support the growth of those strains of H. influenzae and pneumococci described as CO2dependent. We have also found that most pneumococci will grow in an aerobic atmosphere following initial isolation. In the present study, we investigated the CO2 growth requirements of both pathogens on primary culture of sputum specimens and on subculture of stored isolates recovered from various body sites. We also evaluated the effect of CO2 on the results of susceptibility testing of pneumococci by the disc diffusion method. Samples of sputum submitted to the routine diagnostic laboratory were homogenized with dithiothreitol (Oxoid, Basingstoke, UK) at a concentration of 1 g/L, and 10 L aliquots were inoculated on to each of two chocolate agar plates by a standard streak method. After incubation of one plate aerobically and the other in an atmosphere containing 5% CO2 for 24–48 h, the weights of growth of isolates of S. pneumoniae and H. influenzae were assessed semiquantitatively and recorded on a scale of 1 to 4 . In addition, stored isolates of S. pneumoniae and H. influenzae were inoculated on to chocolate agar and, following overnight incubation, colonies were scraped from the plates and suspended in peptone water. Each suspension was then diluted to give a turbidity equivalent to that of a 0.5 McFarland standard and 10 L aliquots were inoculated on to four chocolate agar plates, each of which was incubated under one of the following atmospheric conditions: (i) an aerobic atmosphere without added CO2; (ii) a CO2 incubator (providing an atmosphere containing 5% CO2); (iii) a candle jar; and (iv) a sealed container into which an investigator had breathed, thereby producing low levels of CO2 (the ‘huff jar’). After overnight incubation, weights of growth were assessed semiquantitatively as described above. Finally, the susceptibilities of pneumococcal isolates to oxacillin (1 g disc), erythromycin (5 g disc), trimethoprim (2.5 g disc) and tetracycline (10 g disc) were determined by the comparative method. Each isolate was suspended in peptone water to give a turbidity equivalent to that of a 0.5 McFarland standard and 10 L aliquots of the suspension were then inoculated on to four agar plates containing 5% lysed blood. After the discs had been applied, one plate was incubated under each of the four atmospheric conditions described above; the Oxford strain of Staphylo coccus aureus (NCTC 6571) was used as a control. The diameters of the zones of inhibition were measured following incubation for 18 h. Forty-five strains of S. pneumoniae and 21 of H. influenzae were isolated on primary culture of the samples of sputum; all of the H. influenzae isolates and 44 of the 45 isolates of S. pneumoniae grew aerobically. For only two strains each of H. influenzae and S. pneumoniae were the differences in the weights of growth between samples incubated aerobically and those incubated in an atmosphere containing 5% CO2 greater than two units of the scale used. Of the stored isolates, the weights of growth of 39 of 47 pneumococci and 37 of 52 H. influenzae strains incubated aerobically were within one scale unit of the weights of growth of the same isolates incubated in 5% CO2. Four of 47 pneumococci and seven of 52 strains of H. influenzae required CO2 for growth on subculture, although all grew adequately in the candle and huff jars. The susceptibilities of 42 pneumococcal isolates were determined. Discrepant results were observed in respect of seven isolates when the diameters of the zones of inhibition obtained under the four atmospheres of incubation were compared. For two strains, the diameters of the zones around the oxacillin discs following incubation in the CO2 incubator, the candle jar or the huff jar were 2–5 mm smaller than those obtained under aerobic conditions of incubation. Similarly, the zone diameters produced by erythromycin and tetracycline for