Coisolation of Streptococcus pneumoniae and Haemophilus influenzae from Middle Ear Fluid and Sputum: Effect on MIC Results

Recent development of resistance of Streptococcus pneumoniae to antimicrobial agents, including penicillin, has raised concerns that this bacterium will eventually become resistant to vancomycin. During screening for vancomycin resistance, we have occasionally found S. pneumoniae isolates that appear to be resistant to vancomycin, but the specimens have always been found to be contaminated with a vancomycin-resistant bacterium whose colony morphology is similar to that of the pneumococcus. These contaminants would usually grow within the zone of inhibition around an optochin disk.

Recently an isolate from a middle ear aspirate was reported to be highly resistant to vancomycin (MIC, ≥256 μg/ml). This isolate was alpha-hemolytic, optochin sensitive, and bile soluble; had colony morphology consistent with a pneumococcus; and serotyped as a 19F. After 24 h of growth on a Trypticase soy agar plate containing 5% defibrinated sheep blood (TSA-SB), the isolate appeared to be a pure culture of pneumococcus with a clear zone of inhibition around an optochin disk. The bacterial growth gave off a strong odor, however, that was not typical of a normal pneumococcus. MIC results obtained from this culture confirmed the reported high-level vancomycin resistance, making it appear that this was the first isolation of a vancomycin-resistant S. pneumoniae strain.

After an additional 24 h of growth, white colonies formed around the pneumococcal colonies, and these bacteria were found to be Haemophilus influenzae serotype nontypeable, biotype VII. The isolate was vancomycin resistant, as is generally true of Haemophilus spp. When the S. pneumoniae was purified by growth on a TSA-SB plate containing gentamicin (Becton Dickinson Microbiology Systems, Cockeysville, Md.) and retested for susceptibility to vancomycin, the MIC was found to be 0.25 μg/ml by microplate dilution, which is within the susceptible range as defined by the National Committee for Clinical Laboratory Standards (4).

The isolated haemophilus would only grow around living S. pneumoniae on a TSA-SB plate. Spent culture fluid from a 24-h Todd-Hewitt broth culture of the pneumococcus did not support the growth of the Haemophilus organisms. Because the Haemophilus organisms needed living pneumococci for growth, a mixed culture of the haemophilus and pneumococcus showed a clear zone around an optochin disk on TSA-SB since haemophilus will not grow on a TSA-SB plate without growth factors (2). Serotypes 23F, 6A, 14, 6B, and 9V and a second serotype 19F strain of S. pneumoniae, serotypes reported to be isolated from middle ear fluids and commonly isolated from blood cultures (1), also supported the growth of the original middle ear fluid isolate of haemophilus on TSA-SB. Other Haemophilus biotypes (I to VI and a second serotype VII) and serotypes (a to f) also grew on TSA-SB in association with living S. pneumoniae of the various serotypes. The alpha-hemolysis of the sheep erythrocytes in the TSA-SB by pneumococci probably released the X and V factors required for the growth of H. influenzae (2).

In addition to the sample from the middle ear aspirate, we received a sputum sample from which co-isolation of H. influenzae and S. pneumoniae occurred. The penicillin MIC for the pneumococcal isolate was, by E test, >256 μg/ml on several repeated tests with a single colony of an apparently pure culture. After 48 to 72 h, white colonies of H. influenzae became visible around the S. pneumoniae on TSA-SB agar. When the S. pneumoniae was purified by growth on Columbia CNA agar with 5% sheep blood, it was found to be penicillin susceptible.

The recommended procedure for determining MICs for pneumococci is to incubate the bacteria on TSA-SB for less than 24 h (4). The fact that H. influenzae can be coisolated with pneumococci and may not be visible at 24 h could mean that a pneumococcus would appear to be highly resistant to penicillin or vancomycin. This might increase the risk for error in laboratories that rely only on TSA-SB for culturing pneumococci.

It is not known how frequently these bacteria are coisolated or whether this would have any implications for patient therapy other than inappropriate antimicrobial use, for example, unnecessary and expensive patient isolation procedures, especially in a case of invasive disease, if precautions such as those proposed for antimicrobial-resistant microorganisms are taken (3). Whenever a vancomycin-resistant or highly penicillin-resistant pneumococcus is isolated, the culture must be carefully checked to determine if it is a pure pneumococcus culture. Even cultures that do not have stray bacterial colonies within the clear zone around an optochin disk or do not have distinctly nonpneumococcus colony morphologies may contain penicillin- or vancomycin-resistant bacteria that are not pneumococci.