Emergence of the M Phenotype of Erythromycin-Resistant Pneumococci in South Africa
Figure. Number of erythromycin-resistant blood and cerebro- spinal fluid isolates of pneumococci. DNA was extracted from pneumococcal isolates by using a lysis solution consisting of 0.1% sodium deoxycholate as described in (11), except that we used plate rather than broth cultures.
Seventy-eight MLS strains were probed for the ermAM gene by using dot blots. The probe (supplied by P. Courvalin, Pasteur Institute, Paris, France) (Escherichia coli JM83/pUC19 560bp Ssp1 intragenic fragment of ermB) was labeled with digoxygenin by using random primed labeling (DIG DNA Labeling and Detection Kit; Boeringer, Mannheim, Germany). Hybridization and detection were performed following manufacturer's instructions (DIG DNA Labeling and Detection Kit; Boeringer, Mannheim, Germany). PCR was also used to detect ermAM in 30 strains according to standard conditions, with an annealing temperature of 58°C. We used the following primers: forward primer, 5'-CGAGTGAAAAAGTACTCAACC, reverse primer, 5'-GGCGTGTTTCATTGCTTGATG). Published primers for the mefE gene (5'-AGTATCATTAATCACTAGTGC, and 5'-TTCTTCTGGTACTAAAAGTGG) (12) were used to detect mefE through PCR amplification in 13 M strains. Amplification was performed in a Perkin Elmer Cetus DNA Thermal Cycler under standard reaction conditions, with an annealing temperature of 56oC.