Basis of chloramphenicol resistance in naturally isolated resistant staphylococci.

S. Miyamura (J. Pharm. Sci. 53:604, 1964) noted inactivation of chloramphenicol in cultures of chloramphenicol-resistant Staphylococcus aureus. Our recent findings (S. Okamoto and Y. Suzuki, Nature 208:1301, 1965) on drug-inactivating enzymes of Escherichia coli carrying R factor (E. coli R+) prompted this study. All strains were isolated from patients and supplied by S. Mitsuhashi. They are: S. aureus S 1174 CMrSMrTC', S 1047 CMrSM'TCr, and S 2126 CM8SM8TC8. [S 1174 is resistant to chloramphenicol (CM) and streptomycin (SM), and sensitive to tetracycline (TC). S 1047 is resistant to CM and TC, and sensitive to SM. S 2126 is sensitive to CM, SM, and TC.] Cells were grown in L broth and washed twice with tris(hydroxymethyl)aminomethane (Tris)-Mg-K buffer (S. Okamoto and Y. Suzuki, Nature 208:1301, 1965). The main difficulty was the disruption of staphylococcal cells. They were broken by repeated grindings plus freezings and thawings with silica sand and powdered Dry Ice, extracted by use of buffer, treated with deoxyribonuclease (5 Ag/ml), and centrifuged at 10,000 x g for 15 min. The supernatant fraction was dialyzed over night against the buffer, and was used as the crude extract. Inactivation of chloramphenicol was initially tested by the bioassay method as described in the footnotes to Table 1. A benzene extraction method (see below) was used later. Chloramphenicol was inactivated by crude extracts of S 1174 (Table 1) and S 1047 (data not shown) in the presence of acetyl coenzyme A at rates of from 4 to 60 jig of chloramphenicol per min per mg of protein, depending on the preparation. Cells grown in the presence of chloramphenicol gave a more active preparation than those grown in its absence. Whether this is due to enzyme induction or to selection of the resistant population remains to be tested. The extract of a chloramphenicol-sensitive strain S 2126 failed to inactivate the drug (Table 1). When acetyl co-