RESISTANCE TO BETA-LACTAM ANTIBIOTICS AND AMINOGLYCOSIDES IN GRAM-NEGATIVE BACTERIA .1. MOLECULAR AND GENETIC CHARACTERIZATION OF R-FACTORS

  • 1 January 1976
    • journal article
    • research article
    • Vol. 234  (3) , 371-383
Abstract
With the frequent use of aminoglycoside antimicrobials and .beta.-lactam antibiotics in hospitals in the last few years, the number of bacterial strains resistant to these chemotherapeutics increased. Lately, strains of Escherichia coli, Klebsiella, Enterobacter, Serratia [Ser.], Proteus and Pseudomonas [Ps.] resistant to many antimicrobials (ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamycin, tobramycin, sisomycin, neomycin, paromomycin, kanamycin, streptomycin, spectinomycin, tetracycline, sulfonamides) were isolated from patients of the university hospital in Zuerich [Switzerland]. The resistant phenotype of 2 representative strains (K. pneumoniae 1 and Ser. marcescens 2) could be transferred by mixed cultivation to E. coli K-12. Multiple resistance of strain 1, in addition, could be transferred to Salmonella typhimurium, Ser. marcescens, Providencia, P. mirabilis and K. pneumoniae in varying frequencies. Transfer to Ps. aeruginosa could not be achieved. Spontaneous instability of resistance was observed in 0.15% of the cells of an overnight broth culture and in 90% of the cells of a 3 mo. old culture. Conjugation, instability and the response to the sex phages MS-2 and If-1 suggested that resistance was mediated by a monomolecular R-factor, belonging to the fi+-type. This suggestion was confirmed by molecular characterization of the resistance plasmids. After transfer of the R-factors of K. pneumoniae 1 (R-FK1) and Ser. marcescens 2 (R-FK2) into E. coli K-12, plasmid DNA was labeled with (Methyl-3H)thymidine and isolated by isopycnic centrifugation in cesium chloride-ethidium bromide. Analysis of plasmid DNA then was carried out by sedimentation in a 5-20% neutral sucrose gradient together with reference plasmids of known MW and sedimentation constants. The analysis revealed that R-FK1 had a MW of 54 .times. 106 and R-FK2 of 50 .times. 106 daltons. The values were confirmed by contour length measurements of open circular forms with EM. A comparison of the sedimentation profile of labeled plasmid DNA from strain 1 and 14C-labeled DNA of E. coli K-12 (R-FK1) showed that the wild-type strain contained, besides the large resistance plasmid, at least 2 smaller cryptic plasmids. These smaller plasmid molecules were also found in antibiotic susceptible variants of strain 1, which did not contain the 54 .times. 106 dalton plasmid molecule, responsible for the resistant phenotype. The number of copies of R-FK1 in E. coli K-12 was determined to be 2, indicating stringent control of replication. It is discussed that the growing number of isolations of strains of Escherichia, Klebsiella, Serratia, Proteus, Providencia and Pseudomonas, exhibiting the same resistance phenotype, results from the spread of the R-factor described above among the hospital bacterial flora.

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