Simplified technic of determining the Enterobacteriaceae R-plasmid incompatibility groups suitable for epidemiologic studies
Belokrysenko, S.S.
Antibiotiki 26(7): 509-514
1981
ISSN/ISBN: 0003-5637 PMID: 7023377 Document Number: 175578
An economical technique for classification of newly isolated R plasmids according to the incompatibility groups with the use of a set of standard test-plasmids is described. The plasmid under study is transferred into E. coli P678 nal-r or C600 rif-r and crossed with the carrier strains sensitive to nalidixic acid and rifampicin of the standard test plasmids. The crossed strains are subjected to mixed cultivation in separate sectors of the dishes with full-value agar. After incubation the bacterial growth is transferred from the sectors with a velvet replicator onto the agar containing rifampicin or nalidixic acid and antibiotics for isolation of transconjugants carrying the standard test-plasmids with different resistance markers in addition to the plasmid being studied. The transconjugants are incubated and additionally twice replicated onto an analogous fresh selection medium with incubation each time for 6-16 hours at 37 degrees C. In case of incompatibility with one of the standard plasmids the plasmid being studied segregates in the transconjugant in the respective sectors during the replications and growth. Segregation of the plasmid is detected when the transconjugant is replicated from the sectors of the third selection dish onto the agar with the antibiotics, resistance to which is controlled by the R plasmid being studied. The growth is determined until the 4th-6th hour of growth at 37 degrees C. After that period retardation in the growth of the transconjugants is most evident in the sector where segregation of the plasmid being studied takes place. The transconjugant growth in the other sectors is used as control.