Characterization of a Plasmid Determining Resistance to Erythromycin, Lincomycin, and Vernamycin B _α in a Strain of Streptococcus pyogenes

Abstract

A plasmid determining resistance to erythromycin, lincomycin, and vernamycin Ba was isolated from a strain of Streptococcus pyogenes. The plasmid has a molecular weight of approximately 17 x 108 and is present to the extent of one to two copies per chromosomal genome equivalent.Erythromycin and lincomycin are antibiotics used in the treatment of group A beta-hemolytic streptococcus (Streptococcus pyogenes) infections in patients for whom penicillin is contraindicated. S. pyogenes strains are usually susceptible to these agents, although resistant clinical isolates have been reported (7,8,10,12,13,15). Whether resistance in this species is related to extrachromosomal (plasmid) determinants, as is often the case among drug-resistant staphylococci and enteric bacilli, has, to our knowledge, not been reported. In this communication we report on the isolation and characterization of a plasmid from a clinical isolate of S. pyogenes and show that it determines resistance to erythromycin, lincomycin, and vernamycin Ba (a streptogramin B-type antibiotic).S. pyogenes strain 10535/72 (designated here as AC-1), serotype M22: T12, was originally isolated by J. Dixon in Alberta, Canada. This strain, which was isolated from the inflamed throat of a 17-year-old Cree Indian woman, is resistant to erythromycin and lincomycin (greater than 1 mg/ml in each case), as well as vernamycin B, (no zone of inhibition with susceptibility disks containing 20 ug of drug). The patient had not been treated with antibiotics during the previous 12 months but had been in recent contact with a member of her family who was on erythromycin therapy.Todd-Hewitt broth (THB; Difco) was used as a growth medium in both liquid and agar forms. Cell growth (at 37 C) was followed by measuring turbidity with a Klett-Summerson colorimeter.Materials and methods of plasmid analyses by dye-buoyant density and sucrose density centrifugation were as described in detail previously (3, 4), as were the methods of plasmid elimination with intercalating dyes ("curing") and electron microscopy analysis (5). Extraction of deoxyribonucleic acid (DNA) was performed as follows. A 200-ml log phase culture isotopically labeled with 1 mCi of [methyl-PH]-thymidine (22 Ci/mmol from Amersham/Searle) was washed with 15 ml of 0.03 M tris(hydroxymethyl)aminomethane-0.005 M Na2 ethylenediaminetetraacetic acid (EDTA)-0.05 M NaCl, pH 8.0, (TES) and resuspended in 3.0 ml of 25% glucose (in TES). A 0.5-ml quantity of 0.25 M EDTA (pH 8.0) was added, followed by the addition of 1.0 ml of lysozyme (5 mg/ml in TES). The suspension was allowed to incubate for 3 h at 37 C, at which point 0.5 ml of Pronase (5 mg/ml in TES, preincubated at 37 C for 30 min) was added, followed by another 30-min period of incubation. The cells were then lysed by the addition of 2.0 ml of sodium dodecyl sulfate (a 2% solution in TES). NaClO, (from a 5 M solution) was added to make a final concentration of 1 M. The lysate was then twice extracted with an equal volume of TES-saturated phenolchloroform (2: 1). Two volum...

Characterization of a Small Plasmid Determining Resistance to Erythromycin, Lincomycin, and Vernamycin B α in a Strain of Streptococcus sanguis Isolated from Dental Plaque

“…In several cases, the molecular masses of these plasmids are similar, with plasmid DNA being of a size of approximately 17 megadaltons (6,9,13,26). In one case we showed that plasmid pAM,8l from S. faecalis strain DS-5 was approximately 95% homologous with a plasmid of similar size in S. pyogenes strain AC-1 (36 …”

“…pyogenes (6,25,28), and a group B streptococcus (20). In several cases, the molecular masses of these plasmids are similar, with plasmid DNA being of a size of approximately 17 megadaltons (6,9,13,26).…”

Resistance to Oleandomycin in Streptomyces antibioticus, the Producer Organism

“…Resistance to this antibiotic in Escherichia coli has been found to be due to alterations in either of the ribosomal proteins L4 or L22 (Gale et al, 1981) and, more recently, to enzymic hydrolysis of the antibiotic (Barthelemy ut al., 1984). In clinical isolates of Staphylococcus aureus (Lai & Weisblum, 1971) and of Streptococcus pyogenes (Clewell & Franke, 1974), a relationship has been demonstrated between resistance to erythromycin and a characteristic pattern of coresistance to other macrolides, lincosamides and B-type streptogramins, which was termed the MLS-phenotype. This phenotype has been shown to depend on the presence of N6-dimethyladenine in 23s ribosomal RNA (Lai et al, 1973).…”

Tales of conjugation and sex pheromones

“…31 Another undergraduate student, Art Franke identified an erythromycin resistance plasmid in Streptococcus pyogenes, an important human pathogen. 32 And we eventually found plasmids also in Streptococcus faecalis 33 and S. sanguis. 34 As discussed below there was much to get excited about regarding where some of these new findings would take us, and within a few years our work with ColE1 came to an end.…”