Enzymic hydrolysis of erythromycin by a strain of Escherichia coli. A new mechanism of resistance.

Abstract

Escherichia coil BM2195 is highly resistant to erythromycin by inactivation of the antibiotic. We have determined the structure of the modified antibiotic by physico-chemical techniques including mass spectrometry, infrared spectrophotometry, 13C nuclear magnetic resonance, and circular dichroism.The results obtained indicate that E. coli BM2195 resists erythromycin by the production of an erythromycin esterase which hydrolyzes the lactone ring of the antibiotic.Escherichia coli, like most Gram-negative bacteria, is resistant to "low levels" of macrolides (minimal inhibitory concentrations (MICs) <250 1+g/m1) . We recently described an E. coil strain, BM2195, resistant to high-levels (MIC > 2 mg/ml) of erythromycin and demonstrated that this new resistant phenotype was due to the constitutive synthesis of a plasmid-mediated erythromycin-modifying enzyme1).The genetic information located on the plasmid was transferred to E. coil BM694, and, using this strain, the modified antibiotic was isolated and its structure was determined; the resistance involves the production of an erythromycin esterase which hydrolyzes the lactone ring of the antibiotic. Materials and MethodsBacteria] Strains and Plasmids E. coil BM2506 which is highly resistant to erythromycin (MIC > 1 mg/ml) and does not inactivate the antibiotic was a clinical isolate, identified and kindly provided by A. ANDREMONT, Institut GustaveRoussy, Villejuif. E. coli BM694, prototroph, gyrA2) and E. coli BM694/pAT63 were from our laboratory collection. Hybrid plasmid pAT63 consists of pBR322 (Tra-, Mob-, ApR, TcR) with a 1,660bp Sau3a insert of plasmid pIPI 100 DNA encoding the erythromycin esterase3). MediaBrain-heart infusion broth and agar (Difco) were used. Disc agar diffusion susceptibility tests were done on Mueller-Hinton agar. All incubations were at 37°C. Antibiotic Erythromycin base was provided by Roussel-Uclaf. Inactivation of Erythromycin by Resting CellsCells from 1 liter of an exponential broth culture of E. coil BM694/pAT63, at approximately 3 x 10° bacteria/ml, were harvested, washed once in 0.1 M phosphate buffer (pH 7.0 or 8.0), resuspended in 1 liter of the same buffer containing I mg/mi of erythromycin, and incubated for 3 hours at 37°C. The pH of this suspension, which remained constant, was monitored and inactivation of erythromycin was

Antimicrobial resistance of thermophilic Campylobacter

“…Resistance to macrolides can be based on different mechanisms: target modification by point mutation or methylation of 23S rRNA, thereby inhibiting the binding of macrolides [76], hydrolysis of the lactone ring in the macrolide [5], and efflux pumps removing the macrolide from the bacteria [60]. In Campylobacter it has been shown that resistance is not consistent with the presence of rRNA methylase, with modification of the antibiotic or with efflux [79].…”

Purification and characterization of macrolide 2'-phosphotransferase from a strain of Escherichia coli that is highly resistant to erythromycin

“…Colonization of the intestinal tract by members of the family Enterobacteriaceae that are highly resistant to erythromycin is usually associated with previous long-term oral therapy with the drug (2-4). It has been reported that high-level resistance to erythromycin in members of the family Enterobacteriaceae results from production of erythromycin esterases and rRNA methylases (1,5,(6)(7)(8)24).We reported previously that the structure of inactivated oleandomycin generated by erythromycin-resistant (MIC, 1,600 ,ug/ml) Escherichia coli Tf481A, which was isolated from a patient in Japan in 1983, was oleandomycin 2'-phosphate (22). This paper describes the purification and characterization of a new macrolide-inactivating enzyme, macrolide 2'-phosphotransferase [MPH(2')].…”

“…Colonization of the intestinal tract by members of the family Enterobacteriaceae that are highly resistant to erythromycin is usually associated with previous long-term oral therapy with the drug (2-4). It has been reported that high-level resistance to erythromycin in members of the family Enterobacteriaceae results from production of erythromycin esterases and rRNA methylases (1, 5,[6][7][8]24).…”

Role of an energy-dependent efflux pump in plasmid pNE24-mediated resistance to 14- and 15-membered macrolides in Staphylococcus epidermidis

“…7). Addition of 1 pug of erythromycin per ml had no effect on [14C]niddamycin (4 However, inasmuch as previously published data on strain of medium and suspended in fresh medium at 37°C. 958-2 (20) left open the possibility that ribosome modifica-1 suspension media for ii and iii contained 1 ,ug of tion, intracellular macrolide degradation, and/or reduced ycin per ml.…”