Penicillin tolerance in Streptococcus faecium ATCC 9790

Abstract

Tolerant strains of Streptococcus faecium had higher levels of muramidase 2 and lower levels of trypsinactivable muramidase 1 than did susceptible strains. Susceptible strains lysed faster than did tolerant strains in buffer and at some antibiotic concentrations. The addition of Triton X-100 produced equal lysis rates for susceptible and tolerant cultures.We recently reported that Streptococcus faecium ATCC 9790 was tolerant (T) of the lethal action of penicillin G as well as of that of bacitracin, vancomycin, and D-cyclOserine (8). Single-cell-colony isolates of ATCC 9790 exhibited an approximate 5% loss of tolerance. A lineage analysis over many transfers indicated that the change from tolerance to susceptibility was reversible and that susceptible (S) derivatives became tolerant at the same 5% frequency (8). The T and S cultures studied differed from each other in the extent of survival after exposure to penicillin G or to several other inhibitors of cell wall synthesis and also in the rate of penicillin-induced lysis (8). At concentrations that produced detectable lysis of S cultures, penicillin induced lysis of T cultures at a barely detectable rate. The availability of strains with a high rate of change between tolerant and susceptible states permitted an analysis of the physiological basis for tolerance. S and T derivatives recloned three times were used in this study.Cultures were grown in Todd-Hewitt medium supplemented with 0.15% glucose, and antibiotics were added in the exponential phase of growth when the cultures contained about 108 cells per ml. All glassware was acid washed. For autolysis assay cells were harvested as described previously (14) by filtration on 0.65-p.m-pore membrane filters (Millipore Corp., Bedford, Mass.), washed twice with about 5 ml (each time) of ice-cold distilled water, and suspended in 0.3 M potassium phosphate buffer (pH 6.8) (12). Autolysis rates (h-') were determined from the pseudo first-order reaction rate as described previously (14). Table 1 shows the rates of lysis of exponential cultures exposed to various antibiotics. In the case of penicillin G or D-cycloserine, S cultures lysed faster than did T cultures at antibiotic concentrations below the MIC but not at or above the MIC. For bacitracin, S cultures lysed faster than did T cultures above the MIC. Finally, for vancomycin, rates of lysis were not very different for S and T cultures examined both above and below the MIC. The data shown in Table 1 indicate that in terms of cell lysis, T strains were only relatively tolerant, since high enough concentrations of each antibiotic resulted in lysis. For any one antibiotic, lysis was not necessarily initiated near the MIC, which was determined at a much lower inoculum and after 24 h of incubation. We have shown previously that the MICs of each of the above-mentioned antibiotics for S and T derivatives are essentially indistinguishable but that the MBCs are 16-to 100-fold higher for T derivatives than for S derivatives (8).* Corresponding author. t Present address: CE...

Correlation of penicillin-induced lysis of Enterococcus faecium with saturation of essential penicillin-binding proteins and release of lipoteichoic acid

“…Autolytic mutants that are tolerant to penicillin have also been isolated from E. hirae (35); those mutants lysed slower than did wild-type strains in buffer, grew as long chains, and contained increased amounts of acylated LTA (36). Similar mutants contained less trypsin-activatable muramidase 1 (32). Since all of the strains of E. faecium used in the present study lysed fairly rapidly in buffer (data not shown), it seems unlikely that they are autolysis deficient per se.…”

“…Those investigators postulated that none of the PBPs alone were essential, although it was stated that saturation of PBPs 1, 2, and 3 caused growth inhibition. However, all such studies with E. faecium ATCC 9790 (12,18,25,32,35,36), sometimes referred to as E. faecalis (6)(7)(8)17), are not directly applicable to E. faecium, since strain ATCC 9790 is now clearly identified to the species level as Enterococcus hirae, based on physiological characteristics and PBP profile (11,44), and is referred to here as such.…”

Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin

“…Several authors have suggested that resistance to bactericidal activity of penicillin in enterococci results from a low activity of the endogenous autolytic enzyme system (7,12,16,17,21), but a relationship between autolysins and the paradoxical response to bactericidal activity of penicillin has never been shown or postulated. In our study, strains showing ML enzyme activity only but no or only poor EF enzyme activity lysed very slowly in the presence of penicillin and were PRP positive, whereas strains showing high-level EF enzyme activity lysed more extensively in the presence of penicillin and were PRP negative.…”