Characterization of penicillin-resistant Streptococcus faecium mutants

Fontana, R

doi:10.1016/0378-1097(84)90039-9

Cited by 8 publications

(12 citation statements)

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“…An additional problem with enterococci is that they are typically tolerant to ␤-lactams (i.e., MBC/MIC of Ͼ32). The major mechanism underlying this resistance has been the production of low-affinity PBP (95). Penicillin resistance is directly proportional to the amount of PBP5 (a specific PBP) produced (259).…”

Section: ␤-Lactam Resistancementioning

confidence: 99%

Vancomycin-Resistant Enterococci

Çetinkaya

Falk

Mayhall

2000

Clinical Microbiology Reviews

642

344

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Section: ␤-Lactam Resistancementioning

confidence: 99%

Vancomycin-Resistant Enterococci

Çetinkaya

Falk

Mayhall

2000

Clinical Microbiology Reviews

642

344

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“…Data indicating that spontaneous penicillin-resistant mutants of S. ftieciitm, which were shown to contain increased levels of PBP 5 (12), autolyzed more rapidly than the parent strain were interpreted to suggest that PBP 5 "...possesses both peptidoglycan polymerizing and depolymerizing activities..." (11). Canepari et al (4) proposed that PBP 5 alone is sufficient for cell growth in cells that divide at a slow rate.…”

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confidence: 99%

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

1989

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A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a 0-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a ['4C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.Streptococcus faecium ATCC 9790 (Enterococcius hirae [10]) was previously shown to possess two distinct peptidoglycan hydrolases, which differ from each other in substrate specificity, molecular mass, and, very probably, mechanism of hydrolysis (19). Muramidase-1 was isolated and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose and was shown to occur in a latent 130-kilodalton (kDa) form that can be proteolytically converted to an active 87-kDa form (20, 24). Muramidase-1 was also shown to contain covalently attached monomeric and oligomeric glucose (20) and monomeric 5-mercaptouridine monophosphate (8). In addition, muramidase-1 was shown to processively hydrolyze linear-soluble, un-crosslinked peptidoglycan chains (1).A second peptidoglycan hydrolase (muramidase-2) has been partially purified from culture supernatants by affinity binding to, and elution from, sodium dodecyl sulfate (SDS)-treated cell walls of Micrococcus luteus and has been shown to be a . It was shown to differ from muramidase-1 in substrate specificity, dissolving the cell walls of M. luteus or the acid-insoluble peptidoglycan fraction of S. faecium walls but having little ability to dissolve S. faecium cell walls. Additional data suggest that muramidase-1 and muramidase-2 are separate gene products.We have purified muramidase-2 from the insoluble pellet of disrupted S. faecium cells. Muramidase-2 was extracted with 0.02 N NaOH at 0°C, bound to the acid-insoluble peptidoglycan fraction of S. faecium, and reextracted with alkali. SDS-polyacrylamide gel electrophoresis (PAGE) of fractions highly enriched for muramidase-2 activity showed the presence of two protein bands, at 125 and 75 kDa. Upon elution and renaturation from SDS gels, both protein bands were shown to possess muramidase-2 activity. The two proteins appeared to be biochemically and immunochemically related and may represent different forms ...

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“…Described for the first time in a methicillin-resistant Staphylococcus aureus strain by Brown and Reynolds (1), lowaffinity PBPs are also produced by Streptococcus faecium and other species belonging to the enterococcus group (6,9). S. faecium strains overproducing this protein (PBP 5) show very high penicillin MICs, can grow in the presence of a penicillin concentration saturating all PBPs but the lowaffinity PBP, and stop growing in the presence of the minimal concentration of antibiotic saturating this protein (9).…”

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confidence: 99%

Transition from resistance to hypersusceptibility to beta-lactam antibiotics associated with loss of a low-affinity penicillin-binding protein in a Streptococcus faecium mutant highly resistant to penicillin

Fontana¹,

Grossato²,

Rossi³

et al. 1985

Antimicrob Agents Chemother

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Penicillin-binding protein (PBP) 5 of Streptococcusfaecium has been shown to have a very low affinity for penicillin, and this PBP was suggested to be responsible for both the natural low susceptibility and high resistance to the antibiotic in this species (R. Fontana, R. Cerini, P. Longoni, A. Grossato, and P. Canepari, J. Bacteriol. 155:1343Bacteriol. 155: -1350Bacteriol. 155: , 1983. In this study, an S. faecium mutant (Revl4) hypersusceptible to penicillin was derived from the highly resistant S. faecium R40 treated with novobiocin, and its properties were compared with those of the parent and S. faecium PS, a relatively susceptible strain from which R40 was isolated. The hypersusceptible strain did not synthesize PBP 5, but it did resemble the parent in cell morphology, growth rate, and autolytic activity. In addition, it was highly susceptible to other beta-lactams but remained as susceptible as R40 and PS to antibiotics of a different mechanism of action. The affinity of individual PBPs for the beta-lactams tested was the same in all the strains. This finding suggested that Revl4 hypersusceptibility was due to the lack of PBP 5 and strongly supported the role of this protein in the mechanism of both natural low susceptibility and high-level resistance to beta-lactams in S. faecium.The synthesis of penicillin-binding proteins (PBPs) that show a very low affinity for beta-lactams (low-affinity PBPs) appears to be a newly recognized mechanism of intrinsic resistance to these antibiotics found in several gram-positive species (1, 9, 11).Described for the first time in a methicillin-resistant Staphylococcus aureus strain by Brown and Reynolds (1), lowaffinity PBPs are also produced by Streptococcus faecium and other species belonging to the enterococcus group (6, 9). S. faecium strains overproducing this protein (PBP 5) show very high penicillin MICs, can grow in the presence of a penicillin concentration saturating all PBPs but the lowaffinity PBP, and stop growing in the presence of the minimal concentration of antibiotic saturating this protein (9). These findings have led to the conclusion that low-affinity PBPs may be responsible for the characteristic low susceptibility of enterococci to beta-lactams and, when overproduced, for the capability of these microorganisms to become highly resistant to these antibiotics, a property not shared with other streptococci (9, 12). If this suggestion is correct, S. faecium strains unable to synthesize the low-affinity PBP should demonstrate a susceptibility for beta-lactams similar to that of other streptococci.In this report we describe a mutant of S. faecuium hypersusceptible to penicillin and lacking PBP 5. The comparison of the properties of this mutant with those of strains producing a different amount of PBP S provides fuirther strong evidence of the role of the low-affinity PBP in the mechanism of resistance to beta-lactams in this species.(This work was presented, in part, at the FEMS Symposium: Bacterial morphogenesis, Marseilles, France, 5 to 7 September 198...

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Characterization of penicillin-resistant Streptococcus faecium mutants

Cited by 8 publications

References 0 publications

Vancomycin-Resistant Enterococci

Vancomycin-Resistant Enterococci

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

Transition from resistance to hypersusceptibility to beta-lactam antibiotics associated with loss of a low-affinity penicillin-binding protein in a Streptococcus faecium mutant highly resistant to penicillin

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