Methicillin resistance in staphylococci is determined by mec, composed of 50 kb or more of DNA found only in methicillin-resistant strains. mec contains mecA, the gene for penicillin-binding protein 2a (PBP 2a); mecI and mecR1, regulatory genes controlling mecA expression; and numerous other elements and resistance determinants. A distinctive feature of methicillin resistance is its heterogeneous expression. Borderline resistance, a low-level type of resistance to methicillin exhibited by strains lacking mecA, is associated with modifications in native PBPs, beta-lactamase hyperproduction, or possibly a methicillinase. The resistance phenotype is influenced by numerous factors, including mec and beta-lactamase (bla) regulatory elements, fem factors, and yet to be identified chromosomal loci. The heterogeneous nature of methicillin resistance confounds susceptibility testing. Methodologies based on the detection of mecA are the most accurate. Vancomycin is the drug of choice for treatment of infection caused by methicillin-resistant strains. PBP 2a confers cross-resistance to most currently available beta-lactam antibiotics. Investigational agents that bind PBP 2a at low concentrations appear promising but have not been tested in humans. Alternatives to vancomycin are few due to the multiple drug resistances typical of methicillin-resistant staphylococci.

Methicillin resistance in staphylococci : Molecular and biochemical basis and clinical implications

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

http://digital.csic.es/bitstream/10261/4779/1/Ayala_FemsMicroRev_234.pdf

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

Methicillin resistance in Staphylococcus aureus has been associated with alterations in the penicillin-binding proteins (PBPs). An intriguing property of all methicillin-resistant staphylococci is the dependence of resistance on the pH value of the growth medium. Growth of such bacteria at pH 5.2 completely suppressed the expression of methicillin resistance. We have examined the PBP patterns of methicillin-resistant staphylococci grown at pH 7.0. We detected a high-molecular-weight PBP (PBP-2a; approximate size, 78,000 daltons) that was only present in the resistant bacteria but not in the isogenic sensitive strain. In cultures grown at pH 5.2, the extra PBP was not detectable. Images

Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus.

SUMMARY Staphylococcus aureus, a major human pathogen, has a collection of virulence factors and the ability to acquire resistance to most antibiotics. This ability is further augmented by constant emergence of new clones, making S. aureus a “superbug.” Clinical use of methicillin has led to the appearance of methicillin-resistant S. aureus (MRSA). The past few decades have witnessed the existence of new MRSA clones. Unlike traditional MRSA residing in hospitals, the new clones can invade community settings and infect people without predisposing risk factors. This evolution continues with the buildup of the MRSA reservoir in companion and food animals. This review focuses on imparting a better understanding of MRSA evolution and its molecular characterization and epidemiology. We first describe the origin of MRSA, with emphasis on the diverse nature of staphylococcal cassette chromosome mec (SCCmec). mecA and its new homologues (mecB, mecC, and mecD), SCCmec types (13 SCCmec types have been discovered to date), and their classification criteria are discussed. The review then describes various typing methods applied to study the molecular epidemiology and evolutionary nature of MRSA. Starting with the historical methods and continuing to the advanced whole-genome approaches, typing of collections of MRSA has shed light on the origin, spread, and evolutionary pathways of MRSA clones.

Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology

Antimicrobial resistance of Staphylococcus aureus: genetic basis.

The degree of cross-linking of the peotidoglycan of Staphylococcus aureus H and mutants lacking penicillin-binding proteins 1 and 4 was studies. No major changes were ovserved in organisms lacking protein 1 whereas oloss of protein 4 was accompanied by a marked reduction in the degree of cross-linked and the absence of a membrane-bound ‘model’ transpeptidase activity. A smimlar effect was achieved when cultures of the staphylococci were treated with the β-lactam antibiotic cefoxitin. At low concentrations (0.05μg ml-1) cefoxition shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage.



The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxition and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown.



We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptidoglycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1981.tb05620.x

A Role in vivo for Penicillin-Binding Protein-4 of Staphylococcus aureus

Decreased affinity of a penicillin-binding protein for β-lactam antibiotics in a clinical isolate of Staphylococcus aureus resistant to methicillin

A mutant of Staphylococcus aureus H lacking penicillin-binding protein 4 and transpeptidase activity in vitro

The cell-wall composition and degree of cross-linking of peptidoglycan in a strain of Staphylococcus aureus (strain MR-1) which is highly resistant to methicillin were similar to those of other strains of S. aureus. When the organism was grown in the presence of very low concentrations of methicillin (equivalent to 3 x 10(-4) x minimum growth-inhibitory concentration [MGIC] there was a large decrease in the degree of cross-linking of the peptidoglycan. Increasing concentrations of methicillin (up to 1.25 x 10(-2) x MGIC) caused a further decrease in cross-linkage but thereafter a minimum value was reached. This remained unchanged even after growth of the organisms in much higher concentrations of the antibiotic up to 0.3 x minimum growth-inhibitory concentration. S. aureus MR-1 was able to grow normally for many generations under these conditions and reduction in cross-linkage of peptidoglycan was the only change detected in wall chemistry. Growth in the presence of methicillin (up to 0.3 x MGIC) (or other beta-lactam antibiotics) did not lead to an imbalance in the biosynthesis of peptidoglycan since no soluble polymers were secreted into the growth medium and nucleotide-linked precursors did not accumulate intracellularly. High concentrations of beta-lactam antibiotics (5 x MGIC) were bacteriostatic not bactericidal and this may be related to an apparent deficiency in the endogenous autolytic enzymes of strain MR-1. Studies of the penicillin-binding proteins after growth in the presence of methicillin suggest that one of these proteins remains resistant to very high concentrations of the antibiotic. We propose that this protein acts as the primary transpeptidase responsible for the incorporation of newly synthesised peptidoglycan into the growing wall.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1982.tb06907.x

Synthesis of peptidoglycan in vivo in methicillin-resistant Staphylococcus aureus.

Five penicillin-binding proteins have been isolated from detergent-solubilized membranes of Staphylococcus aureus and have been separated from other membrane proteins by covalent affinity chromatography on 6-aminopenicillanic acid-Sepharose. The PBPs were resolved by electrophoresis on SDS-polyacrylamide gels and their ability to bind radioactive penicillin assayed after transfer from the polyacrylamide gel onto nitrocellulose membrane filters. Synthesis of a β-lactam antibiotic, propionylampicillin and of [2,3-3H]propionylampicillin of high specific activity greatly facilitated the assay of penicillin-binding activity.

Anne W. Wyke

Papers

A Role in vivo for Penicillin-Binding Protein-4 of Staphylococcus aureus

Decreased affinity of a penicillin-binding protein for β-lactam antibiotics in a clinical isolate of Staphylococcus aureus resistant to methicillin

A mutant of Staphylococcus aureus H lacking penicillin-binding protein 4 and transpeptidase activity in vitro

Synthesis of peptidoglycan in vivo in methicillin-resistant Staphylococcus aureus.

Isolation of five penicillin-binding proteins from Staphylococcus aureus