Accuracy of Cefoxitin Disk Testing for Characterization of Oxacillin Resistance Mediated by Penicillin-Binding Protein 2a in Coagulase-Negative Staphylococci

ABSTRACT A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-d-thiogalactopyranoside inducer was determined. The results indicate that mecA—the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.

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Identification and Characterization of the Penicillin-Binding Protein 2a of Streptococcus pneumoniae and Its Possible Role in Resistance to β-Lactam Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1745-1748.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1745-1748 ◽

Cited By ~ 20

Author(s):

Genshi Zhao ◽

Timothy I. Meier ◽

Joann Hoskins ◽

Kelly A. McAllister

Keyword(s):

Streptococcus Pneumoniae ◽

Binding Protein ◽

Penicillin Resistance ◽

Penicillin Binding Protein ◽

Insertional Mutant ◽

Identification And Characterization

ABSTRACT To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2ainsertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for β-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for β-lactam antibiotics in S. pneumoniae.

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Identification of different clonal complexes and diverse amino acid substitutions in penicillin-binding protein 2 (PBP2) associated with borderline oxacillin resistance in Canadian Staphylococcus aureus isolates

Journal of Medical Microbiology ◽

10.1099/jmm.0.46700-0 ◽

2006 ◽

Vol 55 (12) ◽

pp. 1675-1683 ◽

Cited By ~ 29

Author(s):

Jeya Nadarajah ◽

Mark J. S. Lee ◽

Lisa Louie ◽

Latha Jacob ◽

Andrew E. Simor ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Amino Acid ◽

Binding Protein ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Clonal Type ◽

Amino Acid Sequence Variation ◽

Oxacillin Resistance ◽

Relationship Of ◽

The Relationship

Borderline oxacillin-resistant Staphylococcus aureus (BORSA) exhibit oxacillin MIC values of 1–8 μg ml−1, but lack mecA, which encodes the low-affinity penicillin-binding protein (PBP)2a. The relationship of the BORSA phenotype with specific genetic backgrounds was assessed, as well as amino acid sequence variation in the normal PBP2. Among 38 BORSA, 26 had a common PFGE profile of genomic DNA, and were multilocus sequence type (ST)25. The other isolates were genetically diverse. Complete pbp2 sequences were determined for three BORSA, corresponding to ST25, ST1 and ST47, which were selected on the basis of lacking blaZ-encoded β-lactamase. The essential transpeptidase-domain-encoding segment of pbp2 was also sequenced from seven additional ST25 isolates. Amino acid substitutions occurred in the transpeptidase domain of all BORSA, irrespective of clonal type. A Gln629→Pro substitution was common to all ST25 BORSA, but most could be distinguished from one another by additional unique substitutions in the transpeptidase domain. The ST1 and ST47 isolates also possessed unique substitutions in the transpeptidase domain. Plasmid-mediated expression of pbp2 from an ST25 or ST1 isolate in S. aureus RN6390 increased its oxacillin MIC from 0.25 to 4 μg ml−1, while pbp2 from a susceptible strain, ATCC 25923, had no effect. Therefore, different amino acid substitutions in PBP2 of diverse BORSA lineages contribute to borderline resistance. The predominant ST25 lineage was not related to any of the five clonal complexes that contain meticillin-resistant S. aureus (MRSA), suggesting that ST25 cannot readily acquire mecA-mediated resistance.

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Resistance to methicillin of coagulase-negative staphylococci (CNS) isolated from bovine mastitis

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2013-0097 ◽

2013 ◽

Vol 16 (4) ◽

pp. 687-692 ◽

Cited By ~ 4

Author(s):

M. Bochniarz ◽

W. Wawron ◽

M. Szczubiał

Keyword(s):

Binding Protein ◽

Meca Gene ◽

Bovine Mastitis ◽

Penicillin Binding Protein ◽

Coagulase Negative Staphylococci ◽

Methicillin Resistant ◽

Phenotypic Resistance ◽

Staphylococcus Warneri ◽

Staphylococcus Haemolyticus ◽

Lactam Ring

AbstractThe aim of this study was to determine the mechanisms of staphylococcal resistance to methicillin. CNS (n=100 isolates) were prepared from the mammary inflammatory secretions of 86 cows from farms located in the Lublin region.Methicillin-resistant isolates constituted 20.0% of all CNS. Staphylococcus sciuri (n=8) and Staphylococcus xylosus (n=6) were most abundant, followed by Staphylococcus chromogenes (n=3), Staphylococcus haemolyticus (n=2) and Staphylococcus warneri (n=1). The mecA gene was found in 50.0% of MRCNS (10.0% of all CNS isolates) belonging to two species: S. sciuri and S. xylosus. All mecA-positive isolates contained the protein of low affinity to penicillin (penicillin-binding protein 2a - PBP2a). The enzyme hydrolysing the β-lactam ring in antibiotics was detected in 40.0% of MRCNS; 10.0% of MRCNS isolates were characterised by the presence of the mecA gene and ability to produce β-lactamase. The remaining 20.0% of MRCNS isolates showing phenotypic resistance to methicillin were mecA gene-negative and were not able to produce β-lactamase.

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Cloning and characterization of the Pseudomonas aeruginosa pbpB gene encoding penicillin-binding protein 3

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.39.8.1871 ◽

1995 ◽

Vol 39 (8) ◽

pp. 1871-1874 ◽

Cited By ~ 16

Author(s):

X. Liao ◽

R. E. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Gene Encoding

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Cloning and Characterization of a Gene,pbpF, Encoding a New Penicillin-Binding Protein, PBP2B, inStaphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1578 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1578-1583 ◽

Cited By ~ 8

Author(s):

Hitoshi Komatsuzawa ◽

Gil H. Choi ◽

Kouji Ohta ◽

Motoyuki Sugai ◽

Monique T. Tran ◽

...

Keyword(s):

Molecular Mass ◽

Binding Protein ◽

Protein Band ◽

Binding Activity ◽

Amino Acid Residues ◽

Penicillin Binding Protein ◽

Putative Signal Peptide ◽

C Terminus ◽

Histidine Tag

ABSTRACT A previously unrecognized penicillin binding protein (PBP) gene,pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed inEscherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region ofpbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

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