Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus

Wei, M.-Q.; Wang, F U; Grubb, W.B.

doi:10.1099/00222615-36-3-172

Cited by 39 publications

(21 citation statements)

References 29 publications

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“…Contour-clamped homogeneous electric field analysis (32) indicated that the gene mapped to the SmaI A fragment of chromosomal DNA from 209P (data not shown).…”

Section: To 11 In the Translated Protein (T-e-n-k-g-s-s-q-p-k) Werementioning

confidence: 99%

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance

Hackbarth

Kocagoz

Kocagöz

et al. 1995

Antimicrob Agents Chemother

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In Staphylococcus aureus, penicillin-binding protein 2 (PBP 2) has been implicated in non-PBP 2a-mediated methicillin resistance. The PBP 2 gene (pbpB) was cloned from an expression library of a methicillin-susceptible strain of S. aureus (209P), and its entire sequence was compared with that of the pbpB gene from strains BB255, BB255R, and CDC6. Point mutations that resulted in amino acid substitutions near the conserved penicillin-binding motifs were detected in BB255R and CDC6, two low-level methicillin-resistant strains. Penicillin binding to PBP 2 in both BB255R and CDC6 is altered, and kinetic analysis indicated that altered binding of PBP 2 by penicillin was due to both lower binding affinity and more rapid release of bound drug. These structural and biochemical changes may contribute to the strains' resistance to ␤-lactam antibiotics.Penicillin-binding proteins (PBPs) catalyze the cross-linking of the peptidoglycan subunits in the bacterial cell wall, and their transpeptidase activity is essential for the cell's structural integrity (31). With ␤-lactamases, PBPs form a superfamily of penicillin-interacting serine D,D-peptidases. These enzymes have a lysine residue downstream from the active site serine that is required for their catalytic activity (14). This S-X-X-K sequence is a conserved motif in the transpeptidase domain of this class of enzymes. S-X-N and K(H)-T(S)-G sequences downstream of the active site serine are also conserved and are instrumental to the penicillin-binding reaction. ␤-Lactam antibiotics are structural analogs of the PBP substrate, and their mechanism of action involves covalently binding the active site of the enzyme, thus preventing its function. The sensitivity of prokaryotes to these antibiotics is primarily due to their PBPs having a high affinity for the drugs.Seven PBPs have been identified in Escherichia coli, and most have been sequenced. The low-M r E. coli PBPs (no. 4, 5, and 6) appear to have carboxypeptidase, rather than transpeptidase, activity and to be nonessential for growth (29,31). The high-M r PBPs (1A, 1B, 2, and 3) are bifunctional enzymes with a penicillin-insensitive transglycosylase domain as well as a penicillin-sensitive transpeptidase domain (15-17). ␤-Lactams exert their lethal effects by inactivating one or more of these high-M r PBPs.The genes from Streptococcus pneumoniae PBP 1A (21), Streptococcus oralis PBP 1A (21), and Bacillus subtilis PBP F (11, 25) were recently cloned, and the peptides from their translated nucleotide sequences are homologous to the transpeptidase domains of PBPs 1A and 1B from E. coli. Unlike the bifunctional enzymes in E. coli, these high-M r PBPs from gram-positive organisms do not appear to have transglycosylase activity.Four PBPs (no. 1 to 4) with M r s of 85, 81, 75, and 45, respectively, have been identified in susceptible strains of Staphylococcus aureus (8). In addition to PBPs no. 1 to 4, mecA-encoded PBP 2a is produced by methicillin-resistant staphylococci, and its production mediates most methicillin resistance ...

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“…Contour-clamped homogeneous electric field analysis (32) indicated that the gene mapped to the SmaI A fragment of chromosomal DNA from 209P (data not shown).…”

Section: To 11 In the Translated Protein (T-e-n-k-g-s-s-q-p-k) Werementioning

confidence: 99%

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance

Hackbarth

Kocagoz

Kocagöz

et al. 1995

Antimicrob Agents Chemother

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“…Chromosomal DNA was isolated as described previously [10]. The blocks were treated with Sma I restriction endonuclease (New England Biolab) according to the manufacturer's instructions.…”

Section: Chromosomal Dna Isolationmentioning

confidence: 99%

“…Several molecular epidemiological techniques have been used to type MRSA isolates. These include plasmid pro®le [8], restriction fragment-length polymorphism (RFLP) of plasmids [6,9], pulsed-®eld gel electrophoresis (PFGE) of Sma I-digested chromosomal DNA [10,11] and Southern hybridisation with various probes [12]. A combination of different methods can provide good strain discrimination [6,7,13].…”

Section: Introductionmentioning

confidence: 99%

Persistence of a clone of methicillin-resistant Staphylococcus aureus in a burns unit

Al-Haddad¹,

Udo²,

Mokadas³

et al. 2001

Journal of Medical Microbiology

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“…26 Briefly, MRSA were grown to late log phase, washed and resuspended in 50 mM EDTA buffer to a concentration of c. 6 x lo5 cells/ml. The cells were then suspended in agarose 1 YO and lysed.…”

Section: Methodsmentioning

confidence: 99%

Typing of Australian methicillin-resistant Staphylococcus aureus strains by pulsed field gel electrophoresis

Wei

Grubb²

1992

Journal of Medical Microbiology

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Summary. Twenty-six clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected from six Australian hospitals by a National Staphylococcal Study Group were examined by analysis of restriction fragment length polymorphisms (RFLPs) of chromosomal DNA with pulsed field gel electrophoresis. Digestion with the restriction endonuclease SmaI produced 13-17 bands of 7-700 kb. The digestion patterns were easily distinguished and isolates could be classified into 17 groups based on their RFLPs. Isolates giving a pattern associated with one group were from four hospitals in four different states. In another group, the isolates responsible were from three hospitals in two states and in a further group, the isolates were derived from two hospitals in different states. The remaining groups comprised only one member each. The method has promise for typing and studying the epidemiology of MRSA.

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Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus

Cited by 39 publications

References 29 publications

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance

Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance

Persistence of a clone of methicillin-resistant Staphylococcus aureus in a burns unit

Typing of Australian methicillin-resistant Staphylococcus aureus strains by pulsed field gel electrophoresis

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