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Logo of bmcmicrBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Microbiology
 
BMC Microbiol. 2012; 12: 69.
Published online May 8, 2012. doi:  10.1186/1471-2180-12-69
PMCID: PMC3424166
Low level ß-lactamase production in methicillin-resistant staphylococcus aureus strains with ß-lactam antibiotics-induced vancomycin resistance
Yuriko Hirao,1,2,3 Yurika Ikeda-Dantsuji,1 Hidehito Matsui,1 Masaki Yoshida,4 Seiji Hori,4 Keisuke Sunakawa,2 Taiji Nakae,1 and Hideaki Hanakicorresponding author1
1Laboratory for Antimicrobial Agents, Kitasato University, Tokyo, 108-8641, Japan
2Graduate School for Infection Control Sciences, Kitasato University, Tokyo, 108–8641, Japan
3Yamanashi Prefectural University, Kofu, 400-0062, Japan
4Department of Infectious Disease and Infection Control, Jikei University School of Medicine, Tokyo, 105-8461, Japan
corresponding authorCorresponding author.
Yuriko Hirao: y_hirao/at/yamanashi-ken.ac.jp; Yurika Ikeda-Dantsuji: dantsuji/at/insti.kitasato-u.ac.jp; Hidehito Matsui: matsui-h/at/insti.kitasato-u.ac.jp; Masaki Yoshida: myoshida/at/jikei.ac.jp; Seiji Hori: horis/at/jikei.ac.jp; Keisuke Sunakawa: sunakawa/at/med.kitasato-u.ac.jp; Taiji Nakae: nakae-tj/at/insti.kitasato-u.ac.jp; Hideaki Hanaki: hanaki/at/insti.kitasato-u.ac.jp
Received November 17, 2011; Accepted May 8, 2012.
Abstract
Background
A class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of ß-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of ß-lactam antibiotics. This means that ß-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce ß-lactamase. We hypothesised that the BIVR cells either did not harbour the ß-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining ß-lactamase activity and conducting PCR amplification of blaZ.
Results
Five randomly selected laboratory stock BIVR strains showed an undetectable level of ß-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average ß-lactamase activity of 2.59 ± 0.35 U. To test freshly isolated MRSA, 353 clinical isolates were collected from 11 regionally distant hospitals. Among 25 BIVR strains, only 16% and 8% were blaZ positive and ß-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active ß-lactamase, respectively. To know the mechanism of low ß-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of ß-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the ß-lactamase gene in the transformants was tested by PCR amplification of blaZ using 11 pairs of primers covering the entire blaZ sequence. Yield of the PCR products was consistently low compared with that using blaZ-positive non-BIVR cells. Nucleotide sequencing of blaZ in one of the BIVR transformants revealed 10 amino acid substitutions. Thus, it is likely that the ß-lactamase gene was modified in the BIVR cells to downregulate active ß-lactamase production.
Conclusions
We concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of ß-lactamase production, thereby preserving ß-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.
Keywords: Antibiotic resistance, ß-lactam, Vancomycin, Staphylococcus aureus, ß-lactamase, MRSA
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