Low level β-lactamase production in methicillin-resistant staphylococcus aureus strains with β-lactam antibiotics-induced vancomycin resistance

BMC Microbiology

Hideaki Hanaki¹  Taiji Nakae¹  Keisuke Sunakawa⁴  Seiji Hori²  Masaki Yoshida²  Hidehito Matsui¹  Yurika Ikeda-Dantsuji¹  Yuriko Hirao³ 

[1] Laboratory for Antimicrobial Agents, Kitasato University, Tokyo 108-8641, Japan;Department of Infectious Disease and Infection Control, Jikei University School of Medicine, Tokyo 105-8461, Japan;Yamanashi Prefectural University, Kofu 400-0062, Japan;Graduate School for Infection Control Sciences, Kitasato University, Tokyo 108–8641, Japan

关键词: MRSA;    β-lactamase;    Staphylococcus aureus;    Vancomycin;    β-lactam;    Antibiotic resistance;   

Others  :  1221956
DOI  :  10.1186/1471-2180-12-69

 received in 2011-11-17, accepted in 2012-05-08,  发布年份 2012

【 摘 要 】

Background

A class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of β-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of β-lactam antibiotics. This means that β-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce β-lactamase. We hypothesised that the BIVR cells either did not harbour the β-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining β-lactamase activity and conducting PCR amplification of blaZ.

Results

Five randomly selected laboratory stock BIVR strains showed an undetectable level of β-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average β-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 β-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active β-lactamase, respectively. To know the mechanism of low β-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of β-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the β-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 β-lactamase gene was modified in the BIVR cells to downregulate active β-lactamase production.

Conclusions

We concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of β-lactamase production, thereby preserving β-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.

【 授权许可】

   
2012 Hirao et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Sakai F, Hanaki H, Barada K, Hirao Y, Inamatsu T, Nakae T, Sunakawa K: A 25-year trace of methicillin-resistant Staphylococcus aureus dissemination in a geriatric hospital in Japan. Internl J Gen Med 2010, 3:399-405.
• [2]Valaperta R, Tejada MR, Frigerio M, Moroni A, Ciulla E, Cioffi S, Capelli P, Costa E: Staphylococcus aureus nosocomial infections: the role of a rapid and low-cost characterization for the establishment of a surveillance system. New Microbiol 2010, 33:223-232.
• [3]Boucher H, Miller LG, Razonable RR: Serious infection caused by methicillin-resistant Staphylococcus aureus. Clin Infect Dis 2010, 51(Suppl 2):S183-S179.
• [4]Perkins HR: Specificity of combination between mucopeptide precursors and vancomycin or ristocetin. Biochem J 1969, 111:195-205.
• [5]Perkins HR, Nieto M: The chemical basis for the action of the vancomycin group of antibiotics. Ann NY Acad Sci 1974, 235:348-363.
• [6]Cunha BA: Vancomycin revisisted: a reappraisal of clinical use. Crit Care Clin 2008, 24:393-420.
• [7]Totsuka K, Shiseki M, Kikuchi K, Matsui Y: Combined effects of vancomycin and imipenem against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo. J Antimicrob Chemother 1999, 44:455-460.
• [8]Shimizu K, Orizu M, Kanno H, K S, Konishi T, Soma K, Nishitani H, Noguchi Y, Hasegawa S, Hasegawa H, et al.: Clinical studies on vancomycin in the treatment of MRSA infection (article in Japanese). Jpn J Antibiot 1996, 49:782-799.
• [9]Hanaki H, Yamaguchi Y, Barata K, Sakai H, Sunakawa K: Improved method of detection of β-lactam antibiotic-induced VCM-resistant MRSA (BIVR). Intl J Antimicrob Agents 2004, 23:311-313.
• [10]Hanaki H, Yamaguchi Y, Yanagisawa C, Uehara K, Matsui H, Yamaguchi Y, Hososaka YH, Barada K, Sakai F, Itabashi Y, et al.: Investigation of β-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR). J Infect Chemother 2005, 11:104-106.
• [11]Hanaki H, Kuwahara-Arai K, Boyle-Vavra S, Daum RS, Labischinski H, Hiramatsu K: Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J Antimicrob Chemother 1998, 42:199-209.
• [12]Jacobs C, Huang L, Bartowsky E, Normark S, Park JT: Bacterial cell wall recycling provides cytosolic muropeptides as effectors for β-lactamase induction. EMBO J 1994, 13:4684-4694.
• [13]Yanagisawa C, Hanaki H, Matsui H, Ikeda S, Nakae T, Sunakawa K: Rapid depletion of free vancomycin in medium in the presence of β-lactam antibiotics and growth restoration in Staphylococcus aureus strain with β-lactam-induced vancomycin resistance. Antimicrob Agents Chemother 2009, 53:63-68.
• [14]Jacobs C: Life in the Balance:Cell walls and antibiotic resistance. Science 1997, 278:1731-1732.
• [15]Lowy FD: Antimicrobial resistance: the example of Staphylococcus aureus. J Clinl Invest 2003, 111:1265-1273.
• [16]Hartman BJ, Tomasz A: Low-affinity penicillin-binding protein associated with, β-lactam resistance in Staphylococcus aureus. J Bacteriol 1984, 158:513-516.
• [17]Pinho MG, de Lencastre H, Tomasz A: An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci. Proc Nat Acad Sci 2001, 98:10886-10891.
• [18]Utsui Y, Yokota T: Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob Agents Chemother 1985, 28:397-403.
• [19]Martineau F, Picard FJ, Lansac N, Menard C, Roy PH, Ouellette M, Bergeron MG: Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 2000, 44:231-238.
• [20]Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S, Fukuchi Y, Kobayashi I: Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997, 350:1670-1673.
• [21]Kuroda M, Ohota T, Uchiyama I, Baba T, Yuzawa H, Kobayasi I, Cui L, Oguchi A, Aoki K, Nagai Y, et al.: Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 2001, 357:1225-1240.
• [22]Hanaki H, Yamaguchi Y, Nomura S, Haraga I, Nagayama A, Sunakawa K: Method of detecting β-lactam antibiotic induced vancomycin resistant MRSA (BIVR). Intl J Antimicrob Agents 2004, 23:1-5.
• [23]O’Callaghan CH, Morris A, Kirby SM, Shingler AH: Novel method for detection do β-lactamases by using a chromogenic chephalosporin substrate. Antimicrob Agents Chemother 1972, 1:283-288.
• [24]Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing; 15th informantional supplement. CLSI/NCCLS document M100-S15 Wayne, PA. Wayne PA, USA: Clinical and Laboratory Standards Institute; 2005.