金黄色葡菌萄球耐药性监测

目的：监测成都市4家三级甲等医院临床分离的金黄色葡萄球菌（金葡菌）耐药性。方法：用琼脂稀释法测定11种抗菌药物对282株金葡菌的最低抑菌浓度（MIC）修正，并对菌株来源及科室分布进行分析。结果：282株金葡菌共筛出耐甲氧西林金葡菌（MRSA）94株，检出率为33.3％；甲氧西林 敏感金葡菌（MSSA）共188株。MSSA对青霉素和克拉霉素的耐药率分别为71.3％和54.8％，而对其抗菌药物敏感。MRSA对万古霉素、阿米卡星和利福平敏感性高（最强为万古霉素），而对其余多种抗菌药物耐药。MRSA来源最多为痰，其次为各种分泌物；科室分布依次为烧伤科、重症监护病房（ICU）、感染科等。结论：青霉素、大环内酯类药物不宜用于金葡菌感染。MRSA具有多重耐药性。MRSA感染首选糖肽类抗生素治疗。金葡菌是一种重要的病原菌，应长期进行耐药性监测，病室需做好消毒隔离。     

Mechanisms of vancomycin resistance in Staphylococcus aureus

Vancomycin is a glycopeptide antibiotic used for the treatment of Gram-positive bacterial infections. Traditionally, it has been used as a drug of last resort; however, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant S. aureus [VISA]) and more recently with high-level vancomycin resistance (vancomycin-resistant S. aureus [VRSA]) have been described in the clinical literature. The rare VRSA strains carry transposon Tn1546, acquired from vancomycin-resistant Enterococcus faecalis, which is known to alter cell wall structure and metabolism, but the resistance mechanisms in VISA isolates are less well defined. Herein, we review selected mechanistic aspects of resistance in VISA and summarize biochemical studies on cell wall synthesis in a VRSA strain. Finally, we recapitulate a model that integrates common mechanistic features of VRSA and VISA strains and is consistent with the mode of action of vancomycin.     

Triclosan and antibiotic resistance in Staphylococcus aureus

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is an antimicrobial agent used in hygiene products, plastics and kitchenware, and for treating methicillin-resistant Staphylococcus aureus (MRSA) outbreaks. S. aureus strains with low-level resistance to triclosan have emerged. It has been claimed that strains with decreased susceptibility to biocides may also be less susceptible to antibiotics. We tested the susceptibility of S. aureus clinical isolates to triclosan and several antibiotics. Triclosan MICs ranged between 0.025 and 1 mg/L. Some, but not all, strains were resistant to several antibiotics and showed low-level triclosan resistance. S. aureus mutants with enhanced resistance to triclosan (< or =1 mg/L) were isolated. In several cases this resistance was stably inherited in the absence of triclosan. These mutants were not more resistant than the parent strain to several antibiotics. Changes in triclosan MICs associated with the acquisition of a plasmid encoding mupirocin resistance were not observed, suggesting that the triclosan/mupirocin co-resistance seen in a previous study was not the result of a single resistance gene or separate genes on the same plasmid. The continuous exposure of a triclosan-sensitive S. aureus strain to sub-MIC concentrations of triclosan for 1 month did not result in decreased susceptibility to triclosan or to several antibiotics tested. Triclosan-induced potassium leakage and bactericidal effects on a triclosan-sensitive strain, a resistant strain and a strain selected for increased resistance were compared with those of non-growing organisms, exponentially growing organisms and organisms in the stationary phase. No significant differences between the strains were observed under these conditions despite their different MICs. Biocides have multiple target sites and so MICs often do not correlate with bactericidal activities. The ability of S. aureus to develop resistance to triclosan and the current view that triclosan may have a specific target in Escherichia coli, namely enoyl reductase, underline the need for more research on the mechanisms of action and resistance.     

Impact of Glycopeptide Resistance in Staphylococcus aureus on the Dalbavancin In Vivo Pharmacodynamic Target

Dalbavancin is a novel lipoglycopeptide with activity against Staphylococcus aureus, including glycopeptide-resistant isolates. The in vivo investigation reported here tested the effects of this antibiotic against seven S. aureus isolates with higher MICs, including several vancomycin-intermediate strains. Results of 1-log kill and 2-log kill were achieved against seven and six of the isolates, respectively. The mean free-drug area under the concentration-time curve (fAUC)/MIC values for net stasis, 1-log kill, and 2-log kill were 27.1, 53.3, and 111.1, respectively.     

Mechanism of quinolone resistance in Staphylococcus aureus

The resistance mechanisms to fluoroquinolones in Staphylococcus aureus were clarified by analyzing mutations in the genes encoding target enzymes, and examining the expression of the efflux pump, and determining the inhibitory activities of fluoroquinolones against the altered enzymes. Mutations in the grlA and gyrA genes of 344 clinical strains of S. aureus isolated in 1994 in Japan were identified by combinations of methods – single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing – to identify possible relationships with fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA gene in 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA gene in 188 strains (54.7%). Among these mutations, the grlA mutation of TCC → TTC or TAC (Ser-80 → Phe or Tyr) and the gyrA mutation of TCA → TTA (Ser-84 → Leu) were the principal ones, being detected in 137 (39.8%) and 121 (35.2%) isolates, respectively. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≧3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC₅₀ =50 μg/ml). Those strains with the Ser-80 → Phe or Tyr alteration in grlA, but wild type in gyrA showed a lower level of ciprofloxacin resistance (MIC₅₀≦12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of concomitant mutations at codons 73, 84, or 88 in gyrA (MIC ≦6.25 μg/ml). Sitafloxacin (DU-6859a) showed good activity in 186 of 193 isolates (96.4%), with an MIC of ≦6.25 μg/ml. The contribution of membrane-associated multidrug efflux protein (NorA) expression to fluoroquinolone resistance was clarified by the checker-board titration method for determining the MIC of norfloxacin alone and in combination with carbonyl cyanide m-chlorophenylhydrazone. Among 344 clinical isolates, 139 strains (40.4%), in which the MIC of norfloxacin varied from 1.56 to >800 μg/ml, overexpressed the NorA protein. GrlA and GrlB proteins of topoisomerase IV, and GyrA and GyrB proteins of DNA gyrase encoded by genes with or without mutations were purified separately. The inhibitory activities of fluoroquinolones against the topoisomerase IV which contained a single amino acid change (Ser → Phe at codon 80, Glu → Lys at codon 84 of grlA, and Asp → Asn at codon 432 of grlB) were from 5 to 95 times weaker than the inhibitory activities against the non-altered enzyme. These results suggest that the mutations in the corresponding genes may confer quinolone resistance; the active efflux pump, NorA, was considered to be the third quinolone-resistance mechanism. The numerous and complicated mutations seen may explain the rapid and widespread development of quinolone resistance described in S. aureus. Sitafloxacin showed good antibacterial activity against ciprofloxacin- or levofloxacin-resistant mutants because of its high inhibitory activity against both topoisomerase IV and DNA gyrase. Received: March 3, 2000 / Accepted: May 2, 2000     

Pharmacodynamic modeling of ciprofloxacin resistance in Staphylococcus aureus

Three pharmacodynamic models of increasing complexity, designed for two subpopulations of bacteria with different susceptibilities, were developed to describe and predict the evolution of resistance to ciprofloxacin in Staphylococcus aureus by using pharmacokinetic, viable count, subpopulation, and resistance mechanism data obtained from in vitro system experiments. A two-population model with unique growth and killing rate constants for the ciprofloxacin-susceptible and -resistant subpopulations best described the initial killing and subsequent regrowth patterns observed. The model correctly described the enrichment of subpopulations with low-level resistance in the parent cultures but did not identify a relationship between the time ciprofloxacin concentrations were in the mutant selection window (the interval between the MIC and the mutant prevention concentration) and the enrichment of these subpopulations. The model confirmed the importance of resistant variants to the emergence of resistance by successfully predicting that resistant subpopulations would not emerge when a low-density culture, with a low probability of mutants, was exposed to a clinical dosing regimen or when a high-density culture, with a higher probability of mutants, was exposed to a transient high initial concentration designed to rapidly eradicate low-level resistant grlA mutants. The model, however, did not predict or explain the origin of variants with higher levels of resistance that appeared and became the predominant subpopulation during some experiments or the persistence of susceptible bacteria in other experiments where resistance did not emerge. Continued evaluation of the present two-population pharmacodynamic model and development of alternative models is warranted.     

tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.     

Aminoglycosides resistance of methicillin-resistant Staphylococcus aureus]

In the last decade, there has been a dramatic increase in the isolation frequency of methicillin-resistant Staphylococcus aureus (MRSA) in Japan. Especially, a high incidence of multiple resistant MRSA strains has been reported. These strains are resistant not only to beta-lactams but to aminoglycosides and macrolides. About 90% of MRSA strains were resistant to gentamicin (GM) and/or tobramycin (TOB), producing an aminoglycoside modifying enzyme, mainly, APH (2")/AAC (6) and/or AAD (4',4"). Based on these modifying enzymes produced, MRSA strains were classified into three groups; group 1 produced APH (2")/AAC (6), belonging to phage group I and coagulase IV, group 2 produced AAD (4',4"), belonging to phage group III and coagulase II, and group 3 produced APH (2")/AAC (6) and AAD (4',4"), belonging to phage group III and coagulase II. The epidemiological results suggest that MRSA strains changed from group 1 to group 2, and then to group 3. Recently, arbekacin (ABK), a new anti-MRSA aminoglycoside, has been introduced into clinical practice. ABK shows a potent activity to GM-resistant strains, due to poorly modification by APH (2")/AAC (6'). However, there were few ABK- and GM-resistant strains in clinical isolates. These strains produced a higher amount of the enzyme than ABK-susceptible and GM-resistant strains. This observation suggests that ABK-resistant strains might be derived from GM-resistant strains by mutation of the gene coding APH (2")/AAC (6').     

金黄色葡萄球菌的耐药性分析

目的探讨金黄色葡萄球菌的耐药性，为临床治疗选药提供依据。方法应用微量稀释法测试金黄色葡萄球菌对20种抗菌药物的耐药性，并进行统计学分析。结果金黄色葡萄球菌对万古霉素的敏感率为100％，对头孢哌酮、亚胺培南、氨苄西林、哌拉西林、头孢噻吩、头孢曲松、利福平、头孢噻肟、头孢呋辛、头孢地嗪、他唑巴坦的敏感率分别为81．94％、79．17％、77．78％、75．00％、73．61％、68．06％、66．67％、61．11％、59．72％、56．94％、55．56％；对苯唑西林、复方磺胺、头孢米诺的敏感率均为44．44％；对红霉素、头孢他啶、四环素的敏感率分别为41．67％、40．28％和31．94％；对克林霉素、青霉素的耐药率均为25．00％。结论金黄色葡萄球菌对万古霉素最敏感，其次为头孢哌酮、氨苄西林、哌拉西林、利福平。合理监测各病菌的耐药性，做到合理用药，才有助于控制感染，延缓耐药。     

Mupirocin resistance in Staphylococcus aureus in an Indian hospital

Two hundred Staphylococcus aureus strains collected from an Indian hospital were tested for mupirocin susceptibility using disc diffusion method and E-test. High-level and low-level mupirocin resistance was detected in 10 (5%) and 2 (1%) S. aureus strains, respectively. Pulsed-field gel electrophoresis analysis of the high-level mupirocin-resistant methicillin-resistant S. aureus isolates revealed the presence of 2 clones with the majority of strains belonging to 1 clone, suggesting clonal dissemination.