葡萄球菌溶酶对h-VRSA溶菌作用观察

目的从金黄色葡萄球菌临床分离菌株中检测异质性万古霉素耐药金黄色葡萄球菌(heterogeneous vancom yc in-res istan t staphy lococcus aureus,h-VRSA),并考察葡萄球菌溶酶对h-VRSA体外杀菌作用。方法采用E-test法从金黄色葡萄球菌临床分离菌株中筛选h-VRSA,采用棋盘稀释法考察葡萄球菌溶酶单独或联合万古霉素对h-VRSA体外杀菌作用。结果从30株金黄色葡萄球菌临床分离菌株中检测出2株h-VRSA(6.7%),葡萄球菌溶酶联合万古霉素对h-VRSA溶菌作用的部分抑菌浓度指数:F IC<0.5。结论金黄色葡萄球菌临床分离菌株中有h-VRSA存在,其对万古霉素表现出的异质性耐药不容忽视;葡萄球菌溶酶和万古霉素之间有良好的协同作用,其临床应用价值值得进一步探讨。     

金黄色葡萄球菌对万古霉素敏感性试验方法评价

目的比较不同方法检测金黄色葡萄球菌对万古霉素敏感性试验结果。方法将1株从临床标本中分离的异质性耐万古霉素的金黄色葡萄球菌(h-VRSA)在含有不同浓度的万古霉素培养基上连续转种诱导,得到一系列对万古霉素不同程度耐药的菌株,分别用琼脂筛选法、微量肉汤稀释法、E-test法、纸片扩散法和仪器法对万古霉素的敏感性进行检测。结果琼脂筛选法、微量肉汤稀释法和E-test法可以检测出金黄色葡萄球菌对万古霉素中介耐药(VISA),而纸片扩散法和仪器法则不能检出VISA。结论微量肉汤稀释法和E-test法是检测金黄色葡萄球菌对万古霉素敏感性可接受的方法。若以纸片扩散法和仪器法为常规药敏试验的实验室,应增加含6μg／ml的万古霉素脑心浸液琼脂进行筛查,以加强对VRSA和VISA的监测。     

两株对万古霉素敏感性减低金黄色葡萄球菌的透射电镜下超微结构观察

目的 对前期课题筛选出的万古霉素中介金黄色葡萄球菌（vancomycin intermediate Stapylococcus aureus,VISA）及异质性耐万古霉素金黄色葡萄球菌（heterogeneous vancomycin resistant Stapylococcus aureus，hetero-VISA）进行透射电镜下的超微结构观察。方法 用KB法、琼脂稀释法和E-test 法、菌谱分析法对分离出的VISA 和h-VISA 进行确认，并用透射电镜法对其进行超微结构观察。结果 检测到的VISA 和h-VISA 经透射电镜观察发现菌株较标准菌株细胞壁增厚，细胞表面粗糙。结论 VISA 和h-VISA 等万古霉素敏感性减低金黄色葡萄球菌对万古霉素耐药很可能是由于细菌细胞壁增厚所引起的。     

临床分离金黄色葡萄球菌耐药性分析及治疗对策

目的调查金黄色葡萄球菌耐药现状,指导临床合理用药。方法采用ATB分析仪进行细菌鉴定,K—B法做体外药敏试验。结果耐甲氧西林金黄色葡萄球菌（MRsA）检出率为60．5％,药敏结果显示MRSA耐药率明显高于甲氧西林敏感金黄色葡萄球菌（MSSA）,未检出对万古霉素及替考拉宁耐药菌株。结论金黄色葡萄球菌耐药严重,临床应根据药敏结果合理使用抗生素。糖肽类抗生素（万古霉素、替考拉宁）可作为多重耐药金黄色葡萄球菌重症感染的首选抗生素。     

74株溶血葡萄球菌重新鉴定的结果分析

目的 调查近年来临床标本中溶血葡萄球菌迅速增多,而金黄色葡萄球菌显著减少的原因。方法 使用生物化学和分子生物学的方法对临床分离的74株溶血葡萄球菌重新进行鉴定。结果 74株溶血葡萄球菌经传代培养后,有6株的血浆凝固酶、甘露糖和甘露醇等试验由阴性转变为阳性iPCR检测后,有15株细菌显示金黄色葡萄球菌种特异性基因（nuc和coa）阳性。结论 临床标本中葡萄球菌菌种构成比的变迁与部分细菌生化反应改变而导致的菌种鉴定错误有关,使用某些作用于细胞壁的抗生索可能是其原因之一。     

耐万古霉素金黄色葡萄球菌的耐药机制和检测方法

耐甲氧西林金黄色葡萄球菌(MRSA)是引起医院感染的多重耐药菌，其有效的治疗药物为万古霉素。近年来已经发现对万古霉素敏感性降低的葡萄球菌，尤其是异质性万古霉素耐药金黄色葡萄球菌(hetero-VRSA)和万古霉素中介耐药金黄色葡萄球菌(VISA)的检出。在医学界引起关注。一旦herero-VRSA和VISA发展为耐万古霉素金黄色葡萄球菌(VRSA)。临床将面临无药可选择的被动局面。如果应用万古霉素治疗MRSA失败。临床医生应该考虑是否有herero-VRSA或VISA的存在。因此，对VRSA的耐药机制以及hetero-VRSA和VISA的检测方法研究具有重要的意义。     

耐万古霉素金黄色葡萄球菌的耐药机制和检测方法

耐甲氧西林金黄色葡萄球菌 (MRSA)是引起医院感染的多重耐药菌 ,其有效的治疗药物为万古霉素。近年来已经发现对万古霉素敏感性降低的葡萄球菌 ,尤其是异质性万古霉素耐药金黄色葡萄球菌 (hetero VRSA)和万古霉素中介耐药金黄色葡萄球菌 (VISA)的检出 ,在医学界引起关注。一旦hetero VRSA和VISA发展为耐万古霉素金黄色葡萄球菌 (VRSA) ,临床将面临无药可选择的被动局面。如果应用万古霉素治疗MRSA失败 ,临床医生应该考虑是否有hetero VRSA或VISA的存在。因此 ,对VRSA的耐药机制以及hetero VRSA和VISA的检测方法研究具有重要的意义     

异质性万古霉素中介金黄色葡萄球菌的检测与分析

目的了解异质性万古霉素中介耐药金黄色葡萄球菌（h-VISA）在本院的感染情况，探讨不同最低抑菌浓度（MIC）的原代菌株间h-VISA检出率的差异。方法采用琼脂稀释法检测甲氧西林耐药的金黄色葡萄球菌（MRSA）对万古霉素的MIC值，用菌群分析法筛选h—VISA，计算h-VISA的检出率。结果45株MRSA中共检出h-VISA 7株，检出率为15．6％，原代菌对万古霉素MIC值越高，h-VISA检出率越高。结论本院存在h-VISA感染情况，应引起一定重视，系统地监测h-VISA的流行情况，对指导临床正确使用万古霉素，防止VISA和VRSA的出现有重要意义。     

临床常见病原菌分布及耐药性分析

目的分析本院2006年临床常见病原菌分布及其耐药性。方法分离培养得到病原菌,用Micro Scan Walk Away40微生物分析仪进行细菌鉴定和药敏试验。结果革兰阳性球菌占40．2％,革兰阴性杆菌占59．8％;前6位依次为大肠埃希菌、溶血葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、表皮葡萄球菌、金黄色葡萄球菌。葡萄球菌中未发现对万古霉素耐药的菌株;革兰阴性杆菌中未发现对亚胺硫霉素耐药的菌株。结论细菌耐药呈上升趋势,多重耐药日趋严重。临床应重视细菌耐药性问题,根据具体菌种及药敏结果合理使用抗生素。     

6年中院内临床葡萄球菌检出概况和统计

目的了解临床葡萄球菌感染和耐药状况，指导临床治疗。方法采用分离培养基分离培养细菌，用常规鉴定程序鉴定葡萄球菌并做药物敏感实验。结果检出316株葡萄球菌，我院临床近6年表皮葡萄球菌检出最高135株，其次为金黄色葡萄球菌和溶血葡萄球菌。药敏结果显示葡萄球菌对利福平、万古霉素外的大多数抗生素高度耐药（48.1％～70.3％），万古霉素无耐药株，利福平耐药率为10．1％。结论合理、科学应用抗生素应当成为我们治疗细菌感染遵循的原则。     

Vancomycin-intermediate Staphylococcus aureus strains have impaired acetate catabolism: implications for polysaccharide intercellular adhesin synthesis and autolysis

The most common mechanism by which Staphylococcus aureus gains resistance to vancomycin is by adapting its physiology and metabolism to permit growth in the presence of vancomycin. Several studies have examined the adaptive changes occurring during the transition to vancomycin-intermediate resistance, leading to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin. In the present study, we identified metabolic changes common to vancomycin-intermediate S. aureus (VISA) strains by assessing the metabolic and growth characteristics of two VISA strains (vancomycin MICs of 8 microg/ml) and two isogenic derivative strains with vancomycin MICs of 32 microg/ml. Interestingly, we observed the parental strains had impaired catabolism of nonpreferred carbon sources (i.e., acetate), and this impairment became more pronounced as vancomycin resistance increased. To determine if acetate catabolism impairment is common to VISA strains, we assessed the ability of VISA and vancomycin-sensitive S. aureus (VSSA) clinical isolates to catabolize acetate. As expected, a significantly greater percentage of VISA strains (71%) had impaired acetate catabolism relative to VSSA (8%). This is an important observation because staphylococcal acetate catabolism is implicated in growth yield and antibiotic tolerance and in regulating cell death and polysaccharide intercellular adhesin synthesis.     

Mutated response regulator graR is responsible for phenotypic conversion of Staphylococcus aureus from heterogeneous vancomycin-intermediate resistance to vancomycin-intermediate resistance

Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.     

Occurrence of vancomycin-intermediate-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Japan

The Clinical and Laboratory Standards Institute (CLSI) amended the criteria for vancomycin susceptibility and resistance of Staphylococcus aureus in 2006. The earlier criteria had established that S. aureus with minimum inhibitory concentrations (MICs) of vancomycin of < or =4 microg/ml, 8 to 16 microg/ml, and > or =32 microg/ml were vancomycin-susceptible, -intermediate-resistant and -resistant, respectively. The revised recommendation states that bacteria showing vancomycin MICs of < or =2 microg/ml, 4 to 8 microg/ml, and > or =16 microg/ml are -susceptible, -intermediate-resistant, and -resistant, respectively. We examined, based on these new criteria, the vancomycin susceptibility of methicillin-resistant S. aureus (MRSA) strains isolated in Japan from 1978 through 2005 at 17 general hospitals. The results showed that, among 2446 MRSA isolates tested, 8 were classified as intermediate-vancomycin-resistant (VISA). Re-examination of vancomycin susceptibility in these 8 strains in 2006 revealed that 6 strains showed a vancomycin MIC of 4 microg/ml, as tested by the agar dilution method, broth dilution methods, and E-test; the 2 other strains had lost the vancomycin resistance. Pulsed-field gel electrophoresis (PFGE) of the chromosomal DNA of these strains exhibited five unique profiles; 2 strains isolated from the same hospital were identical. These results revealed that at least five different types of VISA strains could be identified in Japan so far according to the new CLSI criteria. All these VISA strains had type II staphylococcal cassette chromosome, mec. This study revealed, for the first time in Japan, the presence of intermediate vancomycin-resistant MRSA in this country.     

Heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) emerged before the clinical introduction of vancomycin in Japan: a retrospective study

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190-6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK, clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.     

Novel mechanism of antibiotic resistance originating in vancomycin-intermediate Staphylococcus aureus

As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41:5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.     

In vitro activities of LTX-109, a synthetic antimicrobial peptide, against methicillin-resistant, vancomycin-intermediate, vancomycin-resistant, daptomycin-nonsusceptible, and linezolid-nonsusceptible Staphylococcus aureus

LTX-109 and eight other antimicrobial agents were evaluated against 155 methicillin-resistant Staphylococcus aureus (MRSA) isolates, including strains resistant to vancomycin and strains with decreased susceptibility to daptomycin and linezolid, by microdilution tests to determine MICs. Time-kill assays were performed against representative MRSA, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus (VRSA) isolates. LTX-109 demonstrated a MIC range of 2 to 4 μg/ml and dose-dependent rapid bactericidal activity against S. aureus. This activity was not influenced by resistance to other antistaphylococcal agents.     

An elevated mutation frequency favors development of vancomycin resistance in Staphylococcus aureus

The emergence of intermediate vancomycin resistance, mainly in methicillin-resistant Staphylococcus aureus strains, has become a great concern. Thorough characterization of clinical and laboratory vancomycin-intermediately resistant S. aureus (VISA) strains identified multiple, resistance-associated changes most probably due to stepwise mutations. We hypothesized that an elevated mutation frequency as found, e.g., in mutator strains defective in DNA mismatch repair could allow rapid acquisition of adaptive mutations in the presence of vancomycin. We therefore subjected S. aureus RN4220 and its isogenic mutator strain, the mutS-knockout mutant RN4220DeltamutS, to a stepwise vancomycin selection procedure. Vancomycin resistance evolved much more quickly in the mutator background than in the wild type (5 versus 19 passages, respectively). In addition, a higher resistance level could be reached (MIC, 32 versus 4 micro g/ml, respectively). The susceptibility to other antibiotics with the exception of teicoplanin remained unchanged. Concomitantly with increasing vancomycin resistance, a loss of phage typeability and differences in growth behavior as well as an improved ability to regrow at high vancomycin concentrations were observed. In conclusion, an elevated mutation rate in S. aureus led to the rapid development of vancomycin resistance, indicating that a high mutation frequency could be one of the factors that favor the emergence of vancomycin resistance in S. aureus.     

Pharmacodynamics of telavancin studied in an in vitro pharmacokinetic model of infection

The antibacterial effects of telavancin, vancomycin, and teicoplanin against six Staphylococcus aureus strains (1 methicillin-susceptible S. aureus [MSSA] strain, 4 methicillin-resistant S. aureus [MRSA] strains, and 1 vancomycin-intermediate S. aureus [VISA] strain) and three Enterococcus sp. strains (1 Enterococcus faecalis strain, 1 Enterococcus faecium strain, and 1 vancomycin-resistant E. faecium [VREF] strain) were compared using an in vitro pharmacokinetic model of infection. Analyzing the data from all five vancomycin-susceptible S. aureus (VSSA) strains or all 4 MRSA strains showed that telavancin was superior in its antibacterial effect as measured by the area under the bacterial kill curve at 24 h (AUBKC(24)) and 48 h (AUBKC(48)) in comparison to vancomycin or teicoplanin (P < 0.05). Telavancin was also superior to vancomycin and teicoplanin in terms of its greater early killing effect (P < 0.05). Against the three Enterococcus spp. tested, telavancin was superior to vancomycin in terms of its AUBKC(24), AUBKC(48), and greater early bactericidal effect (P < 0.05). Dose-ranging studies were performed to provide free-drug area under the concentration-time curve over 24 h in the steady state divided by the MIC (fAUC/MIC) exposures from 0 to 1,617 (7 to 14 exposures per strain) for 5 VSSA, 4 VISA, and the 3 Enterococcus strains. The fAUC/MIC values for a 24-h bacteriostatic effect and a 1-log-unit drop in the viable count were 43.1 ± 38.4 and 50.0 ± 39.0 for VSSA, 3.2 ± 1.3 and 4.3 ± 1.3 for VISA, and 15.1 ± 8.8 and 40.1 ± 29.4 for the Enterococcus spp., respectively. The reason for the paradoxically low fAUC/MIC values for VISA strains is unknown. There was emergence of resistance to telavancin in the dose-ranging studies, as indicated by subpopulations able to grow on plates containing 2× MIC telavancin concentrations compared to the preexposure population analysis profiles. Changes in population analysis profiles were less likely with enterococci than with S. aureus, and the greatest risk of changed profiles occurred for both species at fAUC/MIC ratios of 1 to 10. Maintaining a fAUC/MIC ratio of >50 reduced the risk of subpopulations able to grow on antibiotic-containing media emerging. These data help explain the clinical effectiveness of telavancin against MRSA and indicate that telavancin may have clinically useful activity against Enterococcus spp., and perhaps also VISA, at human doses of 10 mg/kg of body weight/day. In addition, they support a clinical breakpoint of sensitive at ≤1 mg/liter for both S. aureus and Enterococcus spp.