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。     

异质性万古霉素中介金黄色葡萄球菌研究进展

异质性万古霉素中介金黄色葡萄球菌(hVISA)于1997年由日本学者Hiramatsu等首次描述,随后在许多国家和地区报道。研究认为,hVISA感染发生之前存在耐甲氧西林金黄色葡萄球菌(MRSA)感染,是万古霉素中介金黄色葡萄球菌(VISA)的前身,并与万古霉素治疗失败有关。该文主要针对hVISA的流行病学特点、临床重要性及实验室检测等问题进行综述。     

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。     

异源性耐万古霉素金黄色葡萄球菌的研究进展

MRSA是医院内感染的重要致病菌之一 ,万古霉素是唯一有效的药物 ,但万古霉素的应用未能明显降低极高的病死率 ,异源性耐万古霉素金葡菌的存在可能是重要原因。了解该菌的流行状况对监控和治疗MRSA感染具有重要意义。     

金黄色葡萄球菌对万古霉素耐药机制的研究进展

万古霉素是治疗耐甲氧西林的金黄色葡萄球菌（MRSA）感染的首选药物，随着临床万占霉素用量增加而产生的万古霉素敏感性降低的金黄色葡萄球菌感染使治疗变得十分困难，引起医学界的广泛关注。现就金黄色葡萄球菌对万古霉素耐药机制作一综述。     

耐甲氧西林金黄色葡萄球菌对万古霉素敏感性的研究

目的探讨耐甲氧西林金黄色葡萄球菌(MRSA)对万古霉素的敏感性。方法取2007—2014年医院分离的MRSA102株,采用琼脂稀释法检测万古霉素对MRSA的最低抑菌浓度(MIC),采用PCR方法进行SCCmec分型及相关基因检测。结果 2007—2014年万古霉素对MRSA的MIC_(90)分别为1、1、2、2、2、2、2、2 mg/L,MIC_(50)分别为1、1、1、2、1、1、1、1 mg/L。SCCmecⅡ型的MIC_(50)和MIC_(90)都为2 mg/L,SCCmecⅢ型的MIC_(50)和MIC_(90)分别为1 mg/L和2 mg/L,SCCmec V型和Ⅳ型的MIC_(50)和MIC_(90)都为1 mg/L。结论 2007—2014年万古霉素对MRSA的最低抑菌浓度无显著变化,SCCmecⅡ型和Ⅲ型MRSA对万古霉素的敏感性低于SCCmec V型和Ⅳ型菌株。     

耐万古霉素金黄色葡萄球菌生物学特性改变与细菌鉴定

目的 了解金黄色葡萄球菌对万古霉素耐药后生物学特性的变化及其对临床细菌鉴定结果的影响。方法 使用万古霉素体外诱导试验将1株临床分离的异质性万古霉素耐药金黄色葡萄球菌（h-VRSA）逐渐诱导为万古霉素中介耐药金黄色葡萄球菌（VISA）,观察原代菌和诱导菌的生长特征和生化反应,并使用VITEK32和MicroscanWalkAway40进行细菌鉴定。结果 h-VRSA和VISA的主要生物学特性改变为：菌落大小不等、色素由淡黄色变成灰白色、溶血环消失,血浆凝固酶、甘露醇、甘露糖等生化反应由阳性转为阴性;对杆菌肽由敏感变为耐药;仪器鉴定结果为溶血葡萄球菌或模仿葡萄球菌。结论 金黄色葡萄球菌对万古霉素耐药后可表现出培养特征、生化反应等一系列表型特征的改变,并可导致临床细菌鉴定结果错误。     

异质性万古霉素中介金黄色葡萄球菌的分离及检测方法的建立

目的 了解异质性万古霉素中介金黄色葡萄球菌(h-VISA)的分离情况,并筛选简便可行的h-VISA的检测方法.方法 采用琼脂稀释法和E-test法对分离出的113株金黄色葡萄球菌进行检测,筛选出的可疑h-VISA用菌谱分析法进行确认,并对文献推荐的几种方法的筛选效果进行比较.结果 检测到1株万古霉素的MIC为3 μg/mL和2株MIC为4 μg/mL的MRSA,为万古霉素敏感性减低的金黄色葡萄球菌,其中1株MIC为4 μg/mL的MRSA经菌谱分析法证实为h-VISA,本次113株实验菌株h-VISA的检出率为0.88%.结论 通过实验证实K-B法并不是检测h-VISA的理想方法,而菌谱分析法则较为有效,分离率为8.3%,且在本实验中灵敏度可达10-8.本院分离的MRSA中已出现万古霉素敏感性减低菌株,并分离出1株h-VISA,可见,MRSA对万古霉素的异质性中介耐药不容忽视,应引起国内医学界的广泛重视.     

异质性万古霉素中介金黄色葡萄球菌：困扰我们的难题

万古霉素是治疗甲氧西林耐药金葡菌（MRsA）感染的有效药物,也是治疗该菌感染的最后选择,目前该药已在临床上广泛使用。虽然万古霉素耐药金葡菌（VRsA）在全球的检出率很低,但万古霉素低水平耐药的金葡菌,包括异质性万古霉素中介金葡菌（hVIsA）和万古霉素中介金葡菌（VISA）却在多个国家和地区广泛流行。     

High-level vancomycin-resistant Staphylococcus aureus isolates associated with a polymicrobial biofilm

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistant Staphylococcus aureus. This study describes the identification of high-level vancomycin-resistant S. aureus (VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York. S. aureus, Enterococcus faecalis, Enterococcus faecium, Micrococcus species, Morganella morganii, and Pseudomonas aeruginosa were isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 microg/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. The vanA gene was localized to a plasmid of approximately 100 kb in VRSA and E. faecium isolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kb E. faecium plasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genes tet(U) and tet(S), not previously detected in S. aureus isolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster, ermB-aadE-sat4-aphA-3, msrA (macrolide efflux), and the bifunctional aminoglycoside resistance gene aac(6')-aph(2")-Ia. Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, including vanA, tet(S), and tet(U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.     

Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds

A series of 12 Staphylococcus aureus strains of various genetic backgrounds, methicillin resistance levels, and autolytic activities were subjected to selection for the glycopeptide-intermediate S. aureus (GISA) susceptibility phenotype on increasing concentrations of vancomycin. Six strains acquired the phenotype rapidly, two did so slowly, and four failed to do so. The vancomycin MICs for the GISA strains ranged from 4 to 16 microg/ml, were stable to 20 nonselective passages, and expressed resistance homogeneously. Neither ease of acquisition of the GISA phenotype nor the MIC attained correlated with methicillin resistance hetero- versus homogeneity or autolytic deficiency or sufficiency. Oxacillin MICs were generally unchanged between parent and GISA strains, although the mec members of both isogenic methicillin-susceptible and methicillin-resistant pairs acquired the GISA phenotype more rapidly and to higher MICs than did their susceptible counterparts. Transmission electron microscopy revealed that the GISA strains appeared normal in the absence of vancomycin but had thickened and diffuse cell walls when grown with vancomycin at one-half the MIC. Common features among GISAs were reduced doubling times, decreased lysostaphin susceptibilities, and reduced whole-cell and zymographic autolytic activities in the absence of vancomycin. This, with surface hydrophobicity differences, indicated that even in the absence of vancomycin the GISA cell walls differed from those of the parents. Autolytic activities were further reduced by the inclusion of vancomycin in whole-cell and zymographic studies. The six least vancomycin-susceptible GISA strains exhibited an increased capacity to remove vancomycin from the medium versus their parent lines. This study suggests that while some elements of the GISA phenotype are strain specific, many are common to the phenotype although their expression is influenced by genetic background. GISA strains with similar glycopeptide MICs may express individual components of the phenotype to different extents.     

Frequencies of subpopulations of aminoglycoside- and vancomycin-resistant variants in Staphylococcus aureus and Staphylococcus epidermidis.

Selection and regrowth of resistant variants, which are present in low frequencies in the initial inoculum, were seen when large inocula of five strains of Staphylococcus aureus and four strains of Staphylococcus epidermidis were incubated in broth with amikacin, gentamicin, netilmicin and tobramycin. Statistical analysis showed no significant difference between the aminoglycosides in the selective growth of resistant variants (P > 0.5). Vancomycin differed significantly from the aminoglycosides in both the frequency of, and selection of resistant variants (P < 0.001). No bacteria resistant to > 1 x MIC was seen in the vancomycin-exposed cultures of S. aureus and S. epidermidis, while in most aminoglycoside-exposed cultures, bacteria resistant to 4-16 x MIC were seen.     

Hospital infection control measures for methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci

Methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci are major hospital pathogens in hospital settings. These organisms are mainly transmitted by direct skin contact with infected/colonized patients. Air borne transmission may rarely occur in MRSA positive cases with upper respiratory infections. Contamination in immediate environment requires proper disinfection after contact. Screening patients for MRSA and VRE can be restricted against those in/from the high risk areas. The clinical pictures, methods for screening and the precautions were also discussed based on the nature of communicability in both pathogens.     

Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.     

Nosocomial infection of β-lactam antibiotic-induced vancomycin-resistant Staphylococcus aureus (BIVR)

We report here an outbreak of β-lactam-induced vancomycin-resistant methicillin-resistant Staphylococcus aureus (MRSA; BIVR) at one of the Cancer-Institute-affiliated hospitals in Tokyo. We examined a total of 500 strains (100 per year) of clinically isolated MRSA from 1998 to 2002. The detection rates of BIVR in the years 1998, 1999, 2000, 2001, and 2002 were 10%, 9%, 49%, 15%, and 19%, respectively. To investigate the cause of the high incidence of BIVR detection in the year 2000, we carried out pulsed-field gel electrophoresis (PFGE) of the SmaI-digested chromosomal DNA of BIVR and MRSA. The results showed that 96% of the BIVR strains isolated in 2000 were classified as an identical DNA type “A”, while only 47% of the MRSA strains were classified as this type. We concluded, based on these results, that this hospital had a nosocomial infection of BIVR in the year 2000. An important message given by this study would be that nosocomial BIVR infection could occur in any hospital where MRSA infection is treated with vancomycin and β-lactam antibiotics.     

Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center

Staphylococcus aureus strain HMC3 isolated at the Hershey Medical Center, was resistant to vancomycin (VRSA) through the presence of the vanA resistance gene; it also contained mecA, erm(A), erm(B), tet(K) and aac(6')-aph(2"), conferring resistance to licensed beta-lactams, macrolides, tetracycline and aminoglycosides. HMC3 also had alterations in GyrA and GrlB and was resistant to available quinolones. Experimental drugs with low MICs (<2 mg/L) for VRSA HMC3 included cephalosporins BAL9141 and RWJ-54428; glycopeptides oritavancin and dalbavancin; the lipopeptide daptomycin; the glycolipodepsipeptide ramoplanin; new fluoroquinolones WCK 771 A, WCK 1153, DK-507k and sitafloxacin; and the DNA nanobinder GS02-02. These agents were all bactericidal as were trimethoprim/sulfamethoxazole and teicoplanin (MIC 4 mg/L). Oxazolidinones linezolid and ranbezolid; the injectable streptogramin quinupristin/dalfopristin; DNA nanobinders GS2-10547 and GS02-104; peptide deformylase inhibitors NVP-PDF713 and GS02-12; tetracycline derivative tigecycline; the antifolate iclaprim; mupirocin and fusidic acid were all active in vitro but bacteriostatic.     

Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus

The phenotypic expression of methicillin resistance was studied in a number of clinical isolates and laboratory strains of Staphylococcus aureus. The methicillin-resistant S. aureus strains could be divided into three classes, homogeneous, heterogeneous, and thermosensitive heterogeneous methicillin-resistant S. aureus, on the basis of their plating efficiencies at 30 or 37 degrees C on methicillin-containing agar plates. Heterogeneous strains of methicillin-resistant S. aureus were composed of two subpopulations: a small minority of cells (10(-5) to 10(-3); MIC, 600 to 1,000 micrograms/ml) that expressed resistance to high concentrations of methicillin at 37 degrees C, and a majority of cells (MIC, 5 micrograms/ml) that remained susceptible to the drug at 37 degrees C. Cultures of a thermosensitive heterogeneous strain were able to grow in the presence of high concentrations of methicillin, provided that the growth temperature was 30 degrees C. Such cultures lost their phenotypic resistance within 30 min (i.e., in less than one doubling time) after the growth temperature was shifted to the nonpermissive 37 degrees C. Shift of the temperature of the culture in the reverse direction (37 to 30 degrees C) resulted in an equally rapid expression of phenotypic resistance. The majority of the cells in such heterogeneous strains may be considered heat (or salt) conditional in their phenotypic expression of methicillin resistance. Both heterogeneous and thermosensitive heterogeneous strains, irrespective of their temperature of cultivation and degree of phenotypic resistance, contained detectable quantities of the 78-kilodalton penicillin-binding protein 2a (PBP 2a) that previous studies have suggested is a biochemical correlate of methicillin resistance in homogeneous strains of methicillin-resistant S. aureus. However, in contrast to the homogeneous stains, in heterogeneous and thermosensitive heterogeneous isolates the ability to synthesize PBP 2a is apparently not sufficient to provide a resistant phenotype. In these strains some additional, as yet undefined factor(s) is also needed for the expression of methicillin resistance.     

Effect of vancomycin on the cytoplasmic membrane fatty acid profile of vancomycin-resistant and -susceptible isolates of Staphylococcus aureus

This study was designed to analyze the effect of vancomycin on the cytoplasmic membrane fatty acid (FA) composition of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-susceptible S. aureus. One low-level vancomycin-resistant isolate (LLR-VRSA) termed CP2, along with two vancomycin intermediate-resistant S. aureus isolates (VISA-CP1) and Mu50 (ATCC #700699), were studied. The LLR-VRSA isolate CP2, recovered from the blood sample of a postoperative cardiac patient, exhibited vanA type vancomycin resistance [minimum inhibitory concentration (MIC) 16 μg/ml], and the vanA cassette was located on a plasmid. CP1, isolated from the pus sample of the same patient, exhibited vancomycin intermediate resistance (MIC 8 μg/ml) in the absence of the vanA, vanB, or vanC gene. As susceptible controls, we used PSA (vancomycin MIC 2 μg/ml), which was isolated from the pus sample of a neonate, and S. aureus (ATCC# 29213). Membrane FA analysis was carried out using gas chromatography coupled with mass spectrometry. For this purpose, CP1, CP2, Mu50, and the susceptible control isolates were grown in the presence and absence of vancomycin. Comparative analysis showed an increase in the relative proportion of unsaturated FAs during growth under vancomycin stress. The isolate CP2 (LLR-VRSA) exhibited a higher MIC to vancomycin than the other isolates used in present study (16 μg/ml) and under vancomycin stress conditions, quantitatively, it showed a high rate of conversion of saturated to unsaturated membrane FAs than CP1, Mu50 (VISA isolate) and the susceptible control PSA. The rate of saturated-to-unsaturated FA conversion increased as the concentration of vancomycin in the growth media was increased. Therefore, it is concluded that S. aureus tend to modify their membrane lipid chemistry from saturated to unsaturated in order to survive in a vancomycin stress environment.