In vitro Virulenz wundinfizierender Staphylokokken-Isolate von schwerbrandverletzten Patienten

Zusammenfassung 30Staphylococcus aureus Stämme von drittgradig verbrannten Patienten wurden hinsichtlich ihrer Fähigkeit zur Hämolysingenerierung und ihres Vermögens zur Histaminfreisetzung aus Mastzellen (RPMC) untereinander verglichen. Die bakteriellen Kulturüberstande von 8 Stämmen aus Wundbiopsien zeigten signifikant niedrigere in vitro Aktivitäten als die übrigen Staphylokokken-Kulturüberstände. Bei dem Vergleich der in vitro Aktivitäten der gewaschenen Bakterienzellen ergab sich ein umgekehrtes Verhältnis. Diese Befunde zeigen, daß beiS. aureus Isolaten, welche zum Zeitpunkt einer invasiven Brandwundeninfektion isoliert werden können, die mikrobiellen Pathogenitätsfaktoren an die Bakterienzelle gebunden bleiben.

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In vitro measurement of pathogenicity ofStaphylococcus aureus isolated from heavily burned patients

Summary The hemolysin and histamine releasing activity of 30S. aureus strains isolated from third degree burns of heavily burned patients was detected. The culture supernatants (cs) ofStaphylococcus aureus isolated during episodes of invasive burn wound infection displayed significantly lower amounts of hemolysin and histamine releasing activity as compared to cs of bacteria isolated when no sign of septicemia was present. In contrast, when washed bacterial cells were analysed, a reversed ratio could be observed. These data clearly indicate that in strains isolated during invasive burn wound infection pathogenicity factors remain attached to the bacterial surface of the staphylococci investigated.

Gefördert durch das Bundesamt für Zivilschutz (ZS 8-122-42)     

Characterisation of non-multiresistant methicillin-resistant Staphylococcus aureus (including EMRSA-15) in Kuwait Hospitals

This study characterised non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) isolates from Kuwait hospitals to ascertain whether they were community-acquired MRSA (CA-MRSA). Forty-two nmMRSA isolates obtained between July 2001 and October 2003 were analysed by staphylococcal cassette chromosome mec (SCCmec) typing, bacteriophage typing, production of Panton-Valentine leukocidin (PVL), urease and staphylococcal enterotoxins A, B, C and D, TSST-1, and by pulsed-field gel electrophoresis (PFGE). Forty-one isolates were SCCmec type IV, and one isolate was SCCmec type III. The isolates belonged to six PFGE patterns, with two types, A and D, distributed in six and four hospitals, respectively. Most (n = 26; 61.9%) isolates produced urease. These isolates were mainly from wound and skin infections, showed low-level methicillin resistance (MIC 8-48 mg/L), and nine carried genes for PVL. These characteristics, together with their carriage of the type-IV SCCmec, identified the isolates as CA-MRSA. Ten of the 16 urease-negative isolates produced staphylococal enterotoxin C; 12 reacted weakly with phage 75, and were resistant to clindamycin and/or erythromycin, which are characteristics of EMRSA-15. Thus, this study identified the co-existence of two types of nmMRSA, i.e., CA-MRSA and EMRSA-15, in Kuwait hospitals.     

Antibiotic resistance in Staphylococcus aureus colonising the intestines of Swedish infants

Staphylococcus aureus has become a frequent coloniser of the intestinal tract of infants, but the health effects of such colonisation are not clear. In this study, the antibiotic resistance patterns of 116 S. aureus strains from the commensal intestinal microflora were determined. The strains were obtained from 81 Swedish infants who had been followed with regular stool samples and registration of antibiotic usage during their first year of life. The faecal population levels of the individual strains and the duration of their persistence in the microflora had been determined previously. The prevalence of antibiotic resistance among the 116 strains was modest: methicillin, 0%; penicillin G, 78%; erythromycin A, 3%; tetracycline, 2%; clindamycin, 0.9%; and fusidic acid, 0.9%. Colonisation by antibiotic-resistant strains was unrelated to antibiotic consumption by individual infants. Antibiotic-resistant strains were as capable of persisting in the intestinal microflora and reaching high faecal population levels as fully susceptible strains. No strain lost or acquired resistance during the colonisation period. Thus, antibiotic-resistant strains of S. aureus seem to be as fit for competition in the large bowel microflora as susceptible strains, even in the absence of selective pressure from antibiotics. This may aggravate the ecological consequences of antibiotic resistance development.     

Response of body temperature and serum iron concentration to repeated pyrogen injection in rabbits

We measured body temperature and serum iron concentration after five daily consecutive injections of febrile doses of Salmonella typhosa lipopolysaccharide (0.1 g/kg) and two doses of Staphylococcus aureus cell walls (1×10⁷ and 5×10⁷ cells) in rabbits. Tolerance to endotoxin injection, as manifest by a significant attenuation in the body temperature elevation, developed after the first injection of endotoxin. The endotoxin-induced fall in serum iron concentration was attenuated significantly by the 5th day of endotoxin injection. In contrast, no tolerance developed in either the body temperature or serum iron response following repeated daily injections of S. aureus. Rabbits rendered tolerant to endotoxin showed normal febrile and serum iron responses to subsequent S. aureus injection. Rabbits given serial injections of S. aureus, although not tolerant to S. aureus itself, exhibited attenuated body temperature responses but not serum iron responses to endotoxin injection. We suggest that repeated injection of endotoxin diminishes the ability of endotoxin to stimulate endogenous pyrogen (EP) synthesis and/or release, a property not shared by the gram-positive pyrogen S. aureus. However, repeated injection of S. aureus weakens the central endotoxin-EP pathway.     

Cytotoxic effects of ingested Staphylococcus aureus on bovine endothelial cells: Role of S. aureus α-hemolysin

Previously we observed that Staphylococcus aureus phagocytized by cultured bovine endothelial cells do not proliferate intracellularly, but are cytotoxic to bovine endothelial cells. To investigate S. aureus virulence factors which may be produced intracellularly and cause lysis of endothelial cells, we tested S. aureus mutants defective in production of one or more potential virulence factors and corresponding parent strains for cytotoxicity to endothelial cell monolayers subsequent to being ingested. Following incubation of endothelial cell monolayers with S. aureus for 3.5 h, cultures were supplemented with lysostaphin to destroy extracellular but not intracellular S. aureus. At subsequent times, viability of endothelial cells was assayed by retention of ³H-adenine and the number of intracellular S. aureus was measured. The cytotoxic activity of S. aureus culture supernatants was also characterized. The results indicate that S. aureus α-hemolysin is cytotoxic to bovine endothelial cells and plays an important role in the damage suffered by bovine endothelial cell monolayers following ingestion of S. aureus. Ingestion of α-hemolysin-producing S. aureus by endothelial cells in vivo might be expected to result in destruction of endothelium followed by development of platelet-fibrin vegetations. This possible sequence of events is compatible with the frequently fulminant course of S. aureus endocarditis.     

金黄色葡萄球菌 eap 基因的克隆与表达

 目的 构建携带 eap 基因的原核表达载体，诱导表达具有活性的重组 EAP 融合蛋白。 方法 PCR 法扩增金黄色葡萄球菌基因组 DNA，回收、 纯化的扩增产物与 pMD18-T 载体相连接得重组质粒 pMD18-T-EAP，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；未酶切组作为对照组重组质粒 pMD18-T-EAP 和 pET28a（+）表达载体分别用 Nde I 和 Xho I 限制性内切酶双酶切、连接，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；空载体作为对照组。用不同浓度（终浓度 1、2、4、8 mmol/L）和不同诱导时间（1、2、3、4、5、6 h）的异丙基-β-D-硫代半乳糖苷（IPTG）对阳性重组菌进行表达优化，分别取 E.coli上清液和沉淀做电泳分析。应用 MagneHis^TM 蛋白纯化系统纯化重组 EAP 融合蛋白，并通过薄层扫描测定蛋白质的浓度。 结果 所获 eap 基因与 GeneBank 的基因序列同源性 > 99%；氨基酸同源性达 100%。重组质粒经 IPTG 诱导，阳性重组菌转化子均有表达；当吸光度（A ）值等于 0.6 ~ 0.8 时，相对分子质量约 70 000 处出现目的蛋白条带。破碎的重组菌 pET28a-EAP上清液中目的蛋白条带较清楚，沉淀中几乎看不到。终浓度 1 mmol/L 为最佳蛋白表达工作浓度。IPTG 诱导 1 h 重组 EAP 融合蛋白有一定量的表达，随着时间的延长，表达量增加不明显，3 h 时的表达量达最高，之后，蛋白表达量变化不明显。表达的重组 EAP 融合蛋白含量占全菌体蛋白的 29.6%。 结论 成功地克隆和表达了金黄色葡萄球菌重组 EAP 融合蛋白，为进一步研究以 EAP 蛋白作为免疫原预防和治疗由金黄色葡萄球菌引起的疾病奠定基础。     

Spread of old and new clones of epidemic methicillin-resistant Staphylococcus aureus in Poland

Objective: To study the relatedness among methicillin-resistant Staphylococcus aureus (MRSA) isolates originating from two regions of Poland using different epidemiologic typing methods.
Methods: Forty-five MRSA isolates (19 from Warsaw and 26 from the Grajewo region) were collected between 1995 and 1996. For phenotypic epidemiologic analysis, antimicrobial susceptibility testing (AST) with a panel of 19 antibiotics was performed. For genotypic epidemiologic analysis, pulsed-field gel electrophoresis (PFGE) of Smal-digested chromosomal DNA, restriction endonuclease analysis of plasmid (REAP) DNA digested by Hin dIII, random amplification of polymorphic DNA (RAPD) and binary typing (BT) of genomic DNA by hybridization with five different RAPD-generated strain-specific DNA probes, were used.
Results: Six clusters of clonally related strains were found among the MRSA isolates analyzed. Three of these, identified in both regions, were related to previously described Polish epidemic clones, designated HeEMRSA-Pol1 (heterogeneously methicillin resistant—18 isolates) and HoEMRSA-Pol1 (homogeneously resistant—two clones, six isolates each). The remaining three clones, identified in the Grajewo region only, are previously undescribed. One of these, represented by 11 isolates, appears to be new epidemic heterogeneous MRSA clone (HeEMRSA-Pol2). Results of PFGE and BT in general showed good correlation, and, in some cases, RAPD using AP1 and AP7 primers could discriminate between isolates belonging to single PFGE or BT types. Broad AST and REAP can provide useful additional information concerning relatedness.
Conclusion: Evidence for the spread of previously recognized epidemic MRSA clones in Poland and the presence of a new epidemic heterogeneously resistant clone of MRSA in hospitals outside Warsaw is documented.     

Experimental staphylococcal endocarditis and aortitis

Summary The initial colonization, byStaphylococcus aureus, of the catheter damaged aortic valve and aorta of the rabbit, was examined by light and electron microscopy at 15 min, 3 h and 24 h post inoculation (PI). At 15 min PI, the majority of bacteria (80%) were located on the lateral surfaces of the thrombic vegetations while 20% were attached directly to the connective tissue of the aortic valve and aorta in areas where the endothelial lining was disrupted. By 3 h the bacteria on the thrombic vegetations were covered by fibrin. At this time, the bacteria both within the vegetations and on the surface of the vasculature were undergoing multiplication to form small groups. The precipitation of thrombus around the bacteria attached to the surface of the aorta to form microscopic infected vegetations had occurred by 24 h PI. The colonizing bacteria did not elicit any phagocytic response. The colonization of the cardiovasculature byStaph. aureus did not necessarily require pre-existing vegetations.     

应用IRS-PCR对金黄色葡萄球菌分型的研究

目的探讨低频限制性位点聚合酶链反应(infrequent-restriction-site PCR，IRS-PCR)在金黄色葡萄球菌(简称金葡菌)基因分型中的应用价值。方法建立本实验室IRS-PCR方法。同时用IRS-PCR和脉冲场凝胶电泳(PFGE)对金葡菌进行基因分型。根据49株社区感染分离菌的分型结果计算辨别力指数(ID)值估计分辨力。对其中30株社区感染菌重复实验一次估计重复性。比较两种基因分型方法的分型率、分辨力、重复性、结果的一致率及操作特点。结果建立IRS-PCR对金葡菌基因分型的方法。70株金葡菌均可被2种方法分型，分型率100％。IRS-PCR分为38个型，21株院内感染菌分为6个型，49株社区感染菌分为32个型，计算ID值为0．981。PFGE分为40个型，21株院内感染菌分为6个型，49株社区感染菌分为34个型，计算ID值为0．983。两种分型方法的重复性均为100％。对院内感染菌，两种方法分型的一致率为100％；对社区感染菌，两种方法分型的一致率为92％(45／49)。与PFGE相比，IRS-PCR更简单、省时、易于操作、不需特殊昂贵仪器。结论IRS-PCR能对金葡菌简易快速可靠分型，适合检验科对临床标本的快速有效分型，是一种有价值的分子流行病学研究工具。