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1.
AIMS: To describe the epidemiological, clinical, and laboratory features of gentamicin-susceptible methicillin-resistant Staphylococcus aureus (GS-MRSA) seen at a paediatric teaching hospital. METHODS: Patients from whom GS-MRSA was isolated between 1 January 2001 and 31 December 2002 were enrolled. Retrospective chart review was performed. Susceptibility testing was performed with the Vitek2 system; PCR confirmed methicillin resistance. Phage typing and pulsed field gel electrophoresis (PFGE) was performed (utilising MLST/SCCmec-defined control strains). PCR detection of tst, luk-PV, and entA-entE was performed. RESULTS: Eighty-five per cent of all Staphylococcus aureus isolates during the study period were methicillin-sensitive, and 15% were MRSA (9% GS-MRSA, 6% gentamicin resistant-MRSA). 100 GS-MRSA infections in 98 children were identified: 59 cases of skin/soft tissue, four bone and joint, four surgical site infections, three pneumonia, eight other types, and 22 represented colonisation. Ninety-nine isolates were non-multidrug resistant, but 17 strains were resistant to erythromycin, 7 to tetracyclines, 12 to ciprofloxacin, 11 to fusidic acid, 1 each to rifampicin and mupirocin. 44 isolates were Oceania strain (ST30-MRSA-IV), 20 were Queensland strain (ST93-MRSA-IV), ten were UK EMRSA-15 (ST22-MRSA-IV), eight were WA MRSA-1 (ST1-MRSA-IV), two were WA MRSA-5 (ST8-MRSA-IV), one was WA MRSA-2 (ST78slv-MRSA-IV), one was WA MRSA-15 (ST59-MRSA-IV), and the remainder were sporadics. Twenty patients were Pacific Islanders, of whom 12 had the Oceania strain; ten were Aboriginal, of whom eight had the Queensland strain. Sixty-eight isolates possessed luk-PV, including all Queensland strains and 91% of Oceania strains. Enterotoxin genes were detected in 25% of the isolates, and tst was detected in four isolates. CONCLUSIONS: GS-MRSA is a significant paediatric problem in New South Wales: two minority groups are over-represented, multiple epidemic strains were detected, most community strains possess luk-PV, and many isolates are multidrug resistant.  相似文献   

2.
目的:通过基因工程方法获得重组的杀白细胞毒素,为深入研究其致病机制打下基础。方法:利用PCR方法从产杀白细胞毒素金葡菌临床分离株的基因组DNA中克隆PVL-S和PVL-F基因序列,分别将其克隆到载体pET22b中,构建重组pET22b-LuKS和pET22b-LuKF原核表达载体,转化感受态大肠杆菌BL21 (DE3) pLysS, 异丙基硫代β-D 半乳糖苷(sopropyl-beta-D-thiogalactopyranoside,IPTG) 诱导表达,将其可溶性表达经镍离子螯合亲和层析纯化后,通过对外周血粒细胞和多形核白细胞的刺激反应鉴定其活性。结果:以PCR方法获得PVL-S和PVL-F基因片段,与克隆载体连接后测序与文献报道的基本一致,成功构建了pET22b-LuKS和pET22b-LuKF原核表达质粒,并高效诱导表达出相对分子质量约34 kD的S蛋白及相对分子质量约35 kD的F蛋白。重组蛋白(rPVS,rPVF)在37 ℃经 IPTG诱导时均以包涵体形式存在,而30 ℃时部分出现可溶性表达,部分为包涵体。将上清可溶性重组蛋白作用于外周血粒细胞,可导致粒细胞出现凋亡,部分出现坏死。结论:成功构建了表达载体pET22b-LuKS和pET22b-LuKF, 对其编码的S片段和F片段进行了原核表达和鉴定, 高效表达了杀白细胞毒素的S 和F两类蛋白,为进一步对其致病机制和免疫原性的研究奠定基础。  相似文献   

3.
多重PCR对金黄色葡萄球菌杀白细胞素基因的检测   总被引:13,自引:0,他引:13  
目的应用多重PcR检测含Panton-Valentine杀白细胞素(PVL)基因的金黄色葡萄球菌。方法收集我院2005年1月至2006年1月从临床多种标本中分离的金黄色葡萄球菌,用多重PCR同时检测葡萄球菌16SrRNA基因、mecA基因和lukS/F-PV基因。多重PCR检测MRSA的SCCmec基因型及亚型。结果195株金黄色葡萄球菌经多重PCR检测,121株为耐甲氧西林金黄色葡萄球菌(MRSA),74株为甲氧西林敏感金黄色葡萄球菌(MSSA),共检测到26株金黄色葡萄球菌lukS/F-PV基因阳性,阳性率为13.3%(26,195)。其中19株为MRSA,阳性率为15.7%(19/121);7株为MSSA,阳性率为12.2%(7/74)。19株lukS/F-PV基因阳性的MRSA的SCCmec基因型分别为SCCmec Ⅲ型10株、SCCmecⅢA型4株、SCCmecⅣ型4株及SCCmecⅠ型1株。26株lukS/F-PV基因阳性的分离株有11株分离自脓液或创面分泌物,10株分离自痰标本,3株分离自血液标本,2株分离自尿液。结论在温州地区分离的MRSA和MSSA中都能检测到Panton-Valentine杀白细胞素基因,含Panton-Valentine杀白细胞素基因MRSA的SCCmec基因型主要为SCCmec Ⅲ,含PVL基因的金黄色葡萄球菌主要引起化脓性感染和肺部感染。  相似文献   

4.
Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.  相似文献   

5.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) often produce Panton–Valentine leukocin (PVL), which is encoded by two co-transcribed genes located on lysogenized bacteriophages. Six PVL-encoding temperate phages have been described and single nucleotide polymorphisms (SNPs) in the PVL genes have been reported. In the present study, 22 PVL-positive CA-MRSA isolates were chosen to reflect the diversity of multilocus sequence type (MLST) clonal complexes (CC) identified in our hospital. Isolates were characterized by antimicrobial resistance profile, staphylococcal cassette chromosome mec (SCC mec ) and spa type, pulsed-field gel electrophoresis profile and MLST. Primers were designed to sequence the lukSF-PV genes. PVL-encoding phages were characterized using a PCR-based assay. SNPs were identified at seven locations in the lukSF-PV genes, which varied with S. aureus MLST lineage. One SNP was nonsynonymous. All CC80 and some CC1 isolates carried ΦSa2mw; CC8, CC88 and CC154 isolates harboured PVL-encoding elongated head-type phages; and some CC59 isolates harboured a ΦSa2958-like phage. Novel or variant phages were present in CC5 and some CC1 and CC59 isolates. The PVL gene sequence and the PVL-encoding phage varied with lineage. Further work is required to determine whether PVL sequence and/or phage variations result in biological differences.  相似文献   

6.
One hundred and three patients who had previously tested positive for community-acquired methicillin-resistant Staphylococcus aureus (cMRSA) were followed up for a mean time of 32.6 months. Eighty patients had a history of skin or soft tissue infection, and the remainder were mostly asymptomatic carriers. Of 103 patients, only two reported ongoing symptoms with abscess formation. Of 81 nasal swabs available, 30.9% were positive for S. aureus but only four yielded Panton–Valentine leukocidin-positive methicillin-resistant S. aureus. In summary, we were unable to find persistent health issues or nasal colonization with cMRSA in a cohort of previously cMRSA-infected/colonized patients.  相似文献   

7.
The epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in Africa is poorly documented. From January 2007 to March 2008, 555 S. aureus isolates were collected from five African towns in Cameroon, Madagascar, Morocco, Niger, and Senegal; among these, 456 unique isolates were susceptible to methicillin. Approximately 50% of the MSSA isolates from each different participating centre were randomly selected for further molecular analysis. Of the 228 isolates investigated, 132 (58%) belonged to five major multilocus sequence typing (MLST) clonal complexes (CCs) (CC1, CC15, CC30, CC121 and CC152) that were not related to any successful methicillin-resistant S. aureus (MRSA) clones previously identified in the same study population. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 130 isolates overall (57%), were highly prevalent in isolates from Cameroon, Niger, and Senegal (West and Central Africa). This finding is of major concern, with regard to both a source of severe infections and a potential reservoir for PVL genes. This overrepresentation of PVL in MSSA could lead to the emergence and spread of successful, highly virulent PVL-positive MRSA clones, a phenomenon that has already started in Africa.  相似文献   

8.
Our purpose was to determine the dynamics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage and its determinants in persons working at pig farms, in order to identify targets for interventions. This prospective cohort study surveyed 49 pig farms in the Netherlands on six sampling dates in 1 year (2010-11). Nasal and oropharyngeal swabs were collected, as well as environmental surface samples from stables and house. Of 110 pig farmers, 38% were persistent MRSA nasal carriers. The average cross-sectional MRSA prevalence was 63%. Methicillin-susceptible S. aureus (MSSA) nasal carriage was associated with fewer MRSA acquisitions (prevalence rate (PR) = 0.47, p 0.02). In multivariate analysis, an age of 40-49 years (PR = 2.13, p 0.01), a working week of ≥40 h (PR=1.89, p 0.01), giving birth assistance to sows (PR=2.26, p 0.03), removing manure of finisher pigs (PR=0.48, p 0.02), and wearing a facemask (PR = 0.13, p 0.02) were significantly related with persistent MRSA nasal carriage. A higher MRSA exposure in stables was associated with MRSA in pig farmers (p <0.0001). This study describes a very high prevalence of LA-MRSA carriage in pig farmers, reflecting extensive exposure during work. We identified the possible protective effects of MSSA carriage and of continuously wearing a facemask during work.  相似文献   

9.
Methicillin-resistant Staphylococcus aureus (MRSA) is an established nosocomial pathogen, but has recently begun to appear in the community. The clones in the community may not have originated in the hospital setting, and are referred to as community-acquired MRSA (CA-MRSA). Resistance to methicillin is mediated by the gene mecA, which is carried by the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec typing (I-IV) of all clinical isolates of MRSA (n = 92) from 1987 to 2004 in Orebro County, Sweden, was performed by real-time LightCycler PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB. Forty-one isolates harboured type IV SCCmec, of which ten could be classified further as subtype IVa, and 27 as subtype IVc. No isolates belonged to subtype IVb, but four isolates could not be subtyped, and may be examples of novel type IV SCCmec subtypes. Thirty-five MRSA isolates, assigned to six different pulsotypes by pulsed-field gel electrophoresis, did not belong to SCCmec types I-IV. The Panton-Valentine leukocidin (PVL) genes were identified in two of these pulsotypes. Only SCCmec type IV has been associated previously with the PVL toxin, but the results suggest that new PVL-positive clones with novel SCCmec types may be arising and disseminating in the community.  相似文献   

10.
11.
Staphylococcus aureus of sequence type 398 has emerged in Europe, North America and Asia, and has typically been associated with livestock and their human contacts. We analysed two Panton–Valentine leukocidin (PVL)-negative t034-ST398 isolates from humans in contact with pigs and two t034-ST398 PVL-positive isolates from two unrelated, adopted Chinese children, using multistrain microarrays to determine genomic variability between the two sets of isolates. The ST398 isolates clearly belong to the same lineage when compared to other clonal lineages. However, the four isolates cluster into two distinct groups corresponding to differences in epidemiology based on mobile genetic elements and resistance patterns, suggesting that the two groups are epidemiologically distinct.  相似文献   

12.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has not been recognised previously as a cause of MRSA infections in Spain. Nineteen patients carrying Panton–Valentine leukocidin (PVL)-positive MRSA were identified in a Barcelona hospital, of whom 15 were immigrants, mostly from South America. Twelve developed skin and soft-tissue infections. The associated isolates carried the PVL gene and staphylococcal chromosomal cassette (SCC) mec IV. A dominant clone belonging to sequence type (ST)8 and related to the USA300 clone was identified by pulsed-field gel electrophoresis. This clone is emerging in Spain, primarily among immigrants from South America, but dissemination to the native Spanish population could increase.  相似文献   

13.
A prospective study was conducted during an 8-month period, from August 2006 to April 2007, to describe the epidemiology of Staphylococcus aureus-associated infections. In addition, the molecular characteristics, antimicrobial susceptibilities and antibiotic resistance determinants were identified in S. aureus isolates from hospitals and the community in Vladivostok, Russia. Among the 63 S. aureus isolates eligible for this study, methicillin resistance was observed in 48% (n = 30). Hospital-acquired strains accounted for 93% (28/30) of all methicillin-resistant S. aureus (MRSA) isolates. The major MRSA clone (sequence type (ST) 239, staphylococcal cassette chromosome mec (SCCmec) type III, Panton--Valentine leukocidin (PVL)-negative, with two related staphylococcal protein A gene (spa) types (types 3 and 351)) represented 90% of all of the MRSA isolates. This clone was multidrug-resistant, and 41% of isolates showed resistance to rifampicin. Community-acquired MRSA isolates (n = 2) were categorized as ST30, SCCmecIV, spa type 19, and PVL--positive, and as ST8, SCCmecIV, of a novel spa type 826, and PVL-negative. Eight different STs were detected among methicillin-susceptible S. aureus (MSSA) isolates, of which 55% were PVL--positive. One MSSA clone, which was categorized as ST121, spa type 273, and PVL--positive, caused fatal community-acquired pneumonia infections. The strains predominantly isolated in hospitals in Russia belonged to the multidrug-resistant Brazilian/Hungarian ST239 MRSA clone; however, this clone has new antibiotic susceptibilities. Additionally, the emergence of PVL--positive MSSA strains with enhanced virulence was observed, warranting continued surveillance.  相似文献   

14.
In order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol.  相似文献   

15.
The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.  相似文献   

16.
《Immunobiology》2022,227(3):152223
The present study intends to clarify the hypothesis that PVL-positive Methicillin-resistant S. aureus strain (PVL+-MRSA)-infected macrophages regulate autophagy and thus in turn inhibit phagocytosis through the in vitro and in vivo experiments. The autophagy of mouse macrophage cell line RAW264.7 was observed by fluorescence microscopy, and counted based on the number of each cell dot-like structure GFP-LC3. The protein levels of the phagocytic factors associated with autophagy were determined by western blotting. The phagocytosis of RAW264.7 on MRSA was determined by counting the colony. The clinically isolated and identified PVL+-MRSA strain was used to infect BALB/c mice (left nasal drip) to establish a mouse pneumonia model. PVL+-MRSA mice were then treated with 3-MA or linezolid. Bronchoalveolar lavage fluid (BALF) from mice was collected for macrophage counting by Flow cytometry assay. The right lung was aseptically isolated for counting the amount of bacteria. The results showed that PVL+-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA and aggravated by rapamycin. Exogenous rPVL administrated into PVL--MRSA-infected macrophages caused the autophagy of macrophage. Exogenous rPVL, particularly A-Luk S-PV, administrated into macrophages also caused the autophagy of macrophage, which was reversed by PMX53, a C5aR antagonist. In a mouse pneumonia model, PVL+-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA or linezolid. In conclusion, this study indicated PVL+-MRSA regulated macrophage autophagy, which in turns inhibit the phagocytosis of S. aureus by macrophage. This study may provide a potential target against S. aureus infection.  相似文献   

17.
In this study, the use of isothermal microcalorimetry (IMC) for differentiation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) and MIC determination was evaluated. It was possible to differentiate between MRSA and MSSA within 4 h, whereas the standard method required 24 h. The MICs of cefoxitin were successfully determined for MRSA and MSSA by using IMC.  相似文献   

18.
Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVL-positive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.  相似文献   

19.
Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.  相似文献   

20.
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