The nucleotide sequence of a small cryptic plasmid found in Staphylococcus aureus and its relationship to other plasmids

The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

Characterization of staphylococcal plasmids hybridizing with the fosfomycin resistance gene fosB

The distribution of the fosB gene, coding for fosfomycin resistance, in 105 fosfomycin-resistant isolates of Staphylococcus from various geographical areas, was studied by Southern blot hybridization. Nucleotide sequences related to fosB were detected in 36 strains belonging to five species. Plasmids bearing fosB were often of a size similar to that of pIP1842 (2.54 kb) in S. epidermidis, most often small (2.4 to 4.1 kb) in other species including S. aureus where a 2.7-kb plasmid was found in 16 out of the 18 strains studied. The fosB gene was geographically dispersed since it was present in six different locations in France and also in Japan. The weak hybridization observed with plasmid DNA of certain strains of S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. warneri may indicate gene heterogeneity for fosfomycin resistance in Staphylococcus spp.     

A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator

A 2.5 kb plasmid, pA22, isolated from a naturally occurring S. aureus strain confers constitutive MLS-resistance. By restriction enzyme analysis, pA22 is indistinguishable from the S. aureus inducible MLS-resistance conferring plasmid, pT48, apart froma small deletion. DNA sequencing showed that the deletion, is in the leader/attenuator region of the ermC (MLS-resistance) gene and removes some of the complementary repeat regions required by the translational attenuation model in pT48 for inducible ermC expression. The deletion in plasmid pA22 is different from that found in similar 2.5kb constitutive MLS-resistance plasmids in other Gram-positive bacteria. It is suggested that plasmids conferring the constitutive phenotype have evolved from an inducible ancestor on several independent occasions.     

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.     

Conjugal transfer of antibiotic resistances and plasmids from Campylobacter jejuni clinical isolates

Six Campylobacter jejuni clinical isolates were examined for the occurrence of plasmids in association with antibiotic resistances as well as conjugal transfer. All the isolates were found to carry three similar plasmids of 78 kb, 12.6 kb and 3.3 kb in size. Multiple resistance to at least three of the antibiotics tested was observed with resistance to tetracycline most common. En bloc transfer of donor resistances at frequencies ranging from 10(-8) to 10(-4) were seen in all but one of the isolates during conjugation. The conjugal transfer of erythromycin, neomycin and streptomycin were observed to occur at frequencies similar to that of chloramphenicol, kanamycin and tetracycline. In isolate ABA94, three different antibiotic resistance phenotypes of the transconjugants were seen. In addition to en bloc transfer of the donor resistances, in approximately 10% of the transconjugants the streptomycin resistance was lost although these transconjugants carried the donor complement of three plasmids. In a further 1% of the transconjugants, resistance to kanamycin only was detected and these transconjugants did not carry any plasmids.     

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.     

复合生物保鲜剂对松鼠葡萄球菌的抑菌性能及其作用机理研究

以冷藏带鱼中分离出的革兰氏阳性优势菌——松鼠葡萄球菌(Staphylococcus sciuri)为试验菌,研究复合生物保鲜剂(配比浓度为:壳聚糖10.0 g/L,溶菌酶0.3 g/L与茶多酚3.0 g/L)对松鼠葡萄球菌的抑菌效果与作用机理。通过牛津杯法确定复合保鲜剂对松鼠葡萄球菌的最小抑菌浓度(MIC)与最小杀菌浓度(MBC),结合抑菌活力、细菌生长曲线、碱性磷酸酶(AKP)活性、细胞膜完整性、膜通透性与细菌超微结构观察等,综合评价不同浓度复合保鲜剂在不同处理时间内对松鼠葡萄球菌的作用效果。结果表明,复合保鲜剂对松鼠葡萄球菌的MIC与MBC分别为0.8与1.6 mg/mL,随着处理时间的延长,复合生物保鲜剂明显抑制松鼠葡萄球菌的生长,使菌体细胞外的AKP量增多,造成细菌菌体细胞壁通透性增大,细胞结构的完整性受到破坏,菌液电导率值显著升高,菌体电解质等内容物外泄,影响细胞内环境和细胞膜的稳定性,菌体皱缩变形,表面粗糙,细胞壁塌陷,细胞质外泄渗出,导致菌体死亡。     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation

In this paper, the influence of various parameters on plasmid transformation by electroporation of Staphylococcus epidermidis Tü3298 was investigated. Cell growth conditions, various concentrations and forms of plasmid DNA, field strength, pulse duration and media for electroporation and regeneration were tested. In order to obtain optimal transformation efficiency, the cells were incubated for 30 min with DNA before pulsing. With the optimized procedure, other staphylococcal species such as S. aureus, S. staphylolyticus and S. carnosus were transformed with an efficiency up to 3 X 10(5) transformants per micrograms pC194 plasmid DNA.     

2008-2010年舟山某院金黄色葡萄球菌的分布及耐药性变迁

目的 分析舟山医院三年来金黄色葡萄球菌分布及耐药性变迁,并对耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异做对比.方法 用ATB Expression半自动微生物分析仪进行菌株鉴定及药敏试验,用K-B法测红霉素、克林霉素、头孢西丁、苯唑西林直径,比较耐甲氧西林金黄色葡萄球菌(MRSA)与甲氧西林敏感金黄色葡萄球菌(MSSA)的耐药性差异.结果 金黄色葡萄球菌对苯唑西林、庆大霉素、红霉素、四环素和克林霉素的耐药率有上升的趋势;MRSA对苯唑西林、庆大霉素、复方新诺明、克林霉素、红霉素、青霉素、喹奴普汀-达福普汀、利福平和四环素的耐药率都明显高于MSSA的耐药率,二者间差异有统计学意义(P＜0.01),D-试验阳性71株,占72.45％.结论 金黄色葡萄球菌的耐药性逐渐升高,特别是对MRSA应引起临床的重视,检测克林霉素诱导型耐药具有重要的临床应用价值.     

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus

Conjugative transfer, in the apparent absence of plasmid DNA, of high-level vancomycin resistance from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus B111 has been demonstrated in vivo and in vitro. Selection of transconjugants on media containing erythromycin or chloramphenicol may result in the transfer of resistance to erythromycin, chloramphenicol, gentamicin, streptomycin and vancomycin though these are capable of separate transfer. Vancomycin resistance has not been transmitted from staphylococcus to staphylococcus though transfer of erythromycin and of chloramphenicol resistance has been achieved.     

Cloning and sequencing of a plasmid-mediated erythromycin resistance determinant from Staphylococcus xylosus

A 2.3-kb DNA fragment cloned from plasmid pCH200, the largest (52 kb) of four plasmids detected in Staphylococcus xylosus, was found to confer resistance to 14-membered ring macrolides in Bacillus subtilis and Staphylococcus aureus. DNA-sequence analysis of the fragment revealed the presence of an open-reading frame, the deduced product of which was identical to one of the two ATP-binding domains encoded by the macrolide/streptogramin-B-resistance gene msrA of Staphylococcus epidermidis. The observation that a polypeptide homologous to the C-terminus of MsrA is capable of mediating erythromycin resistance in the absence of the N-terminal region is of significance both to the evolution and functional activity of members of the ATP-binding transport super-gene family.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Surfaceome and exoproteome of a clinical sequence type 398 methicillin resistant Staphylococcus aureus strain

For many years Staphylococcus aureus has been recognized as an important human pathogen. In this study, the surfacome and exoproteome of a clinical sample of MRSA was analyzed. The C2355 strain, previously typed as ST398 and spa-t011 and showing a phenotype of multiresistance to antibiotics, has several resistance genes. Using shotgun proteomics and bioinformatics tools, 236 proteins were identified in the surfaceome and 99 proteins in the exoproteome. Although many of these proteins are related to basic cell functions, some are related to virulence and pathogenicity like catalase and isdA, main actors in S. aureus infection, and others are related to antibiotic action or eventually resistance like penicillin binding protein, a cell-wall protein. Studying the proteomes of different subcellular compartments should improve our understanding of this pathogen, a microorganism with several mechanisms of resistance and pathogenicity, and provide valuable data for bioinformatics databases.