Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus

The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.     

Viable Staphylococcus aureus quantitation using ¹⁵N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow

A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled (15)N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the (15)N (input phage) and (14)N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled (15)N bacteriophage peptide internal standard with that of the wild-type (14)N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 × 10(4) colony forming units (CFU) ml(-1) to 2.0 × 10(6) CFU ml(-1), with the (15)N reference phage spiked at a concentration of 1.0 × 10(9) plaque forming units (PFU) ml(-1). Amplification with (15)N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 10(4) CFU ml(-1)), accuracy, and precision for quantitation of S. aureus.     

A trial of the use of mupirocin in the nasal carriage of Staphylococcus aureus in medical personnel]

Many hospital-acquired purulent diseases and wound infections are due to multiresistant hospital strains of Staphylococcus aureus. The role of S. aureus nasal carriage in development of wound infections due to autoinfection is confirmed. Not only inpatients but also hospital staff can be highly colonized with coagulase positive staphylococci. The S. aureus persistence in hospital personnel results in distribution of the microorganisms in the environment. Therefore, detection of S. aureus carriers without signs of the infection among the hospital personnel and eradication of the pathogen make it possible to control outbreaks of S. aureus infection in hospitals. Clinical efficacy of nasal ointment of mupirocin in the treatment of S. aureus carriers among the intensive care personnel of the N. N.Blokhin Cancer Research Center was evaluated. S. aureus nasal carriage was diagnosed in 17 (26 per cent) out of 65 persons. All the isolates were susceptible to oxacillin. 5-7 days after discontinuation of the mupirocin nasal ointment use eradication of S. aureus was stated in 100 per cent of the cases. The effect was still observed in 94 per cent of the cases in 1 month, in 76 per cent of the cases in 5-6 months and in 60 per cent of the cases in 8-9 months. It is believed that mupirocin nasal ointment (Bactroban) is convenient to use, low toxic and highly active in the treatment of persons with S. aureus nasal carriage.     

Self-fertile cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility

Cytoplasmic hybrids (cybrids) in a novel inter-generic combination, Nicotiana tabacum (+ Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and gamma-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated cybrids N. tabacum (+ H. nigrum). In contrast to the cybrids N. tabacum (+ H. nigrum), cybrids N. tabacum (+ H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+ H. nigrum) plants. Therefore, the cybrids N. tabacum (+ H. aureus) present a self-propagating system of Nicotiana (+ Hyoscyamus) nucleo-plastome incompatibility in its pure form.     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

金黄色葡萄球菌凝集因子B的原核表达及其抗血清调理吞噬活性

金黄色葡萄球菌是导致医院院内感染的主要病原。由于金黄色葡萄球菌极易产生抗药性,因此疫苗免疫是预防该细菌感染的主要手段。作为一个粘附分子,凝集因子B(ClfB)的作用是使金黄色葡萄球菌能够在宿主黏膜定植,是预防该菌感染的一个重要的靶分子。本研究成功地在大肠杆菌中表达了可溶的ClfB N1-N3结构域蛋白(Truncated-ClfB),并且利用亲和层析、离子交换层析和凝胶过滤技术对其进行了纯化。用纯化后的Truncated-ClfB免疫新西兰大白兔,收集三免后的血清检测其抗体水平并且利用流式细胞术检测抗血清的调理吞噬活性。检测结果表明,三免后的兔源Truncated-ClfB抗血清抗体效价高达1:640 000;与免疫前兔源血清相比,兔源Truncated-ClfB抗血清能够显著增加多形核白细胞(Polymorphonuclear leukocytes,PMN)对金黄色葡萄球菌的吞噬效率(P0.01)。结果表明Truncated-Clf B有希望作为金黄色葡萄球菌疫苗的候选抗原。     

Immunofluorescence Detection of Enterotoxin B Produced Under CO2 and N2 Atmospheres

The degree of cell fluorescence of Staphylococcus aureus strains 243 and S-6 was found not to correlate with enterotoxin B production under CO(2) and N(2) atmospheres.     

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity.     

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin

Abstract We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus . In the presence of 0.02% Triton X-100, 17 formerly methicillin-resistiant strains exhibited enhanced sensitivity to oxacillin. One homogeneous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N -acetylglucosaminidase and 62 kDa N -acetylmuramoyl-l-alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus by influencing a factor(s) other than PBPs, bacteriolytic enzymes, or femAB products.     

Cross reactions between Staphylococcus aureus and Neisseria meningitidis

The results of the study of rabbit antisera to meningococci A, B, C in the double diffusion in gel, passive hemagglutination test and enzyme immunoassay with antigenic preparations isolated from S. aureus strains are indicative of the presence of common antigenic determinants of protein and polysaccharide nature in S. aureus and N. meningitidis.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.     

Genome sequence of Staphylococcus lugdunensis N920143 allows identification of putative colonization and virulence factors

Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin.     

新型脱氢松香基季铵盐类抗菌剂的合成、结构表征及抗菌活性研究

以天然松香酸衍生物——脱氢松香胺为原料,合成了6种脱氢松香基季铵盐。对合成化合物结构进行了元素分析、紫外、红外光谱及核磁共振谱鉴定。药理实验结果表明,合成产品对金黄色葡萄球菌及大肠杆菌具有一定抗菌活性,其最低抑菌浓度分别为7. 18～31. 25μ/ml和250～500μg/ml。同时对产品结构对抗菌活性影响也进行了比较研究。     

Plasmid profiles of Staphylococcus aureus causing bovine mastitis

A. B aumgartner , J. N icolet & M. E ggimann 1984. Plasmid profiles of Staphylococcus aureus causing bovine mastitis. Journal of Applied Bacteriology 56 , 159–163.
Plasmid profiles of Staphylococcus aureus , isolated from herds affected with chronic bovine mastitis, are heterogeneous, as shown by the study of 85 strains from 18 different farms. The strains isolated within a herd sometimes show related plasmid patterns, although even strains isolated from different quarters of the same udder may show variations in their plasmid content. Strains without antibiotic resistance are frequently free of plasmids. Therefore, the existence of mastitis Staph. aureus virulence plasmids is unlikely. Staphylococcus aureus , resistant to penicillin and streptomycin from 9 different farms show related plasmid patterns. This result confirms a geographic spread of certain mastitis Staph. aureus strains.     

Modification of eremomycin by amine groups

Possible modification of eremomycin, a novel glycopeptide antibiotic by the amine groups with acylating agents such as Ac2O/MeOH and CH3(CH2)7COCl/Et3N and alkylating agents such as CH3CHO, NaBH and NaBH3CN was studied. N-Acetyl, N,N'-diacetyl. N-pelargoil, N-ethyl, N,N'-diethyl and N,N',N"-triethyl derivatives of eremomycin were prepared. Their structure was asserted and the order of the substitute introduction was determined. The antimicrobial activity against Bacillus subtilis and Staphylococcus aureus was assayed. It was found that with introduction of the ethyl substitutes to the eremomycin molecule the antibiotic activity lowered insignificantly whereas the acylation resulted in its decreasing by 1-2 orders.     

A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A

The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.     

Rotational-echo double resonance characterization of the effects of vancomycin on cell wall synthesis in Staphylococcus aureus

Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.