Staphylococcus aureus strains isolated from bovine mastitis: virulence,antibody production and protection from challenge in a mouse model

Septic arthritis in mice was used as a model to evaluate the virulence of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from cases of bovine mastitis. In addition, the model was used to evaluate the cross protection elicited by heterologous antibodies. Mice were intramuscularly inoculated with serial bacterial doses of different strains of S. aureus or CNS, for virulence determination; they were monitored for arthritis, gangrene or death up to 20 days. Antibody response, cross reactivity and resistance to challenge were tested by subcutaneous inoculation with a low dose of one of the S. aureus or CNS strains followed by challenge with two S. aureus strains. S. aureus alpha-hemolysin isolate was the most virulent, followed by alpha+beta-hemolysin and beta-hemolysin isolates. The least virulent isolates were the non-hemolytic S. aureus strains but even they were more virulent than the CNS strains tested. Antibodies against three different S. aureus antigens were detected by the ELISA in all mice that were inoculated with the S. aureus strains but not in any of those with the CNS strains. Immunoblot test against various S. aureus strains as antigens showed high cross-reactivity among the S. aureus strains but only a slight similarity, restricted to the bands above 36 kDa, with the CNS sera. Low-dose inoculation of alpha or alpha+beta strains before challenge with homologous and heterologous strains protected the mice, whereas the two beta strains provided only partial protection. The inoculations of non-hemolytic S. aureus or the CNS strains did not elicit any protection. Our findings demonstrate that pre-exposure of mice to a low dose of certain S. aureus strains could provide protection and that the antibodies produced could have an important protective role.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene

A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively. We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene. DNA replication in these mutants has a fast-stop phenotype, indicating that the S. aureus dnaC gene encodes the replicative DNA helicase required for the elongation step. These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair. The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S. aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects.     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.     

Factors influencing potent antagonistic effects of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures

We examined factors related to the potent antagonistic effect of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures. In the presence of sugars fermentable by E. coli alone or both E. coli and S. aureus, motile E. coli strains exerted a potent antagonistic effect and S. aureus was expelled from the culture vessel within a few days. Conversely, in the presence of a sugar fermentable by S. aureus alone, the antagonistic effect of E. coli was diminished and S. aureus persisted at ca. 5 x 10(5) cfu/mL. B. ovatus alone exerted only a weak antagonistic effect on S. aureus in any culture conditions; however, when B. ovatus was cocultivated with E. coli and S. aureus, even in the presence of a sugar fermentable by S. aureus but not by E. coli, the potent antagonistic effect was restored. Escherichia coli showed the same level of antagonistic effect either in the presence of acetic acid (ca. 32 mM), propionic acid (4 mM), butyric acid (17 mM) and hydrogen sulfide (5 x 10(-1) mM) or when these metabolic products, except for a small amount of acetic acid (1.2 mM) were not present. In these culture conditions, S. aureus populations were lost at rates much higher than theoretical wash out rates of resting cells. These results indicate the presence of some bactericidal factors other than the volatile fatty acids and hydrogen sulfide. The bactericidal factors were not found in cultures of E. coli heated in boiling water for 10 min and in cell-free culture filtrates. Thus, the bactericidal factors seem to be associated with live E. coli cells. The nature of the bactericidal factors is not clear at present.     

利用系统作图(Systems mapping)研究大肠埃希菌和金黄色葡萄球菌的互作遗传机制

【背景】物种间相互作用是物种进化的重要推动力,然而如何将基因型和表型关联以及确定在物种相互作用过程中起重要作用的基因均面临挑战。【目的】通过系统作图(Systems mapping)得到两种微生物在相互作用过程中起重要作用的SNPs (Single nucleotide polymorphism),以及随着时间的变化,这些SNPs是如何相互联系进而影响大肠埃希菌和金黄色葡萄球菌的相互作用。【方法】分别对45株大肠埃希菌、45株金黄色葡萄球菌进行单独培养和混合共培养,通过实时荧光定量PCR(Real-time quantitative PCR,qPCR)进行绝对定量,得到一定时间内各个菌株的生长量,比较相同菌株在不同培养条件下生长情况,以各个菌株重测序结果为基础,结合系统作图得到在相互作用过程中起重要作用的显著SNPs及其相互联系。【结果】通过系统作图分析,获得具有54对显著SNPs组合的三维曼哈顿图,这些组合中41个显著SNPs来自大肠埃希菌,12个显著SNPs来自金黄色葡萄球菌。在上述SNPs中已有6个SNPs所在的候选基因都可以直接或者间接影响微生物的生长量变化,从而影响两种微生物相互作用方式。它们分别是nhaR(E19056)参与生物膜的形成,rhlE(E832164)与核糖体的组装有关,csiD (E2789300)的表达可以使细胞面对恶劣环境,alk B (E2309274)可以参与DNA的损伤修复,sucA(E759230)和yjjW(E4614704)都参与细胞的代谢过程。【结论】系统作图可以检测到物种在相互作用过程中显著SNPs;物种相互作用过程中不同SNPs遗传效应随着时间变化;细菌的相互作用过程是直接遗传效应、间接遗传效应和上位性效应共同产生的结果。     

Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus.

Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

Conjugal transfer of plasmid pWBG637 from Staphylococcus aureus to Staphylococcus epidermidis and Streptococcus faecalis

Plasmids pWBG636 (GmR) and pWBG642 (EmR) derived from the conjugative plasmid pWBG637 were tested for ability to transfer conjugatively to Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis and Escherichia coli. Both plasmids transferred to S. epidermidis and Strept. faecalis but not to B. subtilis and E. coli. Once in S. epidermis and Strept. faecalis they were transferred back to S. aureus. Restriction endonuclease analysis showed that the plasmids were stably maintained in S epidermidis and Strept. faecalis.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

嗜酸乳杆菌S-层蛋白联合抗生素对Escherichia coli和Staphylococcus aureus的抑制作用研究

旨在检测嗜酸乳杆菌S-层蛋白以及S-层蛋白与抗生素联用对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)的抑制作用。采用液体发酵培养法获得嗜酸乳杆菌菌体,LiCl法提取S-层蛋白粗提物,凝胶过滤层析法纯化S-层蛋白,分别用E.coli和S.aureus处理Caco-2细胞2 h后,考察S-层蛋白在不同浓度和不同作用时间条件下对E.coli和S.aureus的抑制作用,并考察S-层蛋白联合抗生素对E.coli和S.aureus的抑制作用,实验分组:(1)空白对照;(2)嗜酸乳杆菌组;(3)S-层蛋白组;(4)抗生素组;(5)嗜酸乳杆菌+抗生素组;(6)S-层蛋白+抗生素组。结果显示,液体发酵得到嗜酸乳杆菌菌体,提取并纯化得到S-层蛋白;S-层蛋白对E.coli和S.aureus有很好的抑制效果,具有浓度依赖性,高浓度下抑制率达到42.2%和31.7%,差异极显著(P<0.01),且在E.coli和S.aureus作用的短时间内干预效果明显,0 h时的抑制率分别达到59.3%和48.4%;S-层蛋白联合抗生素的抑菌率分别达到81.7%和79.3%,差异极显著(P<0.01),效果优于单独使用抗生素。嗜酸乳杆菌S-层蛋白具有较强的抑菌作用,可以与抗生素联用,有望开发称为一种新型的抗菌药物。     

Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus

Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.     

一株金黄色葡萄球菌小菌落突变株的分离鉴定

摘要：【目的】由于金黄色葡萄球菌（金葡菌）小菌落突变株（small colony variants,简称SCVs ）可引起持续复发性感染,且对氨基糖苷类有抗药性,在临床诊断和治疗上造成很大的困扰。我国国内尚无金葡菌SCVs的报道,本研究旨在分离鉴定出金葡菌SCVs菌株,为国内进行SCVs的相关研究提供生物学材料。【方法】通过细菌的形态鉴定、种特异性基因（nuc）的PCR扩增鉴定以及系列生化实验,从人源、动物源及环境源共104株金葡菌分离株中筛选得到金葡菌SCVs,并通过甲萘醌、硫胺素、胸腺嘧啶和血红素等补     

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).