Competence for Transfection in Staphylococcus aureus

Lysogenicity with phage P11 is a requirement for competence in the presence of calcium ions in Staphylococcus aureus 8325N. The wild-type strain 8325N, lysogenic for the phages P11, P12, and P13, is also competent, but strain 8325-4, a nonlysogenic derivative of strain 8325N, as well as strains 8325-4 (P12) and 8325-4 (P13) could not develop competence. Preincubation of strain 8325-4 with culture filtrates from a competent strain can induce competence, but rabbit anti-P11 serum can neutralize the competence factor. Superinfection of competent strain 8325-4 (P11) with phage P11 at high multiplicities increases the transfection frequency. Uptake of deoxyribonucleic acid by competent cells is dependent on calcium ion concentration, pH, and temperature. Inhibition of energy metabolism or protein synthesis before and during incubation with deoxyribonucleic acid affects the binding and uptake. The ability to develop competence during bacterial growth differs between the wild-type strain (8325N) and a nuclease-deficient mutant (8325N nuc). The wild-type strain has a narrow competence maximum in the early exponential growth phase where no extracellular nuclease activity is produced. The nuc strain shows in addition competence maxima later in the exponential growth phase.     

Factors Affecting the Persistence of Staphylococcus aureus on Fabrics

The persistence of Staphylococcus aureus (Smith) on wool blanket, wool gabardine, cotton sheeting, cotton knit jersey, cotton terry cloth, and cotton wash-and-wear fabrics was studied. The fabrics were exposed to bacterial populations by three methods: direct contact, aerosol, and a lyophilized mixture of bacteria and dust having a high content of textile fibers. The contaminated fabrics were held in 35 or 78% relative humidities at 25 C. In general, the persistence time of S. aureus populations on fabrics held in 35% relative humidity was substantially longer when the fabrics were contaminated by exposure to aerosolized cultures or to dust containing bacteria than when contaminated by direct contact. In a 78% relative humidity, bacterial populations on the fabrics persisted for substantially shorter periods of time regardless of the mode of contamination or fabric type. Cotton wash-and-wear fabric (treated with a modified triazone resin) was the material on which populations of S. aureus persisted for the shortest time. This organism retained its virulence for Swiss mice after being recovered from wool gabardine swatches held 4 weeks in 35% relative humidity and 6 weeks in 78% relative humidity.     

Plasmid-Specific Transformation in Staphylococcus aureus

Transformation of Staphylococcus aureus cells with circular duplex deoxyribonucleic acid prepared from plasmid-carrying strains by alkali denaturation and selective renaturation or by dye-buoyant density centrifugation is reported. In all of the transformants tested, the transformed markers became established as autonomous plasmids that were biologically and physically indistinguishable from those carried by the donor strains. Transformation with bulk deoxyribonucleic acid from a strain carrying the penicillinase plasmid, PI(258), gave rise to transformants in which the erythromycin locus, the only plasmid marker transformed, was shown to be integrated into the host chromosome.     

Factors Affecting the Resistance of Staphylococcus aureus to Hydrogen Peroxide Treatments in Milk

Staphylococcus aureus 196E was treated with 0.05% hydrogen peroxide in milk under varying conditions to determine the effects of treatment conditions and characteristics of the culture on bactericidal effectiveness of hydrogen peroxide. Time intervals required for 90 to 99.99% destruction of S. aureus decreased significantly as treatment temperatures increased from 37.8 to 57.2 C. Plots of survivor curves showed extended lags in destruction at 37.8 C, slight lags followed by logarithmic rates of destruction at 48.9 C, and logarithmic rates at 54.4 and 57.2 C except for trials in which there was very rapid initial destruction followed by logarithmic rates. S. aureus 196E was significantly more resistant to heat treatments at 54.4 C without added hydrogen peroxide than to treatment with 0.05% hydrogen peroxide at this temperature. Cultures grown at 37 C for 16 hr in milk were more resistant to hydrogen peroxide than were cultures grown at 35 C. Storage of cultures for 96 hr in milk at 4 C caused a decrease in the resistance of the culture. Numbers of staphylococci being treated had little effect on rates of destruction.     

Induction of Hemolysin Synthesis by Transformation in Staphylococcus aureus

A nonhemolytic strain of Staphylococcus aureus was transformed with deoxyribonucleic acid extracted from two hemolytic strains of S. aureus. In each case the hemolysin pattern after transformation was identical to that of the donor strain. However, bacteriophage type, serotypes, and other biological properties of the recipient strain remained unaffected.     

Transformation of Chromosomal and Plasmid Characters in Staphylococcus aureus

Plasmid and probably also chromosomal characters have been genetically transformed in Staphylococcus aureus. Recipient cells show competence throughout the exponential growth phase with a maximum at early times.     

Factors Affecting Transformation of Micrococcus lysodeikticus

Relatively high transfo mation frequencies were obtained with Micrococcus lysodeikticus cells, previou[ill]sly grown in defined broth, with a tris(hydroxymethyl)-aminomethane-glutamate b uffer containing Mg(++), Ca(++), Ba(++), or Sr(++).     

Factors Affecting Transformation of Pasteurella novicida

The requirements and characteristics of Pasteurella novicida transformations in liquid suspensions were studied. Transformation frequencies of 0.1 to 0.3% were routinely obtained when recipient cells were harvested from 16-hr agar plates and higher than 1% when logarithmic-phase broth-grown cells were used. Calcium ions were essential for transformations. The deoxyribonucleic acid dose response curve, kinetics of transformation, and pH optimum for transformations were similar to those of other bacterial transformation systems. The genetic relatedness of P. novicida and P. tularensis was established by transforming P. novicida mutants with deoxyribonucleic acid extracted from P. tularensis.     

Transformation analysis of three linkage groups in Staphylococcus aureus.

While studying a set of multiply marked mutants of Staphylococcus aureus strain 8325 by transformation, several instances of apparent genetic linkage were encountered. After showing that these linked transformations were readily inactivated by shearing of the deoxyribonucleic acid (DNA) but were resistant to dilution of the DNA, and showing that mixtures of DNA failed to form double transformants, it was concluded that the linkages were legitimate rather than the result of congression. Three linkage groups were defined: thy-101-lys-115-trp-103-thr-106, pyr-141-hisGb15-nov-pur-102, and pur-110-ilv-129. The positions of the previously studied trp and his operons corresponded to the trp-103 and hisGb15 loci. The ilv-129 position adjacent to pur-110 probably corresponds to the ilv-leu gene cluster. The distance over which linkage was detected was greater by transformation than by generalized transduction.     

大肠杆菌感受态细胞转化能力的影响因素

探讨了大肠杆菌菌株、细菌生长状态、转化溶液、抗冻剂及保存时间、质粒长度和纯度对感受态细胞转化能力的影响。结果表明,以100 mmol/L CaCl2为缓冲液,采用经活化培养的A600为0.55的TG1制备的感受态细胞,在冰上放置6h后转化,所得转化率最高,可达2×106-4×107cfu/μg DNA(pUC19)。随着质粒长度增加和纯度降低,转化率有所下降。若感受态细胞要保存备用,以15％甘油为抗冻剂优于7％DMSO,但添加抗冻剂对转化率有抑制作用。贮于甘油的感受态细胞在-70℃冻存两个月后仍有较理想的转化率。     

Identification of Staphylococcus aureus Colony-Spreading Stimulatory Factors from Mammalian Serum

Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.     

Factors affecting sensitivity of Staphylococcus aureus 196E to polyphosphates

The effect of polyphosphates (eight compounds) on growth of Staphylococcus aureus 196E in brain heart infusion broth was studied. The organism was sensitive (in decreasing order) to chain polyphosphates with 21, 3, 13, and 15 PO4 groups, and bactericidal effects were observed with 0.5% of these compounds. No inhibition was effected by PPi or a metaphosphate. The inhibitory effects were pH dependent, and bacterial sensitivity was highest at pH greater than 7.4. Initial populations affected the number of survivors. No growth was observed after 24 h at 35 degrees C when the initial cell population was less than 10(4) CFU/ml, and a 100- to 1,000-fold decline in cell numbers occurred when initial populations were higher than 10(4) CFU/ml. Sodium tripolyphosphate produced less inhibition after heat sterilization (15 min, 121 degrees C) than after filter sterilization, whereas sodium hexametaphosphate (n = 21) retained most of its antimicrobial activity after heat sterilization. Supplementation of broth with Mg2+ was effective in overcoming inhibition by 0.5% sodium tripolyphosphate, and an addition of 0.25 to 1.0 mM cation restored most of the growth. Inhibition was partially eliminated by Ca2+ and Fe2+, but not by Zn2+ or Mn2+.     

Role of the ø11 Phage Genome in Competence of Staphylococcus aureus

Both phage ?11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ?11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ?11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (?11) or 8325-4 (83A) at high multiplicities with phage ?11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (ts?11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ?11 of Kretschmer and Egan and ts?11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ?11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.     

Binary IS typing for Staphylococcus aureus

Background

We present an easily applicable test for rapid binary typing of Staphylococcus aureus: binary interspace (IS) typing. This test is a further development of a previously described molecular typing technique that is based on length polymorphisms of the 16S-23S rDNA interspace region of S. aureus.

Methodology/Principal Findings

A novel approach of IS-typing was performed in which binary profiles are created. 424 human and animal derived MRSA and MSSA isolates were tested and a subset of these isolates was compared with multi locus sequence typing (MLST) and Amplified Fragment Length Polymorphism (AFLP). Binary IS typing had a high discriminatory potential and a good correlation with MLST and AFLP.

Conclusions/Significance

Binary IS typing is easy to perform and binary profiles can be generated in a standardized fashion. These two features, combined with the high correlation with MLST clonal complexes, make the technique applicable for large-scale inter-laboratory molecular epidemiological comparisons.     

Staphylococcus aureus determinants for nasal colonization

Approximately 20% of the healthy human population is persistently colonized in the nasal cavity with Staphylococcus aureus, which constitutes a major risk for infection. S. aureus seems to predominantly colonize the anterior part of the nasal cavity by adhering to nasal surface structures and escaping the host innate and adaptive immune responses. Several bacterial and host factors that play a role in these processes have been identified in the past few years and were in part functionally evaluated in appropriate colonization models. However, the dynamics of host-pathogen crosstalk is only partially understood.