Conspicuous Growth of Intravenously Inoculated Staphylococcus aureus in Subcutaneously Established Ehrlich Ascites Tumor Tissue of Mice

Intratumoral growth of Staphylococcus aureus was compared with the intrarenal growth, to examine the usefulness of the method as a marker of its pathogenicity. When 5 × 10⁷ CFU/mouse of three derivatives from S. aureus Cowan I with different intrarenal growth were intravenously injected into Ehrlich tumor-bearing mice, they lodged in the tumor tissue at approximately 10³ CFU/0.1 g by 30 min after infection, and grew in the range of 10⁶ CFU/0.1 g to 10⁸ CFU/0.1 g by day 4, regardless of their intrarenal growth capacity. In contrast, S. saprophyticus lodged in both tissues to the same degree as S. aureus, but did not grow at all. The time course of the staphylococcal growth was different between tumor tissue and kidney, suggesting differences in the local responses against S. aureus.     

The different actions of normal and supranormal calcium concentrations on the proliferation of BALB/c 3T3 mouse cells

Summary In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. However, the development of such stimulated, calcium-deprived cells stops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later. Issued as NRCC No. 15371.     

Change in drug resistance of staphylococcus aureus

Objective To analyze the change in drug resistance of staphylococcus aureus （SAU） in the PLA general hospital from January 2008 to December 2012, and to provide solid evidence to support the rational use of antibiotics for clinical applications. Methods The SAU strains isolated from clinical samples in the hospital were collected and subjected to the Kirby-Bauer disk diffusion test. The results were assessed based on the 2002 American National Committee for Clinical Laboratory Standards （NCCLS） guidelines. Results SAU strains were mainly isolated from sputum, urine, blood and wound excreta and distributed in penology, neurology wards, orthopedics and surgery ICU wards. Except for glycopeptide drugs, methicillin- resistant staphylococcus aureus （MRSA） had a higher drug resistance rate than those of the other drugs and had significantly more resistance than methicillin-sensitive staphylococcus aureus （MSSA） （P〈0.05）. In the dynamic observation of drug resistance, we discovered a gradual increase in drug resistance to fourteen test drugs during the last five years. Conclusion Drug resistance rate of SAU stayed at a higher level over the last five years; moreover, the detection ratio of MRSA keeps rising year by year. It is crucial for physicians to use antibiotics rationally and monitor the change in drug resistance in a dynamic way.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Moenomycin-resistance is associated with vancomycin-intermediate susceptibility in Staphylococcus aureus

We have previously isolated a vancomycin-intermediate susceptibility mutant from methicillinresistant Staphylococcus aureus(MRSA) strain COL, and demonstrated the increased glycan-chain length and the decreased moenomycin-susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin-resistant mutants (4-16 fold higher compared to the parent) from 5 MRSA and 2 methicillin-sensitive S. aureus(MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2-4 fold), teicoplanin (2-4 fold) and an increased susceptibility to methicillin (2-8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan-chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin-resistance is closely associated with vancomycin-intermediate susceptibility in S. aureus.     

Staphylococcus aureus requires increased level of Ca(2+) or Mn(2+) to grow normally in a high-NaCl/low-Mg(2+) medium.

Mg2+-availability in Staphylococcus aureus cells decreased significantly with increasing NaCl concentration in growth media. Cells grew in a NaCl-free, Chelex resin-treated complex medium only if the medium was supplemented with 50 microM MgCl2, while, growth was limited when the medium was further supplemented with 1.0 M NaCl. Cells grown in such a high-NaCl/low-Mg2+ medium exhibited the morphologic abnormality of larger than normal cells. Both sufficient growth and normal cell morphology were restored by increasing Mg2+ concentration in a high-NaCl medium, or by supplementation with either CaCl2 or MnSO4 in a high-NaCl/low-Mg2+ medium. Supplementing with BaCl2, SrCl2 or FeSO4, however, had no effect. These results indicate that Ca2+ and Mn2+ might play some essential role in the growth of Staphylococcus aureus in a high-NaCl/low-Mg2+ environment.     

Subcutaneous Growth of Staphylococcus aureus Concomitantly Inoculated with Ehrlich Ascites Tumor Cells

Intratumoral growth of Staphylococcus aureus Cowan I-derived AP332 was examined by subcutaneous inoculation of cocci in doses ranging from 18 to 1.8 × 10⁵ CFU with Ehrlich ascites tumor cells. Inoculation of 18 CFU AP332 resulted in staphylococcal growth in one of five mice, and the proportion of mice established intratumoral infection increased with the initial inocula. Six other strains of S. aureus also grew in the tumor tissue, and none of the three strains of coagulase-negative staphylococci grew at all. Ethanol-killed tumor cells did not promote staphylococcal growth as vigorously as the live tumor cells, especially when the initial inoculum of AP332 was smaller than 10⁴ CFU.     

Staphylococcus aureus produces autolysin-susceptible cell walls during growth in a high-NaCl and low-Ca2+ concentration medium

The growth of Staphylococcus aureus 209P becomes unusually sensitive to a high-NaCl concentration by decreasing the Ca2+ concentration in growth media, and cells either autolyze or transform into protoplast-like forms when grown standing in high-NaCl and low-Ca2+ concentration media below 37 C (Ochiai, T., Microbiol. Immunol. 43 (7): 705-709, 1999). To assess the role of Ca2+ in the salt tolerance of this organism, cells grown in the presence of different concentrations of Ca2+ were treated with boiling SDS, and their susceptibilities to crude autolysin (3 M LiCl extract of S. aureus 209P cells) were evaluated by turbidimetric assay and zymographic analysis. Susceptibilities of SDS-treated cells (SDS-cells) to crude autolysin were significantly influenced by Ca2+ concentration in the culture, and SDS-cells prepared from cultures grown in high-NaCl and high-Ca2+ concentration media exhibited marked resistance to crude autolysin when the assay system contained a high concentration of NaCl. On the contrary, SDS-cells prepared from cultures grown in high-NaCl and low-Ca2+ concentration media were rather susceptible to crude autolysin under the same assay conditions. A zymographic analysis revealed that the constitution of cell-associated autolysins was not influenced by the concentration of exogenous Ca2+. These results suggested that at least part of the mechanism of salt-induced autolysis in S. aureus 209P might be related to the synthesis of an autolysin susceptible cell wall.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

Surface protein adhesins of Staphylococcus aureus

Staphylococcus aureus can colonize the host to initiate infection by adhering to components of the extracellular matrix. Adherence is mediated by surface protein adhesins (MSCRAMMs). Ligand binding by these fibronectin-, fibrinogen- and collagen-binding proteins occurs by distinct mechanisms that are being investigated at the molecular level.