The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.     

Effects of Prechilling and Sequential Washing on Enumeration of Microorganisms from Refuse

Techniques were evaluated for formation of a liquid inoculum from shredded municipal refuse, including chilling the refuse at 4°C prior to blending and multiple washing and blending cycles. The average count of cellulolytic bacteria from six different detachment treatments was 5.1 × 10⁴ cells per g (dry weight) of refuse with a range of 0.7 × 10⁴ to 12.7 × 10⁴ cells per g (dry weight). The liquid obtained from blending the refuse in phosphate buffer followed by hand squeezing was the selected detachment procedure. The inoculum formation procedure was validated by the addition of ruminal cellulolytic bacteria to refuse and recovery of the cellulolytic bacteria by most-probable-number enumerations. The ratio of measured to expected cell counts among tests in which different volumes of ruminal fluid were added to refuse ranged from 2.7 to 14.4. There was no evidence of anaerobic cellulolytic fungi in a refuse sample.     

Protein dynamics from chemical shift and dipolar rotational spin-echo 15N NMR

The partial collapse of dipolar and chemical shift tensors for peptide NH and for the amide NH at cross-link sites in cell wall peptidoglycan, of intact lyophilized cells of Aerococcus viridans, indicates NH vector root-mean-square fluctuations of 23 degrees. This result is consistent with the local mobility calculated in typical picosecond regime computer simulations of protein dynamics in the solid state. The experimental root-mean-square angular fluctuations for both types of NH vectors increase to 37 degrees for viable wet cells at 10 degrees C. The similarity in mobilities for both general protein and cell wall peptidoglycan suggests that one additional motion in wet cells involves cooperative fluctuations of segments of cell walls, attached proteins, and associated cytoplasmic proteins.