Virulent and Avirulent Strains of Group B Streptococci from Rio de Janeiro, Brazil: Relationship between Differences in Surface Hydrophobicity, Sialic Acid Content and Macrophage Interaction

Abstract

Bacterial attachment to epithelia is an important mechanism for colonization of mucosal membranes and influences microbial pathogenicity. A number of studies with both Gram-positive and Gram-negative bacteria has indicated that this hostmicro-organism interaction is determined by different factors. The degree of hydrophobicity of the bacterial can influence cell adhesion (GI Geertsema-Doornbusch et al. 1993 J Microbiol Methods 18: 61-68, P Gilbert et al. 1993 74: 67S-78S) and phagocytosis (CJ van Oss 1978 Ann Rev Microbiol 32: 19-39).The streptococci of group B (GBS) are important pathogens involved in human and animal diseases (JL Nancy et al. 1984 J Infect Dis 149: 851-860, GS Tamura et al. 1994 Infect Immun 62: 2450-2458, P Munoz 1997. A wide variety of infections has been described ranging from minor lesions to disseminated diseases. Because hydrophobicity seems to be an important factor that can influence bacterial-eucaryotic cell interactions, in the study presented here we examined the cell surface hydrophobicity of GBS from different clinical sources and examined the role of this surface property on the GBSmacrophage interaction.Two strains of GBS serotype III were used in this study. The strain 80340 was collected from the urethra of an asymptomatic patient and the other strain (90356) from a patient presenting meningitis. Bacteria were stored in the lyophilized state and recovered, when necessary, in broth (Brain Heart Infusion, Difco) supplemented with 5% sheep blood.For the hydrophobicity assay, single (three) colonies were picked from sheep blood plates and grown for 18 hr at 37 o C. The bacteria were then harvested by centrifugation, washed twice and suspended in 5 ml of 0.05M sodium phosphate buffer (pH 7.4) containing 0.15M NaCl to an absorbency of 0.4 at 660 nm. The bacterial suspension was treated with xylene (2.5:1, v/v; Merck), the system was vigorously mixed for 2 min and allowed to settle for 20 min at room temperature. The absorbency of the aqueous phase was then determined at 660 nm against a blank consisting of sodium phosphate buffer. The hydrophobicity indexes (HI) were calculated as previously described (M Rosenberg et al. 1980 FEMS Microbiol Lett 9: 29-33).Peritoneal macrophages were collected from Swiss mice and allowed to adhere to coverslips as previously described (P Valentin-weigand et al. 1996 Infect Immun 64: 2467-2473. The bacteriamacrophage ratio was adjusted to 10:1 and interactions were allowed to proceed for periods of 15 min to 240 min. After interaction, adherent cells were washed, fixed in Bouin for 5 min, stained with Giemsa, and the mean number of attached microrganisms per infected macrophage was determined by counting the number of adherent bacteria in 900 phagocytes by light microscopy.All the procedures for extraction, purification and determination of sialic acid content of GBS were carried out as formally described (JP Kamerling et al. 1982 Biochem Biophys 714: 351-354).The bacteria-macrophage ratios were analyzed by the Kolmogorov-Smirnov test using the "...

CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production

“…As expected, the Δ cpsE mutant displayed increased autoaggregation and surface hydrophobicity and decreased anionic charge. This finding is in agreement with some previous studies that indicated that sialic acid is negatively correlated with surface hydrophobicity and autoaggregation ( 30 , 31 ). Furthermore, we disrupted cpsE in the ΔCRISPR background to determine the relationship between the regulatory role of CRISPR in CPS production and the altered surface properties.…”

Adherence and Biofilm Production of Streptococcus pyogenes

“…Even in this recommended conditions, our isolates have not adhered to hexadecane, even after capsule removal, as suggested by Offek et al [47]. According to experiment performed by Nagao and Benchetritt [48], we used xylene instead of hexadecane, afterwards our isolates adhered to this new hydrocarbon. Hexadecane is low-reactive saturated alkane hydrocarbon in contrast to xylene, which is more reactive non-saturated aromatic hydrocarbon.…”