Oxygen Regulates Invasiveness and Virulence of Group B Streptococcus

Abstract

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5-to 400-fold higher numbers when cultured at a cell mass doubling time (t d ) of 1.8 h than at a slower t d of 11 h. Invasion was optimal when GBS was cultured at a t d of 1.8 h in the presence of >5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t d of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t d of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.

Molecular epidemiology and virulence characteristics of prevalent group A streptococci recovered from patients in northern India

“…The number of intracellular GAS was determined by viable plate counts. The percentage of infecting GAS was calculated with the formula (CFU of intracellular GAS/CFU of original inoculum) x 100 [27]. Each test was done in quadruplicate.…”

“…Next, three mL of fresh RPMI 1640 medium with 10% FBS containing gentamicin (100 µg mL -1 ) and penicillin G (5 µg mL -1 ) (HiMedia Labs, Mumbai, India) was added and plates were incubated in a 5% CO 2 incubator for two hours to kill extracellular bacteria. Monolayers were rinsed five times with PBS (pH 7.4) to remove antibiotics completely [27], and cells were detached by the addition of 0.2 mL of 0.25% trypsin-EDTA (GIBCO); the mixture was incubated for five minutes in a CO 2 incubator. Monolayers were lysed by the addition of Triton X-100 (Sisco Research Laboratories, Mumbai, India) (0.8 mL of 0.025% solution), transferred quantitatively to microtubes, and mixed on a vortex mixer for one minute.…”

“…The cell culture infection assay was performed using a conventional method with some modifications described elsewhere [25,27]. In brief, GAS strains (M1.27 and M49.4 strains) were grown at three different temperatures (25ºC, 30ºC, and 41°C) and two salt concentrations (0.3 and 0.6 M NaCl) to an OD 650 of 0.5 in THB.…”

“…HEp-2 [18] and A549 [27] cell lines have been previously used for invasion experiments. These cell lines were maintained as described [27].…”

“…HEp-2 [18] and A549 [27] cell lines have been previously used for invasion experiments. These cell lines were maintained as described [27]. In brief, both cell lines were maintained in RPMI 1640 culture medium (GIBCO, Invitrogen, Carlsbad, USA) with 1% L-glutamine and 10% fetal bovine serum (FBS) (GIBCO) without antibiotics at 37°C with 5% CO 2 (CO 2 Incubator, Shel Lab, Sheldon Manufacturing Inc., Cornelius, USA ).…”