There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates.

Streptococcus pneumoniae (Spn) and other streptococci produce a greenish halo on blood agar plates referred to as α-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of α-hemolytic streptococci, including Spn, and other bacteria. The α-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply), or to a lesser extent, to lysis of erythrocytes by Spn-produced hydrogen peroxide. We investigated the molecular basis of the α-hemolysis halo produced by Spn. Wild-type strains TIGR4, D39, R6, and EF3030, and isogenic derivative Δply mutants, produced a similar α-hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout ΔspxB/ΔlctO mutants lacked this characteristic halo. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity, and that hydrogen peroxide contributes to the release of Ply, TIGR4 isogenic mutants Δply and ΔspxB/ΔlctO had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB/ΔlctO failed to produce oxidation of oxy-hemoglobin. We demonstrated that the so-called α-hemolysis halo is caused by the oxidation oxy-hemoglobin (Fe+2) to a non-oxygen binding met-hemoglobin (Fe+3) by Spn-produced hydrogen peroxide. Since Spn colonizes the human lung, oxidation of oxy-hemoglobin might have important implications for pathogenesis.ImportanceThere is a misconception that α-hemolysis observed on blood agar plates cultures of Streptococcus pneumoniae (Spn), and other α-hemolytic streptococci is produced by a hemolysin, or alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type Spn strains and hemolysin (e.g., pneumolysin) knockout mutants, produced the α-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide defective mutants prepared in four different strains lacked the characteristic α-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of Spn and other so-called α-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the non-binding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.

Recent studies suggest there may be an environmental exposure component to the development and progression of non-alcoholic fatty liver disease (NAFLD) involving the organochlorine (OC) pesticides or their metabolites. However, the roles of OC compounds in the development of NAFLD has not been fully elucidated. Therefore, the current study was designed to determine if exposure to trans-nonachlor, a prevalent OC compound, could promote hepatocyte lipid accumulation and determine potential pro-steatotic mechanisms. McArdle-RH7777 (McA) hepatoma cells were incubated with trans-nonachlor for 24 h then neutral lipid accumulation was determined by Oil Red O staining. Exposure to trans-nonachlor produced a concentration dependent increase in neutral lipid accumulation. Trans-nonachlor also increased extracellular free fatty acid-induced neutral lipid accumulation which appears to be due at least in part to increased free fatty acid accumulation as evident by increased accumulation of Bodipy labeled dodecanoic acid. Additionally, C-acetate incorporation into total cellular lipids was increased by trans-nonachlor implicating increased de novo lipogenesis (DNL) as a potential mediator of trans-nonachlor-induced neutral lipid accumulation. Taken together, the present data indicate exposure to trans-nonachlor has a direct, pro-steatotic effect on hepatocytes to increase lipid accumulation through the combinatorial actions of extracellular free fatty acid accumulation and increased DNL.

Recent epidemiological studies have revealed significant positive associations between exposure to organochlorine (OC) pesticides and occurrence of the metabolic syndrome and there are a growing number of animal-based studies to support causality. However, the cellular mechanisms linking OC compound exposure and metabolic dysfunction remain elusive. Therefore, the present study was designed to determine if direct exposure to three highly implicated OC compounds promoted hepatic steatosis, the hepatic ramification of the metabolic syndrome. First, the steatotic effect of p,p'-dichlorodiphenyldichloroethylene (DDE), oxychlordane, and trans-nonachlor was determined in freshly isolated rat primary hepatocytes. Exposure to trans-nonachlor significantly increased neutral lipid accumulation as opposed to DDE and oxychlordane. To determine possible mechanisms governing increased fatty acid availability, the effects of trans-nonachlor exposure on fatty acid uptake, de novo lipogenesis, triglyceride secretion, and fatty acid oxidation were explored. Trans-nonachlor did not significantly alter fatty acid uptake. However, insulin-stimulated de novo lipogenesis as well as basal expression of fatty acid synthase, a major regulator of lipogenesis were significantly increased following trans-nonachlor exposure. Interestingly, there was a significant decrease in fatty acid oxidation following trans-nonachlor exposure. This decrease in fatty acid oxidation was accompanied by a slight, but significant increase in oleic acid-induced cellular triglyceride secretion. Therefore, taken together, the present data indicate direct exposure to trans-nonachlor has a more potent pro-steatotic effect than exposure to DDE or oxychlordane. This pro-steatotic effect of trans-nonachlor appears to be predominately mediated via increased de novo lipogenesis and decreased fatty acid oxidation.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite, deoxycholic acid (DoC), affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate Spn colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration DoC (0.5 mg/ml; 1.27 mM) killed all tested Spn strains (N=48) two hours post-inoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by Spn strains as soon as 10 min post-exposure. The mechanism of action did not involve activation of autolysis since the autolysis-defective double mutants Δ lytA Δ lytC and ΔspxBΔlctO were as susceptible to DoC as was the wild-type (WT). Oral streptococcal species (N=20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1x10 −9 , no spontaneous resistance was observed with DoC (srF≥1x10- 12 ). Finally, the efficacy of DoC to eradicate Spn colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with Spn EF3030 carried a median of 4.05x10 5 cfu/ml four days post-inoculation compared to 6.67x10 4 cfu/ml for mice treated with DoC. Mice in the prophylactic group had a ∼99% reduction of the pneumococcal density (median, 2.61 x10 3 cfu/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against Spn.