bPerturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2=-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2=-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2=-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.T he growing problem of antimicrobial resistance has become increasingly difficult to counter by developing new agents for two reasons. First, most large pharmaceutical companies have shelved their antimicrobial development programs due to financial, regulatory, and legal considerations (9, 10). Second, expectations that novel genomic/proteomic approaches would quickly produce many new antimicrobials may not be realistic, since no antimicrobial has materialized from such approaches, despite more than 10 years of effort (8). As an alternative to finding new antimicrobial classes, we have been seeking ways to enhance the efficacy of existing agents. Recent studies indicate that reactive oxygen species (ROS) contribute to antimicrobial-mediated killing of Escherichia coli for three important drug classes (5, 11). If the lethal action of many antimicrobials is stimulated by ROS with many pathogens, it may be possible to develop broad-spectrum, small-molecule enhancers of multiple antimicrobial classes by identifying novel, bacterium-specific targets within oxidative stress pathways. Since work to date has focused primarily on E. coli (only norfloxacin has shown a correlation between killing and hydroxyl radical surge with Staphylococcus aureus [5]), the relevance of ROS-mediated lethality to antimicrobial treatment of Gram-positive bacteria is largely unknown. Below, we describe the effects of inhibitors of ROS accumulation on the ability of three agents to kill two strains of Staphylococcus aureus under laboratory conditions. Two methicillin-susceptible S. aureus strains (RN450 [7] and ATCC 25923) were grown at 37°C on Mueller-Hinton agar or in Mueller-Hinton broth with shaking at 250 rpm. Bacterial growth was measured by diluting an overnight culture into fresh MuellerHinton broth by 100-fold, followed by turbidity determinations at 600 nm with a spectrophotometer. The MIC and minimal bactericidal concentration (MBC) were determined by broth microdilution according to CLSI guidelines (1) for daptomycin (Cubist Pharmaceuticals, Lexington, MA), moxifloxacin (Bayer AG, West Haven, CT), and oxacillin (Sigma, St. Louis, MO) in the presence or absence of subinhibitory concentrations (one-half of the MIC) of 2,2=-bipyridyl plus thiourea, two compounds that interfere with antimicrobial-mediated hydroxyl radical accumulation (5, 11). Concentration-kill curves and killing kinetics were measured by incubating exponentially growing cells with daptomycin, moxifloxacin, or oxacillin in the presence or ab...
Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.
BACKGROUND Unconjugated bilirubin (UCB) is generally considered toxic but has gained recent prominence for its anti-inflammatory properties. However, the effects of it on the interaction between intestinal flora and organisms and how it influences immune responses remain unresolved. AIM To investigate the role of UCB in intestinal barrier function and immune inflammation in mice with dextran-sulfate-sodium-induced colitis. METHODS Acute colitis was induced by 3% ( w / v ) dextran sulfate sodium salt in drinking water for 6 d followed by untreated water for 2 d. Concurrently, mice with colitis were administered 0.2 mL UCB (400 μmol/L) by intra-gastric gavage for 7 d. Disease activity index (DAI) was monitored daily. Mice were sacrificed at the end of the experiment. The length of the colon and weight of the spleen were recorded. Serum level of D-lactate, intestinal digestive proteases activity, and changes to the gut flora were analyzed. In addition, colonic specimens were analyzed by histology and for expression of inflammatory markers and proteins. RESULTS Mice treated with UCB had significantly relieved severity of colitis, including lower DAI, longer colon length, and lower spleen weight (colon length: 4.92 ± 0.09 cm vs 3.9 ± 0.15 cm; spleen weight: 0.33 ± 0.04 vs 0.74 ± 0.04, P < 0.001). UCB administration inactivated digestive proteases (chymotrypsin: 18.70 ± 0.69 U/g vs 44.81 ± 8.60 U/g; trypsin: 1.52 ± 0.23 U/g vs 9.05 ± 1.77 U/g, P < 0.01), increased expression of tight junction (0.99 ± 0.05 vs 0.57 ± 0.03, P < 0.001), decreased serum level of D-lactate (31.76 ± 3.37 μmol/L vs 54.25 ± 1.45 μmol/L, P < 0.001), and lowered histopathological score (4 ± 0.57 vs 7 ± 0.57, P < 0.001) and activity of myeloperoxidase (46.79 ± 2.57 U/g vs 110.32 ± 19.19 U/g, P < 0.001). UCB also regulated the intestinal microbiota, inhibited expression of tumor necrosis factor (TNF) α and interleukin 1β (TNF-α: 52.61 ± 7.81 pg/mg vs 105.04 ± 11.92 pg/mg, interleukin 1β: 13.43 ± 1.68 vs 32.41 ± 4.62 pg/mg, P < 0.001), decreased expression of Toll-like receptor 4 (0.61 ± 0.09 vs 1.07 ± 0.03, P < 0.001) and myeloid differentiation primary response gene 88 (0.73 ± 0.08 vs 1.01 ± 0.07, P < 0.05), and increased expression of TNF-receptor-associated factor 6 (0.79 ± 0.02 vs 0.43 ± 0.09 ...
BackgroundStudies have shown that the absence of bile in the gut lumen, either by bile duct ligation or bile diversion, induces mucosal injury. However, the mechanism remains elusive. In this study, the role of bile pigments in gut barrier function was investigated in a rat model of bile duct ligation.MethodsMale Sprague Dawley (SD) rats were used in this study. After ligation of bile duct, the animals were administrated with free bilirubin, bilirubin ditaurate, or biliverdin by intragastric gavage. 1, 2, or 3 days later, the animals were sacrificed and the damage of mucosa was assessed by histological staining as well as biochemical parameters such as changes of diamine oxidase (DAO) and D-lactate (D-Lac) in the blood. Trypsin and chymotrypsin of the gut were also measured to determine how these digestive proteases may relate to the observed effects of bile pigments.ResultsBile duct ligation (BDL) caused significant increases in gut trypsin and chymotrypsin along with damage of the mucosa as demonstrated by the histological findings under microscope, the reduced expression of tight junction molecules like occludin, and significant changes in DAO and D-lac in the blood. Free bilirubin but not bilirubin ditaurate or biliverdin showed significant inhibitions on trypsin and chymotrypsin as well as alleviated changes of histological and biochemical parameters related to gut barrier disruption.ConclusionBile may protect the gut from damage through inhibiting digestive proteases like trypsin and chymotrypsin by free bilirubin.
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