Oxidative and nitrosative stress in Staphylococcus aureus biofilm

Abstract

Diverse chemical and physical agents can alter cellular functions associated with oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress in biofilm. The present study was designed to address the issues of Staphylococcus aureus biofilm formation with respect to the generation of oxidative and nitrosative stress. We studied three pathogenic S. aureus clinical strains and an ATCC strain exposed to a different range of culture conditions (time, temperature, pH, reduction and atmospheric conditions) using quantitative methods of biofilm detection. We observed that cellular stress could be produced inside biofilms, thereby affecting their growth, resulting in an increase of ROS and RNI production, and a decrease of the extracellular matrix under unfavorable conditions. These radical oxidizers could then accumulate in an extracellular medium and thus affect the matrix. These results contribute to a better understanding of the processes that enable adherent biofilms to grow on inert surfaces and lead to an improved knowledge of ROS and RNI regulation, which may help to clarify the relevance of biofilm formation in medical devices.

Reduction ofCandida tropicalisbiofilm by photoactivation of aHeterophyllaea pustulataextract

“…Reactive oxygen species production ROS were detected by their oxidative action that causes the nitro blue tetrazolium (NBT) reduction to nitroblue diformazan, by following the methodology previously described by us (Paraje et al 2009;Arce Miranda et al 2011;Peralta et al 2015). Since the by-product of this assay is proportional to the ROS generated in biofilms, its absorbance was measured at 540 nm in the same microplate reader used earlier.…”

“…After that, both microplates were incubated at 37 C for 48 h. Then, the supernatant was removed in order to measure the production of ROS and RNI, the SOD activity, and the total antioxidant activity of the biological system by FRAP assay. After removing the supernatant, the biofilm formation was quantified and the results were expressed as BBU (0.1 OD 595 nm ¼ 1 BBU) (O'Toole & Kolter, 1998;Arce Miranda et al 2011;Peralta et al 2015). The percentage of reduction was calculated using the following formula:…”

“…The assay was adjusted to 96-well microtiter plates (Greiner Bio-One, Germany) according to previously published procedure (Arce Miranda et al 2011;Peralta et al 2015). Briefly, plates were pretreated with 50% FBS in SDB at 37 C for 30 min and washed with 10 mM PBS (pH 7.2) twice.…”

“…NO generation (precursor of RNI) was evaluated as nitrite by a microplate assay using the Griess reagent (Paraje et al 2009;Arce Miranda et al 2011). The OD at 540 nm of each well was measured with an automated microplate reader used before.…”

“…Isolates classified as strong biofilm producer, were selected to be used in the in vitro antifungal activity test. The amount of formed biofilm was expressed as biofilm biomass unit (BBU), which was arbitrarily defined as 0.1 OD 595 equal to 1 BBU (Arce Miranda et al 2011;Peralta et al 2015).…”