Staphylococcus aureus Strain Newman Photoinactivation and Cellular Response to Sunlight Exposure

Abstract

Sunlight influences microbial water quality of surface waters. Previous studies have investigated photoinactivation mechanisms and cellular photostress responses of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, but further work is needed to characterize photostress responses of bacterial pathogens. Here we investigate the photoinactivation of Staphylococcus aureus (strain Newman), a pigmented, waterborne pathogen of emerging concern. We measured photodecay using standard culture-based assays and cellular membrane integrity and investigated photostress response by measuring the relative number of mRNA transcripts of select oxidative stress, DNA repair, and metabolism genes. Photoinactivation experiments were performed in both oxic and anoxic systems to further investigate the role of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. S. aureus lost culturability much faster in oxic systems than in anoxic systems, indicating an important role for oxygen in photodecay mechanisms. S. aureus cell membranes were damaged by sunlight exposure in anoxic systems but not in oxic systems, as measured by cell membrane permeability to propidium iodide. After sunlight exposure, S. aureus increased expression of a gene coding for methionine sulfoxide reductase after 12 h of sunlight exposure in the oxic system and after 6 h of sunlight exposure in the anoxic system, suggesting that methionine sulfoxide reductase is an important enzyme for defense against both oxygen-dependent and oxygen-independent photostresses. This research highlights the importance of oxygen in bacterial photoinactivation in environmentally relevant systems and the complexity of the bacterial photostress response with respect to cell structure and transcriptional regulation.IMPORTANCEStaphylococcus aureus is a pathogenic bacterium that causes gastrointestinal, respiratory, and skin infections. In severe cases, S. aureus infection can lead to life-threatening diseases, including pneumonia and sepsis. Cases of community-acquired S. aureus infection have been increasing in recent years, pointing to the importance of considering S. aureus transmission pathways outside the hospital environment. Associations have been observed between recreational water contact and staphylococcal skin infections, suggesting that recreational waters may be an important environmental transmission pathway for S. aureus However, prediction of human health risk in recreational waters is hindered by incomplete knowledge of pathogen sources, fate, and transport in this environment. This study is an in-depth investigation of the inactivation of a representative strain of S. aureus by sunlight exposure, one of the most important factors controlling the fate of microbial contaminants in clear waters, which will improve our ability to predict water quality changes and human health risk in recreational waters.

Keywords: Staphylococcus aureus; coastal waters; enterococci; fecal indicator bacteria; photoinactivation.

FIG 1

Photoinactivation of S. aureus in oxic and anoxic systems with respect to fluence (kilojoules per square meter). C, concentration of S. aureus at a particular fluence level; C₀, initial concentration (both in CFU per milliliter). Dashed lines are modeled biphasic photoinactivation curves based on nonlinear regression.

FIG 2

Effect of sunlight on S. aureus total cells and cell membrane integrity as determined by fluorescence microscopy. (Top) Total cell concentrations of S. aureus with respect to fluence. Concentrations are represented as the natural log-transformed relative concentration. C, concentration of total cells at a certain fluence level; C₀, initial concentration (both in cells per milliliter). (Bottom) Fraction of intact cells with respect to fluence. The fraction of intact cells was calculated as the intact cell concentration divided by the total cell concentration (both in cells per milliliter).

FIG 3

Relative expression of crtN, gapA, msrA, pta, recA, rexA, sodA, and trxB. (Top) Oxic reactors. (Bottom) Anoxic reactors. Note that gapA and recA are not represented in the oxic plot and ftsZ and sodA are not represented in the anoxic plot because these genes were used as reference genes. Error bars represent ± geometric standard deviation. The dashed horizontal lines indicate a relative expression ratio of 1. *, R > 2.

FIG 4

Experimental design for investigating bacterial photoinactivation. Experiments consisted of three phases: bacterial cultivation, microcosm setup, and photoinactivation. Control (“dark”) and experimental (“light”) microcosms were set up and sampled in parallel.