Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

Abstract

Staphylococcal enterotoxin B (SEB), encoded by the seb gene, is a virulence factor produced by Staphylococcus aureus that is involved mainly in food poisoning and is known to act as an aggravating factor in patients with atopic dermatitis. Research results in animal infection models support the concept that superantigens, including SEB contribute to sepsis and skin and soft tissue infections. In contrast to antibiotics, antimicrobial photodynamic inactivation (aPDI) is a promising method to combat both bacterial cells and virulence factors. The main aims of this research were to (1) select the most stable reference genes under sublethal aPDI treatments and (2) evaluate the impact of aPDI on seb. Two aPDI combinations were applied under sublethal conditions: rose bengal (RB) and green light (λmax = 515 nm) and new methylene blue (NMB) and red light (λmax = 632 nm). The stability of ten candidate reference genes (16S rRNA, fabD, ftsZ, gmk, gyrB, proC, pyk, rho, rpoB and tpiA) was evaluated upon aPDI using four software packages—BestKeeper, geNorm, NormFinder and RefFinder. Statistical analyses ranked ftsZ and gmk (RB + green light) and ftsZ, proC, and fabD (NMB + red light) as the most stable reference genes upon photodynamic treatment. Our studies showed downregulation of seb under both aPDI conditions, suggesting that aPDI could decrease the level of virulence factors.

Gallium Mesoporphyrin IX-Mediated Photodestruction: A Pharmacological Trojan Horse Strategy To Eliminate Multidrug-Resistant Staphylococcus aureus

“…However, sublethal doses were defined as a 0.5−2 log 10 reduction in CFU/mL. 14 Light-only treatment and light-independent, 50 min exposure to 10 μM of each compound did not influence the bacterial viability. Ga 3+ MPIX revealed a higher efficiency in bacterial reduction upon green illumination than Ga 3+ PPIX.…”

“…A reduction of more than 3 log 10 units in the number of CFUs (99.9%) was considered a bacterial eradication/lethal dose. However, sublethal doses were defined as a 0.5–2 log 10 reduction in CFU/mL . Light-only treatment and light-independent, 50 min exposure to 10 μM of each compound did not influence the bacterial viability.…”

“…11,12 aPDI has an impact on the production of virulence factors, which cause pathogens to be less virulent. 13,14 Despite our recent studies of aPDI tolerance and an increased stress response upon consecutive cycles of sublethal treatments, 15,16 resistance to photodestruction has not yet been observed. aPDI is efficient in both in vitro 17 and in vivo studies.…”

“…The ideal PS should exhibit low dark toxicity and high phototoxicity, which usually correlates with a high quantum yield of ROS photogeneration and application safety toward eukaryotic cells. Photodynamic inactivation eradicates microbial species efficiently despite their drug resistance profile. − Moreover, recent studies by Woźniak et al revealed a synergy between photodynamic therapy and clinically used antimicrobials. , aPDI has an impact on the production of virulence factors, which cause pathogens to be less virulent. , Despite our recent studies of aPDI tolerance and an increased stress response upon consecutive cycles of sublethal treatments, , resistance to photodestruction has not yet been observed. aPDI is efficient in both in vitro and in vivo studies. , …”

The carbon source-dependent pattern of antimicrobial activity and gene expression in Pseudomonas donghuensis P482

“…The misinterpretation of gene expression data due to the use of one RG instead of two or more for normalisation has already been described in several studies 94 , 95 , as well as in MIQE guidelines 35 , alerting researchers to the shortcomings of this practice. As a result, in the last few years, there has been a growing number of published articles on microbiological research which focus in particular on the validation of RGs for RT-qPCR and the recognition of the MIQE guidelines 96 – 99 . Nevertheless, the thorough, careful selection of RGs is still overlooked in many RT-qPCR studies, not only in microbiological research but also in clinical studies, which ultimately leads to problems with proper diagnostics and therapies of patients, especially those with cancer 100 .…”