It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.
We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Background Staphyloxanthin, a carotenoid pigment found in Staphylococcus aureus, serves not only to impart color but also functions as a crucial antioxidant contributing to virulence. Traditionally, milk agar has been employed to enhance staphyloxanthin production, however, no alternative media have been explored. Objectives This study aims to enhance staphyloxanthin production in Staphylococcus aureus using beetroot and carrot formulations. Methods To assess the efficacy of the media, we utilized filter paper, slide spot tests, and microscopic visualization as preliminary identification techniques. Ultraviolet-visible (UV-Vis) spectroscopy and paper chromatography were employed for characterization. Pigment quantification was conducted using microtiter plate assays, and genotypical detection was performed using Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR). Results Beetroot agar exhibited the highest pigment intensity, followed by beetroot with carrot agar, milk agar, carrot agar, and nutrient agar with the lowest intensity. These novel media formulations increased staphyloxanthin synthesis yield, resulting in spectrum shifts ranging from 450 nm (yellow) of milk agar to 470 nm (carrot agar) /480 nm (orange) of beetroot agar. Conclusion This study demonstrates that beetroot and carrot agar can effectively enhance staphyloxanthin production in Staphylococcus aureus. Furthermore, we propose the potential for large-scale cultivation of these pigments in future studies for various industrial applications, such as integration into paints, fabrics, and sunscreen lotions, due to their antioxidant properties.

Staphylococcus aureus is a highly prevalent and aggressive human pathogen causing a wide range of infections. This study aimed to explore the potential of Patuletin, a rare natural flavone, as an anti-virulence agent against S. aureus. At a sub-inhibitory concentration (1/4 MIC), Patuletin notably reduced biofilm formation by 27 % and 23 %, and decreased staphyloxanthin production by 53 % and 46 % in Staphylococcus aureus isolate SA25923 and clinical isolate SA1, respectively. In order to gain a more comprehensive understanding of the in vitro findings, several in silico analyses were conducted. Initially, a 3D-flexible alignment study demonstrated a favorable structural similarity between Patuletin and B70, the co-crystallized ligand of CrtM, an enzyme that plays a pivotal role in the biosynthesis of staphyloxanthin. Molecular docking highlighted the strong binding of Patuletin to the active site of CrtM, with a high affinity of -20.95 kcal/mol. Subsequent 200 ns molecular dynamics simulations, along with MM-GBSA, ProLIF, PLIP, and PCAT analyses, affirmed the stability of the Patuletin-CrtM complex, revealing no significant changes in CrtM\'s structure upon binding. Key amino acids crucial for binding were also identified. Collectively, this study showcased the effective inhibition of CrtM activity by Patuletin in silico and its attenuation of key virulence factors in vitro, including biofilm formation and staphyloxanthin production. These findings hint at Patuletin\'s potential as a valuable therapeutic agent, especially in combination with antibiotics, to counter antibiotic-resistant Staphylococcus aureus infections.

Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.

Antibiotic-free approaches are more important than ever to address the rapidly growing problem of the antibiotic resistance crisis. The photolysis of the bacterial virulence factor staphyloxanthin using blue light at 460 nm (BL460 nm) has been found to effectively attenuate Staphylococcus aureus to chemical and physical agents. However, phototherapy using BL640 nm still needs to be investigated in detail for its safety in eradicating Staphylococcus aureus in vitro and in vivo. In this study, we employed a 460 nm continuous-wavelength LED source and a low concentration of hydrogen peroxide to treat S. aureus under a culturing condition and a wound abrasion mouse model. The results demonstrated the safety of the combined therapy when it did not modify the bacterial virulence factors or the susceptibility to widely used antibiotics. In addition, the results of the mouse model also showed that the combined therapy was safe to apply to mouse skin since it did not cause adverse skin irritation. More importantly, the therapy can aid in healing S. aureus-infected wounds with an efficacy comparable to that of the topical antibiotic Fucidin. The aforementioned findings indicate that the concurrent application of BL460 nm and hydrogen peroxide can be used safely as an alternative or adjunct to antibiotics in treating S. aureus-infected wounds.

UNASSIGNED: Staphylococcus capitis naturally colonizes the human skin but as an opportunistic pathogen, it can also cause biofilm-associated infections and bloodstream infections in newborns. Previously, we found that two strains from the subspecies S. capitis subsp. capitis produce yellow carotenoids despite the initial species description, reporting this subspecies as non-pigmented. In Staphylococcus aureus, the golden pigment staphyloxanthin is an important virulence factor, protecting cells against reactive oxygen species and modulating membrane fluidity.
UNASSIGNED: In this study, we used two pigmented (DSM 111179 and DSM 113836) and two non-pigmented S. capitis subsp. capitis strains (DSM 20326T and DSM 31028) to identify the pigment, determine conditions under which pigment-production occurs and investigate whether pigmented strains show increased resistance to ROS and temperature stress.
UNASSIGNED: We found that the non-pigmented strains remained colorless regardless of the type of medium, whereas intensity of pigmentation in the two pigmented strains increased under low nutrient conditions and with longer incubation times. We were able to detect and identify staphyloxanthin and its derivates in the two pigmented strains but found that methanol cell extracts from all four strains showed ROS scavenging activity regardless of staphyloxanthin production. Increased survival to cold temperatures (-20°C) was detected in the two pigmented strains only after long-term storage compared to the non-pigmented strains.
UNASSIGNED: The identification of staphyloxanthin in S. capitis is of clinical relevance and could be used, in the same way as in S. aureus, as a possible target for anti-virulence drug design.

Staphylococcus aureus membranes contain carotenoids formed during the biosynthesis of staphyloxanthin. These carotenoids are considered virulence factors due to their activity as scavengers of reactive oxygen species and as inhibitors of antimicrobial peptides. Here, we show that the growth of S. aureus under oxygen-restricting conditions downregulates carotenoid biosynthesis and modifies phospholipid content in biofilms and planktonic cells analyzed using LC-MS. At oxygen-restrictive levels, the staphyloxanthin precursor 4,4-diapophytofluene accumulates, indicating that the dehydrogenation reaction catalyzed by 4,4\'-diapophytoene desaturases (CrtN) is inhibited. An increase in lysyl-phosphatidylglycerol is observed under oxygen-restrictive conditions in planktonic cells, and high levels of cardiolipin are detected in biofilms compared to planktonic cells. Under oxygen-restriction conditions, the biophysical parameters of S. aureus membranes show an increase in lipid headgroup spacing, as measured with Laurdan GP, and decreased bilayer core order, as measured with DPH anisotropy. An increase in the liquid-crystalline to gel phase melting temperature, as measured with FTIR, is also observed. S. aureus membranes are therefore less condensed under oxygen-restriction conditions at 37 °C. However, the lack of carotenoids leads to a highly ordered gel phase at low temperatures, around 15 °C. Carotenoids are therefore likely to be low in S. aureus found in tissues with low oxygen levels, such as abscesses, leading to altered membrane biophysical properties.

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Phenotypic adaptation has been associated with persistent, therapy-resistant Staphylococcus aureus infections. Recently, we described within-host evolution towards a Sigma factor B (SigB)-deficient phenotype in a non-human host, a naturally infected dairy cow with chronic, persistent mastitis. However, to our knowledge, the prevalence of SigB deficiency among clinical S. aureus isolates remains unknown. In this study, we screened a collection of bovine mastitis isolates for phenotypic traits typical for SigB deficiency: decreased carotenoid pigmentation, increased proteolysis, secretion of α-hemolysin and exoproteins. Overall, 8 out of 77 (10.4%) isolates of our bovine mastitis collection exhibited the SigB-deficient phenotype. These isolates were assigned to various clonal complexes (CC8, CC9, CC97, CC151, CC3666). We further demonstrated a strong positive correlation between asp23-expression (a marker of SigB activity) and carotenoid pigmentation (r = 0.6359, p = 0.0008), underlining the role of pigmentation as a valuable predictor of the functional status of SigB. Sequencing of the sigB operon (mazEF-rsbUVW-sigB) indicated the phosphatase domain of the RsbU protein as a primary target of mutations leading to SigB deficiency. Indeed, by exchanging single nucleotides in rsbU, we could either induce SigB deficiency or restore the SigB phenotype, demonstrating the pivotal role of RsbU for SigB functionality. The data presented highlight the clinical relevance of SigB deficiency, and future studies are needed to exploit its role in staphylococcal infections.

Staphylococcus aureus RN4220 has been extensively used by staphylococcal researchers as an intermediate strain for genetic manipulation due to its ability to accept foreign DNA. Despite its wide use in laboratories, its complete genome is not available. In this study, we used a hybrid genome assembly approach using minION long reads and Illumina short reads to sequence the complete genome of S. aureus RN4220. The comparative analysis of the annotated complete genome showed the presence of 39 genes fragmented in the previous assembly, many of which were located near the repeat regions. Using RNA-Seq reads, we showed that a higher number of reads could be mapped to the complete genome than the draft genome and the gene expression profile obtained using the complete genome also differs from that obtained from the draft genome. Furthermore, by comparative transcriptomic analysis, we showed the correlation between expression levels of staphyloxanthin biosynthetic genes and the production of yellow pigment. This study highlighted the importance of long reads in completing microbial genomes, especially those possessing repetitive elements.