Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro

Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.

Rutin, A Natural Inhibitor of IGPD Protein, Partially Inhibits Biofilm Formation in Staphylococcus xylosus ATCC700404 in vitro and in vivo

Staphylococcus xylosus (S. xylosus) has become an emerging opportunistic pathogen due to its strong biofilm formation ability. Simultaneously, the biofilm of bacteria plays an important role in antibiotic resistance and chronic infection. Here, we confirmed that rutin can effectively inhibit biofilm formation in S. xylosus, of which the inhibition mechanism involves its ability to interact with imidazole glycerol phosphate dehydratase (IGPD), a key enzyme in the process of biofilm formation. We designed experiments to target IGPD and inhibited its activities against S. xylosus. Our results indicated that the activity of IGPD and the amount of histidine decreased significantly under the condition of 0.8 mg/ml rutin. Moreover, the expression of IGPD mRNA (hisB) and IGPD protein was significantly down-regulated. Meanwhile, the results from molecular dynamic simulation and Bio-layer interferometry (BLI) technique showed that rutin could bind to IGPD strongly. Additionally, in vivo studies demonstrated that rutin treatment reduced inflammation and protect mice from acute mastitis caused by S. xylosus. In summary, our findings provide new insights into the treatment of biofilm mediated persistent infections and chronic bacterial infections. It could be helpful to design next generation antibiotics to against resistant bacteria.

Molecular Mechanism of Staphylococcus Xylosus Resistance Against Tylosin and Florfenicol

As a result of evolution, certain microbes develop resistance against antimicrobial treatment. The antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of severe infections caused by microbes such as bacteria, viruses, fungi and other parasites. The resistance of Staphylococcus xylosus (S. xylosus) against antibiotic treatment is one of the major causes of the world-wide antibiotic crisis and has remained to be well understood at the molecular level. In order to fill this gap, we investigated various mutations in the sequence of ribosomal proteins involved cross resistance. We discovered that for the mutant containing the insertion L22 97KRTSAIN98 the minimum inhibitory concentration against both tylosin and florfenicol changed dramatically. To understand this effect on a molecular basis and to further elucidate the role of cross resistance, we computationally constructed the 3D model of the large ribosomal subunit from S. xylosus as well as its complexes with both tylosin and florfenicol. Using all-atom molecular dynamics simulations, we found that unique structural changes in the β hairpin of L22 played a central role of this variant in the development of antibiotic resistance in S. xylosus. In addition, the regulation of protein network also played an essential role in the development of cross resistance in S. xylosus. Our work provides insightful views into the mechanism of S. xylosus resistance which could be useful for the development of the next generation of antibiotics.

Contribution of S. xylosus and L. sakei ssp. carnosus Fermentation to the Aroma of Lupin Protein Isolates

Aroma-active compounds of lupin protein isolate and lupin protein isolate fermented with Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus were investigated. The changes in aroma-active compounds were determined by application of aroma extract dilution analysis in combination with gas chromatography-mass spectrometry/olfactometry for identification, and by stable isotope dilution assays for quantification. A total of 30 aroma-active compounds for non-fermented and fermented samples were identified. The aroma profile of LPI fermented with Lactobacillus sakei ssp. carnosus was characterized as roasty and popcorn-like. Staphylococcus xylosus generated cheesy impressions, being in line with the fact that the main aroma compounds acetic acid, butanoic acid, and 2/3-methylbutanoic acid could be identified. Quantification of butanoic acid further confirmed these findings with the highest concentration of 140 mg/kg for LPI fermented with Staphylococcus xylosus. Our study provides insights into how fermentation utilizing different fermentative microbial strains, namely Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus alters the aroma profile of lupin protein isolates. This demonstrates the potential of shaping fermented protein-based foods via targeted microbiological refinement.