The expression of glycosyltransferases sdgA and sdgB in Staphylococcus epidermidis depends on the conditions of biofilm formation.

The Staphylococcus aureus SdrG protein is glycosylated by SdgA and SdgB for protection against its degradation by the neutrophil cathepsin G. So far, there is no information about the role of Staphylococcus epidermidis SdgA or SdgB in biofilm-forming; therefore, the focus of this work was to determine the distribution and expression of the sdrG, sdgA and sdgB genes in S. epidermidis under in vitro and in vivo biofilm conditions. The frequencies of the sdrG, sdgA and sdgB genes were evaluated by PCR in a collection of 75 isolates. Isolates were grown in dynamic (non-biofilm-forming) or static (biofilm-forming) conditions. The expression of sdrG, sdgA and sdgB was determined by RT-qPCR in cells grown under dynamic conditions (CGDC), as well as in planktonic and sessile cells from a biofilm and cells adhered to a catheter implanted in Balb/c mice. The sdrG and sdgB genes were detected in 100% of isolates, while the sdgA gene was detected in 71% of the sample (p < 0.001). CGDC did not express sdrG, sdgA and sdgB mRNAs. Planktonic and sessile cells expressed sdrG and sdgB, and the same was observed in cells adhered to the catheter. In particular, one isolate, capable of inducing a biofilm under treatment with cathepsin G, expressed sdrG and sdgB in planktonic and sessile cells and cells adhering to the catheter. This suggests that bacteria require biofilm conditions as an important factor for the transcription of the sdgA, sdgB and sdrG genes.

Irradiation and parasitism affect the ability of larval hemocytes of Anastrepha obliqua for phagocytosis and the production of reactive oxygen species.

The development of the parasitoid Doryctobracon crawfordi (Viereck) (Hymenoptera: Braconidae) in Anastrepha obliqua (McQuart) (Diptera: Tephritidae) larvae is unviable in nature; however, if the host larva is irradiated at 160 Gy, the parasitoid develops and emerges successfully. This suggests that radiation affects the immune responses of A. obliqua larvae, while the underlying mechanisms remain to be revealed. Using optical and electronic microscopies we determined the number and type of hemocyte populations found inside the A. obliqua larvae, either nonirradiated, irradiated at 160 Gy, parasitized by D. crawfordi, or irradiated and parasitized. Based on flow cytometry, the capacity to produce reactive oxygen species (ROS) was determined by the 123-dihydrorhodamine method in those hemocyte cells. Five cell populations were found in the hemolymph of A. obliqua larvae, two of which (granulocytes and plasmatocytes) can phagocytize and produce ROS. A reduction in the number of cells, mainly of the phagocytic type, was observed, as well as the capacity of these cells to produce ROS, when A. obliqua larvae were irradiated. Both radiation and parasitization decreased the ROS production, and when A. obliqua larvae were irradiated followed by parasitization by D. crawfordi, the reduction of the ROS level was even greater. In contrast, a slight increase in the size of these cells was observed in the hemolymph of the parasitized larvae compared to those in nonparasitized larvae. These results suggest that radiation significantly affects the phagocytic cells of A. obliqua and thus permits the development of the parasitoid D. crawfordi.

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

Non-biofilm-forming Staphylococcus epidermidis planktonic cell supernatant induces alterations in osteoblast biological function.

Staphylococcal biofilms significantly contribute to prosthetic joint infection (PJI). However, 40% of S. epidermidis PJI isolates do not produce biofilms, which does not explain the role of biofilms in these cases. We studied whether the supernatant from planktonic S. epidermidis alters osteoblast function. Non-biofilm-forming S. epidermidis supernatants (PJI- clinical isolate, healthy skin isolate (HS), and ATCC12228 reference strain) and biofilm-forming supernatants (PJI+ clinical isolate, ATCC35984 reference strain, and Staphylococcus aureus USA300 reference strain) were included. Osteoblasts stimulated with supernatants from non-biofilm-forming isolates for 3, 7, and 14 days showed significantly reduced cellular DNA content compared with unstimulated osteoblasts, and apoptosis was induced in these osteoblasts. Similar results were obtained for biofilm-forming isolates, but with a greater reduction in DNA content and higher apoptosis. Alkaline phosphatase activity and mineralization were significantly reduced in osteoblasts treated with supernatants from non-biofilm-forming isolates compared to the control at the same time points. However, the supernatants from biofilm-forming isolates had a greater effect than those from non-biofilm-forming isolates. A significant decrease in the expression of ATF4, RUNX2, ALP, SPARC, and BGLAP, and a significant increase in RANK-L expression were observed in osteoblasts treated with both supernatants. These results demonstrate that the supernatants of the S. epidermidis isolate from the PJI- and HS (commensal) with a non-biofilm-forming phenotype alter the function of osteoblasts (apoptosis induction, failure of cell differentiation, activation of osteoblasts, and induction of bone resorption), similar to biofilm-forming isolates (PJI+, ATCC35984, and S. aureus USA300), suggesting that biofilm status contributes to impaired osteoblast function and that the planktonic state can do so independently of biofilm production.