Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro

Abstract

Communications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.

Keywords: Coagulase-negative staphylococci; Mastitis; Quorum sensing; Staphylococcus aureus; agr.

Figure 1

Example of effect of non-aureus staphylococci isolates on gene expression of Staphylococcus aureus. Tryptone soy agar plates (with erythromycin and X-gal) containing (A) the rnaIII::lacZ (SH101F7; Ery^r), (B) the hla::lacZ (PC322; Ery^r), or (C) the spa::lacZ (PC203; Ery^r) reporter strain of Staphylococcus aureus were exposed to 20 μL of either Supernatant (SP), Supernatant + Proteinase K (SPK), or Cell suspension (CS) obtained from overnight cultures of Staphylococcus chromogenes, Staphylococcus epidermidis, and Staphylococcus simulans, respectively. Staphylococcus schleiferi (strain 2898) [12] and H₂O were used as positive control (P) and negative control (N), respectively. Halos around the wells appeared between 12 and 36 h of incubation at 37 °C, and the diameter (measured in mm) was classified as NE: no effect (≤ 10 mm), SLE: slight effect (11–15 mm), ME: moderate effect (16–20 mm), and SE: severe effect (≥ 25 mm) on gene expression (upregulation for rnaIII and hla, and downregulation of spa). This figure is representative for all β-Galactosidase plate assays.

Figure 2

β-Galactosidase liquid assay: effect of non-aureus staphylococci (NAS) on agr induction of Staphylococcus aureus over time. (A) The β-galactosidase activity of Staphylococcus aureus rnaIII::lacZ reporter strain SH101F7 was monitored for 4 h growing alone (negative control) in TSB, or in co-culture (1:1) with Staphylococcus chromogenes (n = 34), Staphylococcus epidermidis (n = 11), Staphylococcus simulans (n = 14), and Staphylococcus schleiferi strain 2898 (positive control). (B) The rnaIII expression of NAS isolates affected (hatched bars) or not (solid bars) by NAS in vitro growth inhibition of S. aureus [27]. Each bar represents the least square means of three biological replicates and the error bars represent the standard deviation.