Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation

Abstract

Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.

Keywords: Non-aureus staphylococci; Staphylococcus aureus; agr; biofilm; bovine mastitis; quorum sensing.

Figure 1

Effect of the 59 non-aureus Staphylococcus (NAS) isolates on S. aureus (S. a.) biofilm formation (CFU/mL of the biofilm fraction). For dual-species biofilms, S. aureus 8325-4 (agr +) was co-cultured 24 h together with A S. chromogenes (S. c.), S. epidermidis (S. e.) and S. simulans (S. s.); B originating from milk or teat apices (TA); C harboring biofilm-related genes (BG) or not (NBG); D with the capacity to produce biofilm themselves (BP) or not (NBP); E with the capacity to inhibit the growth of S. aureus (GI) or not (NGI) and compared to biofilm formed by S. aureus alone (black bars).

Figure 2

Effect of the 59 non-aureus Staphylococcus (NAS) on biofilm formation (A) and dispersion (B) of S. aureus (CFU/mL of the biofilm and dispersed fraction, respectively) taking into account the capacity of NAS to regulate the agr system of S. aureus [19]. For both assays, S. aureus 8325-4 [S. a. (agr +)] and 8325-4 Δagr [S. a. (agr-)] biofilms (A) or pre-established biofilms (B) were co-cultured together with NAS isolates that are able to repress the agr system of S. aureus (RP) or not (NRP) and compared to biofilm formed by S. aureus alone (black bars).

Figure 3

Effect of the 59 non-aureus staphylococcus (NAS) isolates on S. aureus (S. a.) biofilm dispersion (CFU/mL of the dispersed fraction). For dispersal of biofilms, pre-established (24 h) S. a. 8325-4 (agr +) was co-cultured 24 h together with A S. chromogenes (S. c.), S. epidermidis (S. e.) and S. simulans (S. s.); B originating from milk or teat apices (TA); C harboring biofilm-related genes (BG) or not (NBG); D with the capacity to produce biofilm themselves (BP) or not (NBP); E with the capacity to inhibit the growth of S. aureus (GI) or not (NGI) and compared to biofilm formed by S. aureus alone (black bars).