Staphylococcus aureus sigma B-dependent emergence of small-colony variants and biofilm production following exposure to Pseudomonas aeruginosa 4-hydroxy-2-heptylquinoline-N- oxide

Abstract

BackgroundStaphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.ResultsWe demonstrated that HQNO stimulates S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the α-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background.ConclusionsThese results suggest that S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.

Outcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis

“…The hypothesis that SCVs play a role in the development of chronic infections is well supported by in vitro experiments demonstrating that these variants have an increased ability to adhere to host tissue components (Mitchell et al, 2008;Vaudaux et al, 2002), to form biofilms (Mitchell et al, 2010a(Mitchell et al, , 2010bSingh et al, 2009Singh et al, , 2010 and to infect and persist within non-professional phagocytes (Mitchell et al, 2011b;Sendi & Proctor, 2009). These in vitro characteristics can be explained by the impact of the defective electron transport chain on the expression of virulence factors which seem to be mostly controlled by the activity of SigB rather than by the agr system in SCVs (Moisan et al, 2006;Senn et al, 2005).…”

“…Scale bar is 50 μm. Biofilm formation was evaluated by crystal violet staining as previously decribed (Mitchell et al, 2010a(Mitchell et al, , 2010b.…”

“…Conventional resistance mechanisms include the upregulation of bacterial efflux pumps and mutations of antibiotic target molecules . In addition, the formation of persister cells (Mulcahy et al, 2010) and of small-colony variants (Goerke & Wolz, 2010;Proctor et al, 2006;Schneider et al, 2008) as well as bacterial growth in biofilms Mitchell et al, 2010aMitchell et al, , 2010bWagner & Iglewski, 2008) are mechanisms that are well known for their involvement in difficult-to-treat infections (Galli et al, 2007;Stewart, 2002).…”

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

“…These microenvironments allow for the application of fundamental principles of microbial ecology (Yang et al, 2011;Conrad et al, 2012). For example, synergistic and antagonistic behavior between different CF pathogens has been described (Duan et al, 2003;Harrison, 2007;Sibley et al, 2008;Mitchell et al, 2010;Yang et al, 2011;Venkataraman et al, 2014), and the microbial community has been proposed to contain stable and disturbed states, much like those in classical ecology (Conrad et al, 2012). Known chemical characteristics of the CF lung include lowered pH (Tate et al, 2002;Pezzulo et al, 2012), low oxygen tensions (Worlitzsch et al, 2002) and high levels of amino acids, nitrate, iron and phenazines (Grasemann et al, 1998;Jones et al, 2000;Palmer et al, 2007;Ghio et al, 2012;Hunter et al, 2012).…”

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

“…Further reported mechanisms leading to the formation of SCVs (in vitro and in vivo) include prolonged exposure to subinhibitory concentrations of antibiotics (Massey et al, 2001;Mitchell et al, 2010a;von Eiff et al, 1997a) or to exoproducts from other bacteria, e.g. Pseudomonas aerugenosa (Mitchell et al, 2010b). Moreover, there is growing evidence that the formation of SCVs could also be due to regulatory mechanisms, involving global regulators (e.g.…”