Befriend your competitor: CSI:FingerID identifies metabolite linked to dual-species biofilm pathogenesis in cystic fibrosis

Opportunistic infections in cystic fibrosis

Cystic fibrosis is an inherited disorder that causes severe damage to the lungs, digestive system, and other organs. Opportunistic pathogens like Pseudomonas aeruginosa and Staphylococcus aureus exploit weaknesses in our defense systems to establish infections. Both bacteria are among the most common coinfecting bacteria in human infections, including the cystic fibrosis (CF) lung^​1,2​. Coinfection with these microbes enhances disease severity and antimicrobial tolerance^​3​.

Befriend your competitor: dual-species biofilms

In certain cocultures (e.g. in plankton), the presence of P. aeruginosa reduces the viability of S. aureus^​4​. However, under different conditions, S. aureus can not only survive the presence of P. aeruginosa but the two species can actually thrive together in a dual-species biofilm. Biofilms are highly organized structures that nurture bacteria and protect them from external factors such as the host’s immune system and antibiotic treatments^​5,6​. The cooperation between P. aeruginosa and S. aureus in cystic fibrosis leads to persistent colonization of human airways, increased disease severity and antibiotic resistance. 

Quorum sensing: communicate with your neighbour

So far, only little is known about the chemical communication mechanisms between competing bacteria in multi-species environments that enable both species to adapt and survive, and perhaps even thrive. Quorum sensing is a process of cell–cell communication that allows bacteria to share information about population density through the secretion of signaling molecules and adjust gene expression accordingly^​7​. The secretion of those chemical signals depends on the growth status and environmental conditions surrounding the bacteria. The metabolic profile of bacteria in planktonic cultures, for example, differs significantly from their metabolic profile in biofilms.

Metabolic profiles in mutual biofilms

In a recent study, researchers from the Ben Gurion University of the Negev, Israel, used untargeted mass spectrometry-based metabolomics analyses to identify specific molecules that mediate interactions between P. aeruginosa and S. aureus in dual-species biofilms^​8​. They employed high-resolution tandem mass spectrometry coupled with ultrahigh-performance liquid chromatography (UHPLC-HRMS/MS) to detect and characterize these signaling molecules that may regulate chemical communication between bacteria. 

The researchers found that the relative inoculum size of both species plays a crucial role in the establishment of a successful mutual biofilm. The metabolic profile of the mixed samples resembles P. aeruginosa monocultures more than S. aureus monocultures. But with increased concentration of S. aureus, the metabolic profile of the biofilm drifts away from that of P. aeruginosa. They observed specific changes in P. aeruginosa metabolite secretion in response to the presence of S. aureus. 

Competing for iron

The researchers investigated the molecules secreted by P. aeruginosa, whose abundance depends on S. aureus population density. Several relevant metabolites were identified in GNPS^​9​. One particular metabolite whose increase in concentration is correlated to S. aureus abundance is pyochelin methyl ester (PME). PME is a derivative of pyochelin, a siderophore secreted by P. aeruginosa. Siderophores scavenge iron from the surroundings and import it into the bacterial cells^​10,11​. P. aeruginosa and S. aureus compete for iron, an essential nutrient for bacterial growth. The researchers discovered that pyochelin consistently decreases as the content of S. aureus in the mixed biofilm increases, while the concentration of PME increases. S. aureus possesses an enzyme that converts pyochelin into PME^​12​, which reduces its affinity for iron. This conversion disables P. aeruginosa‘s ability to effectively scavenge iron and may elevate the fitness of S. aureus in the dual-species biofilm. But PME may not only protect S. aureus; it could also serve as a signaling molecule for P. aeruginosa. By recognizing PME, P. aeruginosa may be able to sense the presence of S. aureus and modify its behavior accordingly. This could involve adjusting its virulence toward the potential competitor and either competing or cooperating, depending on the relative population densities of the two species.

Not only a by-product 

PME seems to be more than a by-product of P. aeruginosa – S. aureus encounters. The researchers investigated the influence of PME on P. aeruginosa‘s metabolism and found that it induced the production of a certain metabolite. This metabolite does not appear in the GNPS database, so they used SIRIUS and CSI:FIngerID^​13,14​ to gain more information on its structure. CSI:FingerID annotates this metabolite as methyl phenazine-1-carboxylate (MPCA), a derivative of phenazine-1-carboxylic acid (PCA), with 91% certainty. The annotation was confirmed through preparation of the respective standard and comparison of NMR and UPLC-HRMS data (level I structure identification^​15​). Phenazines play a role in virulence and antimicrobial properties^​16,17​. Some phenazines, such as PCA, are used by P. aeruginosa for iron acquisition under limiting conditions^​18,19​. MPCA was previously found in soil samples containing Streptomyces canus^​20​, and showed antifungal activities, stronger than PCA in some cases^​21,22​. It was also tested for antibacterial activity against MRSA but exhibited only mild inhibitory effects^​23​. The influence of PME on phenazine production could be related to the increased pathogenesis of this dual-species biofilm^​5,6​.