Pyochelin biotransformation by Staphylococcus aureus shapes bacterial competition with Pseudomonas aeruginosa in polymicrobial infections.

Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated bacterial species from polymicrobial infections of patients with cystic fibrosis and chronic wounds. We apply mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S. aureus is equipped with an elegant mechanism to inactivate pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase). Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular reactive oxygen species (ROS) production in S. aureus. In a murine wound co-infection model, an S. aureus mutant unable to methylate pyochelin shows significantly lower fitness compared with its parental strain. Thus, Spm-mediated pyochelin methylation is a mechanism to increase S. aureus survival during in vivo competition with P. aeruginosa.

MBRA-2: a Modified Chemostat System to Culture Biofilms.

Culture-dependent approaches for investigating microbial ecology aim to model the nutrient content of specific environments by simplifying the system for high-resolution molecular analysis. These in vitro systems are enticing due to their increased throughput compared to animal models, flexibility in modulating nutrient content and community composition, scaling of culture volume to isolate biological molecules, and control of environmental parameters, such as temperature, humidity, and nutrient flow. However, different devices are used to investigate homogenous, planktonic microbial communities and heterogeneous biofilms. Here, we present the minibioreactor array 2 (MBRA-2) with media rails, a benchtop multireactor system derived from the MBRA system that enables researchers to use the same system to grow planktonic and biofilm cultures. We simplified flow through the system and reduced contamination, leakage, and time required for array assembly by designing and implementing a reusable media rail to replace the branched tubing traditionally used to convey media through chemostat arrays. Additionally, we altered the structure of the six-bioreactor strip to incorporate a removable lid to provide easy access to the bioreactor wells, enabling biofilm recovery and thorough cleaning for reuse. Using Pseudomonas aeruginosa, a model biofilm-producing organism, we show that the technical improvements of the MBRA-2 for biofilms growth does not disrupt the function of the bioreactor array. IMPORTANCE The MBRA-2 with media rails provides an accessible system for investigators to culture heterogenous, suspended biofilms under constant flow.

Spray-Based Application of Matrix to Agar-Based Microbial Samples for Reproducible Sample Adherence in MALDI MSI.

Microbial mass spectrometry imaging (MSI) is a powerful tool used to generate biological hypotheses about the roles of metabolites in microbial competition based upon their two-dimensional spatial distribution. The most commonly used ionization method for microbial MSI is matrix-assisted laser desorption ionization (MALDI). However, medium components and microbial metabolites influence the adhesion of agar samples to the MALDI target, limiting the applicability of MALDI MSI to microbes grown on specific media. Here, we describe a three-step process using a robotic sprayer for a matrix application that improves the adherence of agar samples to the MALDI target, enabling the use of different media for microbial growth and an MSI analysis of larger sample surface areas.