Identification of a glutathione transporter in A. actinomycetemcomitans.

IMPORTANCE: Microbes produce a large array of extracellular molecules, which serve as signals and cues to promote polymicrobial interactions and alter the function of microbial communities. This has been particularly well studied in the human oral microbiome, where key metabolites have been shown to impact both health and disease. Here, we used an untargeted mass spectrometry approach to comprehensively assess the extracellular metabolome of the pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii during mono- and co-culture. We generated and made publicly available a metabolomic data set that includes hundreds of potential metabolites and leveraged this data set to identify an operon important for glutathione secretion in A. actinomycetemcomitans.

Development of a Versatile, Low-Cost Electrochemical System to Study Biofilm Redox Activity at the Micron Scale.

Spatially resolving chemical landscapes surrounding microbial communities can provide insight into chemical interactions that dictate cellular physiology. Electrochemical techniques provide an attractive option for studying these interactions due to their robustness and high sensitivity. Unfortunately, commercial electrochemical platforms that are capable of measuring chemical activity on the micron scale are often expensive and do not easily perform multiple scanning techniques. Here, we report development of an inexpensive electrochemical system that features a combined micromanipulator and potentiostat component capable of scanning surfaces while measuring molecular concentrations or redox profiles. We validate this experimental platform for biological use with a two-species biofilm model composed of the oral bacterial pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii. We measure consumption of H2O2 by A. actinomycetemcomitans biofilms temporally and spatially, providing new insights into how A. actinomycetemcomitans responds to this S. gordonii-produced metabolite. We advance our platform to spatially measure redox activity above biofilms. Our analysis supports that redox activity surrounding biofilms is species specific, and the region immediately above an S. gordonii biofilm is highly oxidized compared to that above an A. actinomycetemcomitans biofilm. This work provides description and validation of a versatile, quantitative framework for studying bacterial redox-mediated physiology in an integrated and easily adaptable experimental platform. IMPORTANCE Scanning electrochemical probe microscopy methods can provide information of the chemical environment along a spatial surface with micron-scale resolution. These methods often require expensive instruments that perform optimized and highly sensitive niche techniques. Here, we describe a novel system that combines a micromanipulator that scans micron-sized electrodes across the surface of bacterial biofilms and a potentiostat, which performs various electrochemical techniques. This platform allows for spatial measurement of chemical gradients above live bacteria in real time, and as proof of concept, we utilize this setup to map H2O2 detoxification above an oral pathogen biofilm. We increased the versatility of this platform further by mapping redox potentials of biofilms in real time on the micron scale. Together, this system provides a technical framework for studying chemical interactions among microbes.

Micron Scale Spatial Measurement of the O2 Gradient Surrounding a Bacterial Biofilm in Real Time.

Bacteria alter their local chemical environment through both consumption and the production of a variety of molecules, ultimately shaping the local ecology. Molecular oxygen (O2) is a key metabolite that affects the physiology and behavior of virtually all bacteria, and its consumption often results in O2 gradients within sessile bacterial communities (biofilms). O2 plays a critical role in several bacterial phenotypes, including antibiotic tolerance; however, our understanding of O2 levels within and surrounding biofilms has been hampered by the difficulties in measuring O2 levels in real-time for extended durations and at the micron scale. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to quantify the O2 gradients present above a Pseudomonas aeruginosa biofilm. These results reveal that a biofilm produces a hypoxic zone that extends hundreds of microns from the biofilm surface within minutes and that the biofilm consumes O2 at a maximum rate. Treating the biofilm with levels of the antibiotic ciprofloxacin that kill 99% of the bacteria did not affect the O2 gradient, indicating that the biofilm is highly resilient to antimicrobial treatment in regard to O2 consumption.IMPORTANCE O2 is a fundamental environmental metabolite that affects all life on earth. While toxic to many microbes and obligately required by others, those that have appropriate physiological responses survive and can even benefit from various levels of O2, particularly in biofilm communities. Although most studies have focused on measuring O2 within biofilms, little is known about O2 gradients surrounding biofilms. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to measure the O2 gradients surrounding biofilms in real time on the micron scale. Our results reveal that P. aeruginosa biofilms produce a hypoxic zone that can extend hundreds of microns from the biofilm surface and that this gradient remains even after the addition of antibiotic concentrations that eradicated 99% of viable cells. Our results provide a high resolution of the O2 gradients produced by P. aeruginosa biofilms and reveal sustained O2 consumption in the presence of antibiotics.

Bacterial Quorum Sensing During Infection.

Bacteria are highly interactive and possess an extraordinary repertoire of intercellular communication and social behaviors, including quorum sensing (QS). QS has been studied in detail at the molecular level, so mechanistic details are well understood in many species and are often involved in virulence. The use of different animal host models has demonstrated QS-dependent control of virulence determinants and virulence in several human pathogenic bacteria. QS also controls virulence in several plant pathogenic species. Despite the role QS plays in virulence during animal and plant laboratory-engineered infections, QS mutants are frequently isolated from natural infections, demonstrating that the function of QS during infection and its role in pathogenesis remain poorly understood and are fruitful areas for future research. We discuss the role of QS during infection in various organisms and highlight approaches to better understand QS during human infection. This is an important consideration in an era of growing antimicrobial resistance, when we are looking for new ways to target bacterial infections.