Nonsteroidal anti-inflammatory drugs as therapeutic allies of the gut microbiome on chronic inflammation

Our gut harbours around 1014bacteria of more than 1000 species, accounting for approximately 2 kg of biomass. Thegut microbiome plays several vital functions in processes such as the development of the immune system, food digestion and protection against pathogens. For these functions to be beneficial for both host and microbiome, interactions are tightly regulated.Gut and immune cells continuously interact to distinguish among commensal microbiota, harmless foodstuff, and pathogens. A fine balance between inflammatory and anti-inflammatory state is fundamental to protect intestinal homeostasis. Nonsteroidal anti-inflammatories (NSAIDs) are a class of drugs used for management of pain and inflammation. These compounds have heterologous structures but similar therapeutic activities. The target of all NSAIDs are the isoforms of cyclooxygenase enzymes (COX): the primarily constitutive form COX-1, and the inducible from COX-2. Both isoforms catalyse the conversion of arachidonic acid to PGH2, the immediate substrate for specific prostaglandin and thromboxane synthesis. The gut microbiota plays a role in drug metabolism, resulting in altered bioavailability of these compounds. Additionally, complex host-microbiome interactions lead to modified xenobiotic metabolism and altered expression of genes involved in drug metabolism. These effects can be at gut tissue-level, or distant, including in the liver. Besides the gut microbiome influencing drug metabolism, drugs also impact the microbial communities in the gut. As different drugs exert selective pressures on the gut microbiome, understanding this bidirectional relationshipis crucial for developing effective therapies for managing chronic inflammation

NONSTEROIDAL ANTI-INFLAMMATORY DRUGS AS THERAPEUTIC ALLIES OF THE GUT MICROBIOME ON CHRONIC INFLAMMATION

Our gut harbours around 1014 bacteria of more than 1000 species, accounting for approximately 2 kg of biomass. The gut microbiome plays several vital functions in processes such as the development of the immune system, food digestion and protection against pathogens. For these functions to be beneficial for both host and microbiome, interactions are tightly regulated. Gut and immune cells continuously interact to distinguish among commensal microbiota, harmless foodstuff, and pathogens. A fine balance between inflammatory and anti-inflammatory state is fundamental to protect intestinal homeostasis. Nonsteroidal anti-inflammatories (NSAIDs) are a class of drugs used for management of pain and inflammation. These compounds have heterologous structures but similar therapeutic activities. The target of all NSAIDs are the isoforms of cyclooxygenase enzymes (COX): the primarily constitutive form COX-1, and the inducible from COX-2. Both isoforms catalyse the conversion of arachidonic acid to PGH2, the immediate substrate for specific prostaglandin and thromboxane synthesis. The gut microbiota plays a role in drug metabolism,  resulting in altered bioavailability of these compounds. Additionally, complex host-microbiome interactions lead to modified xenobiotic metabolism and altered expression of genes involved in drug metabolism. These effects can be at gut tissue-level, or distant, including in the liver. Besides the gut microbiome influencing drug metabolism, drugs also impact the microbial communities in the gut. As different drugs exert selective pressures on the gut microbiome,  understanding this bidirectional relationship is crucial for developing effective therapies for managing chronic inflammation

Unlocking the mechanism of action: a cost-effective flow cytometry approach for accelerating antimicrobial drug development

ABSTRACTAntimicrobial resistance is one of the greatest challenges to global health. While the development of new antimicrobials can combat resistance, low profitability reduces the number of new compounds brought to market. Elucidating the mechanism of action is crucial for developing new antimicrobials. This can become expensive as there are no universally applicable pipelines. Phenotypic heterogeneity of microbial populations resulting from antimicrobial treatment can be captured through flow cytometric fingerprinting. Since antimicrobials are classified into limited groups, the mechanism of action of known compounds can be used for predictive modeling. We demonstrate a cost-effective flow cytometry approach for determining the mechanism of action of new compounds. Cultures of Actinomyces viscosus and Fusobacterium nucleatum were treated with different antimicrobials and measured by flow cytometry. A Gaussian mixture mask was applied over the data to construct phenotypic fingerprints. Fingerprints were used to assess statistical differences between mechanism of action groups and to train random forest classifiers. Classifiers were then used to predict the mechanism of action of cephalothin. Statistical differences were found among the different mechanisms of action groups. Pairwise comparison showed statistical differences for 35 out of 45 pairs for A. viscosus and for 32 out of 45 pairs for F. nucleatum after 3.5 h of treatment. The best-performing random forest classifier yielded a Matthews correlation coefficient of 0.92 and the mechanism of action of cephalothin could be successfully predicted. These findings suggest that flow cytometry can be a cheap and fast alternative for determining the mechanism of action of new antimicrobials.IMPORTANCEIn the context of the emerging threat of antimicrobial resistance, the development of novel antimicrobials is a commonly employed strategy to combat resistance. Elucidating the mechanism of action of novel compounds is crucial in this development but can become expensive, as no universally applicable pipelines currently exist. We present a novel flow cytometry-based approach capable of determining the mechanism of action swiftly and cost-effectively. The workflow aims to accelerate drug discovery and could help facilitate a more targeted approach for antimicrobial treatment of patients

Real-time flow cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

Antiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disk diffusion assays, both of which are time-consuming, labor-intensive, and provide limited information on the mode of action of the antimicrobials. The abovementioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. In this study, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative responses of two oral pathobionts to different concentrations of chlorhexidine (CHX), cetylpyridinium chloride (CPC), or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprinting method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compound, while at the same time deciphering their mode of action

Antimicrobial potential of known and novel probiotics on in vitro periodontitis biofilms

Abstract Several oral diseases are characterized by a shift within the oral microbiome towards a pathogenic, dysbiotic composition. Broad-spectrum antimicrobials are often part of patient care. However, because of the rising antibiotic resistance, alternatives are increasingly desirable. Alternatively, supplying beneficial species through probiotics is increasingly showing favorable results. Unfortunately, these probiotics are rarely evaluated comparatively. In this study, the in vitro effects of three known and three novel Lactobacillus strains, together with four novel Streptococcus salivarius strains were comparatively evaluated for antagonistic effects on proximal agar growth, antimicrobial properties of probiotic supernatant and the probiotic’s effects on in vitro periodontal biofilms. Strain-specific effects were observed as differences in efficacy between genera and differences within genera. While some of the Lactobacillus candidates were able to reduce the periodontal pathobiont A. actinomycetemcomitans, the S. salivarius strains were not. However, the S. salivarius strains were more effective against periodontal pathobionts P. intermedia, P. gingivalis, and F. nucleatum. Vexingly, most of the Lactobacillus strains also negatively affected the prevalence of commensal species within the biofilms, while this was lower for S. salivarius strains. Both within lactobacilli and streptococci, some strains showed significantly more inhibition of the pathobionts, indicating the importance of proper strain selection. Additionally, some species showed reductions in non-target species, which can result in unexpected and unexplored effects on the whole microbiome

Opportunities in optical and electrical single-cell technologies to study microbial ecosystems

New techniques are revolutionizing single-cell research, allowing us to study microbes at unprecedented scales and in unparalleled depth. This review highlights the state-of-the-art technologies in single-cell analysis in microbial ecology applications, with particular attention to both optical tools, i.e., specialized use of flow cytometry and Raman spectroscopy and emerging electrical techniques. The objectives of this review include showcasing the diversity of single-cell optical approaches for studying microbiological phenomena, highlighting successful applications in understanding microbial systems, discussing emerging techniques, and encouraging the combination of established and novel approaches to address research questions. The review aims to answer key questions such as how single-cell approaches have advanced our understanding of individual and interacting cells, how they have been used to study uncultured microbes, which new analysis tools will become widespread, and how they contribute to our knowledge of ecological interactions