Staphylococcus aureus is an opportunistic bacterium of the human body and a leading cause of nosocomial infections. Methicillin resistant S. aureus (MRSA) infections involving biofilm lead to higher mortality and morbidity in patients. Biofilm causes serious clinical issues, as it mitigates entry of antimicrobials to reach the etiological agents. It plays an important role in resilient chronic infections which place an unnecessary burden on antibiotics and the associated costs. To combat drug-resistant infection involving biofilm, there is a need to discover potential anti-biofilm agents. In this study, activity of polyphenolic flavonoid glabridin against biofilm formation of methicillin resistant clinical isolates of S. aureus is being reported for the first time. Crystal violet assay and scanning electron microscopy evidences shows that glabridin prevents formation of cells clusters and attachment of methicillin resistant clinical isolate (MRSA 4423) of S. aureus to the surface in a dose dependent manner. Gel free proteomic analysis of biofilm matrix by LC-ESI-QTOF confirmed the existence of several proteins known to be involved in cells adhesion. Furthermore, expression analysis of cell surface proteins revealed that glabridin significantly down regulates an abundance of several surface-associated adhesins including fibronectin binding proteins (FnbA, FnbB), serine-aspartate repeat-containing protein D (SdrD), immunoglobulin-binding protein G (Sbi), and other virulence factors which were induced by extracellular glucose in MRSA 4423. In addition, several moonlighting proteins (proteins with multiple functions) such as translation elongation factors (EF-Tu, EF-G), chaperone protein (DnaK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) were detected on the cell surface wherein their abundance was inversely proportional to surface-associated adhesins. This study clearly suggests that glabridin prevents biofilm formation in S. aureus through modulation of the cell surface proteins.

Efficient bioconversion of abundant waste glycerol to value-added chemicals calls for a wider range of fermentative workhorses that can catabolize glycerol. In this study, we used quantitative gene expression and solvent profiling, qualitative metabolite analysis, and enzyme activity assays to investigate the factors that limit glycerol utilization as a sole carbon source by Clostridium beijerinckii NCIMB 8052. C. beijerinckii NCIMB 8052 did not produce acetate, acetone and butanol on glycerol. Congruently, the genes encoding the coenzyme A transferase subunits (ctfAB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE) were down-regulated up to 135- and 21-fold, respectively, at 12 h in glycerol-grown cells compared to glucose-grown cells. Conversely, NADH-dependent butanol dehydrogenase A (bdhA) was upregulated 2-fold. Glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (subunit dhaK) were upregulated up to 5- and 881-fold, respectively. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) showed mostly similar expression profiles at 12 h on glucose and glycerol. At 24 h, gapdh was downregulated 1.5-fold, while NADP⁺-dependent gapdh was upregulated up to 1.9-fold. Glycerol-grown cells showed higher or similar activity profiles for all solventogenic enzymes studied, compared to glucose-grown cells. Butyraldehyde (3 g/L) supplementation led to the production of ~0.1 g/L butanol, whilst butyrate (3.5 g/L) supplementation produced 0.7 and 0.5 g/L acetone and butanol, respectively, with glycerol. Further, the long chain saturated fatty acids cyclopentaneundecanoic acid, methyl ester and hexadecanoic acid, butyl ester were detected in glucose- but not in glycerol-grown cells. Collectively, growth on glycerol appears to disrupt synthesis of saturated long chain fatty acids, as well as solventogenesis in C. beijerinckii NCIMB 8052.

Through biochemical transformation of host-derived bile acids (BAs), gut bacteria mediate host-microbe crosstalk and sit at the interface of nutrition, the microbiome, and disease. BAs play a crucial role in human health by facilitating the absorption of dietary lipophilic nutrients, interacting with hormone receptors to regulate host physiology, and shaping gut microbiota composition through antimicrobial activity. Bile acid deconjugation by bacterial bile salt hydrolase (BSH) has long been recognized as the first necessary BA modification required before further transformations can occur. Here, we show that BSH activity is common among human gut bacterial isolates spanning seven major phyla. We observed variation in both the extent and the specificity of deconjugation of BAs among the tested taxa. Unexpectedly, we discovered that certain strains were capable of directly dehydrogenating conjugated BAs via hydroxysteroid dehydrogenases (HSD) to produce conjugated secondary BAs. These results challenge the prevailing notion that deconjugation is a prerequisite for further BA modifications and lay a foundation for new hypotheses regarding how bacteria act individually or in concert to diversify the BA pool and influence host physiology.

The antibiotic resistance in pathogenic bacteria is a major concern and the emergence of novel multidrug-resistant (MDR) strains are a growing threat worldwide. Bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterial agents and that manifests in all fields of their application. Therefore, novel antimicrobial compounds against new bacterial targets and drug resistance mechanisms are urgently needed. Plants are well-known sources of structurally diverse phytochemicals such as alkaloids, flavonoids, phenolics, and terpenes, which plays important roles in human health. Plant-derived antimicrobial agents are an attractive and ongoing source of new therapeutics. Natural compounds that prevent and treat infections through dual action mechanisms such as oxidative stress against pathogens and antioxidant action in the host cell hold promising potential for developing novel therapeutics. Identification of detailed mechanisms of action of such phytomolecules with both antioxidant and antimicrobial activities may help to develop novel antimicrobial therapeutics and benefit overall human health. The purpose of this chapter is to summarize important antioxidant phytochemicals, and focusing on their potential role in the management of drug-resistant bacterial infections.

In this study, a global response analysis was performed to explore the mechanism of action of Usnic acid and its synergy with Norfloxacin, a well-known quinolone antibiotic to which MRSA clinical isolates showed resistance (MIC, 500 µg/mL). A microdilution assay, a growth kinetics analysis, a microscopic analysis, and cell-based assays consistently showed that Usnic acid possesses strong anti-staphylococcal activity (MIC, 7.8 µg/mL), causes cell leakage, modulates efflux pump activity, and synergizes with Norfloxacin against the multi-drug-resistant clinical isolate MRSA 2071. Whole-cell proteome profiling using gel-free proteomics-based nano-LC-ESI-QTOF-MS/MS revealed several proteins whose expression was significantly modulated by Usnic acid and Norfloxacin alone or in combination. Usnic acid downregulated the abundance of RNA polymerase subunits (RpoB and RpoC), carbamoyl phosphate synthase large subunit (PyrAB), chaperone (GroEL), and adenylosuccinate synthetase (PurA). Interestingly, proteins found to be upregulated in the presence of Usnic acid and Norfloxacin included oxidative-stress-related proteins such as peroxidase (Tpx), alkyl hydroperoxide reductase (AphC), and general stress protein (UspA). This study clearly shows that Usnic acid affects numerous cellular targets and can potentiate the action of Norfloxacin. Furthermore, an in vivo study showed that UA at low concentrations prevents body weight gain, but changes in other tested toxicological parameters were found to be within normal limits. Thus, UA at low doses appears to be a promising candidate for repurposing old antibiotics through combination therapy against MRSA infections.

In this study, global response analysis was performed to explore the mechanism of action of Usnic acid and its synergy with Norfloxacin, a well-known quinolone antibiotic to which MRSA clinical isolates showed resistance (MIC, 500 µg/mL). Microdilution assay, growth kinetics, microscopic analysis, and cell-based assays consistently showed that Usnic acid possess strong anti-staphylococcal activity (MIC, 7.8 µg/ml), causes cell leakage, modulates efflux pump activity, and synergizes with Norfloxacin against multi-drug resistant clinical isolate MRSA 2071. Whole cell proteome profiling using gel-free based nano-LC-ESI-QTOF-MS/MS revealed several proteins whose expression was significantly modulated by Usnic acid and Norfloxacin alone or in combination. Usnic acid downregulated the abundance of RNA polymerase subunits (RpoB, RpoC), carbamoyl phosphate synthase large subunit (PyrAB), chaperone (GroEL), and adenylosuccinate synthetase (PurA). Interestingly, proteins found to be upregulated in the presence of Usnic acid and Norfloxacin included oxidative stress-related proteins such as peroxidase (Tpx), alkyl hydroperoxide reductase (AphC), and general stress protein (UspA). This study clearly showed that Usnic acid affects numerous cellular targets and can potentiate the action of Norfloxacin. Furthermore, an in-vivo study emphasized the use of Usnic acid to develop as a safe phytopharmaceutical to repurpose old antibiotics through combination therapy against MRSA infections.