Library Screening and Focused Multiomics of Antibacterial Action on Vancomycin-Resistant Enterococci faecium

Abstract

Antimicrobial resistance is a major public health threat, and there is an urgent need for new strategies to address this issue. In a gram-positive bacteria, the peptidoglycan layer is thick as compared to gram negatives and is made up of peptide like polysaccharide chains. This peptidoglycan layer is a target for many antibiotics to inhibit bacteria. Enterococcus species are gram-positive bacteria of the intestine in humans and animals that can lead to problematic infections of the gastrointestinal tract and the soft tissues. Vancomycin has been one of most important agents for the treatment of gram-positive bacterial infections. The emergence and spread of vancomycin resistance has become a serious public health issue and vancomycin resistant bacteria are world’s highest priority pathogen according to WHO. Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (vanRSHAXYZ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Alanine-D-Alanine is replaced by D-Alanine-D-Lactate (Lac), to which vancomycin cannot bind effectively. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, vanA gene induction, and changes in the normal and alternative pathway intermediate levels have not been described previously. Part I of my dissertation describes quantifying the cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VRE faecium (VREfm) by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in vanA operon mRNA levels measured by real-time quantitative PCR (RT-qPCR). Normal pathway intermediates in VREfm predominate in the absence of vancomycin, with low basal levels of alternative pathway intermediates. RT-qPCR demonstrated that vanA operon mRNA transcript levels increase rapidly after exposure, reaching maximal levels in 15 minutes. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VRE faecium, vancomycin-sensitive Enterococcus faecium, and methicillin-resistant Staphylococcus aureus after vancomycin exposure demonstrated substantial differences between S. aureus and E. faecium. Part II of my thesis describes developing a two-dimensional chemical compound library screening strategy. The first screening was done using an FDA approved drug library that was screened against vancomycin-resistant Enterococcus faecium (VREfm) in both its original (unmetabolized; UM) and its microsome metabolized (pre-metabolized; PM) forms, and in the absence and presence of vancomycin. This allows the identification of agents with active metabolites and agents that can act synergistically with the resistant-to-antibiotic. 2 x 2 experimental design library screening was also done using NCI diversity set V against both Methicillin-resistant Staphylococcus aureus (MRSA) in absence and presence of cefoxitin and against VRE faecium in absence and presence of vancomycin. The synergistic combinations of all the actives obtained after the screen can be used for in-vitro studies in future as it helps in the reduction the dose when combined. This can help in minimizing the side effects of high concentrations of drugs. Part III of my dissertation describes experiments like active versus active where the active hits obtained after screening results were further combined to look for synergistic and antagonistic combinations. The other experiment was to look at the mechanism of action of various drugs against VRE faecium. As previously described, many resistant genes are involved in the resistance pathway of VREfm. When VREfm was treated with these antibiotics, the resistance genes were induced which showed the presence of resistance in VRE faecium. Therefore, gene induction was observed using both low (1/4th x MIC) and high (4x MIC) of an antibiotic. Furthermore, mutagenesis study was also done using MRSA. In this study, mutants of MRSA were generated on resistant antibiotic plates. It was followed by the extraction of genomic DNA which was later used to study whole genome sequence of MRSA. This study helped us to identify the mutation causing genes. This experiment can further be used for VRE faecium and gram-negatives.