Could antibiotic resistance be linked to the lipidome?

Posted on June 16, 2022

Infection Prevention And Control 2

Antibiotic resistance is a serious threat. The CDC’s Antibiotic Resistance Threats in the United States, 2019 presented data showing more than 2.8 million antibiotic-resistant infections occur annually in the US. And of these 2.8 million infections, more than 35,000 people died as a result. The emergence and spread of new antibiotic-resistant forms is alarming.

The CDC’s report listed several pathogens that they consider threats and broke them down into the following levels of severity: urgent threats, serious threats, concerning threats, and the “watch list”. Pathogens considered to be urgent threats were those such as Carbapenem-resistant Acinetobacter, Candida aureus, Clostridioides difficile, and Drug-resistant Neisseria gonorrhoeae. Listed under the “serious threats” category was Methicillin-resistant Staphylococcus aureus (MRSA).

A previous CDC report from 2018 proposed five core actions to help slow down and prevent antibiotic-resistant pathogens in the future:

Investing in the development of new vaccines, improving existing therapeutics, and discovering better ways of diagnosing infections caused by these pathogens hinge on one underlying principle – understanding the pathogens themselves.

Dr. Kelly Hines, University of Georgia, is interested in doing just that. Dr. Hines and her group of researchers apply bioanalytical methods using ion mobility-mass spectrometry (IM-MS) to enhance lipidomics and metabolomics experiments. They are particularly interested in applying these methods to better understand antibiotic-resistance. With their tools and techniques, they are capable of characterizing metabolic alterations in antibiotic-resistant pathogens. Furthermore, they can use these methods to test for antibiotic susceptibility and small molecule screening.

S. aureus has a complex lipidome comprised of saturated-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs). The lipidome of S. aureus is further complicated by the diversity of branching in BCFAs. Branched-chain amino acids L-valine, L-leucine, and L-isoleucine are used by S. aureus to form even- and odd-carbon fatty acids and iso- and anteiso-BCFA forms. Then these diverse SFCAs and BCFAs are even further complicated by combining with phosphatidylglycerol (PG), diglycosyldiacylglycerol (DGDG), LysylPG, phosphatidic acid (PA), and cardiolipin (CL). So, saying S. aureus has a complex lipidome is really an understatement.

Why do we need to understand BCFAs and SCFAs in S. aureus?

The SCFA to BCFA ratio determines several features of the membrane such as packing, rigidity/fluidity, and net surface charge. Understanding the SCFA-to-BCFA ratio in various lipid species could be key to developing tests for antibiotic susceptibility or screening small molecules. Previously there were two primary methods used to separate and quantify fatty acid isomers, gas chromatography in the form of fatty acid methyl esters (GC-FAME) and reversed-phase liquid chromatography-mass spectrometry (RPLC-MS). But each of these methods require the fatty acids to be freed from the glycerol backbone. This eliminates any information regarding the original distribution of fatty acids among various lipid classes. Another common issue is that eukaryotes do not have high concentrations of BCFAs.

The high concentration of diverse BCFAs in S. aureus presents a perfect opportunity to study alternative methods for the identification and quantification of SCFAs and BCFAs. Dr. Hines and her team at UGA took the opportunity that S. aureus provided and capitalized on it. If you want to read the full article in detail, follow the link!

To summarize, Dr. Hines and her team took a common separation technique, RPLC, and combined it with ion mobility separation to accurately assign glycerol backbone positions of fatty acids. By using isotopically labelled exogenous SCFAs and endogenous BCFAs they were able to bypass another problem in this field – the lack of available standards. They were able to determine that lipids with two BCFAs elute before lipids with one BCFA and one SCFA, and that those elute before lipids containing two SCFAs. This method is also capable of resolving, almost to baseline level, between iso- and anteiso-forms of BCFAs. To cap it all off, this is done without the need to remove the fatty acids from their glycerol backbone via hydrolysis and all the information from the lipid species is retained.

This technique can now be used to study the fatty acid composition of many pathogens. And the better understanding we have of their lipidome and metabolome, the better chance we have of developing vaccines, therapeutics, and diagnostic tools capable of preventing antibiotic resistant infections in the future.