Distribution and Resistance of Enterococcal

Enterococci are Gram-positive, facultative anaerobic oval cocci found in the gastrointestinal tract of virtually all animals, including humans. They are usually harmless but pose a serious threat in immunocompromised patients or patients with dysbiosis caused by antibiotic therapy, such as endocarditis, bacteremia, and intra-abdominal, wound, and urinary tract infections. Enterococci are thus a major cause of infection in hospital intensive care units.

Fig.1 Proposed physiological roles of Enterococcus faecalis polysaccharides in the gut. (Ramos, et al., 2021)

E. faecalis and Enterococcus faecium are the most common clinical opportunistic pathogens. Their pathogenicity is mainly attributed to intrinsic antibiotic resistance, the ability to acquire virulence factors, and the ability to tolerate and grow in harsh environments. Enterococci possess a highly diverse array of glycobiology, and they not only can build essential structural polysaccharides, but also synthesize capsular polysaccharide (CPS), lipoteichoic acid (LTA), and other exopolysaccharides. Antibodies against CPS promote polymorphonuclear neutrophils (PMN)-mediated killing of E. faecalis and E. faecium and protect mice from enterococcus infection, the study showed.

Enterococcal Polysaccharide Antigen Production Service at CD BioGlyco

The growing prevalence of antimicrobial-resistant pathogens has facilitated the identification of new vaccine targets. At CD BioGlyco, we have established a Glyco™ Vaccine Development Platform against multiple pathogens, such as meningococcal, Haemophilus influenzae, and enterococcal. All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division, as well as for resistance to antibiotics. It plays a key role in host-pathogen interactions. Therefore, we provide EPA production development services for our global clients.

Case: E. faecium surface polysaccharide production service

• Routine culture of E. faecium in glucose-supplemented broth at 37°C without aeration.
• After killing the cells by heat treatment, they are centrifuged and the harvested cells are resuspended in Tris/sucrose buffer. Cells are digested with lysozyme and mutanolysin overnight at 37°C to release cell wall-bound polysaccharides. The supernatant after centrifugation is treated with RNase and DNase to degrade nucleic acids and proteins. Crude carbohydrates are obtained after treatment.
• The crude carbohydrates are separated by size exclusion chromatography (SEC) and the fractions are monitored. Each SEC pool is passed through anion exchange column chromatography (AEC) to obtain purified polysaccharides.
• Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) is used to activate carbohydrate hydroxyl groups to prepare E. faecium surface polysaccharide-CRM 197 carrier protein conjugates. And SEC is performed to remove free carrier protein and unconjugated polysaccharide from the conjugate.
• The molecular weight of each purified polysaccharide is determined by SEC-MALLS, the monosaccharide composition is determined by gas chromatography-mass spectrometry (GC-MS), and the structure of the purified polysaccharide is identified by 1H nuclear magnetic resonance (1H NMR) and two-dimensional NMR.

Fig.2 Flowchart of E. faecium polysaccharide production. (CD BioGlyco)

Advantages of Us

• We provide a series of services from bacterial culture, polysaccharide extraction, purification, and structure identification to polysaccharide conjugation with carrier protein.
• There have mature techniques to analyze the molecular weight and monosaccharide composition and characterize the structure of unknown polysaccharides.
• There are various Polysaccharide Conjugation Techniques with different carrier proteins.

As a best-in-class Polysaccharide Vaccine Development company, CD BioGlyco is committed to helping clients solve the difficulties and problems in the development of polysaccharide vaccines against multiple pathogens. If you are interested in our service, please feel free to contact us for more information.

References:

1. Ramos, Y.; et al. Sugarcoating it: Enterococcal polysaccharides as key modulators of host-pathogen interactions. PLoS Pathogens. 2021, 17(9): e1009822.