Development of Vancomycin-resistant-enterococci rapid detection kit for routine clinical care

Integrating rapid diagnostic tests into clinical management has great potential to improve, therapeutic processes, and patient outcomes. For this, we develop an antibody-based lateral flow assay (LFA) for the detection of vancomycin resistant Enterococcus faecium (VREfm). Resistance to vancomycin is disseminating rapidly also in other more virulent bacterial species. Hospitalized patients with gastrointestinal carriage of VREfm appear to be the major reservoir of the pathogen. Two major vancomycin resistance determinants (ligases VanA and VanB) have been described in enterococci. Expression of the ligases, VanA or VanB, results in the synthesis of altered peptidoglycan precursors to which vancomycin binds with markedly lower affinity loosing its bactericidal activity. VanA mediated resistance is characterized by high-level resistance to vancomycin, and was up to recently the most prevalent type in Germany. New epidemiological studies revealed an increasing prevalence of vanB in VREfm and particularly in sequence type ST117 during the last five years in German hospitals. VanB expressing isolates show inducible resistance to more modest levels of vancomycin rendering to false susceptible results in routine phenotypic tests.

We focused primarily on the development of an LFA to detect VanA and VanB resistance determinants with the option to expand it towards a biomarker for VREfm sequence type ST117. We cloned, expressed and purified representative VanA / VanB ligases in E. coli for immunization of mice. Generated monoclonal antibodies (mAbs) by hybridoma technology were screened by ELISA and Westernblot for their binding properties.

We identified 15 mAbs that specifically react with different variants of endogenous VanA or VanB, or both of clinical isolates. In contrast to VanA, VanB expression needs to be induced by vancomycin, which will be considered in the VRE-LFA workflow. The aim is to rapidly detect VREfm in cultivated isolates from patient samples in order to simplify and expedite diagnosis and treatment options. The VRE-LFA will be integrated into a pilot study to analyze its potential impact on clinical management.