M. Mihalache et. al.: Elisa preliminary studies of immobilization and specific detection of bacterial strains Romanian Journal of Ecology & Environmental Chemistry ● Vol.2 ● No.2 ● 2020 64 Elisa preliminary studies of immobilization and specific detection of bacterial strains https://doi.org/10.21698/rjeec.2020.209 Proceedings Paper MADALINA MIHALACHE, ALINA BANCIU*, LUCIAN IONESCU, MIHAI NITA-LAZAR National Research and Development Institute for Industrial Ecology - ECOIND, 71-73 Drumul Podu Dambovitei, 060652, Bucharest, Romania * Corresponding author (e-mail): alina.banciu@incdecoind.ro Abstract The paper aims to emphasize the specific detection of bacterial strains using enzyme-linked immunosorbent assay. The assay is based on the specific binding of polyclonal antibody anti-E. coli tagged with FITC to E.coli and monoclonal antibody anti-Ps. aeruginosa tagged with Alexa Fluor 647 tagged to Ps. aeruginosa and on subsequent enzymatic immunological demonstration of the conjugated enzyme. In this experiment, the negative control was the Salmonella enterica strain. The two antibodies had no interaction with the negative control, instead, they were specific for E. coli and Ps. aeruginosa strains. When both strains were in the same well, the fluorescence intensity given by the presence of E. coli was 2.3 times higher than that given by Ps. aeruginosa, and the intensity of fluorescence decreased if there are both bacterial strains in the wells. Keywords: antigen-antibody, pathogen bacteria, enzyme-linked immunosorbent assay INTRODUCTION The emergence of pollutants and pathogens, mainly antimicrobial-resistant bacteria, in the environment has been an issue in the last decades and continues to be a critical threat worldwide both for the aquatic systems and human health. Nosocomial infections or in-hospital infections affect a large number of patients globally, representing 7% in developed countries and 10% in developing countries. Nosocomial pathogens can be bacteria, viruses, and fungus. WHO estimates that approximately 15% of hospitalized patients suffer from this type of infection [1]. More than 51% of intensive care patients are affected by in-hospital infections, according to the Extended Prevalence of Infection in Intensive Care (EPIC II) study [2]. Some extensive studies in Europe and the USA show that nosocomial infections incidence density ranged from 13,0 to 20,3 episodes per thousand patient-days [3]. The most implicated pathogens in nosocomial infections are E. coli and Pseudomonas aeruginosa. Escherichia coli is a Gram-negative bacillus present in the human and animal intestines and in the feces, which led to this microorganism being considered an indicator of fecal pollution environment, especially for water bodies [4]. Pseudomonas aeruginosa is also a Gram- negative, commensal bacillus of the digestive tract. It is increasingly isolated from the hospital environment on various surfaces or even from instruments, medical devices, antiseptic solutions, or drugs [4]. Water-borne pathogen contamination of water resources and related infectious diseases has been a major water quality concern throughout the world [5-7]. Accelerated pathogens spreading over large areas require fast and specific detection methods for early and successful management of antimicrobial resistant bacteria [8]. Conventional methods of pathogen detection are usually time-consuming, expensive, and often insensitive [9]. These are generally based on metabolic reactions of bacteria, using different types (nutritive and specific) of culture media and requiring more serial replications. One of these methods to assess water body quality has been the use of fecal indicator bacteria (e.g. Escherichia coli) to suggest the presence of harmful fecal pollution [10]. At the present, there is a tendency to switch the conventional detection methods to more advanced molecular methods mainly based on PCR-based methods, next-generation sequencing techniques, and the enzyme-linked immunosorbent assays (ELISA). This immunological technique is highly sensitive, time-effective, and highly specific due to its antigen antibody-like interaction