Rapid detection and identification of bacterial pathogens in drinking water sources using DNA-BASED methods and immunoimmobilization technology

Abstract

Clean water is a basic requirement for the survival of the planet and is essential for human health. Water-borne pathogens and the diseases they cause have been a serious threat to public health. Drinking water is a major route for spreading of such pathogens. Monitoring of microbial community is one of the most significant practices for prevention of water-related diseases and potential outbreaks. Comprehensive analysis of chemical composition and microbiological structure of drinking water reservoirs is essential for understanding the levels of contamination and pathogenic threats. Rapid pathogen detection methods are critical for on-site experiments. This study involves two parts. The first part covers investigation of physicochemical and bacteriological composition o drinking water reservoirs in İstanbul. Physicoche ical quality was monitored in terms of pH, conductivity, total dissolved solids, salinity, dissolved organic carbon, heavy metals, anions and cations. Bacteriological characterization was performed using culture-independent, DNA-based methods such as DGGE, sequencing and qPCR. The second part includes development of a rapid and sensitive detection method based on immunoimmobilization for selected E. coli strains. A recently developed BiyoTra ® technology was used in co ination with immunoimmobilization method for analyzing field samples taken from water reservoirs. Water samples were taken from water intake chambers of drinking water treatment plants. Chemical quality parameters were mostly in agreement with regulation limits. Bacterial diversity was investigated with a combination of DGGE, band excising and sequencing of excised bands. DGGE revealed the presence of 66 OTUs in samples. Similarity research of DGGE band sequences, bacteria available in reservoir samples belong to the members of Bacteria, Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes with abundances of 53%, 24%, 16%, 5%, 3% respectively. According to principal component and correlation analysis, Cr, Zn, Co, F-, and NO3- were found to have a significant impact on bacterial community. qPCR was used for quantification of ETEC, EHEC, Legionella and Salmonella targeting specific genes namely LT, stx, mip and hilA respectively. Maximum gene copy numbers for ETEC, EHEC, Legionella and Salmonella were quantified as 6.74x1011, 1.63x104, 1.48x106 and 1.33x106 per 25 ng DNA/l respectively. Pathogenic E. coli strains were efficiently immobilized on gold surfaces activated with antibodies specific to their fimbrial and LPS antigens. The strains were selectively sorted on antibody microarrays. Minimum antibody requirement for effective immobilization of bacteria was determined as 4 g/ml. Minimum detection limit of immunoimmobilization method was 104 cfu/ml. Pre-concentration o environ ental samples using BiyoTrap® considera ly enhanced the immobilization efficiency of target bacteria on antibody modified chips. Minimum detection limit was improved to 102 c u/ l using BiyoTrap®.