Identification of two aptamers binding toLegionella pneumophilawith high affinity and specificity

Abstract

19Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires' Disease (LD) in 20 humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. 21 The methods currently available require expert skills and are time intensive, thus delaying 22 intervention. In situ detection of Lp by biosensor would allow rapid implementation of control 23 strategies. To this end, a biorecognition element is required. Aptamers are considered promising 24 biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding 25 into a specific structure and are able to bind to specific molecules. Currently no aptamer and thus 26 no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of 27 Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding 28 specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against 29 two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified 30 with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by 31 flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising 32 biorecognition molecules for the detection of Lp in water systems. 33 34 Key words: Legionella pneumophila, aptamers, SELEX, flow cytometry, fluorescence 35 microscopy, Pseudomonas 36 37 38 Legionella pneumophila (Lp) is a pathogenic Gram-negative bacterium responsible for two types 39 of respiratory diseases, namely the severe pneumonia Legionnaires' Disease (LD) and the milder 40 flu-like Pontiac fever 1 . Lp occurs in both natural and engineered water systems and is one of the 41 most prevalent pathogens in man-made, engineered water systems 2 . Infections occur when the 42 bacteria are aerosolized, and the contaminated aerosols are inhaled; Lp can then infect and replicate 43 inside alveolar macrophages 3 . Modern water systems provide optimal transmission conditions for 44 Lp by generating aerosols 4 . Leading sources of infection are cooling towers, hot water distribution 45 systems, humidifiers, misters, showers, fountains, spa pools and evaporative condensers 5 . 46 47 Outbreaks of LD consistently occur globally and have increased in recent years. The average 48 incidence rate is about 10-15 cases per million people 6 . According to the Centre for Disease 49 Control, incidences of legionellosis have increased by four and a half times between 2000 and 50 2016 7 . The rise in LD outbreaks can be attributed to several factors such as aging infrastructures 51and an aging population who is more vulnerable to such infections, as well as increases in diagnosis 52 and reporting 8,9 . Nevertheless, most LD outbreaks are the consequences of management failure of 53 man-made water systems 10 . Examples of these failures for water distribution systems include 54 keeping the temperature of hot water distribution system below 50°C and allowing water to 55 stagnate 10 . For coolin...