The detection of Salmonella species and characterisation of Salmonella Enteritidis and Salmonella Typhimurium from food chain

Izolacija i identifikacija Salmonella spp. ostaje nezamenjiva kao potvrdna metoda u mikrobiologiji hrane i hrane za životinje. Međutim, savremena industrija hrane, ali i javno zdravstvo zahtevaju razvoj novih, brzih metoda za detekciju Salmonella spp., jednog od najvažnijih patogenih mikroorganizama prenosivih hranom. Pored toga, tokom epidemioloških studija, uglavnom nije dovoljno da se izolati Salmonella tipiziraju do nivoa vrste i serotipa, već je neophodna i primena metoda tipizacije koje mogu napraviti razliku između epidemiološki različitih, ali genetski srodnih izolata. Veliki broj alternativnih metoda, uključujući i real-time PCR protokole, razvijen je i validovan u cilju detekcije Salmonella spp. Pored toga, napredak u razvoju molekularnih metoda pružio je alate visoke diskriminatorne moći koji su posebno mesto primene zauzeli pri određivanju izvora kontaminacije hrane, omogućavajući brzu i izuzetno pouzdanu tipizaciju Salmonella spp. Ova doktorska disertacija obuhvata četiri osnovne celine: (1)“In house” validacija real-time PCR protokola za detekciju invA i ttr gena Salmonella spp. u hrani i hrani za životinje; (2) „In house“ validacija real-time PCR protokola za detekciju Salmonella Enteritidis i S. Typhimurium u hrani; (3) Razvoj i „in house“ validacija novog real-time PCR protokola za detekciju invA gena Salmonella spp. u hrani; (4) Molekularna karakterizacija i ispitivanje antimikrobne osetljivosti Salmonella Enteritidis and S. Typhimurium izolovanih iz hrane, hrane za životinje i fecesa. Tokom izrade ove doktorske disertacije formirana je kolekcija od 60 izolata Salmonella Enteritidis i 60 izolata S. Typhimurium (po 12 poreklom ljudi, 24 poreklom iz hrane i 24 poreklom iz fecesa živine), a zatim je izvršena njihova molekularna karakterizacija i ispitivanje antimikrobne osetljivosti. Ispitivanje antimikrobne osetljivosti Salmonella izvršeno je disk difuzionom metodom prema EUCAST protokolu, dok je za određivanje minimalne inhibitorne koncentracije primenjen E test. Molekularna karakterizacija izvršena je primenom PFGE metode (Pulsed Field Gel Electrophoresis) prema „CDC PulseNet“ protokolu...method in the microbiology of food and feed. However, the modern food industry and public health require the development of new, rapid methods for the detection of Salmonella spp., one of the most important foodborne pathogens. In addition, during epidemiological studies, it is generally not sufficient to determine Salmonella isolates up to the species or the serotype level, but it is also necessary to apply subtyping methods that can distinguish between epidemiologically distinct, but genetically related isolates. A number of alternative methods, including real-time PCR protocols, have been developed and validated for the detection of Salmonella spp. In addition, the advances in the development of molecular methods have provided tools of high discriminatory power that have been particularly useful in determining food contamination sources, enabling fast and highly reliable typing of Salmonella spp. This dissertation has four major sections: (1) In-house validation of the real-time PCR protocol for the detection of invA and ttr genes of Salmonella spp. in food and feed; (2) In-house validation of the real-time PCR protocol for the detection of Salmonella Enteritidis and S. Typhimurium in food; (3) Development and in-house validation of new real-time PCR protocol for the detection of the invA gene of Salmonella spp. in food; (4) Molecular characterization and antimicrobial susceptibility testing of Salmonella Enteritidis and S. Typhimurium isolated from food, feed and feces. An extensive validation study, where real-time PCR protocols for the detection of invA and ttr of Salmonella spp. in food and feed were tested, gave excellent results and showed that they can be used as an adequate replacement for the standard method. By comparing different DNA extraction procedures, lower Ct values were achieved by using "Chelex extraction" rather than by extraction based on the thermal lysis of the cell..