Utvrđivanje prisustva Clostridium botulinum u uzorcima meda i medonosnih pčela

Abstract

U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije. Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima, radeno je metodom najverovatnijeg moguceg broja (MPN). Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a, utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pcela 110/kg. U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum. Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih metoda mikrobiologije. Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda. U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne rasporedenosti spora u uzorcima.

In this study, the presence of Clostridium botulinum spores in different types of honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples was included in this investigation and samples originated from different regions of the Republic of Serbia. In addition to classical microbiological methods, after the dilution, enrichment, spinning and membrane filtration, PCR and multiplex PCR methods were used. Determination the number of spores in the PCR positive samples, was done using the most likely possible number (MPN) methodology. According to the results obtained by conventional microbiological methods and multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg. In one honey sample, spores of type B C. botulinum were detected, in one honey sample spores of type E, in two honey samples spores of types A and E, and in one sample spores of three types of C. botulinum (A, B, E) were detected. Type E Clostridium botulinum spores were detected in a sample of honey bees. In the same samples of honey or honey bees, using conventional qualitative and quantitative microbiological methods, spores of Clostridium botulinum were not detected. The detection of Clostridium botulinum spores with PCR, multiplex PCR and conventional microbiological methods in honey and honey bee samples is not possible without preenrichment. Conventional microbiological methods, including SRPS ISO 15213:2011, are not suitable for the detection of Clostridium botulinum spores in honey samples. Due to the low sensitivity of these methods, they can lead to false negative results. In the honey samples which served as a positive control and wich were contaminated with Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of one spore/g of honey. In order to detect Clostridium botulinum spores in honey and honey bees samples using PCR method, due to the small and/or unequal distribution of spores in the samples, the test had to be repeated at least three times for each sample.