Izolacija i karakterizacija Cronobacter sakazakii iz formula za odojčad iz biljnih čajeva

Abstract

Cronobacter sakazakii je patogeni mikroorganizam poreklom iz hrane koji je jako privukao pažnju celokupne javnosti i prehrambene industrije, jer može da izazove smrtne posledice kod odojčadi. Osnovni izvor infekcije su formule za odojčad u prahu u kojima ova bakterija može da se održava duže vreme zbog specifičnih osobina, kao što su: otpornost na nisku aw, termorezistentnost, otpornost na promene pH, visoku osmotsku koncentraciju i sposobnost stvaranja egzopolisaharida. Cilj ovog rada je bio da se istraži prisustvo Cronobacter sakazakii u formulama za odojčad i biljnim čajevima koji se po tradiciji koriste u lečenju svih rizičnih grupa, uključujući novorođenčad, imunokompromitovanu odojčad i odrasle i da odrede genotipske karakteristike izolata Cronobacter sakazakii. Ispitano je 360 uzoraka mleka za odojčad i 520 uzoraka čaja različitog porekla, a posebna pažnja posvećena je čajevima za decu i odojčad, kao i biljnim čajevima koji se prelivaju vodom ispod 72°C. Za izolaciju Cronobacter sakazakii je korišćena standardna metoda ISO/TS 22964. Fenotipizacija izolata Cronobacter sakazakii je urađena primenom komercijalnog testa API 20E. U okviru genetičkih istraživanja izvršen je izbor reprezentativnih izolata i selekcija metoda za izolaciju DNK. Fragmenti DNK su umnožavani metodom lančane reakcije polimeraze - PCR. Ova in vitro tehnika korišćena je za: 1. amplifikaciju različitih delova genoma koji obezbeđuju DNK fingerprinting (rep-PCR i RAPD) i 2. amplifikaciju ciljne sekvence (16S rDNA). Za razdvajanje umnoženih fragmenata DNK molekula na osnovu njihove veličine i naelektrisanja korišćen je metod gel-elektroforeze. Koncentraciju agaroze i uslove pod kojima je rađeno elektroforetsko razdvajanje je prilagođeno broju i veličini fragmenata DNK. Za analizu diverziteta korišćena je rep-PCR metoda sa specifičnim sekvencama - repetitivnim elemenatima DNK: ERIC (enterobacterial repetitive intergenic consensus) i BOX elemenata. Pri ERIC amplifikaciji korišćen je set prajmera ERIC 1R/2. U okviru BOX tipa rep-PCR korišćen je (GTG)5 prajmer (de Bruijn, 1992). Amplifikacija 16S rDNA izvršena je univerzalnim prajmerima fD1/rD1 i amplifikovani fragmenti su prečišćeni i sekvencirani (Weisburg et al, 1991)...

Cronobacter sakazakii is a pathogen originating from food that increased the attention of the general public and the food industry, because it can cause fatal consequences in infants. The main source of infection is powder infant formula (PIF) in which the bacteria can be maintained for a long time due to its specific characteristics, such as resistance to low aw, thermo resistance, resistance to changes in pH, high osmotic concentration and ability to produce exopolysaccharides. The aim of this study was to investigate the presence of Cronobacter sakazakii in infant formulas and herbal teas that are traditionally used in the treatment of high-risk groups, including infants, immunocompromised infants and adults and to determine the genotypic characteristics of isolates of Cronobacter sakazakii. We examined 360 samples of powder infant formula and 520 herbal tea samples of different origins, particular attention is paid to tea for children and infants, as well as herbal teas that are made with water tempered below 72°C. For the isolation of Cronobacter. Cronobacter sakazakii a standard method ISO / TS 22964 has been used. Cronobacter sakazakii phenotyping was performed using a commercial test API 20E. In the area of genetic studies representative isolates have been selected and the selection of methods for isolating the DNA had been made. DNA fragments were multiplied using the polymerase chain reaction - PCR. This in vitro technique was used to: 1. amplify different parts of the genome that provide DNA fingerprinting (rep-PCR and RAPD) and 2. amplify target sequences (16S rDNA). Gel-electrophoresis was used for the separation of the amplified DNA fragments on the basis of their molecular size and charge. Concentration of agarose and conditions, under which the electrophoresis separation was done, was performed according to number and size of DNA fragments. For the analysis of diversity rep-PCR method was used with specific sequences - repetitive DNA elements: ERIC (enterobacterial repetitive intergenic consensus) and BOX elements. In ERIC amplification, we used set of primers ERIC 1R / 2. Within the BOX type, rep-PCR was used (GTG)5 primer (de Bruijn, 1992). Amplification of 16S rDNA was performed using fD1/rD1 universal primers and the amplified fragments were purified and sequenced (Weisburg et al, 1991)...