Izolacija i karakterizacija Cronobacter sakazakii iz formula za odojčad iz biljnih čajeva
Isolation and characterization of Cronobacter sakazakii from power infant formula and herbal teas
Author
Stojanović, Marija M.Mentor
Katić, VeraCommittee members
Jošić, DraganaŠmigić, Nada

Katić, Vera
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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 ma...de 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)...