Detection and Gene expression of selected Virulence Genes of Cronobacter sakazakii from Milk and Milk Products

Abstract

Cronobacter sakazakii is an emerging food-borne pathogen that causes septicemia, meningitis and necrotizing enterocolitis mainly in infants and immunocompromised individuals. C. sakazakii is ubiquitous in nature and it has been isolated from wide range of food, clinical and environmental samples. The present study was conducted to investigate the prevalence of C. sakazakii in milk and milk products of Agra city. A total of 243 samples of cow milk, goat milk, buffalo milk, camel mill, cottage cheese and curd were analyzed in different seasons and total 480 isolates were obtained. These isolates were biochemically characterized and were further confirmed using a C. sakazakii specific PCR assay based on ompA gene. Total twenty seven isolates were confirmed and their in vitro pathogenicity was checked by studying hemolysis on sheep blood agar. Eighteen of the tested isolates show positive hemolysis and out of these isolates five virulence-associated genes (hly, cpa, sip, hha and fim) were detected. Six of the them possess all the 5 virulence-associated genes. These six isolates were further confirmed using 16S rRNA gene sequencing and their 16S rRNA gene sequences were later submitted in NCBI. Their accession numbers obtained are KY971630, KY971631, KY971632, KY971633, KY971634 and KY971635. These isolates were subjected to in vivo pathogenicity testing using mice pubs. The growth profile of these isolates was studied under various stress (acid, alkaline, heat, cold and desiccated) conditions. The relative gene expression of a virulence associated gene (hha gene) was also checked in various stressed and unstressed cells. In order to device a control measure against these isolates, twenty antibiotics were tested and antagonistic activity of eight probiotics was studied against these isolates under stressed and unstressed conditions. Ofloxacin, Ciplofloxacin, Piperacillin, Cefotaxime, Co-trimoxazole and Chloramphenicol were the most effective antibiotics against the unstressed and stressed cells of these isolates. L. s