Molecular analysis of the genes encoding colibactin production in Klebsiella pneumoniae isolated from raw milk by Multiplex-PCR as an agent for colorectal cancer

Subject Areas : Food Microbial Contamination

Marziyeh Radaei Alamoli ¹ , sedigheh mehrabian ² , kumarss amini ³ , parisa mobasseri ⁴

1 - Department of Microbiology, Faculty of Biology Science, North Tehran Branch, Islamic Azad University, Tehran, Iran
2 - Department of Microbiology, Faculty of Biology Science, North Tehran Branch, Islamic Azad University, Tehran, Iran
3 - Department of Microbiology, Faculty of Basic Science, Saveh Branch, Islamic Azad University, Saveh, Iran
4 - Department of Microbiology, Faculty of Biology Science, North Tehran Branch, Islamic Azad University, Tehran, Iran

Received: 2019-06-26 Accepted : 2019-09-27 Published : 2020-06-21

Keywords: Multiplex-PCR, Klebsiella pneumonia, clbB, clbN,

Abstract :

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose-fermenting, facultatively anaerobic, rod-shaped bacterium.It has traditionally been considered an opportunistic pathogen and is a common cause of nosocomial infections. The pks gene cluster encodes enzymes responsible for the synthesis of colibactin. Colibactin is a genotoxin that has been shown to induce DNA damage and contribute to increased virulence. Colibactin is also strongly suspected of being involved in the development of colorectal cancer. The present study investigated the prevalence of pks, clbN, and clbB genes in Klebsiella pneumoniae isolates. In this study, 220 samples were obtained from raw milk. Then, all samples were cultured in the violet red bile agar and then cultured in blood agar, MacConkey agar, and chocolate agar for bacterial isolation. Biochemical and microbiological tests were performed for confirmation of the bacteria. DNA was extracted from all isolates using a genomic DNA extraction kit. Then multiplex polymerase chain reaction (M-PCR) method was performed using specific primers. It was found that 60 K. pneumonia isolates out of 220 samples (27.27%) were confirmed by standard phenotypic tests. The PCR test indicated that 6 (10%) strains carried clbN and clbB genes. The pks positive K. pneumoniae was more prevalent in our samples. Owing to its pleiotropic effects, colibactin profoundly influences cellular physiology, inducing DNA breaks that lead to senescence or apoptosis. It seems that the identification of the pks positive K. pneumoniae in milk is essential to prevent colorectal cancer.