Development of three multiplex-PCR assays for virulence profiling of different iron acquisition systems in Escherichia coli

Abstract

Background and Objectives: Escherichia coli is responsible for various enteric and extraintestinal infections in animals and humans. Iron as an essential nutrient, has a proven role in pathogenicity of E. coli. Pathogenic E. coli benefits of having complicated systems for iron acquisition but our current knowledge is limited because of complexity of these systems. In the present study, three multiplex-PCR assays were developed to screen nine different virulence genes related to diverse iron acquisition systems in E. coli.
 Materials and Methods: The multiplex-PCR systems were designed and optimized in three panels. Each panel includes a triplex-PCR cocktail. The panels are as follow: panel 1: iroN, iutA and fecA; panel 2: fyuA, sitA and irp2; and panel 3: iucD, chuA and tonB. A total of 39 pathogenic E. coli was screened according to the designed multiplex-PCR.
 Results: In total, the top three frequent genes were tonB (100%), fecA (66.6%) and sitA (58.9%). With the exception of fecA and tonB, comparing the prevalence of genes among different origin of isolates (human, cattle, poultry and pigeon) showed significant associations (P < 0.05). Moreover, the iroN, sitA and iucD genes were significantly prevalent (P < 0.05) among members of extraintestinal pathogenic E. coli in comparison with the group of diarrheagenic E. coli.
 Conclusion: The current multiplex-PCR assays could be a valuable, rapid and economic tool to investigate diverse iron acquisition systems in E. coli for more precise virulence typing of pathogenic or commensal strains.

Towards discrimination of mammary pathogenic Escherichia coli (MPEC) in cattle based on possession of different iron acquisition systems

“…Boiling procedure was applied for DNA extraction (34). Briefly, a loop of cultured bacteria was mixed with 500 μl sterile distilled water and was suspended by vortexing for 30 s. Then, the suspension was boiled in 98°C for 10 min and was placed on ice for another 10 min.…”

“…Presence of nine different genes related to diverse iron acquisition systems were investigated through the recently developed multiplex PCR assays which were conducted in three panels. Each panel is capable of simultaneously screening three genes and they are as follows: panel 1 iroN (salmochelin siderophore receptor), iutA (aerobactin siderophore receptor) and fecA (ferric citrate receptor); panel 2: fyuA (yersiniabactin siderophore receptor), sitA (ferrous iron/ manganese transporter substrate-binding) and irp2 (biosynthesis of siderophores yersiniabactin); and panel 3: iucD (biosynthesis of the siderophores aerobactin), chuA (haem receptor) and tonB (energy transducer) (34).…”