Inventors: Assist. Lecturer: Mukhtar Jawad Kazem Al-Imam/Al-Ghazaliya, Baghdad
Prof. Dr. May Talib Flaih/Department of Biology, College of Science, University of Baghdad
Abstract
Two hundred and fifty specimens (stool) from children under five years for both sexes were collected in sterilized containers from some Iraqi hospitals, the result showed 15.7% (33) isolates identified as pathogenic E.coli, and showed 21% (7) isolates were identified as Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 from patients with bloody diarrhea, the identification was confirmed by VITEK 2 system. Monoplex pattern of PCR was used also for detection different genes in (7) Escherichia coli O157:H7 isolates; include 16SrRNA, eae, Stx1,Ler that encoded for ribosomal RNA, intimin, , shiga toxins, regulatory gene, with specific primers. Result showed all isolates of E.coli O157:H7 were positive for 16SrRNA gene with (213bp), eae with (741bp), and Stx1 gene appeared in all isolates with (446bp) amplicon sizes.
Regulatory gene appeared in all isolates with (120bp). Standard strain of Lactobacillus acidophilus was obtained from (American Type Culture Collection ATCC4356). Antimicrobial activity of the cell free culture supernatant (CFCS) from lactobacillus acidophilus was tested against the E.coli O157:H7. Result showed only cell free culture supernatant of Lactobacillus acidophilus has inhibitory activity against E.coli O157:H7 isolates with different dilutions (1/2, 1/4, 1/8, 1/16, 1/32).
Characterization of antimicrobial substances produced by Lactobacillus were performed, Current result showed the bacteriocin was only has inhibitory effect against pathogenic E.coli, while organic acids and hydrogen peroxide hasn’t any role in inhibition. Minimum inhibitory concentration (MIC) of cell free supernatants of Lactobacillus acidophilus was determined by employing different dilutions to detect the concentration of probiotic that will inhibit enterohemorrhagic E.coli serotypesO157:H7 growth, and helped in determination of sub-minimum inhibitory concentration (Sub-MIC) to complete the tests.
Result showed there was significant inhibitory effect (p < 0.05) of Lactobacillus acidophilus against isolates and the findings of this experiment revealed that (1/8) lowest dilution (MIC) was able to inhibit the bacterial growth of E.coli O157:H7 isolates. Gene expression level has been used in study to determinate the behaviors of virulence factor (Stx1) and regulatory factor (Ler) in enterohemorrhagic E.coli (O157:H7) after being under stressed with probiotic strain (Lactobacillus acidophilus), the result showed that expression level of virulence gene (Stx1) and regulatory gene (Ler) were decreased in E.coli (O157:H7).