OPTIMIZATION OF COLONY POLYMERASE CHAIN REACTION FOR THE 16SRRNA OF DIFFERENT STRAINS OF ESCHERICHIA COLI
DOI:
https://doi.org/10.22159/ijls.2023.v11i1.48219Keywords:
16SrRNA, Colony PCR, EMB (Eosin Methylene Blue Agar), Indole, methyl red, voges proskauer, citrateAbstract
Objective: This work aimed to enhance colony polymerase chain reaction (PCR) for the 16S rRNA of several Escherichia coli strains.
Methods: The isolation of E. coli is done from the gut of the chicken and soil. Then, we optimized the condition for colony PCR for the amplification of 16s ribosomal RNA. We successfully designed primer 3 for 16s ribosomal RNA and made the dilution solution with PCR grade water that is 1:10. Moreover, finally, we made a 20 μL solution that contains the master mix of our isolated colony and forward and reverse base primer for amplification. After the conventional PCR, the amplified 16s ribosomal RNA was then run on Gel to obtain the desired bands. And finally saw the bands in the Gel Doc picture.
Results: Our result shows that the technique of colony PCR is an efficient and quick method than other existing methods that are too costly, tedious, and time-consuming procedures that deter their exploitation in various experimentations and for the identification of E. coli strains.
Conclusion: This study concluded that 16s ribosomal RNA can be amplified without the extraction and purification of total genomic DNA from a bacterial colony using colony PCR. Therefore, by designing rRNA primers for E. coli species, we can evaluate their various types of mutations, strain detection, and antibiotic resistance.
References
Lausch KR, Fuursted K, Larsen CS, Storgaard M. Colonisation with multi-resistant Enterobacteriaceae in hospitalised Danish patients with a history of recent travel: A cross-sectional study. Travel Med Infect Dis 2013;11:320-3.
Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004;2:123-40.
Russo TA, Johnson JR. Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J Infect Dis 2000;181:1753-4.
Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998;11:142-201.
Kaper J, Nataro J, Mobley H. Pathogenic Escherichia coli. Nat Rev Microbiol 2004;2:123-40.
Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 1992;56:622-47.
Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev 1997;10:569-84.
Pitari GM, Zingman LV, Hodgson DM, Alekseev AE, Kazerounian S, Bienengraeber M, et al. Bacterial enterotoxins are associated with resistance to colon cancer. Proc Natl Acad Sci U S A 2003;100:2695-9.
Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun 1999;67:5587-96.
Noriega FR, Liao FM, Formal SB, Fasano A, Levine MM. Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes. J Infect Dis 1995;172:1408-10.
Russo TA, Johnson JR. Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J Infect Dis 2000;181:1753-4.
Johnson JR, Russo TA. Extraintestinal pathogenic Escherichia coli: “The other bad E. coli”. J Lab Clin Med 2002;139:155-62.
Anderson GG, Dodson KW, Hooton TM, Hultgren SJ. Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis. Trends Microbiol 2004;12:424-30.
Hooton TM. Clinical practice. Uncomplicated urinary tract infection. N Engl J Med 2012;366:1028-37.
Scholes D, Hooton TM, Roberts PL, Gupta K, Stapleton AE, Stamm WE. Risk factors associated with acute pyelonephritis in healthy women. Ann Intern Med 2005;142:20-7.
Foxman B. Epidemiology of urinary tract infections: Incidence, morbidity, and economic costs. Dis Mon 2003;49:53-70.
Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004;2:123-40.
Schultz M. Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis 2008;14:1012-8.
Tenaillon O, Skurnik D, Picard B, Denamur E. The population genetics of commensal Escherichia coli. Nat Rev Microbial 2010;8:207-17.
Walker WA, Goulet O, Morelli L, Antoine JM. Progress in the science of probiotics: From cellular microbiology and applied immunology to clinical nutrition. Eur J Nutr 2006;45 Suppl 1:1-18.
Fooks LJ, Gibson GR. Probiotics as modulators of the gut flora. Br J Nutr 2002;88 Suppl 1:S39-49.
Schultz M. Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis 2008;14:1012-8.
Hejlnova J, Dobrindt U, Nemcova R, Rusniok C, Bomba A, Frangeul L, et al. Characterization of the flexible genome complement of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). Microbial (Reading) 2005;151:385-98.
Sonnenborn U, Schulze J. The non-pathogenic Escherichia coli strain Nissle 1917-features of a versatile probiotic. Microb Ecol Health Dis 2009;21:122-58.
Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, et al. Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol 2004;186:5432-41.
Andersson P, Engberg I, Lidin-Janson G, Lincoln K, Hull S, Savnborg C. Persistence of Escherichia coli bacteriuria is not determined by bacterial adherence. Infect Immun 1991;59:2915-21.
Karmali MA. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev 1989;2:15-38.
Karmali MA, Petric M, Steele BT, Lim C. Sporadic cases of haemolytic-uraemic syndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet 1983;1:619-20.
Haldane DJ, Damm MA, Anderson JD. Improved biochemical screening procedure for small clinical laboratories for Vero (Shiga-like)-toxin-producing strains of Escherichia coli O157:H7. J Clin Microbiol 1986;24:652-3.
Kleanthous H, Fry NK, Smith HR, Gross RJ, Rowe B. The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O 157. Epidemiol Infect 1988;101:327-35.
Krishnan C, Fitzgerald VA, Dakin SJ, Behme RJ. Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7. J Clin Microbiol 1987;25:1043-7.
March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 1986;23:869-72.
Ratnam S, March SB, Ahmed R, Bezanson GS, Kasatiya S. Characterization of Escherichia coli serotype O157:H7. J Clin Microbiol 1988;26:2006-12.
Doyle MP, Schoeni JL. Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis. Appl Environ Microbiol 1984;48:855-6.
Ratnam SS, March SB, Ahmed R, Bezanson GS, Kasatiya S. Characterization of Escherichia coli serotype O157: H7. J Clin Microbiol 1988;26:2006-12.
Kleanthous H, Fry NK, Smith HR, Gross RJ, Rowe B. The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O 157. Epidemiol Infect 1988;101:327-35.
March SB, Ratnam SA. Sorbitol-MacConkey medium for detection of Escherichia coli O157: H7 associated with hemorrhagic colitis. J Clin Microbiol 1986;23:869-72.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-4.
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 1985;82:6955-9.
Tringe SG, Hugenholtz P. A renaissance for the pioneering 16S rRNA gene. Curr Opin Microbiol 2008;11:442-6.
Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 2003;55:541-55.
Wang Y, Qian PY. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One 2009;4:e7401.
Kramer MF, Coen DM. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr Protoc Mol Biol 2001;15.1.1-15.1.14.
Altenbuchner J, Mattes R. Escherichia coli. In: Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems. Germany: Wiley-VCH; 2004. p. 7-43.
Zhang Z, Gildersleeve J, Yang YY, Xu R, Loo JA, Uryu S, et al. A new strategy for the synthesis of glycoproteins. Science 2004;303:371-3.
Sandig V, Rose T, Winkler K, Brecht R. Mammalian cells. In: Production of Recombinant Proteins. Germany: Wiley-VCH; 2005.
Bettleheim KA, Gyles CL. Escherichia coli in Domestic Animals and Humans. Wallingford, UK: CAB International; 1994.
Leonard FC, Abbott Y, Rossney A, Quinn PJ, O’Mahony R, Markey BK. Methicillin-resistant Staphylococcus aureus isolated from a veterinary surgeon and five dogs in one practice. Vet Rec 2006;158:155-9.
Costa LR, Spier SJ, Hirsh DC. Comparative molecular characterization of Corynebacterium pseudotuberculosis of different origin. Vet Microbiol 1998;62:135-43.
Farmer JJ 3rd, Davis BR, Hickman-Brenner FW, McWhorter A, Huntley-Carter GP, Asbury MA, et al. Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J Clin Microbiol 1985;21:46-76.
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