Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria
DOI:
https://doi.org/10.5219/739Keywords:
putrescine, Gram-negative bacteria, speF, adiA, Real-time PCRAbstract
Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health. The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiA and speF together in Salmonella enterica and Escherichia coli genome by Real-time PCR. These forenamed genes encode enzymes in the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E. coli CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (speF and adiA) in Salmonella enterica and E. coli by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of the standard curves (adiA, speF, gapA). An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R) of adiA and speF genes in E. coli was varied depending on culture conditions. The highest gene expression of adiA and speF was observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF ~11, 10, 9; respectively) after initiation of growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of E. coli.
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Actis, L., Smoot, J., Barancin, C., Findlay, R. 1999. Comparison of differential plating media and two chromatography techniques for the detection of histamine production in bacteria. Journal of Microbiological Methods, vol. 39, no. 1, p. 79-90. https://doi.org/10.1016/S0167-7012(99)00099-8.
Alvarez, M., Moreno-Arribas, M. 2014. The problem of biogenic amines in fermented foods and the use of potential biogenic amine-degrading microorganisms as a solution. Trends in Food Science & Technology, vol. 39, no. 2, p. 146-155. https://doi.org/10.1016/j.tifs.2014.07.007
Bio-Rad Laboratories, 2006. Real-Time PCR Applications Guide. Bio-Rad Laboratories [online] s.a. [cit.2017-01-15] Avaible at: http://www.gene-quantification.de/real-time-pcr-guide-bio-rad.pdf
Carey, C. M., Kostrzynska, M., and Thompson, S. 2009. Escherichia coli O157: H7 stress and virulence gene expression on Romaine lettuce using comparative real-time PCR. Journal of Microbiological Methods, vol. 77, no. 2, p. 235-242. https://doi.org/10.1016/j.mimet.2009.02.010
Coton, M., Romano, A., Spano, G., Romano, A., Spano, G., Ziegler, K., Vetrana, C., Desmarais, C., Lonvaud-Funel, A., Lucas, P., Coton, E. 2010. Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider. Food Microbiology, vol. 27, no. 8, p. 1078-1085. https://doi.org/10.1016/j.fm.2010.07.012
Costa, M. P., Balthazar, C. F., Rodrigues, B. L., Lazaro, C. A., Silva, A. C. O., Cruz, A. G. and Conte Junior, C. A. 2015. Determination of biogenic amines by high-performance liquid chromatography (HPLC-DAD) in probiotic cow's and goat's fermented milks and acceptance. Food Science & Nutrition, vol. 3, no. 3, p. 172-178. https://doi.org/10.1002/fsn3.200
Curiel, J. A., Ruiz-Capillas, C., de las Rivas, B., Carrascosa, A. V., Jiménez-Colmenero, F., Muñoz, R. 2011. Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration. Meat Science, vol. 88, no. 3, p. 368-373. https://doi.org/10.1016/j.meatsci.2011.01.011
De las Rivas B., Marcobal A., Carrascosa A., V., Mu˜noz R. 2006. PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine and cadaverine. Journal of Food Protection, vol. 69, no. 10, p. 2509-2514. https://doi.org/10.1021/jf049340k PMid:15713028
De Las Rivas, B., Marcobal, A. and Muñoz, R. 2006. Gene organization of the ornithine decarboxylase-encoding region in Morganella morganii. Journal of Applied Microbiology, vol. 102, no. 6, p. 1551-1560. https://doi.org/10.1111/j.1365-2672.2006.03188.x
De Las Rivas, B., Marcobal, A., Mu˜noz, R. 2005. Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines. FEMS Microbiology Letters, vol. 244, no. 2, p. 367-372. https://doi.org/10.1016/j.femsle.2005.02.012
Fitzmaurice, J., Glennon, M., Duffy, G., Sheridan, J. J., Carroll, C., Maher, M. 2004. Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E. coli O157:H7. Molecular and Cellular Probes, vol. 18, no. 2, p. 123-132. https://doi.org/10.1016/j.mcp.2003.10.004
Ladero, V., Calles-Enriquez, M., Fernandez, M., Alvarez, M. 2010. Toxicological effects of dietary biogenic amines. Current Nutrition & Food Science, vol. 6, p.145-156. https:// doi.org/10.2174/157340110791233256
Ladero V., Fernandez M., Cuesta I., Alvarez M. A. 2010. Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR. Food Microbiology, vol. 27, no. 7, p. 933-939. https://doi.org/10.1016/j.fm.2010.05.026
Landete, J. M., de las Rivas, B., Marcobal, A., Munoz, R. 2005. Biogenic amines in wines from three Spanish regions. Journal of Agricultural and Food Chemistry, vol. 53, no. 4, p. 258-269. http://pubs.acs.org/doi/full/10.1021/jf049340k
Lavizzari, T., Breccia, M., Bover-Cid, S., Vidal-Carou, M. C., Veciana-Nogués, M. T. 2010. Histamine, cadaverine, and putrescine produced in vitro by Enterobacteriaceae and Pseudomonadaceae isolated from spinach. Journal of Food Protection, vol. 73, no. 2, p. 385-389. https://doi.org/10.4315/0362-028X-73.2.385
Lorencova, E., Bunkova, L., Matoulkova, D., Drab, V., Pleva, P., Kuban, V., Bunka, F. 2012. Production of biogenic amines by lactic acid bacteria and bifidobacteria isolated from dairy products and beer. International Journal of Food Science & Technology, vol. 47, no. 10, p. 2086-2091. https://doi.org/10.1111/j.1365-2621.2012.03074.x
Mackay, I. M. 2007. Real-time PCR in microbiology: from diagnosis to characterisation. Norfolk, UK: Caister Academic, 454 p. ISBN 1904455182. https://doi.org/10.1002/elsc.200890009
Maijala, R. L. 1993. Formation of histamine and tyramine by some lactic acid bacteria in MRS-bro th and modified decarboxylation agar. Letters in Applied Microbiology, vol. 17, no. 1, p. 40-43. https://doi.org/10.1111/j.1472-765X.1993.tb01431.x
Nannelli, F., Claisse, O., Gindreau, E., de Revel, G., Lonvaud-Funel, A., Lucas, P. M. 2008. Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods. Letters in Applied Microbiology, vol. 47, no. 6, p. 594-599. https://doi.org/10.1111/j.1472-765X.2008.02472.x
Shalaby, A. R. 1996. Significance of biogenic amines to food safety and human health. Food Research International, vol. 29, no. 7, p. 675-690. https://doi.org/10.1016/S0963-9969(96)00066-X
Santos, M. 1996. Biogenic amines: their importance in foods. International Journal of Food Microbiology, vol. 29, no. 2-3, p. 213-231. https://doi.org/10.1016/0168-1605(95)00032-1
Suzzi, G., Garnini, F. 2003. Biogenic amines in dry fermented sausages: a review. International Journal of Food Microbiology, vol. 88, no. 1, p. 41-54. https://doi.org/10.1016/S0168-1605(03)00080-1
Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, vol. 29, no. 9, p. 2002-2007. https://doi.org/10.1093/nar/29.9.e45
Pons-Sánchez-Cascado, S., Bover-Cid, S., Veciana-Nogués, M. T., Vidal-Carou, M. C. 2005. Amino acid-decarboxylase activity of bacteria isolated from ice-preserved anchovies. European Food Research and Technology, vol. 220, no. 3, p. 312‑315. https://doi.org/10.1007/s00217-004-1095-y
Postollec, F., Falentin, H., Pavan, S., Combrisson, J., Sohier, D. 2011. Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiology, vol. 28, no. 5, p. 848-861. https://doi.org/10.1016/j.fm.2011.02.008
Thornton, B., Basu, C. 2011. Real-time PCR (qPCR) primer design using free online software education. Biochemistry and Molecular Biology Uducation, vol. 39, p. 145-154. https://doi.org/10.1002/bmb.20461
ROCHE, 2017. High Pure RNA Isolation Kit, [online] s.a. [cit.2017-01-17]. Avaible at: http://netdocs.roche.com/DDM/Effective/0000000000001004022000453_000_12_005_Native.pdf
Roche, 1999-2017. Transcriptor First Strand cDNA Synthesis Kit, [online] s.a. [cit. 2017-01-12]. Avaible at: http://facstaff.bloomu.edu/gdavis/MoBio/Transcriptor%20full%20manual.pdf
Weigel L., M., Steward C., D., Tenover F., C. 1998. gyrA Mutations Associated with Fluoroquinolone Resistance in Eight Species of Enterobacteriaceae. Antimicrobial Agents and Chemotherapy, vol. 42, no. 10, p. 2661-2667. PMid:9756773
Wilhelm, J., Pingoud, A. 2003. Real-time polymerase chain reaction. ChemBioChem, vol. 4, no. 11, p. 1120-1128. https://doi.org/10.1002/cbic.200300662
Wunderlichova, L. 2009. Vývoj nových molekulárně biologických metod detekce putrescin produkujících bakterií : Dissertation theses. Zlín: Univerzita Tomáše Bati ve Zlíně, 123 p.
Wunderlichova, L., Bunkova, L., Koutny, M., Jancova, P., Bunka, F. 2014. Formation, Degradation, and Detoxification of Putrescine by Foodborne Bacteria: A Review. Comprehensive Reviews in Food Science and Food Safety, vol. 13, no. 5, p. 1541-4337. https://doi.org/10.1111/1541-4337.12099
Yin, H., Cao, L., Qiu, G., Wang, D., Kellogg, L., Zhou, J., Liu, X., Dai, Z., Ding, J., Liu, X. 2008. Molecular diversity of 16S rRNA and gyrB genes in copper mines. Archives of Microbiology, vol 189, no. 2, p. 101-110. https://doi.org/10.1007/s00203-007-0298-6 PMid:17957354
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