Abstract
Adherence and invasion to cells are the key processes during infection development by Campylobacter jejuni (C. jejuni). In this study, extracts from the byproducts of Citrus limon, Citrus aurantium, and Citrus medica were added to the cultures of C. jejuni, and the adherence and invasion of C. jejuni to HeLa cells and the expression of cadF and ciaB genes were analyzed. The relative expression of the genes was determined by quantitative reverse transcription PCR (qRT-PCR). The antioxidant activity was determined using spectrophotometric methods. Byproduct extracts at subinhibitory concentrations affected the adherence (reduced 2.3 to 99%) and invasion (reduced 71.3 to 99.2%) to the HeLa cells. The expression of cadF and ciaB was reduced from 66 to 99% and from 81 to 99%, respectively. The total phenolic content of the byproducts varied from 92 to 26 mg GAE/g and the total flavonoids varied from 161 to 29.29 mg QE/g. C. aurantium showed the highest percentage of radical scavenging activity (RSA, 90.1). These extracts can prove as effective alternatives for devising new strategies to control Campylobacter infections.
Keywords: Campylobacter, adhesion, invasion, citrus extract, antimicrobial activity, antioxidant activity
References
- 1.Asakura H, Yamasaki M, Yamamoto S, Igimi S. Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni. FEMS Microbiol. Lett. 2007;275:278–285. doi: 10.1111/j.1574-6968.2007.00893.x. [DOI] [PubMed] [Google Scholar]
- 2.Hänel I, Borrmann E, Müller J, Alter T. Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys. J. Appl. Microbiol. 2007;102:433–441. doi: 10.1111/j.1365-2672.2006.03088.x. [DOI] [PubMed] [Google Scholar]
- 3.Hermans D, van Deun K, Martel A, van Immerseel F, Messens W, Heyndrickx M, Haesebrouck F, Pasmans F. Colonization factors of Campylobacter jejuni in the chicken gut. Vet. Res. 2011;42:82. doi: 10.1186/1297-9716-42-82. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Cróinín T, Backert S. Host epithelial cell invasion by Campylobacter jejuni: Trigger or zipper mechanism? Front. Cell. Infect. Microbiol. 2012;2:25. doi: 10.3389/fcimb.2012.00025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U. Campylobacter jejuni: A brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int. J. Med. Microbiol. 2010;300:205–211. doi: 10.1016/j.ijmm.2009.07.002. [DOI] [PubMed] [Google Scholar]
- 6.Clatworthy AE, Pierson E, Hung DT. Targeting virulence: A new paradigm for antimicrobial therapy. Nat. Chem. Biol. 2007;3:541–548. doi: 10.1038/nchembio.2007.24. [DOI] [PubMed] [Google Scholar]
- 7.Klanènik A, Piskernik S, Jeršek B, Možina SS. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. J. Microbiol. Meth. 2010;81:121–126. doi: 10.1016/j.mimet.2010.02.004. [DOI] [PubMed] [Google Scholar]
- 8.Singh J, Sood S, Muthuraman A. In-vitro evaluation of bioactive compounds, anti-oxidant, lipid peroxidation and lipoxygenase inhibitory potential of Citrus karna L. peel extract. J. Food Sci. Tech. Mys. 2014;51:67–74. doi: 10.1007/s13197-011-0479-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.El-aal HA, Halaweish F. Food preservative activity of phenolic compounds in orange peel extracts (Citrus sinensis L.) Lucrari Stiintifice. 2010;53:233–240. [Google Scholar]
- 10.Chen ZT, Chu HL, Chyau CC, Chu CC, Duh PD. Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress. Food Chem. 2012;135:2119–2127. doi: 10.1016/j.foodchem.2012.07.041. [DOI] [PubMed] [Google Scholar]
- 11.Yusof S, Ghazali HM, King GS. Naringin content in local citrus fruits. Food Chem. 1990;37:113–121. doi: 10.1016/0308-8146(90)90085-I. [DOI] [Google Scholar]
- 12.Nannapaneni R, Muthaiyan A, Crandall PG, Johnson MG, O’Bryan CA, Chalova VI, Callaway TR, Carroll JA, Arthington JD, Nisbet DJ. Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains. Foodborne Pathog. Dis. 2008;5:695–699. doi: 10.1089/fpd.2008.0124. [DOI] [PubMed] [Google Scholar]
- 13.Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 2006;99:191–203. doi: 10.1016/j.foodchem.2005.07.042. [DOI] [Google Scholar]
- 14.Guil-Guerrero J, Ramos L, Moreno C, Zúñiga-Paredes J, Carlosama-Yepez M, Ruales P. Antimicrobial activity of plant-food by-products: A review focusing on the tropics. Livest Sci. 2016;189:32–49. doi: 10.1016/j.livsci.2016.04.021. [DOI] [Google Scholar]
- 15.Nannapaneni R, Chalova VI, Crandall PG, Ricke SC, Johnson MG, O’Bryan CA. Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions. Int. J. Food Microbiol. 2009;129:43–49. doi: 10.1016/j.ijfoodmicro.2008.11.008. [DOI] [PubMed] [Google Scholar]
- 16.Castillo S, Heredia N, Arechiga-Carvajal E, García S. Citrus extracts as inhibitors of quorum sensing, biofilm formation and motility of Campylobacter jejuni. Food Biotechnol. 2014;28:106–122. doi: 10.1080/08905436.2014.895947. [DOI] [Google Scholar]
- 17.Valtierra-Rodríguez D, Heredia NL, García S, Sánchez E. Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts. J. Food Protect. 2010;73:477–482. doi: 10.4315/0362-028X-73.3.477. [DOI] [PubMed] [Google Scholar]
- 18.Castillo SL, Heredia N, Contreras JF, García S. Extracts of edible and medicinal plants in inhibition of growth, adherence, and cytotoxin production of Campylobacter jejuni and Campylobacter coli. J. Food Sci. 2011;76:M421–M426. doi: 10.1111/j.1750-3841.2011.02229.x. [DOI] [PubMed] [Google Scholar]
- 19.Sanchez E, Heredia N, Garcia S. Extracts of edible and medicinal plants damage membranes of Vibrio cholerae. Appl. Environ. Microb. 2010;76:6888–6894. doi: 10.1128/AEM.03052-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Ganan M, Campos G, Muñoz R, Carrascosa AV, de Pascual-Teresa S, Martinez-Rodriguez AJ. Effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by Campylobacter. Int. J. Food Microbiol. 2010;140:14–18. doi: 10.1016/j.ijfoodmicro.2010.02.021. [DOI] [PubMed] [Google Scholar]
- 21.Clark CG, Grant CC, Pollari F, Marshall B, Moses J, Tracz DM, Gilmour MW. Effects of the Campylobacter jejuni CJIE1 prophage homologs on adherence and invasion in culture, patient symptoms, and source of infection. BMC Microbiol. 2012;12:269. doi: 10.1186/1471-2180-12-269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Jeon B, Itoh K, Misawa N, Ryu S. Effects of quorum sensing on flaA transcription and autoagglutination in Campylobacter jejuni. Microbiol. Immunol. 2003;47:833–839. doi: 10.1111/j.1348-0421.2003.tb03449.x. [DOI] [PubMed] [Google Scholar]
- 23.Cloak OM, Solow BT, Briggs CE, Chen CY, Fratamico PM. Quorum sensing and production of autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods. Appl. Environ. Microb. 2002;68:4666–4671. doi: 10.1128/AEM.68.9.4666-4671.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Malik-Kale P, Parker CT, Konkel ME. Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression. J. Bacteriol. 2008;190:2286–2297. doi: 10.1128/JB.01736-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using Real-Time Quantitative PCR and the 2 -?CT Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
- 26.Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Method. Enzymol. 1999;299:152–178. doi: 10.1016/S0076-6879(99)99017-1. [DOI] [Google Scholar]
- 27.Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999;64:555–559. doi: 10.1016/S0308-8146(98)00102-2. [DOI] [Google Scholar]
- 28.Singh S, Singh R. in vitro methods of assay of antioxidants: An overview. Food Rev. Int. 2008;24:392–415. doi: 10.1080/87559120802304269. [DOI] [Google Scholar]
- 29.Kedare SB, Singh R. Genesis and development of DPPH method of antioxidant assay. J. Food. Sci. Tech. Mys. 2011;48:412–422. doi: 10.1007/s13197-011-0251-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Snelling W, Matsuda M, Moore J, Dooley J. Campylobacter jejuni. Lett. Appl. Microbiol. 2005;41:297–302. doi: 10.1111/j.1472-765X.2005.01788.x. [DOI] [PubMed] [Google Scholar]
- 31.Wine E, Gareau MG, Johnson-Henry K, Sherman PM. Strain-specific probiotic (Lactobacillus helveticus) inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells. FEMS Microbiol. Lett. 2009;300:146–152. doi: 10.1111/j.1574-6968.2009.01781.x. [DOI] [PubMed] [Google Scholar]
- 32.Pogaèar M, Klanènik A, Bucar F, Langerholc T, Možina SS. Alpinia katsumadai extracts inhibit adhesion and invasion of Campylobacter jejuni in animal and human foetal small intestine cell lines. Phytother. Res. 2015;29:1585–1589. doi: 10.1002/ptr.5396. [DOI] [PubMed] [Google Scholar]
- 33.Castillo S, Heredia N, García S. 2 (5H)-Furanone, epigallocatechin gallate, and a citric-based disinfectant disturb quorum-sensing activity and reduce motility and biofilm formation of Campylobacter jejuni. Folia Microbiol. 2015;60:89–95. doi: 10.1007/s12223-014-0344-0. [DOI] [PubMed] [Google Scholar]
- 34.Lee KM, Kim WS, Lim J, Nam S, Youn M, Nam SW, Kim Y, Kim SH, Park W, Park S. Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7. J. Food Protect. 2009;72:325–331. doi: 10.4315/0362-028X-72.2.325. [DOI] [PubMed] [Google Scholar]
- 35.Zou Z, Xi W, Hu Y, Nie C, Zhou Z. Antioxidant activity of Citrus fruits. Food Chem. 2016;196:885–896. doi: 10.1016/j.foodchem.2015.09.072. [DOI] [PubMed] [Google Scholar]
- 36.Daglia M. Polyphenols as antimicrobial agents. Curr. Opin. Biotech. 2012;23:174–181. doi: 10.1016/j.copbio.2011.08.007. [DOI] [PubMed] [Google Scholar]
- 37.de Villiers A, Venter P, Pasch H. Recent advances and trends in the liquidchromatography–mass spectrometry analysis of flavonoids. J. Chromatogr. A. 2016;1430:16–78. doi: 10.1016/j.chroma.2015.11.077. [DOI] [PubMed] [Google Scholar]
- 38.Xi W, Fang B, Zhao Q, Jiao B, Zhou Z. Flavonoid composition and antioxidant activities of Chinese local pummelo (Citrus grandis Osbeck.) varieties. Food Chem. 2014;161:230–238. doi: 10.1016/j.foodchem.2014.04.001. [DOI] [PubMed] [Google Scholar]
- 39.Gorinstein S, Martýìn-Belloso O, Park YS, Haruenkit R, Lojek A ý M, Caspi A, Libman I, Trakhtenberg S. Comparison of some biochemical characteristics of different citrus fruits. Food Chem. 2001;74:309–315. doi: 10.1016/S0308-8146(01)00157-1. [DOI] [Google Scholar]
- 40.Ghasemi K, Ghasemi Y, Ebrahimzadeh MA. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Pak. J. Pharm. Sci. 2009;22:277–281. [PubMed] [Google Scholar]
- 41.Nakajima VM, Macedo GA, Macedo JA. Citrus bioactive phenolics: Role in the obesity treatment. LWT-Food Sci. Technol. 2014;59:1205–1212. doi: 10.1016/j.lwt.2014.02.060. [DOI] [Google Scholar]
- 42.Ramful D, Bahorun T, Bourdon E, Tarnus E, Aruoma OI. Bioactive phenolics and antioxidant propensity of flavedo extracts of Mauritian citrus fruits: Potential prophylactic ingredients for functional foods application. Toxicology. 2010;278:75–87. doi: 10.1016/j.tox.2010.01.012. [DOI] [PubMed] [Google Scholar]
- 43.Li B, Smith B, Hossain MM. Extraction of phenolics from citrus peels: I. Solvent extraction method. Sep. Purif. Technol. 2006;48:182–188. doi: 10.1016/j.seppur.2005.07.005. [DOI] [Google Scholar]
- 44.Li B, Smith B, Hossain MM. Extraction of phenolics from citrus peels: II. Enzyme-assisted extraction method. Sep. Purif. Technol. 2006;48:189–196. [Google Scholar]
- 45.Chansiripornchai N, Sasipreeyajan J. PCR detection of four virulenceassociated genes of Campylobacter jejuni isolates from Thai broilers and their abilities of adhesion to and invasion of INT-407 cells. J. Vet. Med. Sci. 2009;71:839–844. doi: 10.1292/jvms.71.839. [DOI] [PubMed] [Google Scholar]
- 46.Preeti M, Craig T, Konkel M. Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression. J. Bacteriol. 2008;190:2286–2297. doi: 10.1128/JB.01736-07. [DOI] [PMC free article] [PubMed] [Google Scholar]