Growth comparison of several Escherichia coli strains exposed to various concentrations of lactoferrin using linear spline regression

Springer Science and Business Media LLC - 2012
Camilla Sekse1, Jon Bohlin2, Eystein Skjerve2, G. Vegarud3
1Norwegian Veterinary Institute, Oslo, Norway
2Norwegian School of Veterinary Science, Epi-Centre, Department of Food Safety and Infection Biolog, Oslo, Norway
3Norwegian University of Life Sciences, Department of Chemistry, Biotechnology and Food Science, Aas, Norway

Tóm tắt

Abstract Background We wanted to compare growth differences between 13 Escherichia coli strains exposed to various concentrations of the growth inhibitor lactoferrin in two different types of broth (Syncase and Luria-Bertani (LB)). To carry this out, we present a simple statistical procedure that separates microbial growth curves that are due to natural random perturbations and growth curves that are more likely caused by biological differences. Bacterial growth was determined using optical density data (OD) recorded for triplicates at 620 nm for 18 hours for each strain. Each resulting growth curve was divided into three equally spaced intervals. We propose a procedure using linear spline regression with two knots to compute the slopes of each interval in the bacterial growth curves. These slopes are subsequently used to estimate a 95% confidence interval based on an appropriate statistical distribution. Slopes outside the confidence interval were considered as significantly different from slopes within. We also demonstrate the use of related, but more advanced methods known collectively as generalized additive models (GAMs) to model growth. In addition to impressive curve fitting capabilities with corresponding confidence intervals, GAM’s allow for the computation of derivatives, i.e. growth rate estimation, with respect to each time point. Results The results from our proposed procedure agreed well with the observed data. The results indicated that there were substantial growth differences between the E. coli strains. Most strains exhibited improved growth in the nutrient rich LB broth compared to Syncase. The inhibiting effect of lactoferrin varied between the different strains. The atypical enteropathogenic aEPEC-2 grew, on average, faster in both broths than the other strains tested while the enteroinvasive strains, EIEC-6 and EIEC-7 grew slower. The enterotoxigenic ETEC-5 strain, exhibited exceptional growth in Syncase broth, but slower growth in LB broth. Conclusions Our results do not indicate clear growth differences between pathogroups or pathogenic versus non-pathogenic E. coli.

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Tài liệu tham khảo

Nataro JP, Kaper JB: Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998, 11: 142-201.

Kaper JB, Nataro JP, Mobley HL: Pathogenic Escherichia coli. Nat Rev Microbiol. 2004, 2: 123-140. 10.1038/nrmicro818.

Farnaud S, Evans RW: Lactoferrin–a multifunctional protein with antimicrobial properties. Mol Immunol. 2003, 40: 395-405. 10.1016/S0161-5890(03)00152-4.

Ochoa TJ, Cleary TG: Effect of lactoferrin on enteric pathogens. Biochimie. 2009, 91: 30-34. 10.1016/j.biochi.2008.04.006.

Arnold RR, Brewer M, Gauthier JJ: Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms. Infect Immun. 1980, 28: 893-898.

Kawasaki Y, Tazume S, Shimizu K, Matsuzawa H, Dosako S, Isoda H, et al: Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells. Biosci Biotechnol Biochem. 2000, 64: 348-354. 10.1271/bbb.64.348.

Ochoa TJ, Clearly TG: Lactoferrin disruption of bacterial type III secretion systems. Biometals. 2004, 17: 257-260.

Ochoa TJ, Brown EL, Guion CE, Chen JZ, McMahon RJ, Cleary TG: Effect of lactoferrin on enteroaggregative E. coli (EAEC). Biochem Cell Biol. 2006, 84: 369-376. 10.1139/o06-053.

Santapaola D, del CF, Bosso P, Morea C, Valenti P, Berlutti F, et al: Effect on bovine lactoferrin on the activation of the enteroinvasive bacterial type III secretion system. Biometals. 2004, 17: 261-265.

Cao R, Francisco-Fernandez M, Quinto EJ: A random effect multiplicative heteroscedastic model for bacterial growth. BMC Bioinformatics. 2010, 11: 77-10.1186/1471-2105-11-77.

Baty F, Delignette-Muller ML: Estimating the bacterial lag time: which model, which precision?. Int J Food Microbiol. 2004, 91: 261-277. 10.1016/j.ijfoodmicro.2003.07.002.

Nelder JA, Wedderburn RWM: Generalized Linear Models. Journal of the Royal Statistical Society Series A (General). 1972, 135: 370-384. 10.2307/2344614.

Zwietering MH, Jongenburger I, Rombouts FM, van’t RK: Modeling of the bacterial growth curve. Appl Environ Microbiol. 1990, 56: 1875-1881.

Dionysius DA, Grieve PA, Milne JM: Forms of lactoferrin: their antibacterial effect on enterotoxigenic Escherichia coli. J Dairy Sci. 1993, 76: 2597-2600. 10.3168/jds.S0022-0302(93)77594-3.

Ellison RT, Giehl TJ: Killing of gram-negative bacteria by lactoferrin and lysozyme. J Clin Invest. 1991, 88: 1080-1091. 10.1172/JCI115407.

Murdock CA, Matthews KR: Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA. J Appl Microbiol. 2002, 93: 850-856. 10.1046/j.1365-2672.2002.01762.x.

Shin K, Yamauchi K, Teraguchi S, Hayasawa H, Tomita M, Otsuka Y, et al: Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7. Lett Appl Microbiol. 1998, 26: 407-411. 10.1046/j.1472-765X.1998.00358.x.

Hastie TJ, Tibshirani RJ: General Additive Models. 1990, Chapman and Hall, London

Augustin JC, Rosso L, Carlier V: Estimation of temperature dependent growth rate and lag time of Listeria monocytogenes by optical density measurements. J Microbiol Methods. 1999, 38: 137-146. 10.1016/S0167-7012(99)00089-5.

Vilar-Fernández JM, González-Manteiga W: Nonparametric comparison of curves with dependent errors. Statistics. 2003, 00: 1-19.

Munk A, Dette H: Nonparametric comparison of several regression functions: Exact and asyptomatic theory. Ann Stat. 1998, 26: 2339-2368.

Osek J, Lebens M, Holmgren J: Improved medium for large-scale production of recombinant cholera toxin B subunit for vaccine purposes. Journal of Microbiological Methods. 1995, 24: 117-124. 10.1016/0167-7012(95)00061-5.

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