Fourier transform infrared spectroscopy, detection and identification of Escherichia coli O157:H7 and Alicyclobacillus strains in apple juice
Introduction
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is one of the most important foodborne pathogens causing inflammation of the colon and gives rise to diarrhea and abdominal pain with bleeding and blood appears in stools, renal failure due to blood clots in the kidney tubules, and internal bleeding due to lack of blood platelets resulting in brain damage that can occur in serious cases (Garbutt, 1997). Despite of the minimal bacterial health risks of fruit juices due to low pH and high organic acid content (Fisher and Golden, 1998, Al-Holy et al., 2006), several foodborne outbreaks of Hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) have been reported due to contamination of apple juice by E. coli O157:H7 (Besser et al., 1993, Centers for Disease Control and Prevention (CDC), 1996, Parish, 1997, Buchanan et al., 1998, Cody et al., 1999). Contamination in certain products is relatively common, in one with 11 out of 314 apple cider samples produced between mid-August and March in Connecticut were contaminated with E. coli (Dingman, 1999, Al-Holy et al., 2006).
Alicyclobacillus spp. are non-pathogenic, thermoacidophilic, spore-forming, Gram-positive to Gram-variable bacteria (Herrmann et al., 1996, Orr and Beuchat, 2000, Lee et al., 2002, Matsubara et al., 2002, Chang and Kang, 2004, Luo et al., 2004, Zierler et al., 2004, Lin et al., 2005). Alicuclobacillus species is distinctive from bacteria of the genus Bacillus due to the difference in 16S rDNA sequences analysis, and the presence of the unique ω-alicyclic fatty acids in its membrane lipid bilayer that support its survival at low pH and high temperature (Wisotzkey et al., 1992, Lee et al., 2002, Sinigaglia et al., 2003, Luo et al., 2004).
The main distinct features of Alicyclobacillus spp. are: i) their ability to survive commercial pasteurization treatments (Pettipher et al., 1997, Sinigaglia et al., 2003, Chang and Kang, 2004); ii) their ability to grow at relatively high temperature and at low pH (Orr and Beuchat, 2000, Sinigaglia et al., 2003, Luo et al., 2004); and iii) for some strains, produce guaiacol and other taint chemicals which create “medicinal” or “antiseptic” off-flavor in fruit juices (Yamazaki et al., 1997, Lee et al., 2002, Sinigaglia et al., 2003, Chang and Kang, 2004, Luo et al., 2004, Lin et al., 2005). These distinctive characteristics that cause Alicyclobacillus spp. to be a major problem in the food industry and particularly create a significant spoilage concern in hot-fill fruit and vegetable juices (Sinigaglia et al., 2003), also make Alicyclobacillus spp. a major quality control target microorganism in the apple juice industry (Chang and Kang, 2004). Recently, Alicyclobacillus acidoterrestris has been reported in fruit juice spoilage incidents in the United States, United Kingdom, and Germany (Lee et al., 2002, Pettipher et al., 1997).
Fourier Transform Infrared (FT-IR) absorbance spectroscopy (4000–400 cm− 1) identifies microbial cells at the strain level (Helm et al., 1991, Beattie et al., 1998, Rodriguez-Saona et al., 2001, Lin et al., 2005). Bacterial spectra represent the total biochemical composition of bacterial cell wall and membrane (phospholipids bilayer, peptidoglycan, and lipopolysaccharides), as well as the composition of cellular cytoplasm (water, fatty acids, proteins, polysaccharides, and nucleic acids) (Goodacre et al., 1996, Kansiz et al., 1999, Lin et al., 2005). The FT-IR spectral features of these biochemical constituents create a ‘fingerprint’ for a bacterial strain (Goodacre et al., 1996).
The main objective of this study was to examine the ability of FT-IR spectroscopy for rapid detection and identification of E. coli O157:H7 and Alicyclobacillus spp. in pure and mixed cultures in apple juice.
Section snippets
Bacterial cultures and growth conditions
The bacterial cells used in this study were obtained from the culture collection in the Department of Food Science and Human Nutrition, Washington State University. Bacterial strains include E. coli O157:H7 ATCC 35150, A. acidoterrestris 1016 (guaiacol-producing), and Alicyclobacillus spp. C-Fugi-6 (non-guaiacol-producing). E. coli O157:H7 ATCC 35150 was activated by transferring bacterial cells from refrigerated slant to tryptic soy agar (TSA) (Difco™, Sparks, MD, USA) and incubation of
Results and discussion
The bacterial tests which were performed on the apple juice before inoculation showed that the apple juice was free from heterotrophic bacterial cells, coliforms, and vegetative Alicyclobacillus spp. Table 1 shows the mean of bacterial loads after inoculation of apple juice and the number of bacterial cells per mm2 of Anodisc membrane filter. There was approximately a 3 log reduction in the count of E. coli O157:H7 ATCC 35150 (original inoculum was ∼ 1.0 × 109 CFU/ml) due to the effect of low pH
Conclusions
FT-IR spectroscopy in the range of 4000–400 cm− 1 with the help of multivariate analysis (PCA and SIMCA) can be used to detect, discriminate and identify bacterial strains. Spectral patterns of the mixed cultures of both Alicyclobacillus spp. strains indicate that the composite spectra obtained are distinct from the spectra of pure cultures. The sensitivity of the FT-IR technique mostly depends on the concentration of harvested bacterial cells on Anodisc membrane and the procedure used for
Acknowledgements
This work was supported by the Washington State University International Marketing Program for Agricultural Commodities and Trade (IMPACT) Center. USDA NRICGP Grants 2002-35201-11683, 2000-01617, 2000-0119, Washington State University, USDA Grant. We gratefully thank Dr. Peter Griffiths from the University of Idaho, Dr. D-H. Kang, Su-Sen Chang and Mr. Peter Gray from Washington State University for the technical support.
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