Fourier transform infrared spectroscopy, detection and identification of Escherichia coli O157:H7 and Alicyclobacillus strains in apple juice

Abstract

Fourier Transform Infrared (FT-IR) spectroscopy (4000–400 cm^− 1) combined with multivariate statistical methods were used to identify and detect Escherichia coli O157:H7 from Alicyclobacillus spp. recovered from apple juice. Four treatments and a control in triplicate experiments (N = 3) were studied; the first three treatments of pasteurized apple juice were inoculated with E. coli O157:H7 ATCC 35150, Alicyclobacillus acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6 respectively. The fourth treatment was a 1:1 (v:v) mixed culture of both A. acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6. The control was uninoculated pasteurized apple juice.

The second derivative transformation and loadings plot over the range of 1800–900 cm⁻¹ highlighted the most distinctive variations among bacterial spectra. Loadings 1 and 2 were distinctively representative of the bacterial spectral data and accounted for 73% of the total variability. Treatments were noticeably segregated with distinct clustering by principal component analysis (PCA). Using soft independent modeling of class analogy (SIMCA) analysis, 88.3% of (E. coli O157:H7 ATCC 35150) spectra, 75.0% of (A. acidoterrestris 1016) spectra, 88.3% of (Alicyclobacillus spp. C-Fuji-6) spectra, and 80.0% of the mixed culture of both Alicyclobacillus strains spectra were correctly classified. Using the spectral features of bacterial cellular constituents such as nucleic acids, proteins, phospholipids, peptidoglycan, and lipopolysaccharides from examined bacterial cells, pure and mixed cultures of Alicyclobacillus spp. cells, and the pathogenic E. coli cells could be detected 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.