Flow‐Cytometry‐Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA‐Based Probe

Yong Xue1, Jon G. Wilkes1, Ted J. Moskal2, Anna J. Williams1, Willie M. Cooper1, Dan A. Buzatu1

1 Division of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 2 Life Sciences Consultant, Jonesboro, Arkansas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.25
DOI:  10.1002/cptx.14
Online Posting Date:  February, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time‐consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow‐cytometry‐based detection method that has greater sensitivity and specificity. This method requires less time‐to‐results (TTR) and can detect a small number of E. coli in the presence of large numbers of other bacteria. Clear step‐by‐step protocols for cell concentration determination, sample preparation, and flow cytometric analysis are provided. © 2017 by John Wiley & Sons, Inc.

Keywords: flow cytometry; foodborne illness; Escherichia coli; microbial contamination; oligonucleotide probe; Shigella; Salmonella

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1:  

  • Brain heart infusion (BHI) medium
  • Nutrient broth (NB) medium
  • Trypticase soy broth (TSB) medium
  • A generic E. coli oligonucleotide probe ES445 (Kenzaka et al., ) with sequence: 5′ CTT TAC TCC CTT CCT CCC 3′ with Alexa Fluor 488 linked to its 5′ end
  • 1× PBS solution
  • 10% buffered formalin
  • 50% ethanol/PBS solution
  • Hybridization buffer (20 mM Tris, pH 7.5, 0.01% SDS, 3 M NaCl; see recipe)
  • Total plate count (TPC) reagent containing thiazole orange (TO) and propidium iodide (PI)
  • 37°C incubator
  • Benchtop centrifuge
  • Thermomixer B
  • Flow cytometer
  • Data analysis software: FCS Express 4 Flow Cytometry software
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., and Stahl, D.A. 1990. Combination of 16S rRNA‐targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919‐1925.
  Besser, R.E., Griffin, P.M., and Slutsker, L. 1999. Escherichia coli O157:H7 gastroenteritis and the hemolytic uremic syndrome: An emerging infectious disease. Annu. Rev. Med. 50:355‐367. doi: 10.1146/annurev.med.50.1.355.
  Buzatu, D.A., Moskal, T.J., Williams, A.J., Cooper, W.M., Mattes, W.B., and Wilkes, J.G. 2014. An integrated flow cytometry‐based system for real‐time, high sensitivity bacterial detection and identification. PLoS One. 9:e94254. doi: 10.1371/journal.pone.0094254.
  Centers for Disease Control and Prevention. 1996. Outbreak of Escherichia coli O157: H7 infections associated with drinking unpasteurized commercial apple juice–British Columbia, California, Colorado, and Washington, October 1996. MMWR Morbidity and Mortality Weekly Report 45.44:975.
  Centers for Disease Control and Prevention. 2011. Reports of Selected E. coli Outbreak Investigations. http://www.cdc.gov/ecoli/outbreaks.html (Accessed July 6, 2016).
  Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single‐cell analyses. Microbiol. Rev. 60:641‐696.
  DeLong, E.F., Wickham, G.S., and Pace, N.R. 1989. Phylogenetic stains: Ribosomal RNA‐based probes for the identification of single cells. Science 243:1360‐1363. doi: 10.1126/science.2466341.
  Feng, P., Weagant, S.D., and Jinneman, K. 2013. BAM: Diarrheagenic Escherichia coli. http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070080.htm (Accessed July 6, 2016).
  Food Safety and Inspection Service. 2014. Recalls and Public Health Alerts. http://www.fsis.usda.gov/wps/portal/fsis/topics/recalls‐and‐public‐health‐alerts (Accessed July 6, 2016).
  Gucker, F.T. Jr., O'Konski, C., Pickard, H.B., and Pitts, J.N. Jr. 1947. A photoelectric counter for colloidal particles. J. Am. Chem. Soc. 69:2422‐2431. doi: 10.1021/ja01202a053.
  Joachimsthal, E., Ivanov, V., Tay, S., and Tay, J. 2003. Quantification of whole‐cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells. World J. Microbiol. Biotechnol. 19:527‐533. doi: 10.1023/A:1025122032762.
  Kenzaka, T., Yamaguchi, N., Prapagdee, B., Mikami, E., and Nasu, M. 2001. Bacterial community composition and activity in urban rivers in Thailand and Malaysia. J. Health Sci. 47:353‐361. doi: 10.1248/jhs.47.353.
  Lan, R. and Reeves, P.R. 2002. Escherichia coli in disguise: Molecular origins of Shigella. Microbes Infect. 4:1125‐1132. doi: 10.1016/S1286‐4579(02)01637‐4.
  Mathusa, E.C., Chen, Y., Enache, E., and Hontz, L. 2010. Non‐O157 Shiga toxin‐producing Escherichia coli in foods. J. Food Prot. 73:1721‐1736.
  Moldavan, A. 1934. Photo‐electric technique for the counting of microscopical cells. Science 80:188‐189. doi: 10.1126/science.80.2069.188.
  Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M., and Swerdlow, D.L. 2005. Epidemiology of Escherichia coli O157: H7 outbreaks, United States, 1982‐2002. Emerging Infect. Dis. 11:603‐609. doi: 10.3201/eid1104.040739.
  Vogt, R.L. and Dippold, L. 2005. Escherichia coli O157: H7 outbreak associated with consumption of ground beef, June‐July 2002. Public Health Rep. 120:174‐178.
  Xue, Y., Wilkes, J.G., Moskal, T.J., Williams, A.J., Cooper, W.M., Nayak, R., Rafii, F., and Buzatu, D.A. 2016. Development of a flow cytometry‐based method for rapid detection of Escherichia coli and Shigella Spp. using an oligonucleotide probe. PLoS One. 11:e0150038. doi: 10.1371/journal.pone.0150038.
PDF or HTML at Wiley Online Library