Flow cytometric based method to detect E. coli O157 in spinach

The Bacteriological Analytical Manual (BAM) method used by the US Food and Drug Administration (FDA) to detect E. coli O157:H7 in spinach, which takes 51 hours to result, was compared to a new flow cytometry based one.

The latest FDA BAM chapter for detection of diarrheagenic E. coli in food (BAM 4a) specifies use of PCR, although earlier methods based on selective media and colony morphology are allowed when PCR instrumentation is not available.

Previous research suggests Enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells but FDA says the dose is unknown.

Flow cytometry method​

The study characterized and validated a new flow cytometry (FC) method developed by the FDA National Center for Toxicological Center (NCTR) for detection of E. coli O157:H7 in 25g samples of spinach compared to the reference Q-PCR method in the BAM Chapter 4a.

“The NCTR flow cytometry-based system was able to detect as few as 1 cell per 100 μL in a 25g amount of spinach and process 20 samples in about nine hours,” ​said the researchers.

“These times suggest use of the system in factory/shift work schedules that would allow for screening of many food samples in a two shift, 16-hour period. The time savings and higher sample monitoring throughput would help to decrease the number of cases of illness resulting from ingestion of contaminated spinach by the food pathogen E. coli O157:H7.”​

Flow cytometry detects individual particles, distinguishing target cells from miscellaneous particles or non-target bacteria based on combination of light signals scattered from or emitted by each cell.

Other methods are based on bioluminescence, immunology (e.g., enzyme-linked immunosorbent assay ELISA), fluorescent in situ hybridization (FISH), and quantitative real-time polymerase chain reaction (Q-PCR).

For an external laboratory level 2 validation study there is a need for 20 samples each for the standard and experimental methods, one inoculated and one non-inoculated, making 40 and for aging of the food matrix (fresh spinach in this study) being validated.

Spinach samples went through several processing steps before being analyzed on the flow cytometer including pulsification to suspend the bacteria in liquid media, photobleaching, large volume centrifugation for concentration of cells with spinach debris, filtration and density gradient centrifugation for spinach debris elimination.

False positives and negatives​

Neither BAM 4a nor the flow cytometry methods reported false positives. BAM 4a reported five positives as negatives and the FC method reported four.

In both BAM 4a and the FC method, false negative results are potentially explained by the low inoculation level: i.e., 100 μL of the nominally positive spiking solutions actually contained no target cells, resulting in a negative result, said the researchers.

The low level positive inoculation was chosen to demonstrate the detection limit for the flow cytometry method, in which 2.0 ± 2.6 viable cells/25 g of spinach could be detected in 25–75% of the inoculated samples.

Optimal growth media and enrichment temperatures for the FC method differed from those of the regulatory method, according to the study.

“This affected experimental design eliminating possible use of samples split after enrichment, necessitating the use of parallel samples,” ​said the researchers. ​ ​

“Since the NCTR and regulatory method were being compared based on parallel samples, comparison of sensitivity was possible only by recovery statistics. That is, the number of samples correctly identified as positive by the two methods were compared out of 10 positive samples per method, with each sample likely to contain at least one CFU E. coli O157:H7.”​

Source: International Journal of Food Microbiology, volume 215, 23 December 2015 pages 1-6

Online ahead of print, DOI: 10.1016/j.ijfoodmicro.2015.08.011​

“Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach”​

Authors: Anna J. Williams, Willie M. Cooper, Christine V. Summage-West, Lillie M. Sims, Robert Woodruff, Jessica Christman, Ted J. Moskal, Shawn Ramsaroop, John B. Sutherland,​Pierre Alusta, Jon G. Wilkes, Dan A. Buzatu