# Presentation: 020355

## Microbiological Findings on Sprout Operations Following FDA Guidance

**Microbiological Findings on Sprout Operations Following FDA Guidance**
**Dr. Jed W. Fahey***
**Johns Hopkins School of Medicine**
**To be presented at Public Meeting:  **
**2005 Sprout Safety **
**Harvey W. Wiley Federal Building Auditorium**
**5100 Paint Branch Parkway**
**College Park, MD  20740**
**Tuesday, May 17, 2005**
**8:30 am – 4:30 pm**
- * _[jfahey@jhmi.edu.](mailto:jfahey@jhmi.edu.)_

## Overview

**  ****Microbial contamination tests were performed on 6839 drums of sprouts, equivalent to about 5 million consumer packages of fresh green sprouts.**
**  ****Only 0.75% of the 3191 sprout samples gave an initial positive test for *****Escherichia coli***** O157:H7 or *****Salmonella***** sp., and when re-tested, 3 drums again tested positive.  **
**  ****Composite testing (e.g., pooling up to 7 drums for pathogen testing) was equally sensitive to single drum testing. **

***Overview***

## Disclosure

***Disclosure***

## Effect of leachate from sprouting broccoli seeds, on the growth of E. coli DH10B in dilute (10%) Luria broth, assessed by spectrophotometry of bacterial broth cultures after 5h of incubation at 37 ̊C.

## Antibiotic Potency of Sulforaphane

**M.I.C. (********g/ml)**

**48 strains of *****H. pylori***

**Antibiotic Potency of Sulforaphane**

**21**
**Bacteria**
**and**
**Fungi**

- Fahey et al., (2002) PNAS 99: 7610-7615

## Introduction

** ****placed in environmentally controlled, hydroponic conditions **
** ****incubated in warm, moist, nutrient-rich conditions [1.5 m. diameter, slowly rotating “drums” charged with ca. 20 kg of seed], which are ideal environments for microbial growth.  **
**  ****If *****Escherichia coli***** or *****Salmonella***** spp. are present on the surface of the seed, it is likely that they will multiply in the sprouting environment.  **

***Introduction***

## Introduction

**  ****They must therefore be prevented from entering the process, or if contamination occurs, affected final product must be identified and destroyed.   **

***Introduction***

## Introduction

**  ****The efficacy of such agents, most notably calcium hypochlorite, has been extensively documented in the laboratories of Beuchat and colleagues as well as others.   **
**  ****When these agents are used correctly, the resulting sprouts are safe to eat.**
**  ****A recommendation to use such a surface disinfection process is now part of a guidance that the FDA issued in 1999.**

***Introduction***

## Introduction

**  ****The ultimate control point for microbial safety is the product, thus incidents of contamination, when they occur, can only be ascertained by testing each lot of sprouts prior to releasing them for sale (hold-and-release testing).  This is best done by testing the spent irrigation water.   **

***Introduction***

## Introduction

**  ****The FDA followed up issuance of this guidance with an inspection of 150 sprouters, and determined that only about half of them were complying with the guidance.  **
**  ****We have examined a subset of sprouters in whose facilities compliance with the FDA guidance was verified based upon inspections by two third party auditors.**

***Introduction***

## Hold-and-Release Testing  

**13 producers or co-packers of broccoli (BroccoSprouts®) and other sprouts for the company Brassica Protection Products (Baltimore, MD, USA) during 2001.  **
**Established companies that grow many different kinds of green sprouts – all had previously agreed to comply with rigid standards of sanitation.  **
**Plant siting and product distribution geographically distributed around the U.S.  **

## Hold-and-Release Testing

**Surface-disinfect all seeds**
**Perform microbial testing of spent irrigation water from each batch of green sprouts produced.**
**Are subject to announced and unannounced third party audits and state and federal inspections of these procedures. **

***Hold-and-Release Testing***

## Seed treatment and sprout production

**Seeds exposed to 20,000 ppm of calcium hypochlorite for 15’, followed by extensive rinsing to remove residual chlorine.  **
**Sprouts grown in trays or drums **
**+ light (fluorescent, incandescent and/or filtered sunlight), **
**+ heat (constant temperature)**
**+ clean water (filtered, well, or municipal-chlorinated)**

## Seed treatment and sprout production

**A sample of the spent irrigation water, typically 1 L, is collected after 48 hours of sprout growth.  **
**Since the sprouts typically are grown for 72 – 120 h, a rapid microbial test permits the sprouters to abort contaminated batches of sprouts prior to packaging, or to shipping and distribution.  **
**All sprouts were held until test results were obtained.**

***Seed treatment and sprout production***

## Data collection 

**All growers directly forwarded their hold-and-release testing results for the calendar year 2001 to Quality Associates Incorporated (QAI; Columbia, MD, USA).**
**In addition to broccoli sprouts, the growers reported growing the following green sprouts: alfalfa, clover, radish, onion, pea, sunflower, and a variety of mixes.**
**Data that was exclusively derived from batches of bean sprouts (e.g. mungbean and soybean) were not used herein.  **
**In some cases pooled water samples that were tested included water from both green sprouts and bean sprouts, and these test results are included in the analyses. **

## Data collection 

**All growers but one used the services of an external microbiological testing laboratory, to which samples could be delivered on the day of collection.  **
**One grower had instead set up an in-house laboratory for these analyses and all laboratory notes were provided to us by this grower. **

## Microbial testing

**All samples were screened for *****Escherichia coli***** O157:H7 and for *****Salmonella***** spp.  **
**Most of the testing for *****E. coli***** O157:H7 (over 75%) was performed using a test kit identified in the FDA Guidance for Industry (*****1999*****).  **
**Only about 10% of the testing for Salmonella was performed using a test kit identified in this FDA Guidance.  **

## Microbial testing

**Most kits were Association of Official Analytical Chemists (AOAC) Official Methods; **
**One kit was an AOAC Performance Tested Method.  **
**One laboratory used the official FDA Bacterial Analytical Method (*****45*****) for *****Salmonella***** testing.  **
**Methods used by two laboratories (both for *****E. coli***** and *****Salmonella*****) and for a third (only for *****Salmonella*****) not specified.  **
**All results entered into a database -- data entries independently verified by an auditor at QAI.**

## Methods used for hold-and-release testing.

| Bacteria | AOAC No. | AOAC Status | Test kit name | Sensitivity (%) | Specificity (%) |
| --- | --- | --- | --- | --- | --- |
| E. coli 0157:H7 | 996.09 a | Official Method | Biocontrol VIP EHEC for E.coli O157:H7 | >98  | >99  |
|  | 996.10 b | Official Method | Biocontrol Assurance EIA EHEC | 100  | >98  |
|  | 2000.14 c | Official Method | Neogen Reveal E.coli O157:H7 | >89  | >98  |
| Salmonella spp. | 999.09 d | Official Method | Biocontrol VIP for Salmonella | >77  | >98  |
|  | 996.08 e | Official Method | bioMerieux VIDAS SLM | >96  | 100  |
|  | 960801 f | Performance
Tested Method | Neogen Reveal Salmonella | >96  | 100  |
|  | 992.11 g | Official Method | Biocontrol Assurance Gold EIA Salmonella | >79  | 100  |
|  | 989.14 h | Official Method | Tecra  Salmonella VIA | >70  | >78  |
|  | -- | -- | Bacteriological Analytical Method Chapter 541 | -- | -- |

*a* Reference (*16*) [Sensitivity and specificity reported in liquid milk and apple cider]
*b* Reference (*1*) [Sensitivity and specificity reported in liquid milk and apple cider]
*c* Reference (*31*) [Sensitivity and specificity reported in apple cider]
*d* Reference (*17*) [Sensitivity and specificity reported in liquid milk]
*e* Reference (*2*) [Sensitivity and specificity reported in milk chocolate]
*f* Reference (*31*) [Sensitivity and specificity reported in orange juice, lettuce rinse, sprout rinse, chicken rinse]
*g* Reference (1*8*) [Sensitivity and specificity reported in apple cider]
*h* Reference (*3*) [Sensitivity and specificity reported in pepper, soy flour, nonfat dry milk, raw ground poultry, chocolate]

## Distribution of number of drums composited into each sample for pathogen testing.

**Distribution of number of drums composited into each sample for pathogen testing.**

**Composite of:         No. samples ****	****     Percent *****a***

**Single drum ****	****1805****	****57**
**2-4 drums ****	****1171****	****37**
**5-7 drums ****	****166****	****5.2**
**8-19 drums ***** ******	*****49****	****1.5**

***a****** *****Expressed as a percent of the 3191 samples **
**for which the number of drums was known.**

## Presumptive positive samples grouped by composite number of drums represented by the water sample tested.

| No. of drums | No. of
presumptive positives | No. of
samples | Total No.
of drums | Presumptive positives
per drum (%) |
| --- | --- | --- | --- | --- |
| 1 | 10 | 1805 | 1805 | 0.554 |
| 2 | 4 | 259 | 518 | 0.772 |
| 3 | 4 | 558 | 1674 | 0.239 |
| 4 | 2 | 354 | 1416 | 0.141 |
| 5 | 2 | 69 | 345 | 0.580 |
| 6 | 0 | 63 | 378 | 0.000 |
| 7 | 2 | 34 | 238 | 0.840 |
| 8-19 | 0 | 49 | 465 | 0.000 |
| Overall | 24 | 3191 | 6839 | 0.351 |

*24 presumptive positives from 3191 samples (6839 drums) = 0.75%*

## Summary

**In most cases of presumptive positives, the water sample was then reanalyzed.  **
**In two of these cases, a follow-up analysis was again positive for *****Salmonella *****spp*****.*****, and the sprouts were destroyed.  **
**In a single case a presumptive detection of *****E. coli *****O157:H7 was assumed by the grower to be real without re-testing; the sprouts were destroyed.   **
**None of these 3 samples included broccoli sprouts.**
**Sprouts were held at the growing facilities until microbial testing results were confirmed.  **
**There were no instances in which contaminated sprouts were released for distribution. **

## ACKNOWLEDGEMENT

**This work was supported in part by generous gifts from the Lewis B. and Dorothy Cullman Foundation. **
**Many colleagues, in particular Philippe J. Ourisson****1****, Katherine K. Stephenson****2****, Douglas L. Archer****3**** & Frederick H. Degnan****4**** contributed to this work.   **
**Special thanks go to the audited sprout companies for permitting us access to their records and for their close cooperation. **
- 1Quality Associates, Inc., Columbia Maryland, USA
- 2Johns Hopkins University, School of Medicine, Department of Pharmacology and Molecular Sciences, Lewis B. and Dorothy Cullman Cancer Chemoprotection Center
- 3University of Florida, Food Science and Human Nutrition Department, Gainesville, FL, USA
- 4King and Spalding LLP, Washington D.C., USA.