# Presentation: 586059

## Testing Methodologies and Sampling

- Mary Lou Tortorello, Ph.D.
- Tong-Jen Fu, Ph. D.
- U. S. Food and Drug Administration
- National Center for Food Safety and Technology
- 6502 S. Archer Rd.
- Summit-Argo, IL  60501

## Overview

- Historical Perspective
- Rationale for the testing recommendations in the 1999 FDA guidance
- Looking Forward
- What has been learned since then?
- New issues to consider

## Why test?

- End-product testing of foods does not ensure their safety
- Low levels of microorganisms
- Sporadic contamination of units
- Sprouts are a unique food
- Natural microbial growth to high levels
- Microorganisms may be uniformly distributed throughout batch

## Pre-guidance questions on microbiological testing

- What should be tested?
- How and when should testing be done?

## What should be tested?

- 1998 FDA Public Meeting:
- Industry advocates the testing of spent irrigation water to indicate  the microbiological condition of the sprouts batch

## Difference in microbial counts between sprouts and water

**Total counts**

***E. coli *****O157 counts***** ***

**mean = 0.55 ********0.47**
**n = 11**

**mean = 0.62 ********0.67**
**n = 39**

**Notes:**

all data from sprouting jar studies
counts in water were lower than those in sprouts, were within 1 log of the counts from the sprouts for 70% of trials.  
Average difference in total counts comparable to average difference in O157:H7 counts;  water analysis may indicate presence of pathogens in sprouts.

## Detection of Salmonella in Sprouts and Water from Outbreak Seeds

- Detection of *Salmonella *in Sprouts and Water from Outbreak Seeds

**Notes:**
had seeds from 3 different outbreaks;  lot X, 4 boxes, lot Y - 4 boxes, lot Z 3 boxes.

Tested by Salmonella Assurance Gold EIA rapid test; and confirmation of colonies from enrichments.  

Confirmed:  sprouts 9/22 samples;  water 9/22 samples
Therefore, water analysis accurately predicted contamination status in every case.

EIA had a 3 false positives in sprouts, 1 false positive in water.  2 false negative in water, but adjustment of EIA cutoff value would have prevented them.

## Pre-guidance questions on microbiological testing

- What should be tested?
- How and when should testing be done?

## How to test:  requirements for guidance testing methods 

- Results 48 hr after sampling
- Validated method
*Salmonella*  Assurance Gold EIA ***** and VIP *****
*E. coli* 0157:H7  VIP *** **and Reveal

***** AOAC Official Method

## Testing of Water from Each Production Batch for :

***Salmonella***

**and**

***E. coli *****O157:H7**

- mBPW + ACV

- BPW + n

- RV / TT

- TSB + DNP +n

## If positive results, then:

** ****discard sprouts and seed;  disinfect **
**	****	****	****or**
** ****run confirmatory tests from enrichment**

**...****.Testing of Water from Each Production Batch**

## Detection of Pathogens in Inoculated Sprouts and Sprout Water by Rapid Test Kits

***Salmonella***

***E. coli***** O157:H7**

## Detection Limit of Rapid Test Kits 

- Detection Limit of Rapid Test Kits
- Without Enrichment

***E. coli *****O157:H7**

**Notes:**
Therefore, sprouters CANNOT run rapid tests on water without enrichment because pathogen may not be present at detectable levels
EHEC data shown; similar results for Salmonella test kits

## Growth of Salmonella During Sprouting of 

- Growth of Salmonella During Sprouting of
- Outbreak Seeds

**Lot A**

**Lot B**

**Notes:**
pathogen at max level by day 1 - 2 (48 h).

## Detection of Salmonella in Sprouts and Water from Outbreak Seeds

- Detection of *Salmonella* in Sprouts and Water from Outbreak Seeds

**Notes:**

Agreement in results of sprout and water testing by EIA.
EIA showed pathogen by day 1 of sprouting, even in batches that were negative on day 0. Improved detection by sprouting seeds, cannot always detect in unsprouted seeds.

## Microbiological testing:  validation of rapid tests

## Validation of Salmonella Detection by 

| Seed Lot C |  | Assurance Gold |  | VIP |  |  |  |  | BAM |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Serotype | Inoc CFU/ml | Positive | Confirmed | Positive |  |  | Confirmed |  | Confirmed |
| Cubana | 0.8 | 5/5 | 5/5 | 5/5 |  |  | 5/5 |  | 5/5 |
| Muenchen | 0.7 | 5/5 | 5/5 | 5/5 |  |  | 5/5 |  | 5/5 |
| Tennessee | 0.6 | 5/5 | 5/5 | 5/5 |  |  | 5/5 |  | 5/5 |
| No inoc | 0 | 0/1 | 0/1 | 0/1 |  |  | 0/1 |  | 0/1 |
|  |  |  |  |  |  |  |  |  |  |
| Totals for 4 seed lots | 0.6-3.6 | 66/66 | 66/66 |  | 66/66 | 66/66 |  | 49/60 |  |

- Validation of *Salmonella* Detection by
- Rapid Tests Assurance Gold*a* and VIP*b*

*a* AOAC Official Method 999.08
*b* AOAC Official Method 999.09

## Selected AOAC Performance Tested Methods

| Test Kit Name | Manufacturer | Cert. Date | Analyte | Matrix |
| --- | --- | --- | --- | --- |
| Gene Trak Salmonella DLP | Neogen | 1996 | Salmonella | Food, animal feed, environmental samples |
| RapidChek Salmonella | Strategic Diagnostics | 2003 | Salmonella | Variety of Foods |
| BAX PCR Assay for Screening E. coli O157:H7 | Dupont Qualicon | 2004 | E. Coli O157:H7 | Apple cider, orange juice, ground beef |
| Pathigen E. coli O157 test | IGEN  | 2003 | E. Coli O157 | Raw ground beef |

## Detection Limit of Rapid Test Kits for Salmonella in Buffer

- Gold EIA – AOAC Official Method
- BAX – unclassified
- GENE-TRAK – AOAC Performance Tested Method

## Detection of Salmonella in Sprouts and Water from Outbreak Seeds by Rapid Test Kits and BAM

*a  *3 false negatives
*b  *5 false positives
*c  *23 false negatives

**Notes:**
Samples for each outbreak lot:  10 sprouts and 10 corresponding waters; sampled, enriched according to manfacturer’s instructions, then ran assay
BAM false negatives:   2 sprouts, 1 water
GOLD EIA:  of 5 false pos, 3 were sprouts

Based on these limited studies, sprouts may be more problematic than water for detection

BAX:  overnite enrichment in lactose broth plus 3 h post enrichment in BHI broth - all non-selective;  probably less successful than other extensive enrichment protocols for increasing Salmonella against high microbial load in sprouts.  Might not normally be a problem, except that PCR inhibitors (not diluted out in short enrichment scheme) also might limit efficiency of reaction.

## Publications on Pathogen Detection Methods in Sprouts

## Method Considerations

- Has the method been tested for sprouts / water?
- Has the entire method been tested against a standard method:
- enrichment – detection - confirmation
- Has the method been evaluated or *validated?*
* *

## Methodologies and SamplingLooking Forward 

## Sampling and Testing: New Issues

- Sampling/testing during sprouting
- Sampling plan
- When to sample
- 24 h vs. 48 h
- How to sample
- 1 L per batch vs. pooling
- Considerations for mung bean sprouts
- Sampling/testing of seeds
- Sampling plan
- How to sample
- Testing
- Testing seeds vs. testing sprouts
- Detection methodologies
- Additional rapid methods are available

## Sampling and Microbial Testing of  Spent Irrigation Water 

## When to Sample: 24 h vs. 48 h 

- To allow earlier determination of presence/absence of pathogens in sprouts
- Additional growth studies have been conducted since 1999

## Growth of pathogens during sprouting of alfalfa seeds

| Apparatus  | Pathogen | 0 h (log CFU/g) | 24 h (log CFU/g) | 48 h (log CFU/g) |
| --- | --- | --- | --- | --- |
| Naturally contaminated seeds |  |  |  |  |
| Mini drum  | Salmonella | -1.5 | 2.0 | 2.0 |
| Mini drum | Salmonella  | -2.4 / -1.5  | -0.6 / 0.5  | -1 / 1.8  |
| Glass jars | Salmonella  | -1 | 1.4 | 2.3 |
| Glass jars | Salmonella  | -1.6 | 2.6 | 3.1 |
| Glass jars | Salmonella | -1.2 | 1 | 2.5 |
| Glass jars | Salmonella | < detection limit | 1.7 to 2.2 | 2 to 4.5 |
| Artificially inoculated seeds |  |  |  |  |
| Plastic box | Salmonella | 3.2 | 6 | 7 |
| Tray | Salmonella | 3.4 | 5 | 5.4 |
| Test tube | Salmonella | 1.8 | 4.7 | 6.3 |
| Easy GreenTM | Salmonella | 0 | 5.7 | 5.7 |
| Screen tray | Salmonella  | 3.6 | 6.9 | 7.9 |
| Plastic box | E. coli O157:H7 | 3.1 | 5.0 | 4.8 |
| Tray | E. coli O157:H7 | 3.1 | 5.7 | 5.7 |
| Glass jars | E. coli O157:H7 | 3.9 / 1.9  | 6 / 4.5  | 5.5 / 5.3  |
| Test tube | E. coli O157:H7 | 2.7 | 3.8 | 4.3 |

**Growth of pathogens during sprouting of alfalfa seeds**

## Summary of Literature DataPathogen growth at 24 h and 48 h of sprouting

|  | Increase at 24 h (log CFU/g) | Increase at  48 h (log CFU/g) | Log increase at 24 h Log increase at 48 h |
| --- | --- | --- | --- |
| Naturally contaminated seeds | 1.8 - 4.2 | 1.4 - 4.7 | 0.85 ± 0.27 |
| Artificially inoculated seeds | 1.1 - 5.7 | 1.6 - 5.7 | 0.88 ± 0.22 |

## How to Sample

- 1999 FDA guidance
- Recommended 1 L of spent irrigation water be collected per production batch
- Pooling was not advised due to concerns of sample dilution
- Microbial counts in mung bean spent irrigation water may be much lower than those of bean sprouts
- Sample pre-concentration
- Allow concentration of mung bean sprout water
- Allow pooled samples to be tested with a single test

## Sample Pre-ConcentrationStandard Membrane Filtration 

**Sample Pre-Concentration****Standard ****Membrane Filt****ration**** **

- Vacuum

- Incubate on selective media at 37oC for 24 h

## Tangential Flow Filtration System

| Volume processed | Up to 10 L  |
| --- | --- |
| Time required | < 2 hours |
| Volume reduction | 100 fold  (10 L – 100 mL) |
| Methods that can be used for analysis | Culture methods, dipstick tests, ELISA, biosensors |

**T****angential Flow Filtration System**

- Determine whether the increase in background microflora in the concentrated samples affects the enrichment and subsequent detection of pathogens by conventional and/or commercially available rapid methods.
- Determine the lowest level of pathogens that the system can recover.

**Evaluation of TFF for ****Concentration of Pathogens**

## Validation of tangential flow filtration as part of sample treatment for detection of Salmonella in spent sprout irrigation water

## Recovery of Salmonella from the TFF system  

**Recovery of *****Salmonella *****from the TFF system  **

## Validation of the TFF system: E. coli 0157:H7 

| Seedlot | Total Counts (CFU/ml) | Inoculum (CFU/ml) | Enrichment media: Reveal 20 h media |  |  | Enrichment media: mBPW+acv  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  | device | confirmed | confirmed with IMS | device | confirmed | confirmed with IMS |
| A | 1.8–6.5 x 107 | 0 | 0/10 | 0/10 | - | 0/10  | 0/10 | - |
|  |  | 1.0 – 2.4 | 5/20 | 5/20 | - | 3/20  | 5/20 | - |
|  |  | 10 – 24 | 20/20 | 14/20 | - | 7/20  | 9/20 | - |
|  |  | 100 – 240 | 20/20 | 20/20 | - | 17/20 | 19/20 | - |
|  |  |  |  |  |  |  |  |  |
| B | 1.3–9.3 x 107 | 0 | 0/10 | 0/10 | - | 0/10  | 0/10 | - |
|  |  | 1.3 – 1.7 | 3/20 | 7/20 | - | 0/20  | 0/20 | - |
|  |  | 13 – 17 | 20/20 | 15/20 | - | 0/20  | 3/20 | - |
|  |  | 130 – 170 | 19/19 | 19/19 | - | 20/20 | 13/20 | - |
|  |  |  |  |  |  |  |  |  |
| C | 1.3–10.7 x 107 | 0 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 |
|  |  | 1.0 - 2.6 | 1/20 | 2/20 | 11/20 | 1/20 | 0/20 | 10/20 |
|  |  | 10 - 26 | 20/20 | 15/20 | 20/20 | 3/20 | 6/20 | 18/20 |
|  |  | 100- 260 | 20/20 | 20/20 | 20/20 | 17/20 | 9/20 | 20/20 |

## Recovery of E. coli O157:H7 from the TFF system  

**Recovery of *****E. coli *****O157:H7 ****from the TFF system****  **

## Seed Sampling and Testing 

- Outbreak investigation
- Screening seeds used for sprout production

## Isolation of pathogens from outbreak associated sprouts/seeds

| Pathogen | Year | Location | Type of Sprout | No. of Cases | Isolated from Sprouts/Seeds |
| --- | --- | --- | --- | --- | --- |
| E. coli O157:NM | 1998 | CA | Alfalfa/Clover | 8 | Yes/No |
| S.  Bovismorbificans | 1994 | Sweden, Finland | Alfalfa | 595 | Yes/No |
| S. Enteritidis | 2000 | CA | Mung | 45 | No/No |
| S. Gold-Coast | 1989 | U.K. | Cress | 31 | Yes/No |
| S. Havana | 1998 | CA, AZ | Alfalfa | 18 | No/Yes |
| S. Havana/Cubana/ Tenn. | 1998 | CA | Alfalfa | 34 | Yes/Yes |
| S. Mbandaka | 1999 | OR,CA, ID,WA | Alfalfa | ~ 68 | Yes/Yes |
| S. Montevideo/ Meleagridis | 1996 | CA | Alfalfa | >500 | Yes/No |
| S. Newport | 1995 | Denmark | Alfalfa | 154 | Yes/Yes |
| S. Newport | 1995-96 | OR, Canada, Denmark | Alfalfa | 133 | Yes/Yes |
| S. Saint-Paul  | 1988 | U.K. | Mung | 143 | Yes/Yes |
| S. Senftenberg | 1997-98 | CA | Alfalfa/Clover  | 52 | Yes/No |
| S. Stanley | 1995 | US, Canada, Finland | Alfalfa | >272 | No/No |

**Isolation of pathogens from outbreak associated sprouts/seeds**

- IFT/FDA Report, 2001

## Detection of Salmonella in Naturally Contaminated Seeds (Inami et al., 2001) 

- Sampling Plan
- Sample size: 500 g
- Sample unit: 100 g
- 60 sample units per lot were analyzed
- Testing methodologies
- Sample processing
- Shredding vs. Sprouting
- Detection
- BAM

## What to Test: Seeds vs. SproutsInami et al., 2001

- Shredding
- 100 g dry seeds blended for 2 min
- Shredded seeds mixed with 900 ml lactose broth
- Followed BAM for sample pre-enrichment, enrichment and confirmation
- Sprouting
- 100 g seeds soaked in 200 ml sterile water for 18-20 h at 25oC.
- Sprouted for 3 days at room temperature
- 100 g germinated sprouts mixed with 900 ml lactose broth and blended for 2 min
- Followed BAM for sample pre-enrichment, enrichment and confirmation

|  | positive / total analyzed |  | # positive shredding                     # positive sprouting  |
| --- | --- | --- | --- |
| Seed lot | Sprouting  | Shredding  |  |
| A | 3/30 | 2/30 | 75% |
| B | 10/30 | 9/30 | 90% |
| C | 27/30 | 24/30 | 89% |

## What to Test: Seeds vs. SproutsFu et al., 2005 (unpublished)

|  | # positive sample
# sample analyzed |  | 
# positive on Day 0                     # positive on Day 2  |
| --- | --- | --- | --- |
|  | Soak water | Day 2 water |  |
| Seedlot CoA98
1999 outbreaks in WI  | 12/18  | 16/18 | 75% |
| Seedlot 36120 
1998 outbreaks in CA  | 2/18 | 5/18 | 40% |
| Seedlot 45197
1999 outbreaks in OR, WA | 2/24   | 6/24 | 33% |

**What to Test: Seeds vs. Sprouts****Fu et al., 2005 (unpublished)**

- Soak water vs. Day 2 water
- 100 g seeds soaked in 500 ml sterile tap water for 3 h. Soak water collected for analysis.
- Soaked seeds were sprouted in glass jars at 25 oC and rinsed with 500 ml sterile water daily. Day 2 water collected for analysis.

## Effect of Sampling Size

|  | Sample size | Soak water 
(# positives / # analyzed)                         |
| --- | --- | --- |
| Seedlot CoA98
1999 outbreaks in WI | 100 g | 12/18 |
|  | 1 kg | 5/5   |
| Seedlot 45197
1999 outbreaks in OR, WA | 100 g | 2/24 |
|  | 1 kg | 5/5 |

## Screening Seeds Used for Sprout Production:Industry Practice

- Six-step procedure developed by ISS and Jonathan’s Sprouts
- Seed sampling
- 25 g subsamples from each bag . At least 3 kg per seed lot
- Seed inspection
- Sprout growing
- Follow normal sprouting procedures
- Spent irrigation water sampling
- 48 h
- Enrichment of sampled water
- Pathogen testing
- Prevented at least one potential outbreak of *E.coli* 0157:H7 and prevented shipment of contaminated seeds to sprouters.

## Detection Methodologies

-   Validation performed in sprout system is critical

## Automated Immunomagnetic Separation System

- AOAC performance certified for detection of* E. coli *O157:H7 and *Salmonella*
- Capable of isolating a single pathogen from 25 g of sample

- The Pathatrix System
- (Matrix MicroScience, CO)

## Evaluation of the Pathatrix IMS system for isolation of 

***E. coli***** O157:H7 from spent irrigation water**

| 
  | Pathatrix protocol 
BPW @ 37 °C w/ shaking |  | Modified protocol
mBPW+ACV @ 37C w/ shaking |  | Modified protocol 
Reveal 8h @ 42°C w/out shaking |  |
| --- | --- | --- | --- | --- | --- | --- |
| Inoc. level (CFU/sample) | Unconc.  | TFF-conc | Unconc.  | TFF-conc | Unconc.  | TFF-conc |
| 0 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| 1 | 0/3 | 0/3 | 2/3 | 0/3 | 3/3 | 3/3 |
| 10 | 0/3 | 0/3 | 3/3 | 0/3 | 3/3 | 3/3 |
| 100 | 0/3 | 0/3 | 3/3 | 2/3 | 3/3 | 3/3 |

**Sample size: 25 ml****; TFF samples ****correspond to 2.5 L of unconcentrated sprout water**

## Evaluation of the Pathatrix IMS system for isolation of 

| 
  | Pathatrix protocol 
BPW at 30°C, plated on XLD/HE/BS |  | Modified protocol 
TT at 42°C, plated on XLD/HE/BS |  |
| --- | --- | --- | --- | --- |
| Inoc. level (CFU/sample) | Unconc.  | TFF-conc | Unconc.  | TFF-conc |
| 0 | 0/3 | 0/3 | 0/3 | 0/3 |
| 1 | 0/3 | 0/3 | 3/3 | 3/3 |
| 10 | 2/3 | 1/3 | 3/3 | 3/3 |
| 100 | 2/3 | 3/3 | 3/3 | 3/3 |

**Evaluation of the Pathatrix IMS system for isolation of **
***Salmonella *****from spent irrigation water**

## Real Time Pathogen Monitoring Fiber-Optic Biosensor

- Analyte 2000

- RAPTOR

## Literature Data

- Analyte 2000 was shown to be able to detect the presence of *E. coli* O157:H7 in ground beef extract at a level of 3-30 CFU/ml  (DeMarco et al., 1999)
- RAPTOR was shown to be able to detect the presence of *Salmonella* in spent irrigation water collected at 67 h of sprouting from seeds inoculated with 50 CFU/g of the pathogen (Kramer et al., 2004).

## Evaluation of the Analyte 2000 Fiber-Optic Biosensor for Detection of E. coli O157:H7 in Spent Sprout Irrigation Water

## Conclusions

- A great deal of information has been accumulated since 1999,  some of which may help to improve the efficacy as well as the compliance of microbiological testing of sprouts.
- Sample pre-concentration by tangential flow filtration would allow 10L of spent irrigation water to be tested with a single test and achieve a detection limit of 1 - 10 CFU/10 L.
- Seed screening provides additional control to prevent sprout associated outbreaks. It also provides additional protection for consumers who produce sprouts at home.
- Testing sprouts/spent irrigation water appears to provide better detection than testing seeds.
- Many new detection methods have been developed.  Evaluation of their efficacy in sprout system is critical.

## Acknowledgements 

**Historical overview**
- Joseph Schlesser, Ph.D., NCFST/FDA
- Diane Stewart, NCFST/FDA
- Karl Reineke, NCFST/FDA
- Jodie Ulaszek, NCFST/IIT
**Validation of the TFF system for sample pre-concentration**
- Karl Reineke, NCFST/FDA
- Olive VanPelt, NCFST/IIT
- Thomas Krupinski, NCFST/IIT
**Evaluation of the automated IMS system**
- Peter Slade, Ph.D., NCFST/IIT
- Nicole Maks, NCFST/IIT
- Brian Parisi, NCFST/IIT
**Evaluation of the fiber optic biosensor **
- Kay Lin, NCFST/IIT
**Seed Sampling **
- Karl Reineke, NCFST/FDA
- Olive Van Pelt, NCFST/IIT
- Thomas Krupinski, NCFST/IIT