# Presentation: 008345

## Pathogen Reduction DialoguePanel 3 Indicators/Surrogates  vs. Pathogens

***Frank F. Busta***
- Professor Emeritus, University of Minnesota
- 7 May 2002

- INSTITUTE OF FOOD TECHNOLOGISTS

- 11 April 2000

## Classic Definitions

**Index Organism** - microorganism or group of microorganisms that is indicative of a specific pathogen
**Indicator organism** - microorganism or group of microorganisms that are indicative that a food has been exposed to conditions that pose an increased risk that the food may be contaminated with  a pathogen or held under conditions conducive for pathogen growth
- 						(Buchanan 2000)

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## What is indicated

- Positive test for **indicator** organism
-  does NOT necessarily indicate the presence of pathogen
-  Detection of **index** organism
- points to the occurrence of a related pathogen
- A **marker** can function both as an index and an indicator organism

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## Other Names

- Model organisms
- Sentinel organisms
- Surrogate organisms

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## Some Preferred Qualities of Ideal Indicators

- History of presence or absence in foods ~ pathogen/or toxin
- Microbial metabolites present initially/or after growth ~ pathogen
- Growth of indicator = target microorganism under all conditions
- Easily detectable, quantifiable, distinguishable, preferably rapid

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## Indicators

- Specific microorganism
- viable colony count, enrichment culture, indirect cell count
- Metabolite
- lactic acid titration
- DNA fragment
- PCR
- Indirect measure
- ATP

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## Traditional Requirements for Indicator of Food Safety

- Easily and rapidly detectable
- Easily distinguishable from food flora
- History of association with pathogen
- Present with pathogen
- Numbers correlate with pathogen
- Growth requirements/rate equal to pathogen
- Die off rate parallels pathogen
- Absent from food free of pathogen

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## Examples of Proposed or Adopted Indicator Organisms

*Enterobacteriaceae*
- Includes collectively to coliform, fecal coliform, *E. coli*
- Coliform*
- Fecal Coliform*
*Escherichia coli**
- Enterococci
*Bifidobacterium*
- Coliphages

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## Enterobacteriaceae

- Facultative anaerobes, G(-) bacilli, mesophilic, ferment gluc  acid, cat +
- (some psychotroph, e.g. *Enterobacter*)
*Citrobacter, Enterobacter, Escherichia, Klebsiella,Salmonella*, and others
- Recommended over coliforms to better assess gluc+, lac- members of food flora
- (i.e. *Salmonella*)
- Problem: Not confined to intestinal tract

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## Coliforms

- Defined by phenotype NOT genotype
- G(-), asporogeneous rods, ferment lactose  acid & gas within 48h @ 35°C and produce dark colonies with a metallic sheen on Endo agar
- Consist of 4 genera:*Citrobacter,Enterobacter, Escherichia *and *Klebsiella*
*E. coli* most indicative of fecal pollution
*E. coli* typeI ( IMViC ++--)

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## Fecal Coliforms

- Coliforms ferment lactose  acid & gas within 48h @ 44.5-45.5°C
- Strains recovered: *E. coli*, *K. pneumoniae*, *Enterobacter* spp., *C. freundii*
- Originally used to assess fecal contamination in water
*E. coli* O157:H7 does not grow well at 44.5 °C

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## E. coli

- For use to determine sanitary significance: must comply with coliform & fecal coliform definitions
- Type I IMViC ++--
- Type II IMViC -+--
- Some strains are neither Type I or II

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## E. coli

- Regarded as most valuable indicator of fecal contamination of raw foods
- Not a reliable indicator of fecal contamination in processed foods
- Grows in environment
- Indicator of inadequate processing

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## Indicator groups that may be or are considered pathogens

*Enterobacteriaceae*
- Includes collectively to coliform, fecal coliform, *E. coli*
- Coliform*
- Fecal Coliform*
*Escherichia coli**
- Enterococci

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## Issues/Use of Coliforms & Fecal Coliforms

- May contain non-enteric members (e.g. *Serratia*, *Aeromonas*)
- Indicator of inadequate sanitation of equipment
- Indicator of inadequate heat-processing /post-pasteurization contamination of RTE foods
- Non-enteric fecal coliforms

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## Limitations of Pathogens as Indicator Organisms

- Concentrations may be very low and         	difficult to relate
- May not compete well with food flora
- Presence may not relate to another pathogen
- Presence may initiate regulatory action –              	may be considered adulteration
- Pathogens require special laboratory skills

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## Advantages Pathogens May Have as Indicator Organisms

- Easily and rapidly detectable
- Easily distinguishable from food flora
- Association with another pathogen
- Present concurrently with another pathogen
- Numbers correlate with another pathogen
- Growth requirements/rate equal to another  pathogen
- Die off rate parallels other pathogen
- Common source with other pathogen

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## Performance Standards

- Intended to effectuate decrease in pathogen with goal to improve public health
- Fecal contamination is major source of enteric pathogens
- May use microorganisms classified as indicator/index organisms
- Pathogen could be used if it meets criteria

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## Indicator in lieu of specific pathogen: Basic Criteria

- Similar survival & growth
- Common source
- Direct relationship between condition influencing pathogen presence & indicator
- Practical methods

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## Performance Standards

***E. coli***** as an indicator/index?**
**Salmonellae as an indicator?**
***Enterobacteriaceae**** ***as an indicator/index?**

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## Ideal/Index Indicator

- Present & rapidly detectable in foods of interest
- History of association/present with pathogen
- Presence and concentrations correlate with pathogen
- Easy to detect/enumerate,and distinguishable
- Growth requirements/rate, and die-off rate equal to pathogen
- Not affected by other food components or microflora
- Resistant to injury from stress of processing
- Non-hazardous to testing personnel

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## Current Status For Indicator/Index Microorganisms

- Indicator and Index organisms used for compliance with the GMPs, hygienic processing and handling of food
*E. coli* Biotype I and coliforms most common
- Stipulated in regulations (e.g.PMO, EPA drinking water standards)
- Vendor agreement for procuring ingredients and raw materials
- Quality assurance, Audits
- Specific pathogens and toxin assays available
- Usefulness of the concept being revisited

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## Surrogate

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## Surrogate

- Microorganism or representative material that serves as an alternate for target pathogen in studies evaluating or validating control or intervention processes such as chemical or physical decontamination procedures.
- Generally taxonomically, physiologically and ecologically related to pathogens or other target microorganisms

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## Surrogate Criteria - 1

- Non-pathogenic
- Inactivation characteristics those of target
- Durability similar to target
- Stable surrogate characteristics
- High concentrations easily prepared
- Stable between preparation and use

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## Surrogate Criteria - 2

- Easily enumerated
- Easily differentiated
- Inactivation kinetics consistent with target
- Genetically stable
- Will not establish as spoilage problem
- Resistant to sub-lethal injury or reversibility

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## Summary & Conclusions

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## Summary & Conclusions

- In use for over 100 years in some situations
- Effective with extensive validation and qualifications.
- Currently no well-established relationship of indicator with the occurrence of emerging water and foodborne pathogens
- Direct, sensitive and specific tests for detection and enumeration of target pathogens/metabolites are available
- Indirect association of markers with food safety and quality may not be reliable for “due diligence”
- May become increasingly useful with new analytical methods
- Challenge : selection and validation of appropriate indicator/surrogate

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## Research Needs and Opportunities

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## Research Needs and Opportunities

_**Identify indicators**_ to determine exposure to conditions permitting contamination or survival/growth of pathogen after  decontamination
_**Identify surrogate microorganisms**_ for use in specific situations to measure effectiveness of intervention decontamination treatments

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- 11 April 2000

## Research Needs and Opportunities

- Develop comprehensive _**standardized and validated protocols for use with surrogate**_ microorganisms in testing efficacy of pathogen control
- Propose, _**design, and test evaluation program(s**_) by systematically assessing possible sources of contamination, number of foodborne outbreaks attributed to the product/category, potential for mishandling, incidence data, and other quantifiable measures.

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## Research Needs and Opportunities

- Identify and _**validate approaches to test the elected indicator(s)**_ against wild and laboratory culture strains in well-controlled pilot plant environments and in open natural commercial conditions
- Develop _**appropriate analytical tools**_ for indicator(s)

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## Research Needs and Opportunities

- Collect _**new survey data with emerging molecular technologies**_ that accurately discriminate between virulent and non-virulent strains
- Identify or develop methodology to _**quantitatively retrieve indicators**_, especially when a stress may result in damaged or VNC organisms.
- Assess existing and new _**testing procedures and sampling plans**_ to verify appropriate stringency with stipulated statistical design

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## Research Needs and Opportunities

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- 11 April 2000