Pathogen Reduction Dialogue Panel 3

Indicators/Surrogates vs. Pathogens Frank F. Busta

Professor Emeritus, University of Minnesota 7 May 2002 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

• •

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) 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Other Names • Model organisms • Sentinel organisms • Surrogate organisms

11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Indicators

• Specific microorganism – viable colony count, enrichment culture, indirect cell count • Metabolite – lactic acid titration • DNA fragment – PCR • Indirect measure – ATP 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Examples of Proposed or Adopted Indicator Organisms

•

Enterobacteriaceae

– Includes collectively to coliform, fecal coliform,

E. coli

• Coliform* • • Fecal Coliform*

Escherichia coli*

• • Enterococci

Bifidobacterium

• Coliphages 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Coliforms

• Defined by phenotype NOT genotype • G(-), asporogeneous rods, ferment lactose  & 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 ++--) acid 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Fecal Coliforms

• Coliforms ferment lactose  within 48h @ 44.5-45.5

° C acid & gas • 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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Indicator groups that may be or are considered pathogens

•

Enterobacteriaceae

– Includes collectively to coliform, fecal coliform,

E. coli

• Coliform* • Fecal Coliform* •

Escherichia coli*

• Enterococci 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Indicator in lieu of specific pathogen: Basic Criteria

• Similar survival & growth • Common source • Direct relationship between condition influencing pathogen presence & indicator • Practical methods 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Performance Standards

•

E. coli as an indicator/index?

•

Salmonellae as an indicator?

•

Enterobacteriaceae as an indicator/index?

11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Surrogate

11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Summary & Conclusions

11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Research Needs and Opportunities 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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. 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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) 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

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 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS

Research Needs and Opportunities 11 April 2000 INSTITUTE OF FOOD TECHNOLOGISTS