11 April 2000INSTITUTE OF FOOD TECHNOLOGISTS Pathogen Reduction Dialogue Panel 3 Indicators/Surrogates vs. Pathogens Frank F. Busta Professor Emeritus, University of Minnesota 7 May 2002

11 April 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Other Names Model organisms Sentinel organisms Surrogate organisms

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

11 April 2000INSTITUTE OF FOOD TECHNOLOGISTS Fecal Coliforms Coliforms ferment lactose  acid & gas within °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

11 April 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Performance Standards E. coli as an indicator/index? Salmonellae as an indicator? Enterobacteriaceae as an indicator/index?

11 April 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Surrogate

11 April 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Summary & Conclusions

11 April 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Research Needs and Opportunities

11 April 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE 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 2000INSTITUTE OF FOOD TECHNOLOGISTS Research Needs and Opportunities