Microbiological Testing of Foods Investigating outbreaks of - PDF document

Importance of detecting microorganisms in food Microbiological Testing of Foods � Investigating outbreaks of foodborne disease � Assessing the safety of the product to consumers. Mehrdad Tajkarimi � Assessing the stability or shelf life of the product under DVM PhD normal storage conditions. University of California-Davis � Determining the level of sanitation during product preparation. VMPHR 250 � Regulatory compliance � Incidence surveys for pathogens Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Bacterial pathogens Indicator or spoilage microorganisms � E. coli O157:H7 � Aerobic/anaerobic plate counts � Salmonella � Coliforms � L. monocytogenes � E. coli , yeast & mold counts � S. aureus � Psychrotrophs Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Toxins and microbial metabolites Bacteriological detection methods Direct enumeration( Microscopic count) � Bacillus cereus enterotoxin (Colony Forming Unit (CFU) count) � Clostridium perfringens toxin � Non-selective media � E. coli O157:H7 enterotoxin � Non-selective differential media � Staphylococcal enterotoxin � Selective media � Aflatoxins and Fumonisin � Selective differential media Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis 1

Bacteriological detection methods Bacteriological detection methods Indirect Determination Pathogen Isolation � Most Probable Number Method (MPN) � Sample does or does not contain microorganism of interest � Enumeration of Injured Cells by Selective � Pre-enrichment step Media Overlay Method � Selective enrichment step � Thin Agar Layer Method � Testing on medium containing selective and/or differential agents Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Testing for bacterial toxins Regulatory compliance testing � USDA-FSIS "Mega-Reg" Testing � Agglutination � Meat and poultry slaughter plant and raw � Radioimmunoassay (RIA) ground products processing facilities are � Enzyme Linked Immunosorbent assay (ELISA) required to test for generic E. coli and � Enzyme Linked Fluorescent Inmmunoassay Salmonella under the provisions of the HACCP (ELFA) program or Pathogen Reduction Final Rule. � Quantitative testing for generic E. coli � Qualitative testing for Salmonella Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Regulatory compliance testing Testing considerations � FDA: Seafood or other food products � Selection of sampling techniques � Examples include microbial analysis for spoilage � Selection of sampling kits microorganisms or pathogens in seafood or cheese. � Use of AOAC-approved methods State Dairy Testing � � Pasteurized Milk Ordinance (PMO) � These tests relate to the quality of various dairy products. � Microbial testing and analysis include coliform counts, standard plate counts (SPC). Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis 2

ISO 17025 Testing methods � Standard Methods for the Examination of Dairy Products � General Requirements for the Competence of Testing and � Standard Methods for the Examination of Water and Calibration Laboratories Wastewater � For international benchmark for approving the � Standard Methods for the Examination of Seawater and competence of the testing and calibration Shellfish � ISO 17025 allows laboratories to carry out procedures in � Compendium of Methods for the Microbiological their own ways, but an auditor may require the laboratory Examination of Food to justify using a particular method � Bacteriological Analytical Manual of Food and Drug � ISO/IEC 17025 is divided into two principal parts: Administration � Management requirements � Technical requirements Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis ISO 17025 Management requirements ISO 17025 Technical requirements include include paragraphs on paragraphs with much detail on � Organization and � General � Equipment � Complaints � Personnel management � Measurement trace ability � Control of non- Accommodation and � � Quality system conformity testing � Sampling environmental conditions � Document control � Corrective action � Handling and transportation of test � Test and calibration � Review of request methods including and calibration items � Preventive action sampling � Subcontracting of � Assuring the quality of test and � Records This includes requirements tests and calibrations calibration results for method validation � Internal audits (laboratory developed, � Purchasing services � Assuring the quality of test and � Management reviews non-standardized, and supplies calibration results standardized but used � Technical Requirements � Service to the client outside of their intended range) and measurement uncertainty Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Microbiological uncertainty Viruses and parasites — how are they “ different ” ? � Cannot multiply other than in specific, living host � It means a method used to estimate the uncertainty cells (rare exception with Giardia ) associated with model inputs, assumptions and structure/form � Cannot multiply in food (no toxins or other metabolites) — either remains infectious or not � Many microbiological laboratories have had procedures available for monitoring variability in � Cannot be enriched for testing duplicate results generated by laboratory analysts for � Usually, qualitative testing at the limit of sensitivity some time � Subjectivity problems � Studies and more complex statistical calculations Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis 3

Sensitivity = concentration method + Detection detection method � Viruses: susceptible hosts unavailable — “ molecular ” methods used � Concentration: start with serving-size sample of food or water? � Most viruses RNA only — reverse transcription (RT) required for PCR � Drinking water samples often 10 – 100 liters � Both RT and PCR are very susceptible to interference by substances in environmental samples; real-time PCR and nucleic acid sequence- � Solid food samples can't be concentrated — separate based amplification (NASBA) agent from food solids into liquid phase � PCR product analysis: gel electrophoresis; biosensors; verification; sequencing � Virus (~30 nm) concentration: adsorption-elution, � Protozoa: larger than bacteria, so microscopy is an option precipitation, or brute force � Staining, fluorescent or otherwise � Concentrating protozoan cysts-oocysts (4 – 20 μ m � Immunofluorescent techniques [larger than bacteria]): filtration, centrifugation (to PCR (multiple chromosomes ) bottom of tube or onto “ cushion ” ) � � Immunomagnetic capture Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Specificity absence of false positives Overview � Methods for microbiological testing of foods are � Detecting only the target organism limited by sampling — spoilage � What if a “ broad-spectrum ” test is wanted? � Detection of noninfectious (inactivated) agent = � Organisms and some indicators may be fairly false positive? homogeneously distributed, but pathogens are � False positives from noninfectious viruses — typically “ spotty ” in distribution and present at look for alternations in the virus that relatively low levels � Accompany inactivation (RNase sensitivity) � Because of distribution and sampling problems, � False positives from noninfectious protozoa — sensitivity (false negatives) and specificity excystation PCR for Cryptosporidium ; in vitro (false positives) present continuing challenges culture of Giardia Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis Overview Thank you ! � The key to detection of bacterial pathogens is usually enrichment (which is not an option with viruses and protozoa), detection and enumeration media may be selective, differential, both, or neither. � Bacterial toxins are usually detected by some adaptation of serology � With viruses and protozoa, sample processing and concentration, as well as a sensitive final detection method, are necessary to a satisfactory outcome, and problems of false positives with noninfectious contaminants remain. Mehrdad Tajkarimi DVM PhD Mehrdad Tajkarimi DVM PhD VMPHR250 UC Davis VMPHR250 UC Davis 4