Post on 30-Apr-2020
Microbiological analysis of food samples - routine and case-linked Investigations
V. Sandalakis School of Medicine University of Crete
Department of Clinical Microbiology and Microbial Pathogenesis - CMMP
Unit of Food, Water and Environmental Microbiology
Unit of Food, Water and
Environmental Microbiology
Unit of Food, Water & Environmental Microbiology
Scientific Supervisor Asst Prof. Anna Psaroulaki
Unit of Food, Water and
Environmental Microbiology
• Established in 1998
• The Unit is an ISO-17025 accredited laboratory.
• ISO procedures and other methodologies for the detection and enumeration of several categories of food and water microbes (Salmonella spp, Listeria spp, E.coli (including STEC strains), Staphylococcus coag +ve etc, Legionella spp etc).
• The laboratory has contributed in more than 20 European and National research programs and has collaborators in Greece, Europe and USA.
• It is an EFSA Focal Point on Hygiene and Food Safety (decision of EFSA 2007057 / 12-3-2007).
Unit of Food, Water, and Environmental Microbiology
Unit of Food, Water and
Environmental Microbiology
Food, Water end Environmental Microbiology
Sample type Test type Test
Human consumption
water samples,
Environmental water
samples,
Bottled water
samples
Counting of microorganisms- colony counting at
36°and 22°C Integration on solid broth
Isolation and counting of Coliforms.
Membrane filtration (MF) and isolation
Isolation and counting of E. coli.
Isolation and counting of Pseudomonas aeruginosa
Isolation and counting of Enterococcae
Isolation and counting of Clostridium perfringens
(spores included)
Isolation and counting of Legionella spp and Legionella
pneumophilla
Food samples
Salmonella spp. detection
Enrichments and isolation
Campylobacter spp detection
Pathogenic Staphylococcae , coaggulase positive,
detection, counting
Detection of Listeria monocytogenes and Listeria spp
Total coliforms counting
Isolation and counting of B- glucuronidase, E coli using
chromogenic media
Counting of Clostridium perfr.
Isolation and counting of Enterobacter sakazakii
Legionella sp
Culture Culture on axenic media
Genotypic detection PCR, Real-Time PCR
Typing MIC on axenic media
Legionella pneumophila
Antibody detection IFA (IgM,IgG)
Serotyping Serotyping
Direct pathogen detection DFA
Antigen production Isolation and cell culture
Antibiotic susceptibility testing MIC on axenic or liquid media
Culture Culture on axenic media
Typing PCR, Real-Time PCR
PCR, MLST, sequencing, SBT
Unit of Food, Water and
Environmental Microbiology
Increasing tendency
• For 2018: – Food Samples vary between 50 to 200 on a weekly basis
– Water Samples vary between 30 to 250 on a weekly basis
– Surface Samples vary between 20 to 50 on a weekly basis
Throughput
2015 2016 2017 2018
Unit of Food, Water and
Environmental Microbiology
• The presented project
– focusing -but NOT limiting to- foodborne pathogens analysis in various food samples
Unit of Food, Water and
Environmental Microbiology
• Unit of Food, Water and Environmental Microbiology
• Unit of Zoonoses and Geographical Medicine
• Unit of Rickettsioses & tick-borne Infectious Diseases
• Unit of Bacteriology
• Unit of Parasitology and Medical Entomology
• Unit of Mycology
• Unit of Pathogenesis and Infectious Diseases Immunology
• Microbiome Unit
• Regional laboratory of Public Health-part of the Hellenic Center for Infectious Diseases Control & Prevention network
We deal with a wide range of pathogens using a variety of methods
Department of Clinical Microbiology and Microbial Pathogenesis - CMMP
Unit of Food, Water and
Environmental Microbiology
It becomes apparent that:
• One of our major fields is Public Health
• What’s the impact of every new technology in relation to Public Health?
• New technologies should serve the concept of One-Health
Department of Clinical Microbiology and Microbial Pathogenesis - CMMP
Unit of Food, Water and
Environmental Microbiology
• Routine control samples
– Hospitals
– Hotels
– Food producing Industries (milk industries included)
– Fast-food and catering units
– Bottling companies
– Intermediate suppliers
– Packing companies
• Food and water Shelf-life analysis
• Research protocols
• Investigations – after results that do not follow the legislation guidelines
– related to Human-cases
Focusing on Food, Water & Environmental samples
Unit of Food, Water and
Environmental Microbiology
• Speed is always crucial but…
• In human case linked incidents … it is essential.
– Can I have my results now?
– Can I have my results within a few hours?
– …at least within the day…?
– How analytical can you get?
Speed
• What can I do?
How fast….is fast enough? How do I NOT compromise accuracy
Unit of Food, Water and
Environmental Microbiology
• Follow the SOPs as described by the related International Standard
• Escherichia coli (ISO 16649)
• Staphylococcus (coag+) (ISO 6888)
• Listeria monocytogenes (ISO 11290)
• Salmonella spp (ISO 6579)
• Escherichia coli / Coliforms (ISO 9308)
• Pseudomonas aeruginosa (ISO 16266)
• Legionella spp (ISO 11731)
• ……….. and so on…..
ISO 17025
I do not compromise accuracy
What about speed?
Unit of Food, Water and
Environmental Microbiology
8.2.2 No treatment/
HCl, Heat treatment
8.2.2 Directly on agar
No Treatment
HCl 50±1oC 30 ±2min 5±0.5min
BCYE & BCYE-cys or Nutrient agar or Blood Agar or TSA
BCYE & BCYE+AB or GVPC or MWY
Suspected colonies
ISO 11731:2017 Annex G
Is the end point
enough?
Up to 10-12 days
Unit of Food, Water and
Environmental Microbiology
L.monocytogenes
Salmonella spp
E.coli Coliforms
E.coli O157:H7 Yeasts/Moulds
h-FR
BPW
Is the end point
enough?
Min of 5 days for –ve
sample
Unit of Food, Water and
Environmental Microbiology
Hypothetical (?) Scenario
Source of infection??
I need to look back approx 12-72 hours
Unit of Food, Water and
Environmental Microbiology
M. 20:00
Tu. 08:00
Tu. 13:00 Tu. 17:00
Tu. 20:30 W. 07:30
W. 12:30
W. 16:30
W. 21:30 Th. 14:30 Th. 08:00
Th. 20:00
How fast is fast enough?
Unit of Food, Water and
Environmental Microbiology
1. Diagnosis in Hospital…fill in Questionnaires
2. Report the incident
3. Inform local authorities
4. Health inspectors - Epidemiologist Questionnaires + search for other human incidents in area hospitals
5. Determine food sources
6. Food sampling
7. Send samples to the Lab
8. Analysis of food samples
9. Results in 5-7 days
10. Compare Food isolate to the clinical isolated strain
11. Match …or No Match
The route of Information
Mobile unit
Results within hours
D A Y S
Unit of Food, Water and
Environmental Microbiology
Speed
Accuracy
Trace-back
Cost-effective
High-throughput
No Restrictions to the type of sample
What we need
Unit of Food, Water and
Environmental Microbiology
Speed Minimum steps procedure
Non specialized personnel
Results within the day ?
Portable instrumentation
Accuracy Sensitivity-Specificity
No cross-reactions
No Cross-contaminations (“safe” SOP)
A non-specialized analyst should be able to evaluate the results
Wish list!
Unit of Food, Water and
Environmental Microbiology
Trace-back A “secure” level of typing would be very
useful
• https://www.compare-europe.eu/Library/Reference-Genomes/Salmonella-enterica
Unit of Food, Water and
Environmental Microbiology
Unit of Food, Water and
Environmental Microbiology
Cost-effective Do I have to comment on this?
High-throughput Many samples – few Analysts
Sample pooling?
No Restrictions to the type of sample
Wish list continued!
Unit of Food, Water and
Environmental Microbiology
Salmonella
Salmonella
Salmonella
Unit of Food, Water and
Environmental Microbiology