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