SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN

FOOD ENVIRONMENTS

Christine Faille

International Biofilm SummitREALCO 24-25 march 2015

INRA, UMET UMR 8207Team « Interface Processes and Hygiene of Materials »

369 rue Jules Guesde, 59651 Villeneuve d’Ascq+33 (0)3 20 43 54 24

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What is a biofilm?

� Collection of microbial communities enclosed by a matrix of extracellular polymeric substance (EPS) and separated by a network of open water channels

� EPS : proteins, carbohydrates, DNA� Within  biofilms,  bacteria  in  ≠  physiological  states  (dead,  cultivable…)

� Different physiological conditions of the microorganism within soils� Viable and cultivable� Viable but non-cultivable� Dead / inactivatedÎ Underestimation of the bacterial load

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Physiological state of the microorganisms

� Presence  of  “injured”  bacteria  (stationary  phase,  stress…)=> Increased sensitivity to environmental conditions (components of the isolation medium / enumeration medium…)

� Colour indicator � Antibiotics� NaCl� Incubation T° (5°C < optimal temperature)

=> Length to recover (resuscitation process)�When bacteria are able to repair injuries => increased duration of the lag phase

.400,511,522,533,54

75 80 85 90 95Température de traitement (20 min)

-log(

1-%

endo

mm

agée

s)

Physiological state of the microorganisms

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Methods to control surface hygiene(to detect biofilms?)

Soil

Biochemical testsMicrobiological tests(viable and cultivable / VNC)

Contact methods

PlatesSlides

Petrifilms

ProteinsCarbohydrates

LipidsFood

EPS ATPNADPH

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Contact methods (microbiological)

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Principle: Adherent bacteria transferred by pressing the agaronto the contaminated surface

� Contact plates, RODAC* plates *Replicate Organism Direct Agar Contact

� PétrifilmTM

� Contact Agar Slides

Contact methods (microbiological)

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Contact methods (microbiological)

Many available growth media� Total mesophilic counts (PCA)� Staphylococcus (Baird Parker)� Coliforms (VRBL)� Faecal streptococci (Slanetz)� Yeasts & moulds� Listeria…+ neutralizers to deactivate residual disinfectants from the surface to be tested

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Contact methods (microbiological)

Contact plates (RODAC)� Surface slightly convex (meniscus of agar extending above the top of the poured plate)

=> contact with the surface to be sampled (ø 56 mm) � Reproducibility of pressure and application time => applicators=> Widely used=> Rather adapted for plane and smooth surfaces

Applicator (500 g, 10 s)

Food Stamp TGSE Agar Staph. aureus

Columbia Agar with Sheep Blood

Staph. / streptococci

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Contact methods (microbiological)PetrifilmTM 3M� Flexible film

• Medium in dry form• Medium protected by a polypropylene membrane

�Moisten the medium before use (30 min to 1-2 h)� Reproducibility of pressure and time => applicator to be adapted on flat surfaces=> Possible testing of curved surfaces (tanks, pipes, door  handles…)

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Contact methods (microbiological)

Incubator

Contact Agar Slides (=dipslides)� Hinged plastic paddle covered with culture medium on both faces (one/two media)� Ready to use�Mini-incubator, no applicator=> Widely used in the food industry=> Rather adapted for plane and smooth surfaces=> Poorly reproducible

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Contact methods (microbiological)

RODAC plates : successively applied to stainless steel coupons contaminated with bacterial spores

Partial recovery of adherent bacteria� Up to 60% spores not detached after the first contact � Great differences between strains

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MICROBIOLOGICAL METHODS• Very long (18 – 72 h)• Accessibility required

• Unable to detect food residues• Only able to detect cultivable microorganisms• Possible identification of the microorganisms

- - - - - - - - - -

ÎNot suited to check the effectiveness of cleaning procedures

Î Suited to check the effectiveness of disinfection procedures OR to validate routine methods

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Biochemical methods

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Biochemical methods

Sampling by swabbing followed by a direct dosage(colorimetric tests…)

� of proteins +/- reducing sugars� of carbohydrates� of biofilm EPS� of food soils� of ATP, NAD(H)

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Biochemical methodsProtein detection� CleanTrace (3M), Clean Test (LiofilChem), PRO-Clean (Hygiena)…

– Biuret test => detection of protein residues + reducing substances

– 1 to 10 min incubation– Detection limit: 50/60 µg on 100 cm² (BSA)– Test reading: visual / luminometer

=> Colour change easy to interpret=> Pro-Clean : very flexibleÎ sometimes, detachmentpoorly efficient (rough materials…)

Luminometer

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Biochemical methodsProtein detection� Clean-Trace Surface Protein (instant) [3M]

– Proteins => green colour– Instant reading of the “swab”

=> Very quick reading required=> Difficult to break up the “capsule” (to activate the reaction)

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Protein detection� Flash [Biocontrol]

– Press device tip into the hydration pad for 2-3 s (device tip color: from blueto yellow)– After swabbing, observation of the device tip (<10 s) (if proteins, colorfrom yellow to blue [Detection limit: 20 µg])

=> Reading sometimes ambiguous on biofilms=> Excessive pressure while swabbing may damage device membrane

Biochemical methods

- +

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Protein detection� Ridacheck (R-Biopharm), Pathcheck (Microgen)…

– Surface sampled = 20 cm²– Detection limit: 20 µg– Result produced in 5 sec to 2 min– Colour change from yellow to blue/green

=> Ease to use=> Colour change easy to interpret

Biochemical methods

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Biochemical methodsCarbohydrate detection� SpotCheck Plus [Hygiena]

– Detection of glucose, lactose– Result obtained in 60 s– Reagent turns green in the presence of either sugar residue– Detection limit: 2.5 μM D-glucose, 5.0 μM lactose

=> Useful for dairy plants, confectionary candy and beverage plants=> Unsuitable for biofilm detection

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Biochemical methodsEPS detection� Biofilm Detector Kit [REALCO]

– Detection of EPS produced by adherent bacteria– Use on stainless steel surfaces– Result obtained in 10-15 min (5 min reactive 1, rinse, 5 min reactive 2)

– Blue colour visible with naked eye in the presence of EPS

=> Direct visualisation of residual biofilm on surfaces=> Unsuitable on polymers

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Biochemical methodsDetection of organic food residues� Vericleen [Charm]

– Detection of organic matter– Surface must be wet(if dry surface => wet with >3 pumps of “wetting Liquid”)– Results obtained in 5-60 s– Development of a purple color in the presence of OM

=> Many false negative results with biofilms=> Unsuitable on rough materials (fragile membrane)

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Biochemical methodsDetection of NAD(P)/NAD(P)H (food/microorganisms)� SpeedCheck [Ecolab]

–Development of a blue colour in the presence of NAD– Results obtained in 5 min

=> Colour change easy to interpret=> Unsuitable on rough materials (fragile membrane )

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Biochemical methodsDetection of ATP (food / microorganisms)� Principle: enzymatic reaction to detect ATPATP found in all living cells (bacteria, animals, vegetables…) = energy source

ATP + Luciferine + O2

Luciferase + Mg

Oxyluciferine + AMP + CO2 + PPi + photons

– Emitted light proportional to the ATP level– Measurement of the emitted light in a luminometer

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Biochemical methodsDetection of ATP (food / microorganisms)� Sampling devices

– Many swabs� Luminometers

– with photodiodes : cost saving, low sensitivity– with photomultiplier tubes : expensive, high sensitivity

=> Detection system can be affected by residual disinfectants– QAC sanitizers known to create false positive readings– Oxidizing sanitizers can create false negatives on freshly sanitized surfaces

=> Great differences in the ATP content in different organisms

=> Few if any detection of dead cells & VNC

=> Quick (≤ 30 min)

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Biochemical methods

Some interesting devices able to detect biofilms (results from PIHM)� Positive tests, easy reading (clear change of colour)

� Speed Check (Ecolab)

� Ridacheck, Pathcheck (R-Biopharm / Microgen)

� Clean trace Plus (3M), Clean Test (LiofilChem)

� Biofilm Detector Kit (REALCO)

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BIOCHEMICAL METHODS• Rapid

• More or less accessible areas

• Minimal training required• Not affected by the physiological state of the bacteria (except ATP, NAD)

• +/- sensitive, +/- reproducible- - - - - - - - - -

Î Not suited to check the effectiveness of disinfection procedures

Î Suited to check the effectiveness of cleaning procedures

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Conclusion

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BIOCHEMICAL METHODS

Could be used routinely in complement to microbiological methods to detect biofilms on

surfaces in food environmentsÎDetection of possible cleaning incidents

ÎImplementation of corrective actions in time