Worst-case biofilm conditions for cleaning evaluations

A recent study carried out by R. Bright, A. Deal, D. Klein and P. Lopolito at the Life Sciences Division of STERIS highlights the importance of testing against both typical and atypical biofilms when evaluating cleaning and disinfection chemistries for biofilm prevention on process equipment

CDC reactor with P. aeruginosa generates standardised biofilm for testing

Process equipment and water system contamination from microbes protected within a biofilm continues to affect production schedules and product quality.

Multiple critical parameters exist for disinfection of hard surfaces. These include: biocidal agent, biocide concentration, contact time, contact temperature, microbial population and type, soil level, surface characteristics, water quality and other factors.

In this series of studies, the authors examined how various surfaces and micro-organism types affected the ability to disinfect and clean biofilms.

Cleaning and disinfection efficacy were evaluated using TOC analysis, visual cleanliness and microbial efficacy testing.

Various CDC bioreactor coupon substrates

The surfaces that were examined included EPDM rubber, PTFE Teflon, Buna rubber and UMHW polyethylene. US Centers for Disease Control (CDC) reactors were used to develop a Pseudomonas aeruginosa biofilm on coupons made of each surface type according to ASTM E2562-12.

The ASTM E2871-13 single tube method was followed for harvesting biofilm and testing biofilm coated coupons using a formulated alkaline detergent at 1% (v/v) and a formulated hydrogen peroxide/peracetic acid ready to use (RTU) sterilant at 20% (v/v).

The cleaning procedure utilised a low action immersion cleaning method and swabbing technique for measurement of total organic carbon (TOC) before and after biocidal agent contact.

The results illustrated that for biofilm contamination, surface type was not the most significant contributing factor in disinfectant efficacy.

Biocidal agents evaluated had similar log reductions independent of surface type, with all trials resulting in a >6 log10 average reduction.

In a similar series of studies using polycarbonate coupons, a Bacillus cereus generated biofilm consisting of vegetative and endospore phenotypes illustrates a challenge in using a single disinfectant step.

The cleaning and disinfectant efficacy results from P. aeruginosa and B. cereus biofilm testing displayed minor variation in surface types but substantial differences between biocidal agents and microbial species tested.

Materials and methods

P. aeruginosa suspension preparation: A fresh culture of P. aeruginosa ATCC 15442 was grown on Tryptic Soy Agar (TSA) for 24 hrs at 36 ± 1°C from a lyophilised stock culture.

An isolated colony was used to inoculate a flask containing 100ml of Tryptic Soy Broth (TSB) (300mg/L). The flask was incubated in an orbital shaker at 130rpm for 24 hrs ± 30 minutes at 36 ± 1°C.

P. aeruginosa biofilm grown in CDC reactor, shown on inverted light micrograph and biofilm stained with crystal violet at 100x magnification

Preparation of B. cereus spore suspension: A broth suspension of B. cereus ATCC 14579 was passed onto nutrient agar supplemented with manganese sulphate monohydrate and incubated for 12–14 days at 36 ± 1°C.

After incubation, bacterial spores were separated from vegetative cells and cellular debris by repeated centrifugation, decanting, and re-suspension in de-ionised (DI) water. After processing, the suspension was checked for high spore titer using phase microscopy.

The spore suspension was stored at 2–4°C.

Coupon preparation

Prior to use, all carrier coupons were inspected for damage, including scratches, chips, or residue that would affect study parameters.

In the event damage was found, the coupon was excluded from testing and replaced by an acceptable carrier.

Coupons were submerged in a laboratory detergent diluted 1:100 in individual 50mL conical tubes and sonicated for 30 seconds.

The coupons were rinsed with DI water and sonicated for an additional 30 seconds in fresh DI water. The rinse/sonication process was repeated until no soap bubbles were remaining.

Coupons were placed in each of the eight CDC Biofilm reactor arms and sterilised.

P. aeruginosa growth phases using the CDC biofilm reactor: A CDC reactor containing sterile TSB (300mg/L) was inoculated with 1.0mL of the P. aeruginosa ATCC 15442 suspension and allowed to constantly stir at 130rpm at 21°C for 24 hrs ± 30 mins.

After the incubation time elapsed, TSB (100mg/L) was continuously flowed through the reactor for 24 hrs ± 30 mins to allow for the circulation of fresh media at a flow rate of 11.7 ± 0.2mL/min at 21°C.

(Note: The flow rate is determined by the working volume of the reactor and a 30 minute residence time. Every reactor has a specific flow rate that needs to be pre-determined before use).

Growth of B. cereus biofilm using the CDC biofilm reactor: B. cereus ATCC 14579 biofilm was prepared following ASTM E2526-12, with modification.

B. cereus ATCC 14579 spores suspended in DI water were passed to TSB (0.3g/L) in a CDC biofilm reactor assembled following ASTM E2562-12 using polycarbonate coupons (RD 128-PC, Biosurfaces Technologies Corporation).

The culture was stirred at 125rpm for 24 hrs at ambient temperature.

After 24 hrs of stirring, the culture was stirred for an additional 24 hrs, at ambient temperature as fresh media (TSB 0.3g/L) was introduced at a constant rate of 11.7ml/min.

The reactor was kept at constant volume, draining from the top of the culture at 11.7ml/min.

Testing by the Single Tube Method

The harvesting and testing of all coupons was performed in accordance with ASTM International E2871-13.

Each reactor rod was gently dipped into a 50.0mL conical tube containing 30.0mL buffered water and gently moved back and forth to remove any planktonic cells.

Efficacy testing

Cleaning evaluation

Care was taken to not touch the sides of the tube. Splash guards were inserted into individual 50mL conical tubes.

A sterile Allen wrench was used to unscrew the coupon from its placeholder in the reactor arm until it dropped to the bottom of the tube.

The splash guard was removed. Any tubes and coupons were discarded from testing if contact with the sides of the tube occurred.

Disinfection efficacy was tested by adding 4.0mL of an alkaline detergent at 1% (v/v) to corresponding coupons.

Product interaction occurred for 10 mins before being neutralised with 36.0mL of LAT broth containing catalase.

The same testing process was carried out with the hydrogen peroxide/peracetic acid RTU sporicide at 20% (v/v).

Each tube containing a neutralised coupon went through a vortex-sonication series to disaggregate the biofilm off the coupons.

The sonicator was degassed for at least 5 mins at 100% power prior to testing samples. Test samples were sonicated at a frequency of 37kHz and 10% power on the sweep function.

The process was vortex-sonicate-vortex-sonicate-vortex, with each step being 30 seconds in duration.

Serial dilutions were performed and representative aliquots pour-plated in LAT agar for incubation for 24 hrs ± 30 minutes at 36 ± 1°C. Enumeration was carried out via aerobic plate counts.

Assessment of surface cleaning on P. aeruginosa CDC biofilm reactor coupons using TOC swab Testing: P. aeruginosa ATCC 15442 biofilm on various coupons were air dried for at least 16 hrs prior to cleaning.

Treatment consisted of submerging the coupons in 1L of 60°C alkaline detergent at 1% (v/v) or ambient temp (18-22°C) hydrogen peroxide/ peracetic acid RTU sporicide at 20% (v/v) and agitated with stirring at 300rpm.

Coupons were cleaned for 5 minutes, rinsed with DI water then swabbed with low TOC polyester swabs. Swabs were then sonicated in 40mL of DI water for 15 mins and analysed for TOC using a Sievers Model 900 TOC lab analyser.

Assessment of surface cleaning of B. cereus CDC biofilm reactor coupons using ATP swab testing: B. cereus ATCC 14579 biofilm on polycarbonate coupons was cleaned by submersion in a stirred, pre-heated volume of formulated alkaline detergent at 1% (v/v) for 5 mins at 30°C or a stirred volume of room temperature hydrogen peroxide/peracetic acid RTU sporicide at 12% (v/v) for 10 mins.

Treated coupons were then rinsed with DI water and either allowed to dry (single treatment), or placed into an additional volume of stirred, room temperature (18-22°C) at 12% (v/v) hydrogen peroxide/ peracetic acid RTU sporicide for 10 mins.

Coupons that were cleaned with the second solution (Two-step treatment) were then rinsed with DI water and allowed to dry.

The dry, treated coupons were then swabbed and those swabs were analysed for adenosine triphosphate content (ATP) (Ultrasnap ATP swabs and SystemSure Plus Lumonometer Hygenia/SS3).


Micrograph of unstained B. cereus biofilm featuring global biofilm architecture (Phase Contrast Illumination. 100x optical magnification)


Micrograph of B. cereus biofilm stained with Live/Dead metabolic stain Composite image of FITC ex/em and Texas Red ex/em illumination. Green coloration: FITC signal. Red coloration: Texas Red signal. ‘Spore’ arrow: bacterial spore morphology. ‘vege.’ Arrow: vegetative cell morphology. 1000x optical magnification.


LOG (base)10 CFU viable colonies per coupon over time. Each data point represents the geometric mean of two determinations. Error bars represent one standard deviation around the mean. Estimated time to complete inactivation (dotted lines) was calculated by fitting the generated survivor curve data to a weibulian inactivation model. A best fit line was determined using a least squares methods via Microsoft Excel with the Microsoft Excel add-on GinaFit.


LOG (base)10 CFU viable colonies per coupon over time. Each data point represents the geometric mean of two determinations. Error bars represent one standard deviation around the mean. Estimated time to complete inactivation (dotted lines) was calculated by fitting the generated survivor curve data to a weibulian inactivation model. A best fit line was determined using a least squares methods via Microsoft Excel with the Microsoft Excel add-on GinaFit.


Each bar represents the geometric mean of six determinations. Error bars represent one standard deviation around the mean. Dark grey bars: Viable colony forming units recovered after treatment. Light Grey Bars: Recovered ATP represented as relative fluorescence units.


Discussion

Biofilms found in processing equipment in the real-world environment of life science product manufacturers are highly varied and very difficult to characterise.

Therefore, it is important when evaluating cleaning and disinfection chemistries for use in these areas that testing is performed against both typical and atypical biofilms.

This includes testing biofilms that are more resistant than those found in normal situations. In this series of studies, we were able to successfully study variables that influenced worst-case biofilm formation conditions by evaluating different surface types and different micro-organisms, P. aeruginosa and B. cereus.

Although surface type and condition will affect many cleaning and disinfection scenarios, we found that the type of micro-organism present and products selected have the greatest impact on remediation success of the most resistant biofilms.

References

1. ASTM E2562-12, Standard Test Method for Quantification of P. aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor, Approved April 1, 2012

2. ASTM E2871-13, Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method, Approved Oct. 1, 2013

3. Deal, A., Klein, D., Lopolito, P. and Schwarz, J. S. PDA J. of Pharma. Sciences and Technology, 2016, doi:10.573/pdajpst.2014.005165.

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