Future techniques for disinfectant efficacy testing

ELSEVIER

INTERNATIONAL BIODETERIORATION& BIODEGRADATION

International Biodeterioration & Biodegradation 41 (1998) 273-279

Future techniques for disinfectant efficacy testing

J. T. Holah*, Amelie Lavaud, Wendy Peters, Kathyn A. Dye Campden and Chorkvwood Food Research Association, Chipping Campden. Glos., GL55 6LD. UK

Accepted; 29 January 1998

Abstract

The current disinfectant testing position for the food industry is that disinfectant manufacturers rely on suspension tests to provide data for recommended in-use concentrations. In the food industry, however, disinfection typically follows a cleaning phase in which microorganisms surviving prior to disinfection will be predominantly surface attached. Simple surface tests are also available to assess the efficacy of disinfectants against surface adhered microorgansims in which test microorganisms are dried onto surfaces, disinfected and then removed for enumeration by traditional techniques. Both current suspension and surface tests are relatively simple and cheap and can thus be undertaken by a range of laboratories. They can also mimic a range of potential in-use conditions e.g. contact times and temperatures, interfering substances and surface properties. They bear little relationship to factory disinfection, however, as the condition of the test microorganisms does not reflect that of environmental growth (e.g. attached cells or biofilms), only a live/dead result can be obtained (not biostasis), the effects of the cleaning phase are not taken into account and, for the surface test, the microorganisms have to be removed from the surface to be enumerated, which is not 100% efficient and may add additional stresses.

This paper reviews some of the available techniques that in the short term may be incorporated into existing methodology to enhance their applicability to the food industry and in the long term may enable laboratory based test methods to more closely mimic the realities of the complete food factory sanitation programme. In particular, techniques which will more closely model surface attachment and disinfectant application strategies will be described, which are at the stage of incorporation into existing surface test methodologies. Future techniques based around vital stains, impedance and bioluminescence methodologies are also indicated that allow surface adhered microorganisms to be enumerated in situ, an assessment of their ability to grow to be made immediately following disinfection and to encompass typical cleaning stressors prior to disinfection. The role of field trials in truly reflecting the efficacy of disinfectants in the food factory environment is also reviewed. 0 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Laboratory and field testing; Determining viability loss and biostasis; Biofilms; Bioluminescence; Impedence; Optical density; ELISA; Lux genes; Flow cytometry.

1. Introduction-The current situation

The requirement for disinfection within the food industry is well understood and the special needs for disinfectant types and how they fit into a joint cleaning and disinfection programme has recently been reviewed (Holah, 1995a, b). Food producers need to be assured that the disinfectants they use, particularly for high risk, short shelflife products, are capable of antimicrobial effects under food manufacturing environmental conditions. Assessment of disinfectants for the food industry by documented, repeatable and reproducible test methods is, therefore, essential.

Traditionally disinfectants have been evaluated by suspension tests and occasionally surface based tests and

* Corresponding author.

S096&8305/98/$19.00 8 1998 Eisevier Science Ltd. All rights reserved. PII: SO964-8305(98)00018-3

these have been well reviewed (e.g. Reybrouk, 1982, 1990). Currently CEN/TC 216/WG3, which is respon- sible for preparing harmonized test methods suitable for food hygiene, institutional, industrial and domestic applications in Europe (Holah, 1996), has prepared bactericidal and fungicidal suspension tests, which should become European standards in late 1997, and are at the first draft stage of a surface test based on the method developed by CEN/TC 216/STG (Surface Test Group) and described by Van Klingeren et al. (1998).

Suspension tests have a number of benefits. They are relatively simple and do not require specialised or expensive pieces of laboratory equipment and, other than labour costs, are cheap to perform. They are also well defined and are thus within normal microbiological limits, repeatable and reproducible. Within the test methodology it is also possible to test a wide range of variables including

274 J.T. Holah et al./lnternational Biodeterioration & Biodegradation 41 (1998) 273-279

contact time, temperature, microorganism type and interfering substance (i.e. food product residues). Their major problem, however, is that they do not necessarily reflect in-use conditions. In food factory situations where cleaning chemicals are used first, for example, microorganisms remaining to be disinfected will be predominantly surface attached. The typical application of disinfectants by mist spraying or immersion is also not likely to bring these organisms into suspension.

Suspension tests are best used, therefore, as screening tests to evaluate whether disinfectants show efficacy under a range of environmental conditions which could be expected in food processing facilities. Because microorganisms will be predominantly surface attached after cleaning. this review only considers the future devel- opment of surface based tests.

Surface tests are used to mimic some aspects of microbial attachment to surfaces including short term attachment and drying. They have similar advantages to suspension tests in that they are also relatively simple, do not require expensive pieces of specialised equipment and are reproducible (Bloomfield et al., 1994). They can also assess a range of variables including contact time, microorganism type, surface type and interfering substance. Temperature is a possible variable but this requires a large test chamber or a laboratory that can operate under controlled climatic conditions. The major problem of the test method is that the microorganisms have to be removed from the surface to be enumerated which in itself may induce lethal stresses. The test also only mimics non-mechanical disinfectant application methods, e.g. mist spraying or immersion and does not include the mechanical effects of brushing or wiping.

Both the suspension test and the surface test also only assess the action of the disinfectant in the total sanitation programme and record the viability of the microorganisms not at the point of time immediately after disinfection but after a period of time in which cell recovery is possible.

2. Short term future techniques

2.1. Mechanical cleaning

The majority of disinfection in the food industry is undertaken by mist spraying of surfaces where contact time due to run-off is of the order of a few minutes. For more critical disinfection, smaller pieces of dismantled equipment and utensils are also often disinfected in soak tanks where both contact time and solution temperature can be increased. Manual methods of disinfectant application, which inherently apply a mechanical action, are uncommon because they are time consuming and labour intensive. If, however, mechanical application could be demonstrated to have efficacy benefits, the food industry

would welcome such improvements, particularly in the disinfection of equipment in high risk food processing areas.

Initial studies conducted at the authors laboratory have focused on trying to mimic wiping and brushing actions and these have been incorporated into the current CEN/TC 216/WG3 surface test. Five comparisons have been made using Pseudomonas aeruginosa (ATCC 15442) at 20C on 2 cm stainless steel discs (AISI 3 16, grade 2B), for a 5 min contact time under clean conditions (0.03% bovine albumin), against 4 disinfectants tested at the manufacturers recommended in-use conditions, as follows:-

a) Standard test conditions. Application of 100 ~1 disinfectant.

b) Pre-soaked swabto mimic a disinfectant soaked cloth. A standard swab was immersed in disinfectant until saturated (no volume measurement) and then wiped across the disc surface 4 times (twice in one direction and then twice after rotating the disc through 90).

c) Disinfectant application followed by pre-soaked swab-to mimic a disinfectant spray and wipe action. Application of 100~1 disinfectant for 5min followed by the application of a pre-soaked swab as above.

d) Disinfectant application and interdental brush-to mimic a traditional brushing application. Application of 100 1.11 disinfectant for 5 min followed by the application of a small interdental brush across the surface 4 times (twice in one direction and then twice after rotating the disc through 90).

e) Suspension test. As a control, the efficacy of the surface tests were compared with suspension test conditions using the protocol of the CEN/TC 216/WG3 suspension test under the same parameters (Holah, 1996).

The results, the mean of a minimum of three replicates undertaken on different days, are shown in Table 1 and in all cases are expressed as the log reduction achieved by the disinfectant minus any log reduction recorded from a water control. All the work was undertaken by one operator to minimise person to person repeatability errors. The results indicate that disinfectant wiping (pre- soaked swab) is probably less effective than disinfectant application only, possibly because of a short contact time and, on a volume basis, little bacteria/disinfectant inter- action. The disinfectant spray and wipe action generally increased disinfectant efficacy whilst in all cases, brushing resulted in increased log reductions. In some cases e.g. for the quaternary ammonium (QUAT)/amphoteric product, results suggested that a disinfectant with no efficacy under traditional testing conditions can be made into an acceptable disinfectant if applied with a brushing action.

Similar results have been observed with respect to improvement in disinfectant efficacy by a mechanical wiping action by Spicher and Peters (1997). who suggest

J.T. Holah et aLlInternational Biodeterioration & Biodegradation 41 (1998) 273-279 275

Table 1 Mechanical cleaning effects

Disinfectant product Log reduction

Disinfectant Disinfectant soaked Disinfectant and Disinfectant and Suspension only swab

QUAT Biguanide QUAT -Amphoteric QUAT

1.46 1.05 1.90 1.29 0.60 I .oo 2.48 0.95

that mechanical action may increase the contact potential between disinfectant and test microorganism. Initial results appear promising in this area but much additional work is required before the implications of mechanical action can be adopted by the food industry.

2.2. Surface attachment

Within the food processing environment, microorganisms will adhere to surfaces in a number of ways. These are primarily related to time, temperature and water availability and can be regarded as short term attached or dried on (approximately l-2 hours), mid term attached (approximately the length of a production shift e.g. several hours) and long term attached (longer than the duration of a production shift) which is often termed biofilm growth. Fortunately true biofilms are rare in the food industry and can be readily controlled by sanitation programmes (Gibson et al., 1995a, b). The length of time of microbial attachment has been reported to affect both attachment strength (Eginton et al., 1995) and thus cleanability, and resistance to disinfection (Hugo et al., 1985; Lechevallier et al., 1988; Holah et al., 1990; Frank and Koffi, 1990; Wright et al., 1991 and Dhaliwal et al., 1992).

The effect of surface attachment on disinfectant resistance is shown in Table 1 and Table 2. The test parameters

Table 2 Effect of surface adhesion on disinfectant efficacy

disinfectant swab brushing

2.02 2.67 1.59 2.30 2.73 3.87 3.49 2.87

test

>5 5 >5

for Table 1 have already been described. In Table 2 the results were generated for a different range of disinfectant products using the impedance method as described in Holah et al., 1990. For the 1 hour adhesion, P. aeruginosa was allowed to attach to stainless steel discs (as above) for one hour in a phosphate buffer (0.1 mol/l KH2P0,, (BDH AnalaR) adjusted to pH7.0 with NaOH). For the 5 hour adhesion, to simulate growth through the production period. the organism followed the same 1 hour attachment phase followed by 4 hours incubation in a growth medium (0.07% Yeast Extract (Oxoid L21), 0.1% Bacteriological Peptone (Oxoid L37)). The concentrations of disinfectants used were selected to demonstrate differences in resistance between the three states of attachment as at the higher concentrations for the peracetic acid, amphoteric and quaternary ammonium product, the recommended in-use conditions were sufficient to render 100% reduction compared to water controls. For the iodine based product, the manufacturers recommended in-use condition only gave a 100% reduction when tested in suspension.

The combined results of Table 1 and Table 2 indicate that disinfectant products differ in their ability to demonstrate efficacy against organisms attached or dried onto surfaces and in suspension. As a general conclusion it can be suggested that disinfectants are less active against

Disinfectant product Cont. (%) Log reduction

Suspension 1 hour adhesion 5 hours adhesion

Peracetic acid 0.1 6.2* ?.6* 5.6 0.5 6.2* 7.6* 6.3*

Amphoteric 0.3 6.F 8.0* 2.6 0.75 6.7* 8.0* 6.3*

QUAT 0.1 7.4* 4.1 1.2 0.5 6.9* 7.8* 6.1*

Iodine 1.0 7.0* 4.8 2.1

* indicates 100% reduction of organisms as compared to a water control

216 J.T. Holah et al./International Biodeterioration & Biodegradation 41 (1998) 273-279

surface attached microorganisms and that the longer the attachment, the greater the microorganisms resistance.

In conclusion, the mechanical action and surface attachment short term future techniques are regarded as developments of current testing methodology to increase their relevance to the assessment of disinfectant efficacy in the food industry. They could be incorporated into existing test methodologies with little further devel- opment and are capable of being used by most disinfectant testing laboratories. The mechanical surface test still records the viability of the microorganisms not at the point of time immediately after disinfection but after a period of time in which cell recovery is possible and induces removal stresses on the attached organisms to allow enumeration; both techniques only assess the action of the disinfectant in the total sanitation programme.

3. Long term/research techniques

3.1. In-place viability detection

Removal of microorganisms from surfaces for enumeration following disinfection is not ideal as it involves loss of cells in the process (recovery processes are not 100% efficient) and may induce viability stresses. An assessment of organism viability in situ is preferable. This may be accomplished by microscopic examination of the cells with vital stains, by allowing the cells to grow until such growth can be detected over a time sequence which can be related back to initial starting numbers, or by assessing viability by visible metabolism markers e.g. light.

In situ viability assessment of attached microorganisms can be undertaken microscopically using a number of live-dead staining techniques, e.g. acridine orange (Holah et al., 1988), 5-cyano-2,3-ditolyl tetrazolium chloride (Schaule et al., 1993), green fluorescent protein (Chalfie, 1995) and many vital stains (Wirtanen, 1995). In addition. Yu et al. (1993) describe a direct viable count technique for the assessment of biofilm disinfection. Alternatively, viability techniques in which microorganisms are encour- aged to grow in determined formats e.g. extended length due to restricted division via naladixic acid (described in Colwell, 1987) could be used to assess cell viability. However, despite extensive vital stain studies, there is still a need to further develop simple methods of in situ disinfectant efficacy testing.

Impediametric techniques measure microbial growth by detecting electrical conductivity changes in the surrounding incubation medium such that the rate of electrical change can be correlated with microbial numbers. The unique advantage of impedance techniques is that they may be used whether the microorganisms are in suspension or attached to surfaces; provided that surface adhered organisms are viable, they can change the elec-

trical properties of the surrounding medium. Dis- infectants have been successfully evaluated against surface attached microorganisms through the use of impediametric techniques (Holah et al., 1990; Dhaliwal et al., 1992; Mosteller and Bishop, 1993 and Johnston and Jones, 1995). These techniques are excellent in that they assess the viability of the attached microorganisms in an unchanged morphological state. They are, however, very expensive techniques and would not be acceptable for routine disinfectant testing laboratories.

Measurement of light, both natural and from 1uxAB recombinant microorganisms, has been used to assess the viability of microorganisms. Wirtanen (1995) has used the naturally bioluminescent Photobacterium leiognathi for assessing the cleanability of food processing equipment and a lux recombinant strain of Listeria mono- rytogenes has been used for assessing disinfectant resistance by Walker et al. (1992a, b). In addition, it is possible to assess the viability of the recombinant L. monocytogenes strain in disinfectant tests when surface attached (Walker et al., 1993) although detection is lim- ited by the sensitivity of typical photomultiplier tube luminometers. It is not known whether it is possible to detect P. leiognathi when surfaced adhered and at this stage, the use of light detection to assess cell viability as part of a disinfectant test procedure is still at the research stage.

Some initial work has been undertaken by Das (1996) in which biofilms are grown on the base of ELISA plates and, after subsequent disinfectant tests, survivors are enumerated via change in optical density. Whilst it is only possible to mimic one surface, the flexibility of the numerous wells allows a range of variables to be exam- ined at the same time.

3.2. Biostasis

Traditional disinfectant test procedures allow a recovery time for cells sustaining injury (but not death) in the disinfection process. An assessment of the total number of cells that have been killed is very useful in determining the biocidal capability of the biocide. In practice, however, it is less useful in many food production scen- arios when food manufacturers are trying to reduce the number of microorganisms and control their growth potential only in the window between the finish of disinfection and the start of production as they know that microorganisms will soon recontaminate food processing surfaces during production. It may be possible, therefore, to use lower concentrations of existing disinfectants or alternative products if it can be shown that a biostatic reaction, rather than a biocidal action as determined by traditional disinfectant tests, is occurring.

Currently, the only test procedure that determines viability in situ and immediately after disinfection is the bioluminescent test of Walker et al. (1993). It may be

J.T. Holah et aLlInternational Biodeterioration d Biodegradation 41 (1998) 273-279 271

possible to use vital stain methods to assess immediate does demonstrate, however, the potential for the use of viability levels after disinfection although this would be impedance techniques to reflect the conditions of micro- a once only assessment. In the case of bioluminescence biological growth during the window between sanitation readings, the light output could be measured over a time and production by observing changes in detection times. period after disinfection, though the time frame in which The assessment of microbial viability by bioluminescence the microorganism can sustain light output is unknown. during this window also shows promise for the future.

The influence of the effect of cell recovery in determining the extent of biostatic and biocidal effects has been determined with analyses for cell viability by traditional enumeration and via impedance. A biguanide, an amphoteric. an iodophore and a sodium hypochlorite based disinfectant were assessed for a 5 min contact time against Pseudomonas aeruginosa only. Using the protocol of EN 1040 (Anon, 1997) from the bacterial test suspension, validation controls and the test neutralisation medium, in addition to the two 1 ml aliquots pour plated as per the test procedure, two 0.5ml samples were also trans- ferred into Malthus tubes containing 4 ml of SPYE broth. These were connected to a Malthus 2000 Microbiological Growth Analyser (Malthus Instruments, Crawley, UK) and incubated until a change in conductance occurred and a detection time (DT) was obtained (up to a maximum of 48 hours). Detection times were correlated to microorganisms numbers from a calibration curve with a regression equation (log TVC = 9.16 -0.357 DT) such that the longer the detection time the fewer microorganisms capable of growth.

3.3. Cleaning stressers

Of concern to both disinfectant manufacturers and food producers is the sometimes large difference in disinfectant resistance to microorganisms in suspension and attached to surfaces (e.g. Table 1) with often a l&100 times increase in concentration required for equivalent surface based results (Holah et al., 1990). This has led to doubts over whether food producers are using disinfectants at the right concentration as disinfectant manufacturers recommended in-use concentrations have been determined traditionally from suspension tests. The consequences of the use of higher disinfectant concentrations include increased costs, enhanced taint potential, toxicological considerations and environmental pollution. when currently the only concerns that the food industry has over the success of disinfectants in practice is low temperature performance on chilled food production surfaces.

Results, the mean of three replicates, (Table 3) clearly show that enumeration by impedance reflects a similar efficacy performance as the traditional colony counts using this disinfectant test protocol. Closer examination reveals, however, that the impedance technique is better able to reflect the significance of the extent of disinfectant action as increasing concentrations of the biguanide, amphoteric and iodophore products show no difference in colony counts but large differences in detection times. Much of this is due to the inherent inability of this test protocol to precisely enumerate the remaining cells after disinfection as only one dilution is counted (to simply identify whether a 5 log reduction has or has not been reached).

The comparison in Table 3, together with the use of impedance techniques for surface assessment (Table 2).

What is clearly required is some understanding of the condition that microorganisms are in following the cleaning phase of a typical food industry sanitation programme. At present both standardised suspension and surface tests use healthy test strains whereas in reality, prior to disinfection, microorganisms on food processing surfaces will have been affected by a whole range of cleaning stressers including the action of detergents, high temperatures, pH and mechanical actions from brushing, wiping or spray jets etc. Very little work has been reported on the combined effects, synergistic or otherwise, of cleaning stressers and disinfectant performance but initial studies by Gibson et al. (1995b) have indicated that disinfectant performance can be significantly enhanced by pre-soaking the test microorganisms in detergent solu- tions.

It would be very unwise to assume that the results of

Table 3 Comparison of impedance and traditional techniques in the detection of biocidal and biostatic actions

Disinfectant product Colony count Detection time (hours)

Product concentration (%) 0.01 0.1 1.0 0.10 0.1 1 .o

Biguanide > 300 >300 0 13.7 20.3 >48 Amphoteric > 300 >300 0 9.7 16.6 >48 Iodophore >300 >300 0 12.6 20.1 >48 Hypochlorite >300 0 0 13.3 48 48

278 J.T. Holah et aLlInternational Biodeterioration & Biodegradation 41 (1998) 273-279

surface tests reflect truly the likely appropriate recommended in-use concentration any more than the results of suspension tests. Both tests can only be seen as indicators of the relative performance of a disinfectant under simulated suspension and surface attached test conditions. Future work must, therefore, focus on the relationship between the cleaning and disinfection stages of the sanitation programme before we can reliably pre- dict in-use concentrations from laboratory based test protocols. If such test protocols also include an understanding of the effect of the disinfectant on the growth potential of the test microorganism immediately after disinfectant application we can also begin to manipulate the stages of the sanitation programme to the real benefit of the food manufacturer.

4. Field trials

In principle, the testing of disinfectants in the field is the ultimate reality. The test conditions incorporate attached and suspended microorganisms, a variety of microorganism types with environmentally acquired resistances and which have been subjected to cleaning stressers and a whole range of variables including temperature, contact times, surface materials and interfering substances.

The results in Table 4 indicate the mean counts remaining on equipment surfaces before cleaning, after cleaning and after disinfection following 8 field trials. Field trials were only undertaken in factory environments in which levels of microorganisms of approximately lo6 per swab were present prior to cleaning as it was felt that only with such high starting numbers could an assessment of disinfectant performance be realistically assessed. Each field trial was of approximately 10 weeks duration and consisted of swabbing the same 10 sites on a chosen piece of equipment 3 times a week after disinfection and approximately once per week before and after cleaning to monitor cleaning performance. Throughout the 10 week period the cleaning and disinfection procedures were undertaken to a strict schedule which was as close as possible to the schedule routinely undertaken by the factory cleaning operatives and was undertaken as much

Table 4 Mean total viable counts remaining after various sanitation programme stages over 8 separate field trials

Count per swab

Mean Sample No.

Before cleaning

1.3 x lo6 498

After cleaning

8.7 x lo4 1090

After disinfection

2.5 x 10 3147

as possible by the same trained cleaning operative at each site. The time taken between sample collection and enumeration was also kept within a close time frame. The 10 week duration of the trial was chosen to more closely reflect normal sanitation programme performance as it had been noted in previous factory studies that trials of short duration were undertaken by factory operatives in an over conscientious manner.

The results of the field trials have proved useful in that they provide a good indication of the relative roles of the cleaning and disinfection operations performance in carefully controlled factory sanitation programmes. In addition, they provide a measure of the likely numbers of microorganisms to be found after well structured and controlled sanitation programmes. They also, however, reflect the true realities of undertaking field trials for the assessment of disinfectant performance. They are very difficult to establish and manage, take a very long time and are very expensive. The range of variables that the trial could encompass has also to be strictly controlled and the results only reflect the number of microorganisms that are viable after a recovery period, not their growth potential immediately after disinfection and before production recommences. In addition each trial is significantly different and could not be repeated at another factory site.

Field trials undoubtably have their uses but due to their complete lack of repeatability we can only look to CEN/TC 216/WG3 to devise guidelines on the principles and practices of undertaking meaningful field trials.

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Future techniques for disinfectant efficacy testing