International Journal of Microbiological Research 3 (3): 208-215, 2012 ISSN 2079-2093 IDOSI Publications, 2012 DOI: 10.5829/idosi.ijmr.2012.3.3.

64231

A Novel Improved Bioburden Recovery Method Using Swabbing Technique

Mostafa E.A. Eissa and Ahmed M. Mahmoud Quality Control Supervisor, Hikma Pharmaceutical Company, 6th October City, Egypt
Abstract: The validation of surface-recovery methods is a pre-requisite for residual determination of cleaning effectiveness in process validation studies. These methods should be challenged in the laboratory using pilot-scale controlled conditions in order to evaluate the suitability for their intended use. Different swabbing techniques have been studied and compared with rinse method for 7 microorganisms (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Kocuria rosea, Candida albicans and Aspergillus niger) on 5 different coupon surfaces (Stainless steel, Glass, Rubber, TEFLON and Plexiglass) representing machine materials in production area. Only one swabbing technique gave good results in comparison to both rinsing method and previously reported Nylon flocked QUANTISWAB. The new swabbing technique is based on ordinary Rayon fiber which is known of low microbial recovery using traditional method. However simple modification in the recovery procedure using simple device improved the bioburden recovery to become over 80%. Moreover this novel method has good flexibility to be customized to enhance recovery according to cleaning validation condition and study. Key words: Swabbing Technique INTRODUCTION The objective of the cleaning validation is to verify the effectiveness of the cleaning procedure for removal of product residues, degradation products, preservatives, excipients and/or cleaning agents as well as the control of potential microbial contaminants. In addition one needs to ensure there is no risk associated with cross-contamination of active ingredients [1]. It is therefore not surprising that cleaning validation undergoes extensive regulatory review in the pharmaceutical industry during inspections. In fact, during the past few years, cleaning validation has ranked among the top 10 areas of concern in warning letters issued by the FDA. According to Kristen Evans, leader of the Guidance and Policy Team in the Division of Manufacturing and Product Quality in FDAs Center for Drug Evaluation and Research, equipment cleaning and maintenance ranked second in warning letters and GMP citations for the fiscal year (FY) 2004-2005 [2]. Although not all cleaning issues relate to microbial contamination, controlling bioburden through adequate cleaning processes is a regulatory expectation enforced by the FDA. Rinsing Method Rayon Fiber Bioburden Recovery Coupon

Validation of cleaning methods is required to ensure that the equipment-cleaning cycle consistently provides results that meet acceptable levels of cleanliness. Guidelines for cleaning requirements are provided to the pharmaceutical industry in the cGMP documents in the United States, Europe and other countries. Equipment cleaning requirements are also addressed in the Pharmaceutical Inspection Convention (PIC) recommendations and the Parenteral Drug Association (PDA) Technical Report No. 29, Points to Consider for Cleaning Validation [3]. Cleaning should be carried out as soon as practical after the end of processing and should leave the plant in a suitable condition for next use. In developing the sampling plan for a validation study, it makes scientific sense to incorporate an understanding of the limitations of the sampling method relative to the surface to be sampled. The two methods of sampling generally employed are swab and / or rinse sampling. The selection of either of these techniques must be consistent with sound scientific judgment and must support the objective of the study [4]. Equipment swabbing must be performed by qualified personnel and sterile swabs made from materials that do not interfere with the test should be used. There are

Corresponding Author: Mostafa E.A. Eissa, Quality Control Supervisor, Hikma Pharmaceutical Company, 6 th October City, Egypt. Tel: +20224705461 - +201006154853.

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various types of swabs used to monitor flat or hard-to-reach surfaces such as the bottom of a tank, O-rings, traps, transfer lines and U-bends. Swab sampling should be carried out wearing sterile gloves to minimize adventitious contamination. For bioburden recovery, after swabbing is complete, the swab may be streaked onto an agar medium or broken into a diluent, vortexed for about 30s and the liquid sample preparation is then tested by pour-plate or membrane filtration method [5]. Equipment rinse is performed using a solvent that will not interfere with recovery. Sometimes placebo is used, although this approach is not typical for collection of bioburden. Most rinse samples are collected using purified water or water for injection (WFI). For bioburden recovery, after the rinse sample is collected, it is processed via the membrane filtration technique [5]. Swabbing technique although has its special advantage over rinsing method yet its major disadvantage is its low recovery of collected bioburden of microorganisms which is related to the swab fiber matrix that hinders the release of microbial cells. In this work, a special but simple technique was looked for to release gathered microbial cells from swab material. This new method was compared with ordinary, modified swabbing and rinse techniques. The importance of swabbing technique is extended to include other practical fields such as inanimate surfaces of medical facility [6], medical instruments [7], clinical specimen [8] and food and veterinary industry [9, 10]. MATERIALS AND METHODS Representative Challenge Organisms: Challenge microorganism used for the validation studies Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Escherichia coli Kocuria rosea Candida albicans Aspergillus niger Sterilized coupons (small pieces of material representing equipment to be sampled e.g. Stainless steel, Glass, Rubber, TEFLON and Plexiglass). Preparation of the Working Cultures: Fresh transfers using agar medium or culture broth were prepared and the bacterial cultures were grown using Tryptone soya medium (agar or broth) and incubated at 30-35C for 18-24 h. Yeast cultures were grown using Sabouraud's dextrose agar (SDA) medium (agar or broth) and incubated at 20-25C for 2-3 d. Cultures of filamentous fungi (mold) were inoculated onto SDA and incubated at 20-25C for 5-7 d or until good sporulation was achieved. Transfers were prepared on solid media, the bacteria and yeast cultures were harvested by washing each slant or plate with approximately 2 mL of sterile USP Saline test solution (TS), pH 7.0 buffered sodium chloride solution or pH 7.2 phosphate buffer. Transfers prepared in liquid 209 Vendor (number) ATCC 6538 ATCC 6633 ATCC 9027 ATCC 8739 EM isolate ATCC 10231 ATCC 16404 Organism Type Gram-positive coccus Gram-positive spore-forming rod Non-fermenting, Gram-negative rod Fermenting, Gram-negative rod Gram-positive coccus Yeast Filamentous fungus

media were centrifuged, the supernatant was removed and the microbial pellet was re-suspended in sterile USP saline TS. Bacterial suspensions were adjusted with the buffer diluent to an optical density of 0.1-0.3 at a wavelength of 550 nm, using a spectrophotometer; yeast suspensions were adjusted with the buffer diluent to a 5.0 McFarland turbidity standard [11, 12]. As a guideline, a 1-mL aliquot of the 10 5 or 10 6 dilutions of these recommended standardized suspensions of bacteria and a 1-mL aliquot of the 104 dilution of the recommended standardized suspension of yeasts would yield counts in the range of 10-100 CFU. If the standardized inocula of bacteria and yeasts werent used promptly (within 2 h), the suspensions should be stored under refrigeration. Suspensions of vegetative organisms prepared in USP saline TS or a buffer solution remain viable and stable for 7-10 d if maintained under refrigerated conditions.

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Spore Suspension Preparation: Mold spores were harvested by washing the agar surface with sterile USP saline TS or a buffer solution containing 0.05% polysorbate 80. A sterile inoculating loop or some sterile glass beads were used to loosen the spores and combine the washings in a sterile container. The bacterial spore suspension (e.g., Bacillus subtilis), was prepared by harvesting the inoculated agar plates with sterile water and heat shock the suspension for 15 min at 65-70C, starting the timing when the temperature reaches 65C [11, 12]. Then the suspension was cooled rapidly in an ice bath (0-4C) and stored under refrigeration. Bioburden Recovery Methods for Cleaning Validation Procedure: Bioburden recovery methods under study were grouped into: Rinse method. Swab method (wet and dry) with different modification for comparison of best recovery: Direct swabbing technique: Usual normal method of sampling. Agar Streaking Technique: After sampling swab was streaked on solid agar media. Five Minutes Sonication after Swabbing: Tubes of swabs in diluents were sonicated before filtration. Prolonged Vortexing: 2 minutes vortexing instead of 30 seconds before filtration. Glycerol Swabbing: Normal swabbing technique except using glycerol as diluent for sampling. (at which the swab was immersed in liquid glycerol just prior sampling). Outward Flushing Technique: Swab after vortexing was subjected to washing by flushing diluent from inside of the swab hollow stick to outside. Recovery Study Using the Wet Method: This procedure was performed for vegetative cells (bacteria and fungi) to prevent loss of viability due to desiccation. Each type of coupon surface was inoculated with 25-100 CFU of the inoculum's suspension prepared in sterile saline solution using micropipette and a contact time of less than 1 min was allowed. The swab method was used to recover the test organisms by using a dry swab to remove the liquid inoculum's from the surface of the coupon [5, 13, 14]. 210

The swab was streaked onto an agar medium or broken into a diluent, vortexed for about 30 s and the liquid sample preparation was processed by the membrane filtration method and inoculated onto TSA with incubation conditions at 30-35C for 3-5 d. Results were reported as number of CFU per swab and the percent recovery for each test organism was calculated. Swab sampling was done in triplicate, for each challenge organism. A test-negative control for each swab set was applied to verify aseptic manipulations by carrying out the procedure just described but with uninoculated coupons. The inoculums level counts exceeded 100 CFU and microbial growth was recovered from the negative control samples. Recovery Study Using the Dry Method: The dry method was used for spore-forming bacteria (e.g. Bacillus subtilis) only because vegetative cells suffer desiccation and, therefore, would not be viable on dry surfaces [5, 15]. Each type of coupon surface was inoculated with 25-100 CFU of the inoculum's suspension prepared in sterile saline solution using the micropipette, the inoculum was allowed to evaporate to dryness under laminar flow conditions. The swab method was used to recover the test organisms. Using a wet swab the dried inoculum was removed from the surface of the coupon. The swab was streaked onto an agar medium or broken into a diluent, vortexed for about 30 s and the liquid sample preparation was processed by the membrane filtration method and inoculated onto TSA with incubation conditions at 30-35C for 3-5 d. Results were reported as number of CFU per swab and the percent recovery for each challenge spore former organism was calculated. Swab sampling was done in triplicate, for each challenge organism. A test-negative control for each swab set was applied to verify aseptic manipulations by carrying out the procedure just described but with uninoculated coupons. Test should be repeated if the inoculums level counts exceeded 100 CFU or less than 25 CFU and microbial growth was recovered from the negative control samples. In Recovery study using the wet method by swab technique,the inoculum's volume on the surface should not be less than 100 ul for Rayon sterile swab used in this study. Acceptance criteria for the bioburden recovery percent from coupon surface should not be less than 50 % of the inoculum's control.

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Recovery Study Using the Rinse Method: The inoculated coupon was placed in an aliquot of sterile diluents (e.g., purified water) contained in a sterile vessel, which would be used to rinse the given piece of equipment [5, 16]. The vessel was shaked well and then the coupon was removed aseptically. The rinse solution was filtered through a 0.45-m membrane filter. Then, aseptically the filter was removed and placed onto a TSA agar plate. The prepared TSA plates were incubated at 30-35C for 3-5 d. The recovered colonies were enumerated and results were reported. This procedure (rinse sampling) was performed in triplicate, for each challenge organism. A test-negative control for each swab set was applied to verify aseptic manipulations by carrying out the procedure just described but with uninoculated coupons. Test should be repeated if the inoculums level counts exceeded 100 CFU and microbial growth was recovered from the negative control samples. RESULTS Results demonstrated in Figure 1 showed that generally all microorganisms were recovered from all surfaces using the rinsing method; with observation that Staphylococcus aureus and Bacillus subtilis showed the highest recovery while Escherichia coli followed by Pseudomonas aeruginosa gave the lowest recoveries from all surface materials. Results illustrated in Figure 2 revealed that; all microorganisms generally failed to be recovered using direct swabbing method, except for Aspergillus niger. Aspergillus niger although failed for the rubber surface, yet passed for all other surfaces. The highest recovery was 78% for Aspergillus niger from glass surface while the lowest was 12% for Candida albicans from glass surface material. Modifications were done on the sampling or testing techniques to increase the bioburden recovery from various surfaces using cotton swabs. Results shown in Figure 3 indicated that all microorganisms failed to be recovered using direct agar streaking method. The highest recovery was 25% for Aspergillus niger from stainless steel surface while the lowest was 1% for Kocuria rosea from glass surface material. Results illustrated in Figure 4 revealed that in general all microorganisms failed to be recovered after 5 minutes sonication swabbing technique. The highest recovery was 71% for Aspergillus niger from stainless steel surface while the lowest was 0% for Staphylococcus aureus from stainless steel and glass surface material. 211

Results demonstrated in Figure 5 showed that in general all microorganisms failed to be recovered using 2 minutes vortexing swabbing technique except 89% for Aspergillus niger from stainless steel surface while the lowest was 5% for Staphylococcus aureus from plexiglass. Results shown in Figure 6 indicated that all microorganisms failed to be recovered using glycerol swabbing method (Glycerol immersion) with exception of Staphylococcus aureus and Eschericia coli from stainless steel surfaces. While Candida albicans, Kocuria rosea, Pseudomonas aeruginosa and Bacillus subtilis gave 0% recovery from stainless steel surfaces. Results illustrated in Figure 7 revealed that all microorganisms were recovered from all surfaces using swab filtration - after outward flushing of swabs using buffer pH 7 with Escherichia coli giving highest recovery followed by Kocuria rosea from rubber surface material. Pseudomonas aeruginosa followed by Bacillus subtilis gave the lowest recovery from all surfaces if compared with other microorganisms. The swabbing technique (with outward flushing) is valid to be used in cleaning validation studies. DISCUSSION Validation studies are usually performed using representative surfaces identified on the production areas. In the current study material of coupons was carefully chosen to represent the majority of equipments and machines in the production area of pharmaceutical facility. Among the six methods of swabbing technique only one method gave promising results. The remaining five methods failed -with few exceptions with some microorganisms on some surfaces- to achieve 50% recovery. Rinsing method was found to be superior in terms of recovery if compared to traditional swabbing technique however new modification in swabbing process rendered both methods nearly equal (approximate overall recovery 79% for rinsing versus about 80% for swabbing technique). Both methods were nearly the same since in either technique a process of flushing with diluent was done as method of extraction of microorganisms. The aim of using glycerol in one of the swabbing method was to form a thin layer around swab material that can take microorganisms off the surface however results obtained were variable in which Staphylococcus aureus and Escherichia coli gave the highest results among the studied group on stainless steel surface while the remaining failed to give acceptable results.

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Fig. 1: The recovery percentage of bioburden from rinsing technique for all tested surfaces microorganisms

Fig. 2: The recovery percentage of bioburden from direct swabbing technique for all tested surfaces and all microorganisms

Fig. 3: The recovery percentage of bioburden from agar streaking technique for all tested surfaces and all microorganisms 212

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Fig. 4: The recovery percentage of bioburden from filtration after 5 minutes sonication swabbing technique for all tested surfaces and all microorganisms

Fig. 5: The recovery percentage of bioburden from filtration after 2 minutes vortexing swabbing technique for all tested surfaces and all microorganisms

Fig. 6: The recovery percentage of bioburden from glycerol swabbing technique for most of the tested surfaces and microorganisms 213

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Fig. 7: The recovery percentage of bioburden from swab outward flushing -applying pressurized flushing by sterile pumping device with buffer pH 7 at the internal side of the swab- criteria for all tested surfaces and all microorganisms It should be noted that Aspergillus niger spores gave the highest recovery in comparison with some microorganisms with some surfaces. This can be attributed to the large size of fungal spore which does not allow for it to be trapped in microstructure of swab matrix. Vortexing for 30 seconds followed by 2 minutes was found to be superior over sonication for 5 minutes and overall recovery for all microorganisms from all surfaces was 32, 22 and 20% respectively. In the study of Dalmaso et al. [17] the Nylon flocked QUANTISWAB was able to recover 60% of microorganisms, which is considerably higher than traditional Rayon swabs with only 20% recovery. Our study is in agreement in the part of using traditional Rayon swabs with ordinary technique which gave recovery about 8% using streaking on agar method and approximately 32% using membrane filtration technique for all microorganisms on all used surfaces while using modified swabbing with outward flushing technique gave overall recovery above 80% for the seven microorganisms used with the five surfaces. The advantage of this new technique is that it can be modified by several ways to maximize the recovery. This can be achieved with either change volume of washing diluent, change number of washes or type of diluent. Dalmaso et al. [17] suggested that Rayon classical swabs can absorb microorganisms but have 214 difficulties to release viable micro-organisms. Our study (unpublished data) revealed that Rayon swabs can pick up to about 96% of microorganisms from the surfaces used in the study but the recovery on the media was low suggesting that the remaining cells were trapped in the swab itself. Since number of microorganisms left on the surfaces under study was very small it could be concluded that the role of cell to surface interaction plays a minor role in swabbing process. Possible explanation for the high bioburden recovery from Rayon swabs using outward flushing technique is that during the flow of the diluent from inside of the swab to outside it dragged trapped microorganisms which were held between fibers of the swab and mechanically carried them out through fluid stream a process which could not be achieved by other physical means such as vortexing and sonication. Finally, it could be concluded that, in general, the best method for microbial recovery is the rinse method and swab-filtration method (internal flushing of swabs using buffer pH 7) while direct filtration, agar streaking, 5 minutes sonication, 2 minutes vortexing and swabfiltration method (Glycerol immersion) failed in giving acceptable recovery. It is obvious that microbial recovery from Rayon swabs is dependent mainly on the direction of flow of extraction diluents through swab matrix and the number of washes which is not normally achieved using only simple vortexing technique.

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