Bioburden Method Suitability for Cleaning and Sanitation Monitoring: How Far Do We Have to Go? - Pharmaceutical Technology

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Bioburden Method Suitability for Cleaning and Sanitation Monitoring: How Far Do We Have to Go?
The author reviews test methods for microbiological cleaning processes and suggests ways to improve microbial bioburden method suitability studies.


Pharmaceutical Technology
Volume 34, Issue 8

Surface microbial bioburden monitoring methods are described in Standard Methods for the Examination of Dairy Products (1). A review of the literature shows that few studies have been conducted to examine actual swab-recovery efficiencies and limits of detection (2–6). Niskanen and Pohja indicated that the contact-plate method is suitable for flat, firm surfaces, (considering both recovery and repeatability), whereas swabbing is better for flexible and uneven surfaces and for heavily contaminated surfaces (7, 8).

Previous studies reported a poor correlation with the amount of microbial contamination on surfaces and the recovery obtained. For example, a collaborative study comparing surface monitoring methods showed that artificially contaminated stainless steel at a theoretical level of 1.4 colony forming units (cfu) per cm2 (about 35 cfu per sample) gave recoveries of 25 to 30% when bacterial spores were employed (9). As reported by Kang, Eifert, and Williams, recovery of Listeria monocytogenes from stainless-steel inoculated test areas, or coupons, using a sonicating brush head (contact or noncontact) yielded a recovery level of about 60% compared with swab and direct agar contact methods (about 20%) (10). Many factors may contribute to this poor correlation, including differences in materials used (e.g., cotton, polyester, rayon, calcium alginate), the organisms targeted for culture, variations in surface, and differences in the personnel collecting and processing samples (11). Additional sources of error are the potential for nonhomogenous surface deposition of test microorganisms resulting in unequal or incomplete removal of microorganisms from the test surface (4). Based on these studies it is widely accepted that positive swab samples are indicative of high surface concentration of microbes, whereas negative swab samples do not assure that microorganisms are absent from the surface sampled (4).

As stated by Moore and Griffith, in the use of swabs for recovery of microorganisms from surfaces, there is a lack of standardization of both the swabbing pattern and the pressure applied to the swab during sampling. This means that technician-to-technician variation in the sampling procedure may potentially play a significant role in the recovery and enumeration of the sampled surface. This can lead to biased results for the initial cleanliness of the surface or the effectiveness of the cleaning procedure used. Although many surface-sampling techniques are available, their effectiveness as surface cleanliness indicators may vary. The bioburden recovery methods using swabs can be also influenced by the experimental design. Bioburden recovery results using swabs may not be as sensitive as other sampling techniques (i.e., replicate organism detection and counting [RODAC]). In addition, results may be invalidated due to the presence of excessive, uncontrolled variability and data scattering due to technician-to-technician variability (12, 13).

In microbiological studies evaluating cleaning and/or sanitization in the pharmaceutical industry, the removal and enumeration of viable microorganisms from material surfaces is a critical point for environmental control. The data obtained pertaining to survival and enumeration of microorganisms would affect validation (11). Variables affecting the accuracy of the detection and enumeration using the swabbing technique initially include the ability of the swab to remove the microflora from the surface as well as its effectiveness to release removed microorganisms from the swab and their subsequent recovery and cultivation (12, 13). The proportion of attached microflora on surfaces that are trapped or tenaciously bound to the interwoven fibers of a swab head are unknown. Sampling techniques that preserve the underlying surface as well as the viability of the detached microflora will detach only a portion of the total population. Adherent bacteria on surfaces become increasingly difficult to remove by use of swabs, especially if they become associated with a biofilm (11, 12–14). Studies conducted under controlled conditions have demonstrated that recovery is low. Kusumaningrum, et al. reported that in evaluating the survival and recovery of Bacillus cereus, Salmonella enteriditis, Campylobacter jejuni, and Staphylococcus aureus on stainless-steel surfaces, the direct contact method using solidified agars recovered 18% of Bacillus cereus, 23% of Salmonella enteriditis, 7% of Campylobacter jejuni, and 46% of Staphylococcus aureus from the initial concentration applied to the surface (15). A validation and comparative study on recovery of microorganisms using swabs, Hygicult TPC dipslide, and contact agar plates yielded similar results and did not differ in precision, with recoveries ranging from 16 to 30% of the microbial load applied to the surface (9).

Cleaning validation

Inadequate cleaning procedures may result in a number of contaminants present in future batches manufactured on the contaminated equipment such as:

  • The previous product, including active pharmaceutical ingredients and product intermediate
  • Solvents and other materials employed during the manufacturing process
  • Microorganisms—particularly where microbial growth may be sustained by the product or ingredients
  • Cleaning agents and lubricants.

Process validation is a series of interrelated functions and activities using a variety of specified actions that are designed to produce a predetermined result. This activity examines a process under normal operating conditions to prove that the process is in control. Cleaning in process validation is the documented study that demonstrates the reproducibility and consistency of the cleaning process to reduce undesired material from contact surfaces to a safe level. These studies apply appropriate analytical recovery methods designed to determine the presence of selected materials or entities (16, 17).

Cleaning validation should include studies using appropriate bioburden recovery methods where the possibility of microbial contamination of subsequent product is deemed possible and presents a product-quality risk. Validation studies are performed using coupons of the representative surfaces inoculated with the test microorganisms. The test microorganisms, which are usually known laboratory-adapted strains, are spread onto a space that is approximately 25 cm2 and allowed to air dry. After air drying, the test microorganisms are recovered by either swabs or contact plates. The test samples, along with positive and negative controls, are treated and/or incubated. Results are analyzed based on the percent of test microorganisms that grow after recovery compared with an inoculation control (1).


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