Who Needs Environmental Monitoring and Process Water Testing?

Introduction 

Controlling manufacturing environmental conditions is not only a regulatory requirement but also part of protecting and producing a quality product. Environmental monitoring (EM) of manufacturing facilities provides assurance that the environment is both adequately controlled and in compliance. There is substantial evidence establishing a direct relationship between the level of environmental control and the final quality of the product. 

EM serves a critical role in product safety by ensuring that the environment in maintained properly. Swabs are often used for sampling irregular or hard-to-reach surfaces and critical surfaces where contact plates are not practical. In addition, cleaning hold-time studies are often performed using swabs. In general, the purpose of a Microbial EM Program is to: provide crucial information on the quality of the work environment during manufacturing; prevent future microbial contamination by detecting and reacting to adverse trends; prevent the release of a potentially contaminated batch if the appropriate standards are not fulfilled; prevent the risk of contamination of the product; ensure there are environmental controls in the production areas; and provide a profile of the microbial cleanliness of the manufacturing environment. 

Current Methodology 

Most EM is done by plate counting of colonies which is both simple and inexpensive. However, plate counting methods are slow requiring two to seven days to complete, thereby causing a delay in the detection of contamination, which can increase product loss, plant downtime and result in expensive clean up. The delay in obtaining results impacts reaction to contamination issues and can make investigations very difficult. For example, the rooms in question typically have been cleaned numerous times, so re- sampling results are almost always meaningless and determining the root cause of the contamination is difficult. Since real-time response is not possible, batches are jeopardized. The plate count methodology is also labor intensive and requires manual data entry and documentation. Such documentation is prone to human errors and compliance issues. 

Available RMM Methods 

Methods are available to measure total particles in the air, including Total Organic Carbon (TOC), and ATP (Adenosine Tri-phosphate). These methods are very fast to perform but do not correlate well with total bacterial count or any specific group of organisms and do not measure viable organisms (Carrick et. al. 2001 and Easter 2010). Therefore, these results do not measure viable organisms in the environment or on production lines. The standard plating methodologies can take several days. Rapid microbiological methods (RMM) can provide rapid and efficient solutions over traditional plating methodologies. Therefore, both manufacturers and regulators are motivated to develop initiatives and help in the implementation of rapid testing methods (FDA 2004). 

On June 8th the conference on Contamination Control ( http://www.pdamidwest.org/) the data from the validation of BioLumix growth based system for EM and water testing will be presented. The BioLumix Optical System is based upon the detection of microorganisms due to color or fluorescence changes caused by the growth and metabolic activity of microorganism in the test vials. 

Study Design: 10×10 cm surface coupons made out of 5 various materials (Stainless Steel, Aluminum Alloy, High Density Polyethylene, Silicone Rubber, and Perspex, Plexiglas) were inoculated with different organisms (Bacillus spizizenii var subtilis ; Escherichia coli; Pseudomonas aeruginosa; Staphylococcus aureus; Citrobacter freundii; Candida albicans; and A. brasiliensis formerly Aspergillis niger). The coupons were then swabbed and testing using three assays: (i) Total aerobic count; (ii) Yeast and Molds and (iii) Gram Negative Bile Tolerant Bacteria. 

In total, 550 coupons were tested, 290 coupons were inoculated above the specified levels while 260 coupons had counts below the specified levels. There was very good correlation between the BioLumix results and the plate count results, with an overall agreement for samples above spec of 97.2%. None of the 260 un-inoculated coupons detected in the BioLumix system or had plate counts above the specified level. Consequently there was 100% agreement between the two methods. The overall agreement between the two methods was 98.5%. 

Total Aerobic Count: A total of 129 swabs were analyzed using the BioLumix TAC vial and the standard plate count method with TSA. All the swabs with count above specified level signaled as being above the specified level in the vials. Five marginal samples detected in the vials and had counts just below the specified level. The agreement between the methods was 96.1%. 

Yeast and Molds: A total of 85 coupons were analyzed using the BioLumix YM vial and the standard plate count method with SDA (Sabouraud Dextrose Agar W/ Chloramphenicol). All the swabs with count above specified level signaled as being above the specified level in the vials. A few coupons with count very close to the specified level (e. g. 50-80 cfu/swab for a specified level of < 50 cfu/swab) did detect in the vials. One coupon that had a count of 40 cfu/swab, while technically found to be below the specified level, was a very marginal result being so close to the specified level of 50 cfu/swab, did detect in the vial. The agreement between the two methods was 98.8%. 

Gram Negative Bile Tolerant Bacteria: A total of 75 coupons were analyzed using the BioLumix ENT vial and the standard plate count method with VRBGA (Violet Red Bile Glucose Agar). One swab with a marginal count of 310 cfu/swab did not detect in the vial. A few coupons with count very close to the specified level (e. g.300-400 cfu/swab for a specified level of < 300 cfu/swab) did detect in the vials. One coupon that had a count of 190 cfu/swab did detect in the vial. The agreement between the two methods was 97.3%. 

Conclusion: The BioLumix system was validated as an alternative to the plate count method for EM. The study involved a total of 550 surface coupons representing five diverse types of surface material. These five surfaces represent those encountered in manufacturing, including metal, plastics and rubber. Some of the coupons were inoculated with bacteria or yeast or mold. There was 100 % agreement between BioLumix assay and the plate count assay for the 260 coupons that were determined to be below the specified level by the plate count method. There was an overall agreement of 97.2 % between the two methods when swabs containing counts above the specified level were used. 

The advantages offered by the BioLumix system include: automation of results, great speed to results, paperless EM, direct detection of viable microorganisms, detection of multiple types of organisms, increased operation efficiency and consequently an improvement in product quality, reduction in costs, and both enhanced reporting and ability to track trends. 

Water Testing 

Water is widely used as a raw material, ingredient, and a solvent in the processing, formulation, and manufacture of pharmaceutical products, active pharmaceutical ingredients and intermediates. As such, all water purification systems must be monitored regularly to verify the quality of the water produced. Monitoring of water for microbiological quality may include testing for total heterotrophic plate count, coliforms/E. coli, or by checking for the presence of other organisms suspected to be present in a water sample. The relevant standards relating to pharmaceutical grade water are USP <1231> Water for Pharmaceutical purposes. 

The BioLumix system is capable of testing water for heterotrophic bacteria, coliforms, E. coli, and Pseudomonas. For levels of < 1 cfu/ml the water can be inserted directly into the vial. To test for levels such as < 1/100 ml the water is filtered and then the filter is added directly to the vial. 

Water study summary: Ninety- two water samples were analyzed with two specified levels (10 cfu/ ml and 100 cfu/ml). Sixty samples were below the specified level by both methods while 28 samples were above the specified level by both methods. Four samples were below the specified level by the BioLumix method, but above by the plate count method. All these samples had very low counts (1-3 colonies on the plate). There was 96.9% agreement between the two methods. 

BioLumix advantage for water testing: Final results were seen in the BioLumix system roughly 13 hours earlier than the plate count method using Standard Methods Agar. The BioLumix method can detect organisms at a level of < 1 cfu/mL of water. The BioLumix system is faster, less labor-intensive, and more sensitive than the plate count method.

Rapid Microbiological Testing of E. coli with the BioLumix Vial

Escherichia coli are Gram negative rod-shaped organisms found naturally in the lower intestines of warm blooded organisms. Most serotypes of this organism are relatively harmless, making up a small percentage of bacterial colonization in the gut. These serotypes prevent the establishment of pathogenic strains.

E. coli is one of the most common bacteria found in the gut of animals. This includes humans. There are other animal species that contain this organism in the gut including reptiles and fish. E. coli colonizes the gut and can cause infection in the urinary tract and brain stem (meningitis) as well as intestinal diseases referred to as gastroenteritis. There are five classes of E. coli that produce disease. The most serious disease is the Enterohemorrhagic (EHEC) class. These organisms can cause diarrhea distinct from some others (including Shigella) in that there is copious bloody discharge and no fever. The life threatening situation is its toxic effects on the kidneys (hemolytic uremia).

Why Test for the Presence of E. coli?

E. coli is often used as indicator organisms to test the effectiveness of effluent disinfection in a wastewater treatment plant, on animal products as well as in nutraceutical and pharmaceutical products. While these organisms are generally harmless, they do live under the same conditions that human pathogens live. Since we cannot test for every pathogen, we test for easily detectable indicator organisms. The assumption is that if we kill the indicator organisms then we most likely kill the pathogens during effluent disinfection. E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Current Methodology

The current methodology can take anywhere from 3-7 days and includes multiple broths, agars, transfers and temperatures. Different methodologies are utilized by the various industries. Below are some examples:

Nutraceutical and Dietary supplements: The protocol described in USP <2022> requires absence of the organisms in 10 grams of product. A 1:10 dilution of product is made into TSB or another enrichment medium. This mixture is incubated for 24 to 48 hours at 30°C to 35°C, and then 1.0 mL is transferred to 10 mL of MacConkey broth. This mixture is incubated for 24 to 48 hours at 42°C to 44°C. A loop from the MacConkey broth is transferred to MacConkey agar and the plate is incubated for 18 to 24 hours at 30°C to 35°C. If typical colonies appear, these colonies are then transferred to Levine Eosin Methylene Blue agar and incubated 24 to 48 hours at 30°C to 35°C. If none of the colonies exhibit green metallic sheen under reflected light or if none of the colonies exhibit a blue-black appearance under transmitted light, the sample meets the requirement for the absence of Escherichia coli. Because results can sometimes be misread due to interpretation of plates, identification may be run on the sample(s) adding another 2-3 days of testing.

Pharmaceutical: The protocol described in USP <62> again requires the absence of E. coli in 10 grams of product. A 1:10 dilution is made and incubated for 18 to 24 hours at 30°C to 35°C. From this mixture, 1.0 mL is transferred to 100 mL of MacConkey broth and incubated 24 to 48 hours at 42°C to 44°C. A loop is transferred to MacConkey agar and this plate is incubated 18 to 72 hours at 30°C to 35°C. Growth of typical colonies indicates the presence of E. coli which is confirmed with an identification test.

Food Testing: The food industry follows Bacteriological Analytical Manual (BAM). In most cases, the Most Probable Number (MPN) method is utilized. This is labor intensive, multi-step assay consists of presumptive (in LST tubes), confirmed (in BGLB tubes), and completed phases (in EC tubes). In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed and then uses the combinations of positive results to consult a statistical table. From this table, the analyst is able to estimate the number of organisms present. The 3-tube MPN test is used for testing most foods. The 5-tube MPN is used for water, shellfish and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated. Positive EC tubes must be transferred to L-EMB agar and if typical colonies are observed, they must be further identified.

The BioLumix E. coli Vial

The E. coli vial, or EC vial, is a membrane vial that is monitor by the fluorescent signal in the instrument. The membrane separates the incubation zone from the reading zone. If E. coli is present, it utilizes MUG (4-Methylumbelliferyl-3-D-Glucuronide) through an enzymatic reaction to create fluorescence. A 1:10 dilution is made in TSB or a similar growth medium and this mixture is incubated for 18-24 hours at 35°C. From the TSB dilution, 0.1 mL is added to an EC vial containing the MUG supplement and then tested in the BioLumix instrument. The EC assay runs for 18 hours in the instrument. An example of E. coli curves is shown in the graph. If detection occurs, a confirmation test is performed. From the time the sample is prepared to the time the confirmation is complete, the EC test takes only 2 days, saving valuable time.

The BioLumix assay is much simpler to perform, requiring less labor and disposables as any to the standard method. It is faster (completed in 30 hours), accurate, automated, and it eliminates any product interference due to the two zone vial.