Case Study of a New Growth-Based Rapid Microbiological Method (RMM) That Detects

the Presence of Specific Organisms and Provides an Estimation of Viable Cell Count AUTHORS- Ruth Eden, President, BioLumix, Inc.; Michael J. Miller, President, Microbiology Consultants, LLC

ABSTRACT

Currently available growth-based rapid microbiological methods (RMM) provide either a quantitative cell count, estimation of viable cell concentration, information regarding the presence of a specific microorganism, or a microbial identification. However, the ability of these RMM technologies are limited in scope in that they cannot be used to perform all the required assays using a single technology platform.

A technology overview and validation case studies for the BioLumix System, a new RMM technology that is capable of simultaneously detecting microbial growth, providing an estimation of viable cell counts, and identifying the presence of specific microorganisms, using a novel and automated growth-based format is presented. The case studies demonstrate the ability of the technology to detect total aerobic microorganisms, total yeast and molds, and the presence of E. coli, Staphylococcus, Pseudomonas, Salmonella, as well as additional microorganisms.

Technology The BioLumix system is based upon detection of color or fluorescence variations due to microbial metabolism in liquid medium within a novel two-zone test vial. An optical sensor monitors changes in color and fluorescence within the vial’s reading zone, which is physically separated from the incubation zone. This two-zone approach prevents masking of the optical pathway by product or microbial turbidity and therefore, eliminates product interference. Changes in color or fluorescence, expressed as light intensity units, are monitored 10 times per hour and recorded in the computer. Separate test vials are also used to automatically detect the presence of viable microorganisms and/or to estimate the concentration of viable counts by monitoring changes in CO2 production during cellular growth. Each of these applications can be simultaneously performed using the same instrumentation and at the same time. The sensitivity of the BioLumix system is a single viable cell per sample vial; when a single cell replicates to a specific detection threshold level, a positive response is recorded. The threshold level is ~ 100,000 cells/ml for bacteria and ~ 10,000 cells/ml for yeast and molds. Additionally, the system yields significantly faster results than the plate count method; one bacterial cell is usually detected within 8-18 hours, a single yeast cell is detected in 20-30 hours, and mold cells require 35-48 hours. The system creates dynamic patterns as the microorganisms grow in the medium. As shown in Figure 1, the green curve shows a pattern where no growth had occurred. The curve is flat without any significant increase in the signal and no detection time (DT) is observed. The blue curve shows the pattern generated when the microorganism grows in the vial (a DT of 11 hours is observed). Highly contaminated samples rapidly detect bacteria (typically in 8-12 hours), yeast (16-24 hours) and mold (24-35 hours), providing timely warning of contamination.

Dilute-to Spec Protocol: BioLumix uses the Dilute-to-Specification protocol, which requires diluting the sample to product release specifications or in-process action levels. If growth is detected, the sample fails; if there is no detection, the sample passes (i.e., the counts are below the specification limit). For example, this protocol can be used for samples with an action level of not more than 10 cfu/g for yeast and molds and not more than 100 cfu/g for total aerobic count. If the system detects growth in a 1:10 sample dilution (1 mL of sample is added to the BioLumix vial), then the counts are >10cfu/g; if there is no detection of growth, the sample had <10cfu/g. Different dilutions can be used depending on the sample’s specification level (e.g., 0.1 mL is added to a vial when the spec is <100 cfu/g).

Instrument: Each BioLumix instrument (Figure 2) has a capacity of 32 sample locations with a single incubating temperature.

Figure 1. Typical curves with microbial growth (blue) and no growth (green)

Figure 2. The BioLumix System

Software: A personal computer with a Windows®-based program controls the operation of the BioLumix instrument(s) and is barcode capable. The software is validated to meet 21 CFR Part 11 requirements, provides an audit trail, operator identification (log in and log out), trend analysis, and provides various data reports. Detection events are automatically displayed.

Vials: A critical element of the technology is the two-zone detection vial:

� An upper incubation zone where the sample is added. � A lower reading zone that remains optically clear and free of turbidity from

microorganisms and sample components. This two-zone vial design eliminates interference of the optical pathway during color and fluorescence monitoring by the sample and microbial growth. There are two types of vials: a membrane vial and a CO2 vial.

Membrane vials: The patent-pending vial has an embedded 0.2 micron filter that separates the incubation from the reading zone.

Carbon Dioxide Vials: CO2 is a universal metabolite produced by all microorganisms. The transparent solid sensor located at the bottom of the vial detects CO2, resulting in a color change. Only gases can penetrate into the sensor; liquids, microorganisms and particulates are blocked.

Available Vials: Total Aerobic Microbial Count (TAMC), Total Combined Mold and Yeast Count (TCMY), Enterobacterial Count (Bile Tolerant Gram Negative Bacteria), and a variety of objectionable organisms, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella.

Validation Data Comparison to USP <61> USP <61> provides a method for the enumeration of mesophilic bacteria and fungi. This is to determine whether a product complies with an established specification for microbiological quality. The data generated for this comparison is summarized in Table 1. The table shows the specification ranges tested, the number of samples and the % agreement between the BioLumix method and the USP plate count method using TSA.

Total Aerobic Count: (i) Naturally contaminated samples: 201 non-sterile OTC medicine samples such as vitamins, antacids, suppositories, laxatives, Ibuproferin, aspirin, etc, were tested. All un-inoculated samples were below the action level by both methods. There was 100% agreement between the two methods in classifying samples as above or below the specification levels. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method when detecting growth over

the range of 10 cfu/g to 1,000 cfu/g. (ii) Inoculated products: 59 products were inoculated with all the organisms cited in USP <61>. Ten inoculated samples that had counts below the specification level and all were correctly classified as the challenge organism by the BioLumix system. There was 100% agreement between the two methods in determining if samples were above or below the specification level.

Table 1. Comparison Study

Figure 3. The BioLumix Vials

Yeast and Molds: (i) Naturally contaminated samples: 147 non-sterile product samples were analyzed for Yeast and Mold Count at a range of specification levels. All un-inoculated samples were below the specification level by both methods. There was 100% agreement between the two methods. This indicates the ability of the BioLumix system to yield equivalent results to the plate

count method over the range of 10-1,000 cfu/g. (ii) Inoculated products: 33 samples were inoculated with Aspergillus niger, Candida albicans, or Saccharomyces cerevisiae at various levels. E. coli was tested as a challenge organism that should not grow in this medium. 15 samples had counts below the specification level and all were correctly classified as the challenge organism by the BioLumix system. E. coli did not grow in the medium and was not detected by the instrument. There was 100% agreement between the two methods in determining that the number of organisms found in each sample were either above or below the specification level of <10-1,000 cfu/ml.

Gram Negative Bile Tolerant (Enterobacterial counts): (i) Naturally contaminated samples: 147 non-sterile product samples were analyzed for Enterobacterial Count at two specification levels. All un-inoculated samples were below the specification level by both methods. There was 100% agreement between the two methods. This indicates the ability of the BioLumix system to yield equivalent results to the plate

count method when detecting growth over the range of 10-100 cfu/g. (ii) Inoculated products: 36 samples were inoculated with Citrobacter freundii, Escherichia coli, or Salmonella enteritidis. Staphylococcus aureus was tested as a challenge organism that should not grow in this medium. There was 100% agreement between the two methods in determining that the number of organisms found in each sample were either above or below the specification level of <10-100 cfu/ml. S. aureus was not detect by the instrument.

Specificity (Inclusivity and Exclusivity)

This section relates to the ability of the vial to detect a wide range of organisms belonging to the target group, and not

detecting non-target organisms. Target organisms were inoculated at a level of 10 to 1,000 cfu/vial, while interfering

organisms were inoculated at 10,000 to 100,000 cfu/vial. The results of the specificity tests are summarized in Table 2:

Total Aerobic Count: 125 well diverse bacteria were inoculated into the TAC vial. All organisms tested were detected in the TAC vials and DT data were provided by the BioLumix instrument and software.

Yeast and Molds: 42 yeast and molds were tested in the YM vial. All detected in the vial. 25 species of various bacteria were also tested in the YM vial and none of them detected in the vial. Therefore, inclusivity and exclusivity has been demonstrated for the YM vial.

Gram Negative Bile Tolerant (Enterobacterial counts): 52 bacteria belonging to the Enterobacteriaceae family were tested. All of the organisms were detected in the ENT vial. 23 Gram-negative species and 28 Gram-positive species of bacteria that do not belong to the family Enterobacteriaceae were also tested in the Enterobacteriaceae vial and none of them were detected in the vial. Therefore, inclusivity and exclusivity have been demonstrated for the ENT vial.

Similar results were obtained for all other BioLumix vials, including as E. coli, S. aureus, Salmonella and Pseudomonas aeruginosa (data not shown).

Detection Limit

USP <1223> states that “the limit of detection is the lowest number of microorganisms in a sample that can be detected under the stated experimental conditions. A microbiological limit test determines the presence or absence of microorganisms.” The Limit of Detection was evaluated by inoculation with a low number of challenge microorganisms followed by a measurement of recovery by both the plate count and BioLumix methods. Test organisms diluted to achieve counts in the range of 1-10 cfu/g. Products were inoculated and detection of the presence of microorganisms was evaluated. Statistical analyses used the Fisher’s exact test rather than Chi square because it always gives an exact P value and works well with small sample sizes.

Table 2. Specificity Data

Total count:

The Fisher’s exact test yields a p=0.300, indicating that the Limit of Detection for the BioLumix system is equivalent to or slightly more sensitive (although not statistically significant) in detecting low numbers of microorganisms as directly compared to the plate count method. The Limit of Detection is approximately 1-3 organisms per sample vial.

Yeast and Mold:

The data showed that the Limit of Detection for the BioLumix system is equivalent to the plate method in detecting low numbers of yeast or mold as directly compared to the plate count method. The Fisher’s exact test yields a p=1.00, indicating that there is no difference between the two methods in detecting low numbers of yeast and molds.

Gram Negative Bile Tolerant (Enterobacterial Count) The data showed that the Limit of Detection for the BioLumix system is equivalent to the plate method in detecting low numbers of Enterobacteriaceae as directly compared to the plate count method. The Fisher’s exact test resulted in a p=1.00, indicating equivalence.

Table 3. Examples of two Total Count Experiments

Table 4. Examples of two Yeast and Mold

Table 5. Examples of two Enterobacterial Count

Precision or Repeatability of Data

The precision of a quantitative microbiological method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of suspensions of laboratory microorganisms across the range of the test. For each set of data the Standard Deviation (SD), Coefficient of Variation (CV) or Relative Standard Deviation (RSD) was calculated. USP <1223> states that a RSD of 15%-35% is acceptable. Vials were inoculated in quadruplicate with each test organism.

Total count: 71 quadruplicate sets were tested in the BioLumix system, with seven different types of bacteria. The average SD for the total count medium was 0.27 hours, the average RSD was 7.07%.

Yeast and Mold Count: Seven yeast strains and eight mold stains were tested in quadruplicate. Figure 3 shows some examples of the replicate curves obtained. The detection times found for individual microorganisms tested using quadruplicate vials were found to be similar within the YM test. The average SD for the yeast and mold was 0.39 hours. Molds had longer detection times and an average SD of 0.66 hours and the average RSD was 4.72%.

Gram Negative Bile Tolerant: 23 Enterobacteriaceae strains were tested in quadruplicate. The detection times found for individual microorganisms were found to be similar within this test. The average SD was 0.17 hours, the average SD was 2.94%. Similar results were obtained for other BioLumix vials such as E. coli, S. aureus, P. aeruginosa, and Salmonella.

Summary The data shown in the study indicates that the BioLumix system can be used as a RMM with comparable results to USP <61>. The method has good specificity in detecting target organisms and excluding non-target flora. The detection limit for the BioLumix system equals or is slightly better that the limit for the plate count method. High precision or repeatability was obtained for all three assays tested. The system is capable of performing multiple assays in a streamlined protocol as shown in Fig 5. The system can be used to detect presence or absence of organisms of Total aerobic count, yeast and mold, Enterobacterial count and absence of objectionable organisms in 10 grams of product, such as E. coli, S. aureus, and Salmonella.

Figure 4 Quadruplicate curves

Figure 5 Flow Chart