**3.5 RMDS applications**

106 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

phosphatase, have been described and used to remove efficiently ATP (Askgaard et al., 1995; Thore et al., 1975). Combination of apyrase and adenosine phosphate deaminase showed a good reduction of extracellular ATP and was applied to successfully detect E. coli and *S. aureus* in media broth and biological specimens (Sakakibara et al., 1997). When the objective of the assay is to detect and quantify bacterial contamination from a mixed population containing eukaryotic cells and bacteria, a differential lysis can be applied to selectively remove eukaryotic cells from the sample. This approach was used to separate bacterial ATP from biological fluids by lysing somatic cells with detergent as Triton X 100 at low concentration and combining this step with an enzymatic degradation of ATP released from lysed cells (Chapelle et al., 1978). RMDS protocol is based on sample filtration through membrane which naturally helps to eliminate extracellular ATP. If background ATP remains after filtration, rinsing the membrane with physiological serum or sterile water contributes to removal of residual ATP and allows bacterial detection. The figure 5 shows

the impact of adding rinsing steps to reduce background on beverage products.

Fig. 5. Example of 2D and 3D views given by RMDS software for flavored water analysis with and without rinsing with sterile water. Picture A shows light spots corresponding to ATP present naturally in the filtered sample. Picture B shows the impact of rinsing water to

A

B

A protocol was developed to use RMDS to detect and quantify bacterial contamination from a mixture of mammalian cells and bacteria. The filtration of mammalian cells and bioluminescence detection through RMDS protocol shows (see Fig.6A) a high amount of light produced by mammalian cells preventing any bacterial detection. The sample treatment with a combination of a mammalian cells lysis solution and with apyrase contributes to efficiently remove the bioluminescent background and the figure 6B demonstrates that light spots remain detectable. These spots correspond to light emitted by bacteria in the mixture. Results obtained show that ATP-bioluminescent assay could be a

powerful tool to microbiologically and quickly monitor eukaryotic cell cultures.

remove background.

## **3.5.1 Use of Bioluminescence for microorganisms detection in water**

Water is a key raw material utilized in the manufacturing of products within the food and beverage, healthcare, microelectronics and pharmaceutical industries. Within each industry, different regulatory requirements exist for microbial contamination in the water used for the manufacturing of a product for a specific application. The microorganisms found in these water systems are mainly stressed, slow-growing strains characterized by long incubation times before growth can be detected using traditional microbiology methods such as membrane filtration or pour plates. The time it takes before contamination can be detected in water can cause delays in product release, and extend the storage time of products. Using a rapid Bioluminescence based detection method allows manufacturers to identify microbial

Use of ATP Bioluminescence for Rapid Detection and Enumeration

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 109

An easy protocol was developed to quickly enumerate spore contamination in artificially inoculated products with RMDS. Physiological water was inoculated with a calibrated concentration of *Bacillus subtilis* spores. After a heat shock at 80°C for 10 min, the inoculated product followed the protocol described in section 3.3. The incubation was performed with R2A medium at 32.5°C+/- 2.5°C. Results show that ATP bioluminescent signal start to be detected after 4h of incubation and that the reliable detection and enumeration of spores was achieved after 5 hours. Results given by RMDS are consistent with the expected inoculation level of the product and exhibit a recovery of almost 100% (tests performed in triplicate) compared with the control plate incubated 48h. Figure 7 gives an example of spore detection after 5h of incubation with R2A medium. RMDS protocol provides an

alternative approach to perform rapid detection of spores in filterable matrix.

Fig. 7. RMDS 2D and 3D views showing Bacillus spore detection and enumeration.

**3.5.3 Use of RMDS for the rapid detection of contaminants in bioreactor samples** 

RMDS has also been evaluated to detect contaminants in complex matrix containing mammalian cells. Mammalian cells including hybridoma are widely used in the biotechnology industry. Cell culture batches as well as consecutive downstream processes must be thoroughly monitored for microbial contamination. The ATP-bioluminescence technology is not selective of microbial ATP. The mammalian ATP released from the cells produces an interfering signal that must be eliminated to allow accurate counting of cfus.

Triton X100 combined with ATPase was already described to selectively extract and degrade ATP from blood products and urine samples enabling specific bacterial detection (Thore et

A simple and fast pretreatment method based on a selective lysis of mammalian cells and ATP removal has been developed. The harmlessness of this treatment for microorganisms was demonstrated, allowing the use of the RMDS to monitor mammalian cell samples. The protocol is as fellow: 1. Differential lysis of the mammalian cells (Chinese hamster ovary [CHO]-K1, ATCC CCL-61) using the selective mammalian cell lysis solution (Millipore MSP010053); 2. removal of mammalian ATP using 5U apyrase (Sigma Aldrich); 3. Milliflex Rapid membrane filtration using Milliflex funnel; 4. phosphate buffered saline rinsing to remove remaining mammalian ATP; 5. membrane incubation for bacteria growth; and 6.

detection and counting of bacteria using the RMDS as described in the protocol.

al., 1985).

contamination earlier, which provides them with better process control, product yield, and shortens time to market.

The following table 3 provides the incubation times for detectable growth, by organism, for the traditional microbiology method and RMDS. The detection time is significantly reduced using RMDS. Detection of growth is on average 4.5 times faster than traditional microbiology, and up to 6 times faster for the very slow growers tested (*Methylobacterium mesophilicum* ATCC 29983, stressed strain of *Methylobacterium* and a mix of various slowgrowing strains). RMDS allows for overnight detection of the industrial-stressed microorganisms tested. The incubation temperature also has an influence on time-to-result. Incubating at 25 °C showed that longer incubation times were required (data not shown). The mean recovery between RMDS and the traditional microbiology method in these experiments was 92.7%, which shows the equivalence of the two methods.


Table 3. Detection time of reference strains and water isolates in traditional method and RMDS using either R2A agar, Tryptic Soy Agar (TSA) or Plate Count Agar (PCA).

#### **3.5.2 Rapid detection of spores**

Spores are major food spoilages and are also a concern in pharmaceutical samples. The classical microbiological method to enumerate spore contamination combines heat shock and on average 5 days incubation into sterile and molten specific medium Agar (Wayne et al., 1990). The amount of ATP in spores is very low and germination is necessary to increase ATP content and develop a rapid detection method based on ATP-bioluminescence (Kodata et al., 1996). ATP-bioluminescence rapid screening assay has been described showing that after germination, spore containing powder has been detected in a short time with a detection limit of 100 spores (Lee & Deininger, 2004). Fujinami et al (2004) also showed that short incubation of the sample in nutrient broth medium containing L-alanine increased RLU from spores and optimize the ATP- bioluminescent assay.

contamination earlier, which provides them with better process control, product yield, and

The following table 3 provides the incubation times for detectable growth, by organism, for the traditional microbiology method and RMDS. The detection time is significantly reduced using RMDS. Detection of growth is on average 4.5 times faster than traditional microbiology, and up to 6 times faster for the very slow growers tested (*Methylobacterium mesophilicum* ATCC 29983, stressed strain of *Methylobacterium* and a mix of various slowgrowing strains). RMDS allows for overnight detection of the industrial-stressed microorganisms tested. The incubation temperature also has an influence on time-to-result. Incubating at 25 °C showed that longer incubation times were required (data not shown). The mean recovery between RMDS and the traditional microbiology method in these

30°C

R2A PCA TSA R2A PCA TSA

*P. aeruginosa* ATCC 9207 1 day 1 day 1 day 9 hrs 9 hrs 9 hrs *M. mesophilicum* ATCC 29983 6 days 6 days MNA 26 hrs 26 hrs MNA *E. coli* ATCC 8739 1 day 1 day 1 day 6 hrs 6 hrs 6 hrs *B. cepacia* ATCC 25416 ND ND 2 days ND ND 16 hrs *S. epidermidis* ATCC 12228 ND ND 1 day ND ND 9 hrs

strains 6 days 6 days MNA 24 hrs 24 hrs MNA

*Methylobacterium* 6 days 6 days MNA 24 hrs 24 hrs MNA

*pickettii* 2 days 2 days ND 11 hrs 11 hrs ND

Table 3. Detection time of reference strains and water isolates in traditional method and RMDS using either R2A agar, Tryptic Soy Agar (TSA) or Plate Count Agar (PCA).

Spores are major food spoilages and are also a concern in pharmaceutical samples. The classical microbiological method to enumerate spore contamination combines heat shock and on average 5 days incubation into sterile and molten specific medium Agar (Wayne et al., 1990). The amount of ATP in spores is very low and germination is necessary to increase ATP content and develop a rapid detection method based on ATP-bioluminescence (Kodata et al., 1996). ATP-bioluminescence rapid screening assay has been described showing that after germination, spore containing powder has been detected in a short time with a detection limit of 100 spores (Lee & Deininger, 2004). Fujinami et al (2004) also showed that short incubation of the sample in nutrient broth medium containing L-alanine increased

MNA : Medium Not Appropriate for growth of microorganism, ND : Not Done

RLU from spores and optimize the ATP- bioluminescent assay.

Milliflex Rapid Detection System 30°C

experiments was 92.7%, which shows the equivalence of the two methods.

Microorganisms Traditionnal Microbiology

shortens time to market.

**ATCC Strains** 

Stressed strain of

**Industrial-Stressed Microorganisms** 

Mix of various slow-growing

Environmental isolate of *R.* 

**3.5.2 Rapid detection of spores** 

An easy protocol was developed to quickly enumerate spore contamination in artificially inoculated products with RMDS. Physiological water was inoculated with a calibrated concentration of *Bacillus subtilis* spores. After a heat shock at 80°C for 10 min, the inoculated product followed the protocol described in section 3.3. The incubation was performed with R2A medium at 32.5°C+/- 2.5°C. Results show that ATP bioluminescent signal start to be detected after 4h of incubation and that the reliable detection and enumeration of spores was achieved after 5 hours. Results given by RMDS are consistent with the expected inoculation level of the product and exhibit a recovery of almost 100% (tests performed in triplicate) compared with the control plate incubated 48h. Figure 7 gives an example of spore detection after 5h of incubation with R2A medium. RMDS protocol provides an alternative approach to perform rapid detection of spores in filterable matrix.

Fig. 7. RMDS 2D and 3D views showing Bacillus spore detection and enumeration.

## **3.5.3 Use of RMDS for the rapid detection of contaminants in bioreactor samples**

RMDS has also been evaluated to detect contaminants in complex matrix containing mammalian cells. Mammalian cells including hybridoma are widely used in the biotechnology industry. Cell culture batches as well as consecutive downstream processes must be thoroughly monitored for microbial contamination. The ATP-bioluminescence technology is not selective of microbial ATP. The mammalian ATP released from the cells produces an interfering signal that must be eliminated to allow accurate counting of cfus.

Triton X100 combined with ATPase was already described to selectively extract and degrade ATP from blood products and urine samples enabling specific bacterial detection (Thore et al., 1985).

A simple and fast pretreatment method based on a selective lysis of mammalian cells and ATP removal has been developed. The harmlessness of this treatment for microorganisms was demonstrated, allowing the use of the RMDS to monitor mammalian cell samples. The protocol is as fellow: 1. Differential lysis of the mammalian cells (Chinese hamster ovary [CHO]-K1, ATCC CCL-61) using the selective mammalian cell lysis solution (Millipore MSP010053); 2. removal of mammalian ATP using 5U apyrase (Sigma Aldrich); 3. Milliflex Rapid membrane filtration using Milliflex funnel; 4. phosphate buffered saline rinsing to remove remaining mammalian ATP; 5. membrane incubation for bacteria growth; and 6. detection and counting of bacteria using the RMDS as described in the protocol.

Use of ATP Bioluminescence for Rapid Detection and Enumeration

**3.5.5.1 Specific detection protocol** 

**3.5.5 Use of RMDS for specific detection of** *Pseudomonas aeruginosa*

the bioluminescent reaction is recorded by the CCD camera of RMDS.

detect and enumerate *P. aeruginosa* with RMDS (Fig 8):

Fig. 8. *P. aeruginosa* specific detection and enumeration protocol

This procedure was also used to obtain both total viable count (TVC) and specific detection and enumeration of *P. aeruginosa* using the same membrane sample. The adapted procedure

pour 50 mL of saline solution into a Milliflex funnel. Spike the appropriate dilution of each microorganism into the funnel (10–100 CFUs). Add 50 mL of saline solution into the funnel

performed inside a laminar flow hood.

is as fellow:

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 111

RMDS was used with specific hybridization probes targeting *P. aeruginosa* and coupled to Soy Bean Peroxydase. A new and unique permeabilization solution was developed and is based on polyethylimine (PEI). Cells are fixed on the membrane using a formaldehyde mix. Hybridization was performed using Peptide Nucleic Acid probes targeting 16S RNA conjugated to Soybean peroxydase diluted in Hybridization buffer. Free probes are washed with a Tween buffer. SBP catalyzes conversion of Luminol into photons and light activity of

The following procedure was used to determine the minimum incubation time necessary to

1. Pour 50 mL of saline solution into a Milliflex funnel; 2. Spike the appropriate dilution of each microorganism into the funnel (10–100 CFUs); 3.Add 50 mL of saline solution into the funnel to homogenize the content; 4. Filter and transfer the membrane onto a prefilled TSA Milliflex cassette. Incubate at 32.5 °C ± 2.5 °C for the appropriate time; 5. Once incubation is complete, separate the membrane from the cassette and let the membrane dry; 6. Follow the Milliflex Rapid *P. aeruginosa* detection procedure described before; 7. Spray the specific detection reagents using the Milliflex Rapid AutoSpray Station; 8. Read the sample with the Milliflex Rapid Detection and Enumeration System. Steps 1 through 4, 6 and 8 were

This method enabled the detection of microorganisms in the presence of up to 5.107 eukaryotic cells, and involved a single pre-treatment step of the sample prior to filtration. Figure 3 (paragraphe 3.2) demonstrates that in E. coli-contaminated CHO cells, the pretreatment removed specifically mammalian ATP and enabled the enumeration of contaminants.

The harmlessness of the cells treatment toward microorganisms was also demonstrated using *B. subtilis*, *S. aureus*, *P. aeruginosa* and *Candida albicans* spiked at approximately 50 cfus with a recovery ranging from 80% to 109.7% compared with traditional microbiology counts. During the filtration step, mycoplasma, unlike bacteria, will pass through a 0.45 µm filter (Baseman & Tully, 1997). Moreover, mycoplasma membranes are easily solubilized by detergents, and the lysis of mammalian cells simultaneously affects mycoplasma viability.

The specific mammalian cell lysis solution coupled with the RMDS method allowed fast detection of contaminating microorganisms in high value cell samples. Using RMDS to detect and quickly enumerate microbial contamination in biotechnology samples such as eukaryotic cells will allow better control throughout the process.

## **3.5.4 Rapid sterility testing based on ATP-Bioluminescence**

In pharmaceutical companies, products are released based on microbiological quality. The Sterility test is a mandatory and critical step to ensure that the product is free of microorganism. The test takes 14 days of incubation before getting results. Time is the main reason why there is a need for an alternative and rapid method. Du to its universality and high sensitivity, the ATP-bioluminescence technology represents an alternative to ease sterility testing and shorten incubation time (Bussey & Tsuji, 1986). In addition to reduce time to detection, ATP-bioluminescence brings a solution to one drawback of current methods. Light detection replaces the subjectivity of visual determination of turbidity. Bioluminescence test that uses adenylate kinase reaction to convert ADP in ATP to significantly amplify the signal is described as a rapid sterility alternative method with results below or equal to 7 days (Albright, 2008).

Sterility testing based on RMDS follows the protocol described in section 3.2 with the major difference that the filtration step is performed under an isolator or a sterile chamber to ensure a sterile environment throughout the test. Reducing the incubation time from 14 days to 5 days is an achievable goal which benefits pharmaceutical companies. As RMDS is based on filtration, this method is compatible with complexe matrices. A comparative study was performed between RMDS and technologies based on CO2 detection. Peptone water and biological matrix such as inactivated influenza vaccines were inoculated with low concentration of microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi. Results showed that RMDS detected all microorganisms significantly faster than the compendial method (Parveen et al., 2011). RMDS using incubation onto Schaedler Blood Agar detected all tested microorganisms in 5 days in the presence of a matrix containing preservative 0.01% thimerosal and was also compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants (Parveen et al., 2011). RMDS is likewise used as rapid sterility testing by other pharmaceutical company and shows no interference with bioluminescence mechanism and a detection in 5 days of stressed and reference strains including worst microorganism such as *Propionibacterium acnes* (Gray et al., 2010).
