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

### **3.5.5.1 Specific detection protocol**

110 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

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

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

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

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).

eukaryotic cells will allow better control throughout the process.

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

results below or equal to 7 days (Albright, 2008).

contaminants.

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 bioluminescent reaction is recorded by the CCD camera of RMDS.

The following procedure was used to determine the minimum incubation time necessary to detect and enumerate *P. aeruginosa* with RMDS (Fig 8):

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 performed inside a laminar flow hood.

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 is as fellow:

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

Use of ATP Bioluminescence for Rapid Detection and Enumeration

detection count data were then analyzed (fig.10).

CFUs were detected in each assay.

**Rapid microbiology detection of TVC (A) and**  *P.aeruginosa* **specific detection (B) using pure culture of**  *P.aeruginosa* **ATCC 9027**

[TSA, 32.5 ± 2.5°C]

contaminants. The limit of the sensitivity is 1 CFU (data not shown).

**Rapid Microbiology Count 15 CFUs Specific Detection Count 15 CFUs Recovery 100% Incubation time 9 h Overall procedure 11 h 30** 

culture of *P. aeruginosa* ATCC 9027 incubated on TSA at 32.5°C+/-2.5°C.

Fig. 10. Specific detection of *P .aeruginosa* after TVC on the same membrane using pure

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 113

Using the specific detection procedure described above *P. aeruginosa* was detected and enumerated in 8 hours in a water sample. The specificity of the method has been assessed against numerous microorganisms and only *P. aeruginosa* was detected in this panel of

The objective of this experiment was to first obtain the TVC in CFUs using the total viable count assay, followed by the specific detection assay for *P. aeruginosa*. After performing the TVC analysis, the results were stored on the Milliflex Rapid system and the same membrane was then treated following the specific detection procedure. The TVC and the specific

Figure 10 provides results for both TVC and specific detection of *P. aeruginosa* using the same membrane. The images below show that the position of each colony forming unit is identical when using the TVC and specific detection assay. One hundred percent of the

> **2D view 3D view A**

> > **B**

to homogenize the content. Filter and transfer the membrane onto a pre-filled TSA Milliflex cassette. Incubate at 32.5 °C ± 2.5 °C for the appropriate time. Once incubation is complete, separate the membrane from the cassette and let the membrane dry. Spray the ATP releasing and bioluminescence reagents using the Milliflex Rapid AutoSpray Station. Read the sample with the RMDS. Then, follow the Milliflex Rapid P. aeruginosa detection procedure starting from fixation step. Spray the specific detection reagents using the Milliflex Rapid AutoSpray Station and read the sample with the Milliflex Rapid Detection and Enumeration System.

## **3.5.5.2 Specific** *Pseudomonas aeruginosa* **detection and total viable count results**

The Milliflex Rapid system is a proven automated solution for the rapid detection and enumeration of total viable count (TVC) in purified water and Water For Injection. Based on membrane filtration and image analysis together with an adenosine triphosphate (ATP) bioluminescence reagent, the Milliflex Rapid System delivers TVC test results faster than traditional methods. We have developed a hybridization assay that enables the Milliflex Rapid system to specifically detect and enumerate *P. aeruginosa*. The hybridization assay is performed with a peroxidase-conjugated DNA-oligonucleotide probe targeted to a specific RNA-sequence of *P. aeruginosa*. Applying luminol and peroxide substrates to the membrane filtration sample generates light that is detected by the Milliflex Rapid system.

In order to determine the minimal incubation time to detect *P. aeruginosa*, a pure culture of *P. aeruginosa* ATCC 9027 was spiked into Milliflex and incubated on TSA for 6 hours for the alternative method and on R2A for 24 hours for the compendial method. Results are presented in Figure 9.


Fig. 9. Specific Detection and enumeration of *P. aeruginosa*

to homogenize the content. Filter and transfer the membrane onto a pre-filled TSA Milliflex cassette. Incubate at 32.5 °C ± 2.5 °C for the appropriate time. Once incubation is complete, separate the membrane from the cassette and let the membrane dry. Spray the ATP releasing and bioluminescence reagents using the Milliflex Rapid AutoSpray Station. Read the sample with the RMDS. Then, follow the Milliflex Rapid P. aeruginosa detection procedure starting from fixation step. Spray the specific detection reagents using the Milliflex Rapid AutoSpray Station and read the sample with the Milliflex Rapid Detection

The Milliflex Rapid system is a proven automated solution for the rapid detection and enumeration of total viable count (TVC) in purified water and Water For Injection. Based on membrane filtration and image analysis together with an adenosine triphosphate (ATP) bioluminescence reagent, the Milliflex Rapid System delivers TVC test results faster than traditional methods. We have developed a hybridization assay that enables the Milliflex Rapid system to specifically detect and enumerate *P. aeruginosa*. The hybridization assay is performed with a peroxidase-conjugated DNA-oligonucleotide probe targeted to a specific RNA-sequence of *P. aeruginosa*. Applying luminol and peroxide substrates to the membrane

In order to determine the minimal incubation time to detect *P. aeruginosa*, a pure culture of *P. aeruginosa* ATCC 9027 was spiked into Milliflex and incubated on TSA for 6 hours for the alternative method and on R2A for 24 hours for the compendial method. Results are

**2D view 3D view** 

**10 CFUs** 

**11 CFUs** 

**3.5.5.2 Specific** *Pseudomonas aeruginosa* **detection and total viable count results** 

filtration sample generates light that is detected by the Milliflex Rapid system.

**Recovery 91% Incubation Time 6h** 

Fig. 9. Specific Detection and enumeration of *P. aeruginosa*

and Enumeration System.

presented in Figure 9.

**Rapid Milliflex Microbiology Detection of P.aeruginosa** 

> **Specific Detection Count [TSA, 32.5 ± 2.5°C]**

**Traditional Microbiology Count [R2A, 25 ± 2.5°C]** 

Using the specific detection procedure described above *P. aeruginosa* was detected and enumerated in 8 hours in a water sample. The specificity of the method has been assessed against numerous microorganisms and only *P. aeruginosa* was detected in this panel of contaminants. The limit of the sensitivity is 1 CFU (data not shown).

The objective of this experiment was to first obtain the TVC in CFUs using the total viable count assay, followed by the specific detection assay for *P. aeruginosa*. After performing the TVC analysis, the results were stored on the Milliflex Rapid system and the same membrane was then treated following the specific detection procedure. The TVC and the specific detection count data were then analyzed (fig.10).

Figure 10 provides results for both TVC and specific detection of *P. aeruginosa* using the same membrane. The images below show that the position of each colony forming unit is identical when using the TVC and specific detection assay. One hundred percent of the CFUs were detected in each assay.


Fig. 10. Specific detection of *P .aeruginosa* after TVC on the same membrane using pure culture of *P. aeruginosa* ATCC 9027 incubated on TSA at 32.5°C+/-2.5°C.

Use of ATP Bioluminescence for Rapid Detection and Enumeration

matrices containing high concentrations of eukaryotic cells.

followed by specific detection of this very specific bacterium.

systems usually deliver after the treatment.

applications.

**6. References** 

**5. Acknowledgment** 

R&D laboratory (Molsheim, France).

Bioluminescence Test.

*Biologicals*, 23(1), 55-60.

*Health,* 209(2), 203-206.

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 115

The association of Bioluminescence to sensitive sensors such as RMDS provides a result in colony forming units equivalent to the standard plate count but is 4 times faster than classical microbiology. This method can be used in samples from industrial water, to food and beverage samples for the detection of any type of bacteria, yeasts and molds including spores. We also showed that it can be used to detect bacterial contamination in cell culture

Interestingly, Bioluminescence was also coupled to molecular biology through the use of 16S RNA probes for specific detection of bacteria. The example presented here allowed not only the detection of *P. aeruginosa* but also the total viable count using Luciferin and luciferase

Finally, the development of the method in a pharmaceutical environment allowed sterility testing of drug products 3 times faster than the compendial method. This recent developments in the pharmaceutical field show that the method is also able to help patients taking drugs usually associated with a very short shelf life (gene therapy products, cell therapies...) as the result is delivered before the injection of the product while the traditional

In conclusion, the use of Bioluminescence either in its "classical" or molecular format allows for a number of developments in the field of microorganisms detection. The flexibility of the method and its ease of use coupled to the considerable savings in time compared to the traditional method make it a valuable tool for life scientists as well as for other clinical

Authors would like to thanks colleagues from Merck-Millipore Application group, Development group and Predevelopment - Technology – Collaboration for their technical collaboration. The research described in this paper was carried out at the Merck-Millipore

Albright, J. (2009). Implementing Rapid Sterility using the Celsis Enhanced ATP

 www.celsis.com/media/pdf/rdpdfs/Poster\_RapidSterilityTesting\_PDA0904.pdf Andreotti, P. E. & Berthold, F. (1999). Application of a new high sensitivity luminometer for industrial microbiology and molecular biology. *Luminescence*, 14(1), 19-22. Askgaard, D. S.; Gottschau, A; Knudsen, K. & Bennedsen, J. (1995). Firefly luciferase assay of

Aycicek, K., Oguz, U. & Karci, K. (2006). Comparison of results of ATP bioluminescence

Baseman, J. B. & Tully, J.G. (1997). Mycoplasmas: Sophisticated, reemerging and burdened

by their notoriety. *Emerging Infectious Disease*, 3, 21-32.

adenosine triphosphate as a tool of quantitation of the viability of BCG vaccines.

and traditional hygiene swabbing methods for the determination of surface cleanliness at a hospital kitchen. *International Journal of Hygiene and Environmental* 

In a second assay, a mixed microbial population composed of *P. aeruginosa, Burkholderia cepacia* and *E. coli* were spiked and analyzed with the procedure described in "Combination of Total Viable Count and Specific Detection of P. aeruginosa." Results are presented in the figure 11. After 9 hours growth at 35 °C, 24 CFUs were detected after the TVC procedure and 8 CFUs were detected using the *P. aeruginosa* specific detection procedure.

This demonstrates that the system is able to make TVC and specific detection even in a mixed population of microorganisms.

Fig. 11. Specific detection of *P. aeruginosa* after TVC on the same membrane using a mixed population of *P. aeruginosa* ATCC 9027, B. cepacia ATCC 25416 and E.coli ATCC 25922 incubated on TSA at 32.5°C+/-2.5°C.
