May 1, 2014 (Vol. 34, No. 9)

Getting to Pathogens before They Get to You

Adventitious microorganisms—the bane of bioprocessing—may mingle with raw ingredients, light upon exterior surfaces, or settle into bioreactors. To deal with such organisms, bioprocessors need to understand them, the better to deter such unwelcome guests, avoiding all the problems associated with microbiological contamination.

Numerous techniques have emerged that allow bioprocessors to detect contaminants without having to tolerate lengthy delays—the days or weeks that would be required to culture organisms formally. Yet, one way or another, all these techniques rely on concentrating samples.

The BioLumix platform consists of an incubating/reading instrument and proprietary vials. Organism expansion occurs in the top zone, and reading takes place within a separate bottom zone.

The system detects microorganisms by monitoring changes in color or fluorescence changes in a microbial liquid growth medium. The color changes resulting from microorganism metabolism are checked every six minutes. By separating the growth and read zones, the system eliminates interference from sample matrix and microbial turbidity.

Signal is relatively constant until microorganisms reach a threshold value, after which it accelerates rapidly. Thresholds are 10,000 and 100,000 cells per milliliter for yeast/mold and bacteria, respectively.

According to Ruth Eden, Ph.D., president of BioLumix, the system’s sensitivity is a single viable bacterium or fungus per sample vial. “Bacterial cells are detected in 8 to 24 hours,” notes Dr. Eden, “while yeast are detected in less than 48 hours.”

Time to detection depends on the initial microorganism concentration: higher equals faster. Using conventional culture, bacterial counts take between two and three days; yeast/mold, up to five days.

BioLumix operates under USP <61> and USP <62> for testing nonsterile raw materials and surfaces.

BioLumix is unsuitable for sterile injectable products, whose purity is guaranteed by kill steps involving heat or chemicals (and sterilizing filtration) and confirmed post-production through lengthy culture-based assays. Rather, its strength lies in testing of raw materials and laboratory or process area surfaces.

In any event, volumes required for testing sterile batches would be too high for the BioLumix system, Dr. Eden explains: “It’s used before the bioreactor. People could in theory pre-enrich the sample and then go into the vial, but most would prefer a system that simply accepts much larger volumes than we do, in the 100 to 500 milliliter range.”

Automated, on-site microbiology testing has the advantages of rapid results and walk-away operation. “The process is fully automated and paperless,” Dr. Eden tells GEN. “And you don’t need a microbiologist to run it, although you should probably have one nearby to interpret the results.”

BioLumix technology is based on monitoring changes in a broth medium in which the target organisms grow. In the main image, a technician enters samples into the incubating/reading instrument. In the inset, the disposable two-zone vial is shown. The vial includes an incubation zone (top) and a reading zone (bottom). The reagents at the bottom of the vial change their color/fluorescence due to microbial metabolism. These changes are continuously monitored by a single optical sensor.

ATP Bioluminescence

Like the Biolumix system, many rapid tests employ indicators of viable and actively multiplying organisms.

For example Pall’s Pallchek™ rapid microbiology system uses ATP bioluminescence to detect living, growing organisms. Unlike the Biolumix system, however, PallChek encompasses sterility testing for finished products. PallChek’s underlying rationale is the Pharmaceutical Inspection Co-Operation Scheme (PIC/S) document PI 012, Recommendation on Sterility Testing, which states that “sterility of a product is defined by the absence of viable and actively multiplying microorganisms when tested in specified culture media.”

“ATP is the fuel that energizes all cellular activity,” explains Tricia Griffiths, global product manager, microbiology at Pall Life Sciences. “However it is a very unstable molecule.” ATP chemical bonds break easily, which releases energy. Once a cell dies, ATP levels decrease drastically, becoming undetectable in a matter of hours.

The Pallchek protocol involves concentration of the sample on a membrane filter; washing to remove potential interferences; addition of extract reagent followed by luciferin/luciferase enzyme/substrate; and light measurement. Cross-contamination by the operator is the leading cause of a false positives, but this can be easily avoided by wearing sterile gloves and working inside of an appropriate enclosure.

Users of rapid microbiological assays often need to confirm the identity of the contaminating organism. Genome Sequence Scanning™ (GSS) by PathoGenetix claims to provide molecular serotyping and bacterial strain typing from complex samples in five hours, which according to John Czajka, Ph.D., vp for business development, is “days faster and less costly than current techniques.”

“Our system creates a genetic fingerprint for the target pathogen,” Dr. Czajka asserts. “We’re positioning it as a secondary analysis, after achieving a positive result through screening.”

Suitable samples include biopharmaceutical ingredients and products as well as environmental swabs. GSS does not work directly with those samples, but from the enriched, processed samples required by most rapid screens. In some instances, the technique requires secondary enrichment that increases pathogen counts while diluting matrix.

GSS confirms pathogen species and serotype based on genomic signature, and indicates if the sample matches a species in the GSS database. If not, it will indicate the strain most closely related to the actual contaminant.

“Because we use single molecule analysis, we can identify pathogens in mixed culture, and even multiple strains of the same organism,” Dr. Czajka adds. “Other serotyping methods require pure culture isolates.”

Can You Cheat with MALDI?

All rapid microbiology tests require some level of enrichment of target pathogens or surrogate molecules, and all tests must somehow match practical sample size and pathogen concentration with the test’s physical and detection capabilities.

Even new methods such as mass spectroscopy (MS) encounter these issues. Interest in MS as a pathogen identifier has exploded of late, particularly in clinical laboratories. Gentle ionization methods such as MALDI (matrix-assisted laser desorption ionization) digest bacteria and provide identification based on fragmentation of signature proteins. Theoretically, MALDI requires little or no sample preparation. In practice, microorganisms must be present at detectable concentrations.

“It all comes down to sensitivity,” Dr. Eden says. “What kind of volumes can you inject into a mass spectrometer?”

MS also has difficulty with mixed cultures, an issue that many rapid microbiology tests have overcome. MS only works by comparing an organism’s fragmentation pattern with that of a known reference, and the instrumentation is expensive.

“Ideally, you want the organism to exist at detectable levels in a small volume,” Dr. Eden adds. “Which means you still have to grow the organism before doing the analysis.”

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