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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Microbial identity data can be critical for determining contamination sources.
In bio/pharmaceutical manufacturing, monitoring the bioburden of raw materials, intermediates, drug substances, formulated drug products, and processing environments is essential for ensuring patient safety. Successful bioburden monitoring requires knowledge of both the quantity and identity of detected microbes. The level of information and extent of microbe characterization, and thus the testing protocols, required depend on the specific sample and situation. In cases of potential contamination, knowledge of the identity of a contaminant can help determine its source and thus an appropriate course of action. It is essential, therefore, to implement a microbial identification strategy as part of an effective microbial control program.
Microbial identification is an important and often overlooked component of bioburden monitoring programs, according to Phil Tuckett, study director at Nelson Laboratories. The intent is to characterize microbes to differentiate one type from another. Identification allows placement of the microbe, depending on the required level of identification and the type of testing employed, into a specific family (genus), species, and/or strain.
Microbial identifications can be used to provide a platform for thorough investigations, such as for determining the nature of specific contamination events, according to Poonam Bhende, assistant manager at SGS Life Sciences. Microbial identity determination can also be used in a broader manner to provide a rough estimate of the bioburden in a dose of product as an indication of its sterility.
“It is important to understand not only the numbers of microorganisms present in a product, but also the types of microorganisms they are. Particularly with regard to bioburden reduction strategies, the identity of a microorganism can dictate the best practice for eliminating it,” says Tuckett. Indeed, Bhende notes that through detailed and accurate microbial identification, it is possible to narrow down the source of contamination and take appropriate measures to mitigate the risk for future contamination.
There are several methods available for microbial identification. They are generally classified as phenotypic or genotypic techniques.
Phenotypic testing provides data on the physical properties (i.e., morphology, reaction to different chemicals, behavior under certain conditions) that are indicative of a microbe’s genus and in some cases species. “Phenotypic methods, which focus on outward characteristics of an organism-appearance, staining characteristics, biochemical utilization, metabolic requirements, protein analysis-are important components of the microbial characterization level. Given enough of these tests, along with a high level of expertise, a genus/species ID may be obtained,” observes Tuckett. Currently, these methods are most widely used because they tend to be lower in cost and easier to implement. They are, however, generally culture-based and growth dependent, and results can vary with the media and growth conditions that are used. In addition, because many phenotypic tests involve studying the response to treatment with biochemical reagents, repeatability can be an issue.
Automated systems have been developed to overcome some of these limitations, including Fourier-transform infrared spectroscopy, matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry, and flow cytometry.
The industry is, however, moving toward genotypic identification methods due to the growing number of species that are being described every year, according to Tuckett. Genotypic methods involve analysis of the genetic makeup and provide information on the genus, species, and in some cases, the strain of the microbe.
Analysis of the genome is achieved either through hybridization or sequencing. In hybridization, the extent to which the microbe’s DNA binds with known DNA strands provides information on its structure. Sequencing, generally of the 16S rRNA region, a highly conserved, sufficiently large region present in most bacteria (or the large subunit ribosomal gene in yeasts and molds) is achieved using automated blot technology or polymerase chain reaction (PCR) approaches. Importantly, genotypic testing is not affected by culture or media conditions. “As technology becomes cheaper and more available, whole genome sequencing may provide accurate species and strain identifications,” Tuckett states.
Separately, the detection and identification of endotoxins indicates the presence of gram-negative bacteria. “Contamination with high levels of endotoxin can be fatal, so accurate results are vital,” asserts Bhende.
Microbial identification, according to Pia Darker, global senior product manager for the pharmaceutical analytics division of Thermo Fisher Scientific, is most often the end point of different microbiological tests, such as out-of-specification bioburden, failed sterility tests, excursions from environmental monitoring, etc.
The strategy for identification will depend on the origin of the test samples, as well as the microbial identification method, both of which depend on the overall microbial test strategy. “The first thing to consider is what level of identification is appropriate, and that depends on what the data will be used for,” adds Tuckett.
There are three basic levels of identification: microbial characterization, genus/species identification, and bacterial strain typing. “For tracking and trending of bioburden levels only, microbial characterization may be sufficient, such as descriptions of the colonies and cells as well as gram staining and other descriptive microbiological assays,” he notes. Such characterization requires a certain level of expertise because appearances can be variable and many characteristics of microorganisms change over time. Given the inherent subjectivity of some of these tests, Tuckett strongly recommends that genus/species identification be performed for at least the overall three to five most common organisms.
In situations where action/alert levels are exceeded or when contamination events are encountered, genus/species level identification is more appropriate. “Since microbial identification is used to understand the source of contamination, it is important for the identification method to give an accurate species identification in order to implement appropriate corrective actions and preventative actions (CAPA). Implementation of the appropriate CAPA will prevent the re-occurrence of microbial contamination and reduce the risk of quality issues in the manufacturing process,” Darker comments.
Species identification is also an essential part of testing for objectionable organisms (United States Pharmacopeia 62 testing), according to Tuckett, because mere characterization can sometimes be insufficient to rule out specific species.
At SGS, most clients ask for species-level identification. “Species-level identification helps us to eliminate the risk of an antibiotic-resistant pathogen becoming prevalent if a new strain is observed that cannot be eliminated through cleaning by disinfectant. Additionally, seasonal change can see a change in bioburden levels which need to be identified,” says Bhende.
When investigations are being conducted to determine if multiple contaminants are of the same source, bacterial strain typing is necessary. “This testing reveals if different isolates come from the same strain or source, which cannot be determined from species level identification methods,” Tuckett explains.
An effective microbial identification strategy, according to Darker, results in an appropriate CAPA to reduce any risk of microbial contamination and in the event of microbial contamination enables the determination of the root cause of the contamination. “In essence, if CAPA has been put in place and it mitigates any further risk to product quality and patient safety, then the microbial identification strategy was effective,” she states.
For Tuckett, an effective microbial identification strategy is one that provides meaningful data pertinent to the given situation and draws upon sufficient resources to ensure the identification is as accurate as possible. “The basic principle behind microbial identification is comparison of the characteristics of an unknown organism to those of a known organism. The more that is known about the known organism, the better the comparisons can be. When genotypic data [are] analyzed, [they are] generally compared to a library or database of known DNA sequences and [are] therefore only as accurate as the database [they draw] upon. An extremely limited database may provide inaccurate identifications,” he explains. In addition, methods based on automated identification software may be inadequate for the intended use if a high number of “unidentified” results are obtained.
An ineffective strategy is also one for which a CAPA cannot be implemented to stop the reoccurrence of contamination by an identified microbe, according to Bhende. Therefore, as with bioburden and environmental monitoring programs, continuous evaluation and assessment of microbial identification strategies are essential. “As compendial requirements change, and as new technologies become available, identification procedures should be revaluated to ensure they are sufficient for their intended purpose. If a specific identification method doesn’t yield adequate results, alternate methods should be considered,” asserts Tuckett.
For clients of SGS, Bhende also recommends for critical microbial identification applications the establishment and maintenance of a database logging the trending data for locations and accurate identifications. “This information can be used to identify early on any potential patterns of contamination that need to be addressed,” she says.
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