Microorganisms, if detected in drug substances, excipients, water for pharmaceutical use, the manufacturing environment, intermediates, and finished drug products, typically undergo characterization. This may include identification and strain typing, as appropriate.

The need for microbial identification is specifically cited in several U.S.P. general test chapters such as Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (62). This chapter indicates a requirement for confirmatory identification tests for organisms that grow on selective or diagnostic media and demonstrate defined morphological characteristics.

U.S.P. general test chapter Sterility Tests (71) allows for invalidation of the test, if after identification of the microorganisms isolated from the test, the growth of this (or these) species may be unequivocally ascribed to faults with respect to the material and/or the technique used in conducting the sterility test procedure. U.S.P. general information chapter.

The manufacturing area is also a concern for microbial identification. Environmental monitoring of cleanrooms provides an indication of the state of control of a facility. Control is assessed in terms of trends analysis. There are two important types of trend analysis: microbial count and the types of microorganisms isolated. Microbiological Control and Monitoring of Aseptic Processing Environments (1116) recommends that microbial isolates be identified at a rate sufficient to support the environmental monitoring program.

Furthermore, there is also considerable cGMP emphasis upon screening for objectionable microorganisms. The impact of microorganisms upon product quality attributes will depend on the product, its intended or potential application, the method of manufacture, and subsequent treatment.

Identification Methods
Microorganisms are present in a variety of milieu in the pharmaceutical manufacturing environment. The first step in identification is to isolate a pure colony for analysis. This purification is normally accomplished by subculturing one or more times on solid media to ensure purity, each time streaking for single colonies. For many types of investigations and routine surveying of manufacturing environmental bioburden, few tests such as Gram-staining, Spore staining and biochemical screening tests such as oxidase, coagulase and catalase tests, can provide sufficient information for ongoing evaluation. However, when circumstances dictate greater in-depth assessment, identification to the genus, species, or strain level can yield valuable insights about the nature and source of the bioburden. Also, microbial identification to the species and even strain level can be critical in assessing and mitigating risk from microbial contamination.

Identification methods can be divided into two groups: phenotypic and genotypic. Phenotypic methods use expressed gene products to distinguish among different microorganisms. Examples include methods based on carbon utilization and biochemical reaction, as well as fatty acid profiles by gas–liquid chromatography and wholecell composition by MALDI–TOF mass spectrometry. Generally, these methods require a relatively large number of cells in pure, monoclonal culture. Recovery and growth methods for microbial enumeration and identification are limited by the length of incubation and the fact that many organisms present in the environment are not recovered by general microbiological growth media. Phenotypic microbial identification methods are successfully used in food, water, clinical, and pharmaceutical microbiological testing laboratories. Phenotypic microbial identification methods provide information that enables microbiologists to make informed decisions regarding product risk and to recognize changes in environmental microflora. In many quality control investigations, phenotypic identification alone is sufficient and will enable scientists to conduct a thorough investigation and to recommend appropriate corrective actions as needed.

Genotypic microbial identification methods are theoretically more reliable because nucleic acid sequences are highly conserved in most microbial species. Applicable genotypic methods include DNA–DNA hybridization, PCR, 16S and 23S rRNA sequencing, multilocus sequence typing (MLST), pyrosequencing, DNA probes, and analytical ribotyping.
They generally require more expensive analytical equipment and supplies. Often these analyses are conducted by contract laboratories, government laboratories, universities, research institutes, or specialized laboratories within industrial firms. Therefore, the use of genotypic identification methods is typically limited to critical microbiological investigations such as product failure investigations.

Genotypic or Nucleic acid-based methods can also be used to screen for specific microorganisms. The steps associated with this activity are sample collection, nucleic acid extraction, target amplification, hybridization, and detection. The problem of amplifying DNA from nonviable bacterial cells can be overcome by using reverse transcription to convert rRNA that is transitional, hence related to viability, to DNA for PCR amplification. Issues include the detection of microbial variants, limits of detection, matrix effects, positive cutoff verification, instrument and system carry-over, diagnostic accuracy, and reproducibility.

Strain Typing is an integral part of epidemiological investigations in clinical and public health microbiology. Methods including pulsedfield gel electrophoresis, riboprinting, arbitrarily primed polymerized chain reaction, and whole genome ordered restriction or optical mapping can be used to demonstrate that microbial species are the same strain and most likely are from a common source.
With identification systems, verification of the identity of the species should be evaluated and the level of agreement should be considered. Typically greater than 90% agreement can be achieved with samples of microorganisms that are appropriate for the identification system. The hierarchy of microbial identification errors in descending order of impact is (1) misidentification to genera, (2) misidentification to species, and (3) no identification. Misidentification could lead to inappropriate corrective and preventive actions and product disposition.
A microbial identification system may not be able to identify an isolate because the organism is not included in the database, the system parameters are not sufficiently comprehensive to identify the organism, the isolate may be nonreactive in the system, or the species may not have been taxonomically described. Such isolates can be sent to the supplier of the microbial identification system for additional study and, if appropriate, added to the database. Alternatively, genotypic identification tests can be conducted, and the species can be added to an in-house database. Misidentification is more difficult to determine, but any microbial identification should be reviewed for reasonableness in terms of the microorganism’s morphology, physiological requirements, and source of isolation. The most important verification tests are accuracy and reproducibility. Other measurements are sensitivity, specificity, and positive and negative predictive value.

The concept of Polyphasic Taxonomy that refers to assembly and use of many levels of information, e.g., microbial characterization, phenotypic and genotypic data, and origin of the microorganisms, can be successfully applied to microbial identification. This avoids decisions made solely using genotypic data that make no sense when the microbial characteristics, testing history, and source of isolation are considered.