Current USP Perspectives on Microbial Identification

Microbial identification is the determination of the broad group (eg, bacteria, yeast, or mold) or narrow group (eg, genus and / or species) to a qui microorganism belongs to.
Microbial characterization is the use of colony growth, cellular morphology, differential staining, and key diagnostic features to Characterize a laboratory isolate for trending and investigative Purposes without identification, example, nonpathogenic Staphylococci.

Microorganisms, if detected in drug substances, excipients, water for pharmaceutical use, the manufacturing environment, intermediates, and finished drug products, UNDERGO Typically characterization. This May include identification and strain typing, as considers.

The need for microbial identification is specifically Cited in several general chapters USP test Such As Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (62). This chapter indicates a requirement for confirmatory identification tests for organisms grow on selective gold That diagnosis media and morphological characteristics defined Demonstrate.

USP general chapter test Sterility Tests (71) Allows for invalidation of the test, if after-identification of the microorganisms isolated from the test, the growth of this (or thesis) species May be unequivocally ascribed to faults with respect to the material and / or technical the used in Conducting the sterility testing procedure. USP General Information chapter.

The manufacturing area est aussi 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 major kinds of trend analysis: microbial count and the kinds of microorganisms isolated. Microbiological Monitoring and Control of Aseptic Processing Environments (1116) recommends That microbial isolates APPROBATION be at a rate Sufficient to Support the environmental monitoring program.

Furthermore, There est aussi cGMP considerable emphasis upon screening for objectionable microorganisms. The impact of microorganisms upon product quality attributes Will depends on the product, icts Intended or potential application, the method of manufacture, and Subsequent treatment.

Identification Methods
Microorganisms are present in a variety of environment in the pharmaceutical manufacturing environment. The first step in identification is to isolate a pure colony for analysis. This purification is subculturing Normally Accomplished by one or more times to Ensure media is solid purity contents, each time streaking for single colonies. For Many kinds of investigations and routine manufacturing of surveying environmental bioburden, Such As Few tests gram staining, spore staining and biochemical screening tests Such As oxidase, catalase and coagulase tests, can Provide Ongoing Sufficient information for evaluation. HOWEVER, When Circumstances dictate Greater in-depth assessment, identification to the genus, species, or strain level can yield valuable insights about the type and source of the bioburden. Also, microbial identification to the species level and Even strain can be critical in Assessing and Mitigating risk from microbial contamination.

Identification methods can be divided into two groups: phenotypic and genotypic. Phenotypic methods Expressed use gene products to Distinguish Among different microorganisms. Examples include methods are based carbon utilization and biochemical reaction, as well as fatty acid profiles by gas-liquid chromatography and wholecell composition by MALDI-TOF mass spectrometry. Generally, methods require a thesis Relatively wide 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, microbiological testing and pharmaceutical laboratories. Phenotypic microbial identification methods Provide Information That Enables Microbiologists to make Informed Decisions Regarding product to Recognize risk and exchange in environmental microflora. Many in quality control investigations, phenotypic identification alone is Sufficient and Will enable scientists to conduite has Thorough investigation and to recommend corrective action as needed Appropriate.

Genotypic Theoretically microbial identification methods are more reliable nucleic acid sequences Because are highly conserved microbial species in Most. Applicable genotypic methods include DNA-DNA hybridization, PCR, and 16S 23S rRNA sequencing, multilocus sequence typing (MLST), pyrosequencing, DNA probes, analytical and ribotyping.
They Generally require more expensive analytical equipment and supplies. Often thesis analyzes are Conducted By contract laboratories, government laboratories, universities, research institutes, laboratories or Specialized Within industrial firms. The Therefore, the use of genotypic identification methods is limited to critical Typically microbiological investigations Such as product failure investigations.

Genotypic or Nucleic acid-based methods can be used to screen aussi 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, tool and system carry-over, diagnostic accuracy, and reproducibility.

Strain Typing is an integral portion 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 and strain are the Sami are Most Likely from a common source.
With identification systems, verification of the identity of the species shoulds be Evaluated and the level of agreement shoulds be regarded. Typically 90% Greater Than Can Be Achieved agreement with samples of microorganisms That are Appropriate for the identification system. The hierarchy of microbial identification errors in descending order of impact is (1) to misidentification genera (2) to species misidentification, and (3) No identification. Could lead to misidentification corrective and preventive action Inappropriate and product disposal.
A microbial identification system May not be reliable 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 begging 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 difficulty to determine, purpose Any microbial identification shoulds be reviewed for reasonableness in terms of the microorganism's morphology, physiological requirements, and source of insulation. Most importantly the verification tests are accuracy and reproducibility. Other measurements are sensitivity, specificity, and positive and negative predictive value.

The concept of Polyphasic Taxonomy Microbial characterization, phenotypic and genotypic data, and origin of the microorganisms, can be successfully applied to microbial identification. This guidance is based on the assumption that the microbial characteristics, testing history, and source of isolation are considered.



1) USP <1113> Microbial Characterization, Identification and Strain Typing. USP 38-NF 33 (2015), 1180 page
2) Bergey's Manual of Systematic Bacteriology, 2nd Edition, 2003.
3) O'Hara, CM, MP Weinstein, and JM Miller. Manual and automated systems for the detection and identification of microorganisms. ASM Manual of Clinical Microbiology, 8th Edition, 2003.
4) Cumitech 31. Verification and Validation of Procedures in the Clinical Microbiology Laboratory. Elder, BL, Hansen SA, JA Kellogg, Marsik FJ, and RJ Zabransky, ASM, February 1997.
5) JE Clarridge III. The Impact of 16S rRNA Gene Sequencing Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases, Clin. Microbiol. Rev. 17 (2) 840-862, 2004.
6) Gillis, M., P. Vandamme, P. De Vos, J. Swings and K. Kersters. Polyphasic Taxonomy. Bergey's Manual of Systematic Bacteriology, 2nd Edition, 2003.