reverting to type

The identification of microorganisms is an essential part of quality control in the pharmaceutical, personal care and food industries. Recent improvements in PCR, sequencing and software have greatly reduced the time to obtain results from genotype-based bacterial identification.  

Staphylococcus aureus

IDENTIFICATION of microorganisms is important during routine monitoring and testing, but it becomes critical if microbial numbers exceed alert and action levels or a sterility failure is encountered, requiring corrective action that may differ depending on the species. Therefore, a highly accurate, rapid, reproducible and simple-to-perform identification method is essential.

Phenotypic methods of microbial identification, which rely on the determination of cellular morphology, gram reaction, enzymatic activity, antigenicity and other biochemical reactions, have traditionally been widely used1. These methods require a relatively large number of cells in pure culture, which can be time-consuming and labour intensive to prepare. Phenotypic testing also has the disadvantage of relying on the isolated microorganisms to exhibit full expression of their respective phenotypic properties. Within the highly controlled environment of manufacturing facilities, microorganisms are often stressed, which can greatly influence the outcome of the results by phenotypic methods. In addition, during culture, factors such as temperature, media composition and age of culture affect the expression of phenotypic characteristics, and can influence the identification of bacteria and fungi.

In contrast, genotypic methods are based on DNA analyses, typically using PCR, DNA-DNA hybridisation and/or 16S and 23S rRNA gene sequencing. The genotype is independent of both the environment from which the microorganism was isolated and the culture conditions used prior to identification. A recent publication2 confirmed the stability of repetitive sequence PCR patterns, irrespective of age of culture or following five, 10, or 15 subcultures, in six species of bacteria, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, S. epidermidis and Acinetobacter baumannii.

Isolation of genomic DNA from pure colonies was performed using PrepMan Ultra Sample Preparation reagent (Applied Biosystems), which uses a simple boil-and-spin protocol. The extracted DNA was diluted and added directly to a PCR master mix. The bacteria-specific PCR primers, in the ready-to-use master mix from the Fast MicroSeq Bacterial Identification Kit (Applied Biosystems), amplify either the first ~500 nucleotides or the full-length sequence of the 16S ribosomal DNA. PCR products were sequenced on Applied Biosystems 3130xl Genetic Analyser, and sequence files were analysed by MicroSeq ID Analysis Software v2.0 (see figure 1 for a process overview).

The time taken for the PCR step was reduced to just 20 minutes by introducing new, fast-PCR technology with hot-start chemistry. The speed of sequencing, both in cycle sequencing time and capillary electrophoresis run, has also been improved, further reducing processing time. In the new version of the software, improvements include an auto-analysis function that automates the process, from DNA sequencing to final reporting, and an extended library covering over 300 new species. With the enhancements in the MicroSeq Microbial Identification System, the overall process time of bacterial identification using 16S rDNA sequencing has been greatly reduced from approximately 10 hours to five hours. Challenged by a panel of approximately 50 organisms, covering a broad range of species including hard-to-detect organisms, the new MicroSeq Identification system was able to rapidly and accurately identify all species on the panel.

An efficient process for accurate bacterial identification in five hours was developed by applying improved methods of PCR, sequencing and software analysis. A validated database of full-length 16S rDNA sequences provides quality control that can be strictly maintained to ensure accuracy of the bacterial identification process.

Genetic-based identification methods that provide a high level of accuracy and reproducibility can be achieved by rDNA sequencing1. The Fast MicroSeq Microbial Identification System provides rapid time to results3, provides a simple workflow and is an excellent alternative for the routine identification of bacteria and fungi in the industrial setting by rDNA sequencing.


References
1. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral J-P, Raoult D (2000) 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J. Clin. Microbiol. 38: 3623-3630.

2. Kang HP, Dunne WM (2003) Stability of repetitive-sequence PCR patterns with respect to culture age and subculture frequency. J. Clin. Microbiol. 41: 2694-2696.

3. Fontana C, Favaro M, Pelliccioni M, Pistoia ES, Favalli C (2005) Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J. Clin. Microbiol. 43: 615-619.

By James Bruce and Dr Thomas Schild. James is Senior Product Manager in Applied Markets Division at the Applied Biosystems headquarters at Foster City, CA. Dr  Schild has been Marketing Manager of Applied Markets Division at Foster City, and is now Molecular and Cell Biology Marketing Manager of Real-Time PCR Systems in Europe.