Real-time PCR for characterisation of Culture Collection organisms

KIRSTIN EDWARDS, SAYEH SABERSHEIKH & NICK SAUNDERS**

National Collection of Type Cultures and Molecular Biology Unit, Central Public Health Laboratory, 61 Colindale Avenue, LONDON NW9 5HT

Tel 020 8200 4400 (ext 3070) Fax: 020 8200 1569 email: nsaunders@phls.nhs.uk

SUMMARY

The purpose of the study was to show how real-time PCR could be used to enhance the characterisation of strains supplied by the UK Culture Collections. A series of demonstration projects were chosen to illustrate the concept and the possible scope of enhancements.

Messenger RNA plays a key role in the regulation of protein synthesis. When the level of a particular mRNA is raised synthesis of the corresponding protein is generally also increased. It is thought that key phenotypic features of particular strains are controlled in this way. Initially, methods for the quantification of specific bacterial mRNAs by real-time PCR using the LightCycler^TM were developed.

In the first demonstration project the regulation of genes controlling the production of alginates in Pseudomonas aeruginosa was investigated. Strains that produce large quantities of alginate have a typical mucoid appearence when grown on solid culture media and are tolerant of conditions prevailing in the lungs of patients with the inherited disease cystic fibrosis. We were able to confirm the central role of the algD gene in the regulation of the biosynthetic pathway leading to alginate. A proposed mechanism for induction of algD via algU and its down regulators mucA and mucB was investigated. It was found that this pathway did not operate in our strains.

In the second demonstration project the regulation of virulence-associated factors in Staphylococcus aureus strains was investigated. This pathogen expresses a series of proteins including toxins, haemolysins and binding factors in a growth phase dependent manner. Real-time quantitative PCR was used to investigate some of the mechanisms controlling these factors in UK methicillin resistant strains. Large differences between strains were found in the levels and patterns of expression of RNAIII a product of the agr (accessory gene regulator) operon thought to play a key role in virulence factor regulation. Levels of some of the key virulence factors and their mRNAs were also measured. The results indicate that some of the successful disease-causing S. aureus clones employ radically different mechanisms to establish infection and avoid host defences.

Strain identification and verification is an essential concern of the Culture Collections. The third demonstration project was done to show that the LightCycler^TM real-time PCR machine could be used for rapid detection of species-specific gene sequences. Coagulase-negative staphylococci represent a particular problem in their identification, but in most cases they may be reliably assigned to species by analysis of their 16S gene sequences. A real-time PCR probe hybridisation system was developed that allowed colonies to be assigned to one of 14 different staphylococcal species within 30-40 minutes. This identification system was found to be accurate and reproducible.

The final demonstration project was done to show that single nucleotide polymorphisms (SNPs) can be readily identified by real-time PCR. SNPs may be important strain characteristics for Culture Collections to provide information on. For example, SNPs in the Mycobacterium tuberculosis gene encoding RNA polymerase (rpoB) result in resistance to one of the key drugs used to treat tuberculosis. A LightCycler^TM PCR/probe hybridisation test for SNPs in rpoB was developed. The assay was shown to be reproducible and accurately detected the wide range of SNPs that may be present.

Acknowledgements:

This work was funded by the BBSRC