Bacterial taxonomy is in a continual process of refinement as microbial classification is rapidly moving from phenotypic-based grouping to genetically-based relationships. Both specific gene sequencing (such as 16S rRNA, rpo B, gyr A etc) and complete genome sequencing have finally laid out a foundation for microbial classification and tools for bacterial identification based on genetically stable and universal components. Among these, comparative ribosomal RNA sequence analysis is pivotal for identifying bacteria to both genus and species level and is now the basis for species proposals and taxonomical analysis. This is reflected in the International Journal of Systematic and Evolutionary Microbiology papers and reports of various taxonomic subcommittees and integrated in Bergey's Manual (2007). Once the 16S rDNA foundation is used, clusters of organisms can be further analysed for subtle and close relationships using additional genes such as rpo B, gyr A, ATPases etc).

Services:

The Molecular Identification Services is a Unit of the Centre for Infections (CfI, HPA) that provides microbial identification services and complementary developmental activities. Bacterial identification is undertaken both independently and jointly with other disease-specialist laboratories (including LHCAI, STBRL & GEZI).
In particular it is responsible for the molecular identification of atypical or rarely isolated bacteria using 16S rDNA sequencing. These include:

• Atypical and rarely isolated aerobic/capnophilic bacterial isolates
• Aerobic actinomycetes; a broad group of gram positive rods/filaments that encompass such taxa as Mycobacterium (non-TB), Nocardia, Rhodococcus, Gordonia, Tsukamurella, Corynebacterium etc.
• Aerobic spore-forming bacilli
• Aerobic gram negative cocci, tentatively identified as Neisseria gonorrhoeae.

MISU has established an identification approach based upon comparative sequence analysis of the small ribosomal subunit encoding gene for a range of pathogens and to date some 10,000 sequences (comprising over 100 genera and 400 species) have been analysed. The service is provided by a specialist team and utilises the sequencing service of the Applied and Functional Genomics Unit of CfI.

For information regarding Identification Services please refer to our DBHT User Manual (PDF, 254 KB) .

For the submission of single isolates please use the Molecular Identification Request Form M1:

 M1 Microbial identification form (PDF, 318 KB).

 

Research and Development:

The research and development activities extend from analysis of conserved genes to comparative proteomic approaches to complement biomarker identification. Gene products such as toxins and unique biomarker proteins are characterised by various forms of mass spectrometry. CfI aims to develop applications using a range of structural and functional high-resolution analytical tools.

Some examples include:

• Intact Cell Analysis - Rapid (few minutes) analysis for identification of human pathogens. A database of over 5,000 mass spectral profiles have been developed and is currently in use. ( Waters Inc.-Manchester Metropolitan University collaboration)
• Rapid profiling of the low-mass proteins/peptides of bacteria using SELDI TOF MS and the ProteinChip arrays. The highly discriminatory fingerprints created using this method combined with data mining approaches such as Artificial Neural Network analysis (with Nottingham Trent University) is being utilised to build predictive models for the identification of pathogens such as Neisseria gonorrhoeae, Staphylococcus aureus etc.
• Comparative and quantitative Proteomics using 1D and 2D gel electrophoresis. For high-accuracy detection of expression changes Difference in Gel Electrophoresis (DIGE) is utilised. Pre-labelling with fluorescent dyes allows for multiplex analysis of different samples on the same gel, which increases throughput and provides accurate quantitative data. Comparative analysis using DIGE can reveal subtle changes in protein expression related to pathogenicity and potentially useful in vaccine development.
• Mass spectrometry for protein identification. Proteins of interest are identified using accurate mass-measure of their peptide fragments (Peptide Mass Fingerprinting) carried out using MALDI TOF MS equipped with a reflecting analyser or using the state of the art Orbitrap mass spectrometer (make a link to figures below) .ThehybridOrbitrapinstrument,incombinationwithnano-HPLC,allowsproteins(>50) in complex mixtures and at a very low concentrations (sub-femtomole levels) to be analysed. It also provides sequence data together with high accuracy and resolution generating confidence in protein identifications (see Figure 1).
  
  What it looks like inside...
  
  
  Figure 1: The Orbitrap contains two separate mass analysers initially a linear trap provides fast MS/MS capabilities for high sensitivity and structural characterisation, secondly ions are passed in to the Orbitrap where there masses can be measured to an accuracy of within 2 parts per million.
  Each peptide eluting from the HPLC is ionised, accelerated and stored in the Orbitrap, here the accurate mass of this peptide is measured before it undergoes collision induced dissociation. The mass of fragments produced are also measured. Using the masses of both peptide and fragments the sequence is pieced back together.

• Identification of Microbes using MALDI-TOF-MS
  Added/updated: 24 August 2009
  
• Proteome Analysis and the Search for Biomarkers
  Added/updated: 26 August 2008
  
• Proteome Biomarker Discovery using Surface Enhanced Laser Desorption/Ionisation Time of Flight Mass Spectrometry (SELDI-TOF-MS)
  Added/updated: 26 August 2008