What is typing?

 

In microbiological terms, typing may be defined as strain-specific identification. The objective of typing studies is to ascertain if two or more strains are derived from a single parent organism. Such investigations are usually performed to clarify whether two isolates from different sources represent the same strain or distinct ones, in order to provide essential data for epidemiological investigations. Typing studies are most commonly initiated when a sudden increase in the incidence of a particular pathogen in a geographically limited area is noted (i.e. an outbreak). In such examples, strain typing can help in identifying the source of infection - a particular batch of foodstuffs, perhaps - and thereby assist in limiting the further spread of infection. There are a wide range of phenotypic and genotypic typing methods available for epidemiological studies of bacteria.

Campylobacters present a special, well-recognised problem in that most cases of infection are reported sporadically and over a wide geographic area. The bacteria are broadly distributed in nature (various food animals, the environment) and thus accurate identification of the source of infection is extremely difficult. The problem is exacerbated by the lack of standardised typing methods that can be widely used and provide adequate strain discrimination for good quality epidemiological analyses.

 Although a range of phenotypic methods have been described for typing campylobacters (including biotyping, serotyping and phage typing schemata), these are insufficiently discriminatory and, in the case of phage- and serotyping schemes, not widely available. As a consequence, they are of limited value for accurate surveillance and identification of infectious sources even at a national level.

The inadequacies in phenotypic typing methods have led to the wider application of genetic methods for typing campylobacters. These include plasmid profiling, ribotyping, and various polymerase chain reaction (PCR)-based techniques. Most of these methods provide a higher level of discrimination than the phenotypic techniques, but without exception they suffer from a lack of standardisation. Results from one laboratory cannot be easily compared with those from another and this significantly restricts their usage for investigating the complex issue of campylobacter epidemiology at national and international levels.

The Campynet project seeks to assist in solving this problem by recommending standard methodologies for performing high-resolution molecular typing of C. jejuni and C. coli. Three key methods have been selected for standardisation. These are:

  1. Flagellin gene restriction fragment length polymorphism analysis (fla-PCR). This method involves amplification of the flagellin gene by PCR and subsequent digestion of the PCR product with a restriction enzyme. Strain differences are detected when the fragments are separated by electrophoresis and visualised, since differences in restriction site distribution in the fla genes between strains will generate different sized fragments and thus a different band pattern.


  2. Flagellin gene profiles of C. jejuni Penner reference strains

  3. Pulsed-field gel electrophoresis (PFGE)-DNA profiling. This method involves the digestion of whole-cell DNA with so-called "rare cutting" restriction enzymes, generating relatively few genomic fragments of a comparatively large size. The DNA fragments are separated by a special electrophoretic method, PFGE, which involves the co-ordinated application of pulsed electric fields from different positions in a specially-designed electrophoresis cell, such that the large DNA restriction fragments are gently oriented through the agarose gel matrix.


  4. KpnI-based PFGE-DNA profiles of Danish C. jejuni serotype 1,44 strains

  5. Amplified Fragment Length Polymorphism (AFLP) analysis. This method, like PFGE-DNA profiling, also determines whole-genome polymorphisms but by a different approach. Briefly, target DNA is digested with two different restriction enzymes and specially designed "adaptor oligonucleotides" ligated to the sticky-ended DNA fragments. A PCR method is then used to amplify a subset of these fragments, which are then separated and detected by a suitable (usually sequencer-based) system.


  6. BglII-Csp6-A based AFLP profiles of various C. jejuni strains. Red bands denote internal molecular weight markers; blue bands are bacterial AFLP fragments

     In addition, Campynet considers newly developed, and complimentary methods for typing campylobacters. Present methods of interest include Riboprinting (automated ribotyping) and standardised Randomly Amplified Polymorphic DNA (RAPD) profiling. Let us know if you have developed a new Campylobacter typing method!

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