Genetic Methods for Detecting and Characterizing Bacteria

Introduction

Traditional biochemical and morphological identification techniques do not provide enough information about microorganisms for today's needs.

Genetic methods can much more rapidly determine the species of a particular isolate, often without the need to isolate a pure culture. These tests can provide information as to whether a particular gene is present which is important in determining if a particular isolation is pathogenic.

Genetic tests can also provide increased differentiation between isolates within the same species. This subtyping is useful in the study of outbreaks of disease: are the isolates from the patients all the same and is there a common source from which the same subtype can be isolated?

Basic DNA Structure

Most genetic methods are based on the ability of DNA strands to reform a double helix if they have been separated. Learning how to use this feature of DNA molecules was important for the development of methods such as DNA hybridization and the Polymerase Chain Reaction.

Important features of DNA.

DNA hybridization

After the strands of the double helix are separated (denatured), lowering the pH or temperature will cause them to reform the double helix (renatured). If each of the strands happens to be from a different organism, this process is called DNA hybridization. The similarity of the nucleotide sequences in separate strands determines if these strands will be able to renature.

One application of DNA hybridization is called DNA colony hybridization. To detect particular genes that might be present in the cells of a bacterial colony, the DNA is tested by hybridization to a gene probe . An array of colonies growing on an agar service is lysed so the DNA is gently released and then it is immobilized on a membrane. The gene probe is used to test the DNA on the membrane to determine if it contains similar sequences by using DNA hybridization. If the sequences hybridize, the "reporter" groups on the probe will be associated with the DNA on the membrane (target DNA).

For example, cells of pathogenic Yersinia enterocolitica were seeded into foods that were then spread-plated and incubated until colonies were formed. Colony hybridization was carried out to determine the efficiency of detection and the effect of various foods. Detection levels were between 50 and 100 % of the cells added and the different types of food had little effect on the efficiency of detection.

The Polymerase Chain Reaction (PCR):

PCR is a procedure for selectively replicating a specific segment of DNA. It consists of adding two primers (single stranded DNA, oligonucleotides, about 20 basepairs in length) which will serve as sites for the initiation of DNA synthesis. The basic steps are three temperature changes to allow primers to bind to the single stranded DNA to be copied (the template). Each cycle of temperature changes allows the DNA strands (including the newly made ones) to serve as templates so the amount of DNA doubles every cycle. At the end of the second cycle the newly made strands are bounded by the primers. These fixed length molecules increase exponentially for 30 or 40 cycles (a 2³⁰ to 2⁴⁰-fold increase) and because they are the same length will appear as a band when examined by agarose gel electrophoresis.

PCR Example:

The schematic map of the toxin genes sltI and sltII of Escherichia coli O157:H7 shows the locations of the primers and the predicted size of the amplified products. In this example, two sets of primers are used to amplify two different regions (multiplex PCR). The predicted size of each region is different so that the PCR products can be separated by gel electrophoresis as shown. During an outbreak caused by E. coli O157:H7, isolates from patients can be characterized and compared with those recovered from a food suspected to be contaminated as part of the epidemiological investigation. The isolates for hamburger are the same as some of the patient isolates suggestion but not proving a link between the disease and the food.

Genetic Subtyping and Molecular Epidemiology:

PCR is only of limited use for subtyping because only a few bands (generally 1 or 2) are generated. Other subtyping techniques such as ribotyping and pulsed-field gel electrophoresis can produce many bands from each isolate so that many comparisons can be made.

Ribotyping:

All organisms carry genes for encoding ribosomal RNA (rRNA), a principle component of the ribosome. The ribosome is the site of cellular protein synthesis. Because all organisms are evolutionarily related, they share some similarity in their sequence of these genes. In bacteria, there are usually several copies of these genes that may be located at different sites in the bacterial chromosome. This can be revealed by isolating chromosomal DNA, digesting it with a restriction endonuclease, separating the fragments by gel electrophoresis and then transferring the pattern of fragments to a solid support (often called a Southern blot). Next, hybridization using labeled rRNA, usually from Escherichia coli, is carried out. Only fragments containing sequences complementary to rRNA will be seen. This pattern of chromosomal fragments is called a ribotype and is a measure of the genetic similarity between two organisms. . Ribotypes of Listeria monocytogenes have been used to differentiate strains from each another. Note that the pattern obtained from the isolate from Patient C does not match the strain isolated from the mussels suggesting that the illness probably was caused by another source.

Pulsed-Field Gel Electrophoresis (PFGE):

Ordinary gel electrophoresis techniques are not capable of separating molecules larger than 50-100 kilobase pairs, but by precisely varying the direction and duration of the electric current, small differences between large molecules can be resolved. Newly developed, very gentle DNA isolation techniques to obtain intact bacterial chromosomes are essential. The similarity of the restriction endonuclease fragment patterns of chromosomal DNA is a measure of the genetic similarity between two organisms. Strains of E. coli from an outbreak have been characterized by using this technique. Notice the differences between strains that harbor sltII only and those that have both the sltI and sltII genes.

PCR-RFLP:

When PCR is used to amplify closely related organisms the PCR products may be identical in size. However, there may be some differences in nucleotide sequence which can be exploited by cleaving (digesting) the PCR product with a restriction endonuclease. When restriction enzyme digests of amplified Cyclospora and Eimeria DNA are separated by electrophoresis, different patterns are observed.

Analysis of Similarities:

As an example, let's consider the 16S rRNA gene sequences of some strains of bacteria. These can be aligned by computer and displayed so that only the sequence differences are shown, that is, the locations where each sequence matches the consensus sequence has been left blank. The sequences can be compared with one another and a number assigned to reflect the similarity. Where two sequences are identical, the similarity is equal to 1.0; If the sequences are only half the same, the similarity is equal to 0.50. When all sequences are compared to every other sequence, a similarity matrix can be constructed. The significance of this data is hard to grasp in this format but a two-dimensional graphical representation called a dendrogram allows us to visualize similar groups as clusters. Finally, the relationships between the groups can be illustrated in three dimensions using principal component analysis. Thus, rather complex mathematical relationships of similarity data can be easily visualized graphically.