8.3: Introduction to bacterial identification by genotypic methods (2023)

Phenotypic methods

During this semester we learned many methods to characterize and identify bacteria. These methods include characterizing cell shape (cell morphology), identifying Gram status or specialized cell characteristics by staining, growth requirements (oxygen, pH, temperature, etc.), colony appearance (colony morphology), and reactions biochemical (enterotubes). , selective and/or diverse media, etc.). All of that comes first.phenotypicAnalysis and characterization methods, that is, the results are the product ofExpressionyour genes

• cell morphology:Cell shape - by microscopy

• Coloring properties:Gram status, cellular structures such as flagella, endospores --- microscopy

• Growth features:Culture requirements such as oxygen, osmotic pressure, temperature, colony morphology ---- culture techniques

• Biochemical properties:biochemical tests such as enterotubes, oxidase, catalase tests, selective/differential media.

genotypic method

The final method for identifying bacteria isgenetic analysisusing nucleic acid probes or other molecular techniques.

Molecular techniques are widely used to detect and identify pathogens. Especially in surveillance studies, these methods provide reliable epidemiological data to trace the source of human infections, e.g. B. Outbreak of foodborne illness. A wide range of molecular techniques were used (including pulsed field gel electrophoresis, multilocus sequence typing, randomly amplified deoxyribonucleic acid polymorphism, extragenic palindromic repeats, deoxyribonucleic acid sequencing, multiplex polymerase chain reaction, and much more) for detection. , speciation, typing and classification and/or characterization of pathogens of great importance to humans.

The advent of the "age of molecular biology" has provided a wealth of tools and techniques for detection, identification, characterization and typing of bacteria for a variety of clinical and research purposes. Historically, identification and characterization of bacterial species has largely been done by phenotypic and biochemical methods (eg, by selective/differential media and biochemical assays, which we discuss in the last two modules) that rely on preliminary isolation and culture.

While these methods continue to have their place in specific settings, molecular techniques have provided unprecedented insight into the identification and typing of bacteria. To cite just a few examples, genotypic methods have allowed the identification of a wide variety of hitherto unknown taxa, the characterization of non-culturable bacteria, and have facilitated metagenomic studies on large and diverse bacterial communities. Clinical and research settings provided detailed information on bacterial virulence, pathogenesis, antibiotic resistance and epidemiological typing, as well as the identification of new, emerging and re-emerging species. Furthermore, the widespread use and availability of molecular tools for bacterial genotyping has resulted in high-throughput assays, more sensitive and discriminatory results, and fast turnaround times that are likely to be improved by automated tools and data analysis pipelines.

Most molecular methods for identifying bacteria rely on some variation of DNA analysis, either amplification or sequencing. These methods range from relatively simple approaches based on DNA amplification (PCR, real-time PCR, RAPD-PCR) to more complex methods based on restriction fragment analysis, whole genome and gene sequencing, objective and mass spectrometry. Furthermore, approaches based on unique protein signatures, such as

polymerase chain reaction

Many of these molecular techniques use thePolymerase Chain Reaction or PCR.A technique you've probably heard about in other classes. The polymerase chain reaction (PCR) allows researchers to make millions of copies of a given DNA sequence in about two hours. This automated process avoids the need to use bacteria to amplify the DNA. During the course of a bacterial infection, rapid identification of pathogens is required to determine effective treatment options, so molecular methods such as PCR are a popular choice due to their speed. You may already know that RT-PCR is widely used to detect the SARS-CoV2 virus.

PCR allows amplification of DNA, allowing specific segments of DNA to be copied multiple times and then separated and analyzed using gel electrophoresis. The presence of a specific fragment of amplified DNA can be used to identify an organism or specific trait, such as antibiotic resistance. We often use theGeneration of 16S rRNAfor bacterial identification purposes, as present in all bacteria, are highly conserved sequences (large regions of nucleotide similarity) interspersed with genus- or species-specific variable regions. Bacteria can be identified by analyzing the nucleotide sequence of the 16S rRNA PCR product and comparing it to a database of known sequences.

Illustration 1:Schematic PCR primers that bind to the 16S rRNA gene for amplification of a bacterial chromosome.

A series of steps occur in a PCR reaction. Normally, dsDNA is denatured to ssDNA. At 55-58°C, a pair of synthesized oligonucleotide primers hybridize to the ssDNA flanking the sequence of interest. At 72°C, a thermostable DNA polymerase replicates ssDNA sequences into dsDNA. The cycle is repeated 20-40x to amplify the DNA.

Figure 2:PCR reaction. Image by Erica Suchman, Colorado State University, Ft. collins, co.

The order of events in PCR is as follows. The temperature rises to 92-98^oC, causing the DNA strands to separate. Two sequences of primers of approximately 20 nucleotides each hybridize to opposite strands of DNA. (The RNA requires an initial reverse transcription step to generate a double-stranded cDNA template.) The temperature is raised to the optimum for a polymerase from a thermophilic bacterium; generallyaquatic thermos(Taq)used in 72^oC. Replication continues from the 3'-OH of the primers, generating two copies of the DNA. Temperature rises back to 92-98^oC, causing the DNA strands to separate. The temperature is then lowered to allow new primers to bind to each of the four strands generated in the above reaction. The temperature used for primer annealing must be optimized for each individual set of primers. HeTaqFortunately, the polymerase is stable during the DNA fusion step and can start a new round of synthesis. The process is repeated for 20 to 40 cycles so that more copies are created exponentially, i.e. in a chain reaction.

Watch Video 1: How to set up a PCR reaction

See video 1:how to set up a PCR reaction. Practical DNA video. (4:38)URL:https://youtu.be/95qOSslefMM

Visualization of PCR products

After amplification, the PCR product, sometimes called an amplicon, is visualized and analyzed on an agarose gel and is abundant enough to be detected with ethidium bromide or SYBRsafestain (see figure below). The amplicon is compared to molecular markers of known size to generate bands of the correct size.

Image 1:Agarose gel electrophoresis of 16S rRNA PCR products. Note the 4 bands on the 1600 bp marker.

The 16S rRNA gene is often a target for those interested in identifying the genera or species of a bacterium. Amplification of the entire gene (~1500 bp) is more than enough to obtain a sequencing result that indicates the identity of the bacterium.

Watch Video 2: How to run an agarose gel

See video 2:how to load and run an agarose gel. Bio-Rad Explorer video. (4:06)URL:https://youtu.be/uAttNVEEEwY

target other genes

In some cases, it is not necessary to identify the genus or species of bacteria, but some other characteristics, such as B. Resistance to antibiotics. In this case, you design your primers to hit a specific target.Antibiotic-Gen.In the gel below, themecThe target was a methicillin resistance gene.

image2: Ethidium bromide-stained agarose minigel visualizing the products of multiple PCR reactions that amplify part of themecA gene that encodes methicillin resistancestaphylococcus aureus("MRSA"). Distinct 533 base pair bands are clearly visible in samples containing the resistance gene. Less pronounced bands indicating smaller primer dimers and unincorporated primers are also visible. (Commented view) (Rebecca Buxton, University of Utah, Salt Lake City, UT)

Watch Video 3: How to interpret a DNA gel

See video 3:How to interpret a DNA gel. Video by Nicole Lantz. (2:30)URL:https://youtu.be/eDmaBtxym30