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Genetic Technology and

Bacterial Diagnosis

•How to identify bacteria in patient specimens or in samples from nature? Or the MM project;)

-phenotypic: considers macroscopic and microscopic morphology, physiology,

and biochemistry

-genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria

-immunologic: serological analysis

•Data from these methods can provide a unique profile for any bacterium

Survey of Microbial Diseases

•Direct observation of fresh or stained specimen

•Stains most often used-Gram stain-acid-fast stain

Phenotypic Methods: Direct Examination of Specimen

• Isolation Media and Morphological Testing

-Selective media: encourage the growth of only the suspected pathogen

-Differential media: used to identify definitive characteristics and fermentation patterns

Survey of Microbial Diseases: Phenotypic Methods

MacConkey

Mannitol Salts

Physiological/Biochemical Characteristics•Traditional mainstay of bacterial identification

•Enzyme production and other biochemical properties are reliable ways to ID microbes

•Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities:

-fermentation of sugars-capacity to digest complex

polymers-production of gas-sensitivity to antibiotics-nutrient sources

Survey of Microbial Diseases: Phenotypic Methods

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Unknownmicrobe +differentsubstrates

Results (+/–)

• Enzyme-mediated metabolic reactions often visualized by a color change

-microbe is cultured in a medium with a special substrate, then tested for a particular end

product-microbial expression of the enzyme is made

visible by a colored dye

Phenotypic Methods: Biochemical Testing

Flowchart: We will one to ID our MM!

Gram (–)

Cocci

Anaerobic,oxidase (–),catalase (–)

VeillonellaNeisseriaBranhamella

Moraxella

Aerobic,oxidase (+),catalase (+)

Catalase (–),pairs, chainarrangement

Gram (+)

Catalase (+),irregular clusters,

tetrads

Strictlyaerobic

Micrococcus StaphylococcusPlanococcus

Facultativeanaerobic

Streptococcus

-Testing for sensitivity to various phage groups

-a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block

-cleared areas corresponding to lysed cells indicate sensitivity to that phage

-Ex. S. aureus Phage Group I vs. Group II

Phenotypic Methods: Phage Typing

Bacterial Tools in the Lab and Genotypic Diagnosis

Tools of the Lab: Electrophoresis• DNA is separated

according to size by running it trough a gel.

• The gel is made of agarose

• The gel is placed in an electrical field that pushes the negatively charged DNA towards the positive electrode.

Tools of the Lab: Restriction Endonucleases

• Enzymes capable of recognizing foreign DNA and breaking the bonds between adjacent nucleotides on both strands of DNA

• Protects bacteria against incompatible DNA of bacteriophages

• Allows biotechnologists to cleave DNA at desired sites

• Necessary for recombinant DNA technology

• Recognize and clip at palindromes

• cut four to five bases on the 3’ strand and on the 5’ strand, leaving overhangs on each end

• adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids

Tools of the Lab: Restriction Fragment Length Polymorphisms

• Restriction fragment length polymorphisms (RFLPs): differences in the cutting patterns of specific restriction endonucleases

• RFLP allows the comparison of different cutting sites at specific areas in the genome

• Specific DNA Replication of a particular portion of the DNA• Rapidly increases the amount of DNA in a sample without the need

for making cultures or carrying out complex purification techniques• Sensitive enough to detect cancer from a single cell or diagnose an

infection from a single bacteria• Rapid enough to replicate target DNA from a few copies to billions of

copies in a few hours

Tools of the Lab: Polymerase Chain Reaction

Tools of the Lab: Polymerase Chain Reaction

• First the DNA is heated so that it will open up it’s A’s, T’s, G’s, and C’s.

• This is called denaturing the DNA

Tools of the Lab: Polymerase Chain Reaction

• Second the DNA is cooled a small amount so that primers which match a portion of the target DNA can bind to the template DNA.

• Primers: DNA strands 15 – 30 bases long that serve as landmarks where DNA amplification should begin

• This is called annealing

Tools of the Lab: Polymerase Chain Reaction

• Third the DNA Polymerase uses free nucleotides in the solution to build the desired DNA sequence.

• This is called DNA polymerization

Tools of the Lab: Polymerase Chain Reaction

• One cycle of denaturation, annealing, and polymerization will double the DNA fragments between the primers

• Since the reaction goes from 50°C to 95°C and there are about 35 cycles total there is a need for a DNA polymerase that can be heated to 95°C and not denature.

• We get this DNA polymerase from thermophilic bacteria Thermus aquaticus!

Morning Glory hot springs, habitat for Thermus aquaticus

Genotypic Methods of Identifying Bacteria: PCR

• Bacterial DNA from a sample can be PCRed at a particular area in the genome

• Using restriction endonucleases the PCR producted are cut and an RFLP emerges

• This is a DNA fragment of that bacteria

Genotypic Methods of Identifying Bacteria: Genetic Probes

Hybridization:

•Used to identify bacterial species by analyzing the sequences of nitrogenous bases in DNA

•Probes: small fragments of single-stranded DNA or RNA complementary to the specific DNA sequence of a particular microbe

•Probes with florescent or radioactive tags are added to the RFLP to visible changes in the DNA sequence in that area

After PCR all of the banding patterns look the same, but are all of the bands the same sequence?

•Remove genetic material from one organism and combine it with that of a different organism

•Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods

Tools of the Lab: Recombinant DNA Technology

Tools of the Lab: Recombinant DNA Technology

Genetic clones and cloning:

•involves removal of a selected gene from an animal, plant, or microorganism and grow it in a host microorganism•gene must be inserted into a vector (usually a plasmid or a virus)•vector inserts the gene into the cloning host•cloning host is usually a bacterium or yeast which can translate the gene into the desired protein

• Small, well characterized, easy to manipulate

• Readily accepted DNA by the cloning host

• Contain an origin of replication

• Contain a selective antibiotic resistant gene

• Ex. Plasmids, phages

Tools of the Lab: Cloning Vectors

Producing Recombinant DNA• Start with a cloning vector (special plasmid) and DNA with your gene

of choice.• Cut the cloning vector and your desired gene out of the parent

chromosome with specific enzymes.

Producing Recombinant DNA• Mix the vector and the gene together with a ligase enzyme which

“seals” the DNA together.• Use various techniques to insert the vector +gene into a new cell.

Producing Recombinant DNA• Grow cell on selective or differential media to find out which cells

possess the recombinant plasmid.

Transgenic Bacteria• Pseudomonas syringae

– Natural bacteria that grow on plants but promote frost crystals– Alteration of the normal frost gene now prevents frost crystals

from forming on plants (applied with crop duster to compete with the natural bacteria in the field)

• Pseudomonas fluorescens– This bacteria was engineered to contain an insecticide gene. The

bacteria is sprayed on fields with crop dusting planes. The bacteria grow on the plants and when the insects start to eat the plant they will also eat some bacteria with the insecticide. The ingestion of insecticide kills the insects.

Transgenic Animals

• Pharmaceutical production• Knockout mouse

– Tailor-made genetic defects• Cystic fibrosis• Gaucher’s disease• Alzheimer’s disease• Sickle-cell anemia• Allow us to research cures to these diseases

Nkx2.2 KO

Genotypic Methods of Identifying Bacteria: Nucleic Acid Sequencing

• Best way to identify a bacterial species is to determine the 16s rRNA sequence of that bacteria

• 16s rRNA is part of the 30s subunit of the bacterial ribosome

• 16s rRNA is highly conserved across species and evolutionary time

• perfectly suited for bacterial identification and diagnosis of infection

Genotypic Methods of Identifying Bacteria: Fluorescent in situ hybridization

• Fluorescent in situ hybridization (FISH) rapidly identifies 16s RNA sequences without first culturing the organism

• Relies on dyes to emit visible light in response to UV radiation

• Turnaround time for identifying suspect pathogens present in blood cultures has been reduced from 24 hours to 90 minutes