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6: Microbial Genetics and the Evolution of Antibiotic Resistance

The optimism of the 1960’s over the “defeat” of infectious diseases was short-lived.  For example, in less than a decade, pathogenic Staphylococcus aureus had developed resistance to penicillin, once thought of as a miracle drug. By the late 1980’s, a select group of scientists gathered to provide evidence that the “disease causing microbes of the planet, far from being defeated, were posing ever growing threats to humanity” (Garrett, The Coming Plague p5-6)

“ ‘Nature isn’t benign’ (Joshua) Lederberg said at the meeting’s opening. The bottom lines: the units of natural selection-DNA, sometimes RNA elements-are by no means neatly packaged in discrete organisms. They all share the entire biosphere. The survival of the human species is not a preordained evolutionary process. Abundant sources of genetic variation exist for viruses (and other pathogens) to learn new tricks, not necessarily confined to what happens routinely, or even frequently.’” (Garrett p6)

What Lederberg appreciated was the contribution of microbial genetics to the evolution of pathogens which could “outwit” antimicrobial drugs and vaccines and to the evolution of new pathogens.

DNA as the genetic information of cells

Oswald Avery and colleagues proved that DNA was the genetic information of  bacteria in 1944 . DNA structure was revealed by Watson and Crick (using info provided by Rosalind Franklin and Maurice Wilkins) in 1953. Eventually it was revealed that DNA is the genetic information of all cellular organisms. However not all pathogens use DNA as their genetic information. Below are important exceptions:

  1. Prions: lack genetic information
  2. Viruses: use either DNA or RNA as genetic information. RNA viruses change     or mutate rapidly as the enzymes which copy their RNA genomes make lots of   mistakes (example influenza virus)

 

 All cellular organisms (prokaryotes and eukaryotes) use DNA as their genetic information     (see fig 2.15 p 38-40 Belk’s Biology)

DNA is made of chains of nucleotides which carry 1 of 4 different bases (note: see Appendix 1 for description of nucleotide structure). The order of the bases determines the order of amino acids in a gene’s protein product (see Appendix 2 for description of transcription and translation, the processes involved in converting DNA base sequence into the amino acid sequence of proteins). The amino acid sequence of a protein determines the protein’s shape and therefore the protein’s function. If you change the DNA base sequence, you can change the amino acid sequence of the gene’s protein which may change the shape of the protein and the function of the protein.  Changes in DNA base sequence are called mutations.  Mutations occur naturally/spontaneously in nature because the enzymes responsible for copying DNA sometimes make mistakes. Mutations can be beneficial for the bacteria for example mutations may permit a bacterium to resist killing by an antibiotic (“antibiotic resistance”)

Replication in bacteria

 Bacteria reproduce by growing larger then splitting in two, a process called binary fission.

 

                        O à   O  +  O

       1 bacterium divides into 2 bacteria= “binary fission”  

(see fig 1.2 p469 Belk’s Biology)Belk’s Biology)

 

Prior to splitting in two, the parent cell copies its chromosomal DNA so that each daughter cell receives a copy. Since the offspring are genetic clones of the parent cell, this process is called “asexual  (“without sex”) reproduction”.

In theory, the 2 “daughter cells” produced in binary fission should be genetic clones of the original  “parent”  cell . however the enzymes copying the parent DNA make mistakes which are passed on to the daughter cells, thus each daughter cell will carry these “spontaneous mutations”, the “clay” of genetic diversity, natural selection and evolution.

Mutations and mechanisms leading to antibiotic resistance

Evolution, Natural and Artificial Selection                                    

Unity and Diversity of Life

All cellular organisms share certain traits as a result of sharing (or “descending from”) a common ancestor (the first primitive prokaryotic cells). Yet the incredible diversity of living organisms is astounding. How did such diversity arise?

  • The diversity of life is explained by evolution, a change in the genetic makeup of a population of organisms.
  • The “raw material” of evolution is genetic variability, the genetic differences between individuals of a population of organisms. How does genetic variability arise?
  • Genetic variability arises through random mutations, changes in the nucleic acid sequence of organisms. These mutations usually occur when the enzymes which copy genetic information before a cell divides make a mistake. As an example, the enzyme DNA polymerase should copy the DNA sequence below precisely.

9Note; complementary base pair rules permit precise replicationof DNA and transcription. The base pairing rules are: adenine pairs with thymine (or uracil in RNA) and cytosine pairs with guanine.

                        A:::T  and    C:::G  where ::: represent hydrogen bonds

 The “pairing” consists of formation of hydrogen bonds which can stabilize the DNA “double helix”.  In DNA replication, one of the “old parent strands” of DNA acts a a guide or template for synthesis of a “complementary” new strand of DNA. Very kool!

DNA base abbreviations:

  • A=adenine      
  • T=Thymine   
  • G=Guanine 
  • C=cytosine

 

Original genetic info, DNA base sequence of double stranded DNA

 

            A-T- C- G- G

            T- A- G- C- C

            Original DNA

DNA polymerase should make a precise copy of the DNA before the cell divides such that each “daughter” cell receives a precise copy of genetic information from the parent cell:

                                                                                    A-T- C- G- G

                                                                                    T- A- G- C- C                                                                                                                 copy 1

 

A-T- C- G- G                                                           /

            T- A- G- C- C   ---à DNA polymerase copies

Original DNA                                                           \      A-T- C- G- G

                                                                                    T- A- G- C- C

                                                                                     copy 2

                                                                                                               

When the original cell divides, both “daughter” cells will receive one copy of the DNA, exact copies of the DNA from the “parent” cell

 

Genetic variability occurs when DNA polymerase makes a mistake copying the original DNA as shown below:

 

A-T- C- G- G

                                                                        T- A- G- C- G* ,<-mistake=mutation                                                                                                                                copy 1

 

A-T- C- G- G                                                           /

            T- A- G- C- C   ---à DNA polymerase copies

Original DNA                                                           \      A-T- C- G- G

                                                                                    T- A- G- C- C

                                                                                     copy 2

In this case, DNA polymerase made a mistake in copy #1 (instead of a “C”, it used a “G”). This is a mutation, a change in the DNA sequence.  As a result, the mutated gene might encode information for a different variety of protein. This different protein could change some feature of the daughter cell which receives the mutant DNA copy.  This mutant organism might be a better competitor compared to “normal, non-mutant” member of its population (or the mutant might be  less able to compete with its “normal” colleagues). 

If  resources are limited, “variants” or mutants which are better competitors will survive in higher numbers than their non-mutant neighbors. The mutants will have more offspring than the  non-mutant members of the population which are weaker competitors. The mutation which permitted the variant to compete better will be passed on to its offspring. Over time, the mutant form of the genetic information will be carried by a greater proportion of the surviving population, another way of saying the frequency of the mutant gene increases in the population. Thus the gene pool or genetic makeup the population has changed over time and the population of organisms has “evolved” (and the population becomes better adapted to the environment over time)

This mechanism describing “how” evolution occurs is called “natural selection”, a concept developed by Charles Darwin and Alfred Wallace in the 1800’s. (Darwin and Wallace were not the first people to describe evolution, but they were the first describe the process of natural selection).

Darwin and Wallace’s explanation of evolution by natural selection is based on 5 assumption (source: Keeton and Gould’s Biology 4th edition, Norton Publishers)

  1. Many more individuals are born in each generation than will survive and reproduce (limiting environmental resources, competition for resources)
  2. There is variation among individuals of a population.
  3. Individuals with certain characteristics have a better chance of surviving and reproducing than individuals with other characteristics.
  4. Some of the differences resulting in differential survival and reproduction are heritable
  5. Vast spans of time have been available for change

Natural Selection in a “nutshell”

  1. Each species produces more offspring than can survive
  2. The offspring compete with one another for limited resources
  3. Organisms in every population vary
  4. Organisms with most favorable traits/variations are most likely to survive and produce more offspring

Result:…..consequently variant genes “spread” through the population over time, genetic makeup of population changes and population changes/evolves over time=”Evolution

Natural selection vs Artificial selection

When “nature” chooses which variants are best at competing for natural resources,  and thus will survive and reproduce at higher rates than other variants , “natural selection” occurs. However if humans selectively breed organisms for specific traits, or if humans purposely change the environment (for example through overuse of antibiotics), “artificial selection” occurs.  The overuse/misuse of antibiotics worldwide has artificially selected for a growing number of antibiotic resistant bacteria.

 

Evolution of antibiotic resistant bacteria

As bacteria reproduce so quickly, it does not take  “vast spans of time” for their populations to evolve. Antibiotic resistance can evolve within a few years (even within a few weeks) within some populations of bacteria.

Mechanisms of antibiotic resistance in bacteria:

  1. Mutations may permit evolution of protein enzymes which destroy antibiotics. An example is the bacterial enzyme  beta-lactamase which destroys beta-lactam antibiotics such as penicillin and ampicillin. Most Staphylococcus aureus carry genes for production of beta-lactamases and therefore are not killed in the presence of penicillin.
  2. Mutations may permit evolution of protein enzymes which chemically modify antibiotics or targets, inhibiting action of antibiotics
  3. Mutations change the target of the antibiotic so the antibiotic can no longer bind to and inhibit function of the protein
  4. Mutations permit evolution of  bacterial “pumps” which specifically pump out antibiotics if they enter the bacterium

Horizontal gene transfer in bacteria

Although bacteria reproduce asexually, they are very promiscuous-they love to share genetic information with their neighbors. In this way bacteria can share their mutations with one another. This sharing of genetic information with neighbors is called “horizontal gene transfer” and is another reason antibiotic resistance has spread so rapidly among bacteria.

3 different ways bacteria share genetic information.

  1. Conjugation: bacteria make physical contact with each other (some using conjugation/sex pili) and transfer DNA from a donor bacterium to a recipient bacterium.
  2. Transduction:  a bacterial virus called a “bacteriophage” transfers DNA from a donor bacterium to a recipient bacterium.
  3. Transformation: transfer of “naked DNA” from a donor to a recipient bacterium. Some bacteria make DNA binding proteins on their surfaces. These DNA binding proteins can bind “naked DNA” in the environment, then pass the DNA into the interior of the bacterium. Bacterial cells which can be transformed are called “competent cells”.

Additional concerns regarding antibiotic resistant bacteria and the selection for and spread of antibiotic resistance genes

“R” plasmids and multi-drug resistant bacterial pathogens: Plasmids are circular DNA structures outside of the chromosome (thus described as “extrachromosomal”) which carry extra genetic information. Of great concern are plasmids carrying antibiotic resistance genes. Such plasmids  are called “resistance” plasmids or “R” plasmids. Some R plasmids may carry multiple genes (up to 8) for antibiotic resistance to several different classes of antibiotics.  R plasmids may be rapidly shared between bacteria, even between different species of bacteria.  Chronic (long-term) exposure to low levels of antibiotics may select for normal bacterial microbiota carrying R plasmids. Consequently these R plasmids can be transferred to pathogenic bacteria such as Salmonella or Shigella.

Nosocomial infections and antibiotic resistance: Nosocomial infections are infections acquired at a health care facility such as a hospital. Many bacterial nosocomial infections are caused by antibiotic resistant bacteria. Why?

Recall from the discussion of evolution, natural and artificial selection (Appendix 3) that use of antibiotics can select for antibiotic resistant bacteria. Consequently we would predict that environments in which high levels of antibiotics are used would select for high prevalence of antibiotic resistant bacteria.

Problems with low level antibiotics in livestock/poultry feed: Often low levels of antibiotics are added to animal feeds to improve “weight gain efficiency” (the amount of weight gained per pound of feed).  This practice selects for antibiotic resistant bacteria among the normal microbiota of these animals. The antibiotic resistance genes may then be transferred to bacterial pathogens which subsequently contaminate meat/milk/poultry products consumed by humans. Humans can become ill with these resistant bacteria, making successful treatment difficult. European nations which have outlawed the use of antibiotics in animal feeds have subsequently noted a decrease in antibiotic resistance among human bacterial pathogens

Problems with inappropriate broad-spectrum antibiotic therapy of humans: Broad spectrum antibiotics kill a wide range of bacteria, both “good” and “bad” bacteria.  The body’s “good bacteria” compete with invading pathogens for attachment sites and nutrients and thus are part of our natural defenses against invading pathogens. If people lose this line of  defense through broad spectrum antibiotic use/abuse, they are at higher risk for infections with antibiotic resistant bacterial pathogens (reduces infectious dose when normal microbiota disrupted) or fungal infections (ex yeast infections by Candida albicans, a fungus not killed by antibiotics-vaginal yeast infections, oral infections/”thrush”, “diaper rash”, anal infections)

Antibacterial Mania: Triclosan and “Triclo-insanity”: Triclosan is a poplar antibacterial agent incorporated into toys, cutting boards and kitchen utensils (even some toothpastes!). It belongs to the phenolic group of antimicrobials and inhibits an enzyme involved in bacterial fatty acid synthesis; consequently bacterial cell membranes become “leaky” leading to bacterial death.

Public health scientists are concerned that the overabundance of triclosan in our environment will select for resistant bacteria-some fear multi-resistant bacteria will be selected .

Ideally, when possible, warm soap and water is still the best way to rid body surfaces of pathogens.

 Triclosan will not kill all pathogens but washing can wash away all pathogens.

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Class discussion: Strategies to reduce selection of antibiotic resistant bacteria:

1.

2.

3.

4.

5.

 

 

Footnote on mutations and viruses:

Evolution of variant strains of RNA viruses:  HIV and influenza

Cellular organisms all have DNA as their genetic information. The enzyme which copies DNA, the DNA polymerases, have “high fidelity”, they make relatively few mistakes because they have the ability to “edit or proofread” their work and correct many of their mistakes. Mistake rates fro DNA polymerase are approximately one incorrect nucleotide per 108-109 nucleotides.

In contrast, some types of viruses (acellular pathogens) use RNA as their genetic information. The enzymes which  copy RNA, the RNA polymerases, lack the ability to correct mistakes, therefore RNA polymerases have a very high mistake rate (one incorrect nucleotide every104-105 nucleotides) . Consequently many RNA viruses such as influenza virus and HIV, have very high mutation rates thus “populations” of HIV and influenza viruses evolve quickly. The high mutation rate of these viruses results in rapid drug resistance and huge challenges in vaccine production.

Study guide microbial genetics

1. What were 2 reasons for the tremendous optimism of western physicians in the 1950’s-1960’s?

2. When was the structure of DNA first described and by whom?

3. What is the function of DNA?

4. The DNA base/nucleotide sequence determines the specific amino acid sequence of proteins, thus the shape and function of proteins.

            -what is a change in the DNA sequence called?

            -mutations may lead to evolution of which beneficial traits for bacterial    pathogens (from the pathogens “point of view”)?

5. Describe 3 different ways bacteria can resist antibiotics

6. In bacteria, what is “horizontal gene transfer”?

            -be able to recognize definitions for the following:

                        conjugation

                        transduction

                        transformation

7. Why does antibiotic resistance spread so rapidly amongst bacteria?

8. Why does over-use or inappropriate use of antibiotics contribute to the spread of antibiotic resistance among bacteria? Provide examples

Appendix 1 Nucleic acid structure of cells: DNA and RNA

DNA structure, nucleotide structure

The DNA of chromosomes is a beautiful “double helix”; 2 chains of DNA are held together by chemical attractions

Each chain of DNA is made of nucleotides linked by strong chemical bonds.

Each nucleotide of DNA consists of 3 parts (figure 1 below):

            a. a sugar called deoxyribose

            b. a phosphate group

            c.  one of 4  nitrogenous bases:

                        -the nitrogenous bases of DNA are adenine (A), thymine (T), guanine                                 (G) and cytosine (C)

                        -adenine can form  hydrogen bonds with thymine  A:T   and

                          guanine can form hydrogen bonds with cytosine   G:C

  • the above “pairing rules” are officially called “complementary base                                  pairing rules”
  • hydrogen bonds between “complementary bases” on the 2 sister DNA strands  holds the 2 strands or chains of DNA together in the chromosome
  • complementary base pairing also permits the precise replication of DNA, and its transcription

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Figure 2: The* “double helix” of chromosomal DNA. A single strand of DNA consists of a chain or polymer of individual nucleotides joined by strong chemical bonds. Each nucleotide carries one of 4 “bases”, the “letters” of the DNA alphabet. 3 specific bases make up the “words” (or “codons”) of DNA language. Each DNA word or codon specifies one of 20 amino acids the cell uses to build proteins. Therefore the DNA base sequence determines the amino acid sequence of cellular proteins. In turn, the amino acid sequence of proteins determines how the protein will fold and the protein’s ultimate shape. The shape/structure of the protein determines its function. 

                *(The 2 strands of DNA are held together by weak attractive forces between “complementary” bases; adenine to thymine A:T and guanine to cytosine, G:C)

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RNA, second nucleic acid of cells

RNA is the second nucleic acid found in cells. It plays an important role in the process in which DNA information eventually is processed into the information of proteins. RNA plays at least 3 roles in cells.

  1. RNA acts as a cellular “messenger” as mRNA, to deliver info encoded in DNA   to ribosomes for translation into proteins
  2. RNA acts as the cell “translator” as transfer RNA or tRNA, delivering the  correct amino acid to the ribosome as the mRNA is “read”
  3. RNA is a component of ribosomes as rRNA  or ribosomal RNA ( in fact it is rRNA which catalyzes formation of chemical bonds between the amino acids as the ribosomes read mRNA and produces proteins).

RNA is similar to DNA in that RNA is formed by a chain of nucleotides. However, RNA in cells is found as a single strand and the parts of RNA nucleotides differ from DNA nucleotides.

 RNA nucleotides consist of:

            a. a sugar called ribose

            b. a phosphate

            c. a nitrogenous base: the nitrogenous bases of RNA are the same as for DNA       EXCEPT in RNA,  uracil replaces thymine.

                        RNA complementary base pairing:

                        A:U  and G:C

 

RNA as genetic information in some viruses

 

Recall that acellular viruses contain either DNA or RNA but not both. Some viruses use RNA as their genetic information and thus are called “RNA viruses”. Examples of RNA viruses include influenza viruses, West Nile virus and HIV. Unfortunately the enzymes which copy RNA make many more mistakes than the enzymes which copy DNA. Consequently some RNA viruses have very high mutation rates as seen with influenza viruses and HIV. As a consequence, it is challenging to develop vaccines against al the mutant strains and the viruses may quickly evolve resistance to antiviral drugs making treatment very difficult.

 

 

 

 

 

 

 

 

 

Appendix 2: Gene expression from DNA to Proteins: Transcription and Translation

The DNA bases are the genetic alphabet of the cell. Each combination of 3 specific bases (called codons) are the genetic “words” of the cell. These codons or genetic words encode information for a specific amino acid. Therefore a specific sequence of DNA bases in a gene encodes information for the specific sequence of amino acids in a protein. The specific sequence of amino acids determines how a protein will fold and ultimately the protein’s shape/structure. The protein shape/structure in turn determines the protein’s function.  In this way, DNA encodes information for making all the proteins within a cell.  Many of the cell’s proteins will function as enzymes which will make ALL the other components of the cell (carbohydrates, lipids, nucleic acids).

 

Cells however cannot directly translate information from a  DNA base sequence into the amino acid sequence of proteins. Instead, a messenger carries information from DNA to the protein synthesis factories, the cell ribosomes. These messengers are called messenger RNA or “mRNA”. 

 

Transcription

                                                 DNA----à mRNA

                                                 “transcription

 

The process by which complementary mRNA is formed using DNA as a template is called “transcription” and is performed by an enzyme called “RNA polymerase”. It is important to note that RNA polymerases make many mistakes when they copy DNA into mRNA, mistakes which they can not “fix”. 

 

Transcription is possible because of complementary base pairing rules between DNA bases and RNA bases

 

                DNA base pairs with RNA base:

                                A:U

                                T:A

                                G:C

Note that transcription is conversion from one nucleic acid language (DNA language) into another nucleic acid language (RNA language)

 

Translation

 

                                                                mRNA--à protein

                                                    “translation

 

Once mRNA is formed, ribosomes will attach at specific sites on the mRNA and begin “reading” or translating the mRNA into proteins. (translation because we are changing from nucleic acid language in which nucleotides or bases form “words” into protein language in which amino acids form “words”-new alphabet!)

 

Gene expression or the process of using DNA to make proteins can be summarized as below:

DNA-à mRNA---à protein

              transcriptionà translation

 

 

Translation and the “Genetic Code”

 

The Genetic Code is the relationship between nucleic acid nitrogenous base sequence and specific amino acids.

 

Nucleic acid base sequences are read as “triplets”, each “triplet” is called a codon.

 

When DNA is transcribed into RNA, a special RNA called messenger RNA or mRNA carries the codon sequences to the ribosomes, the “biochemical workbench” (as described by Prof Naganuma) at which the mRNA will be translated into a specific chain of amino acids. The actual translator molecule of the cell is another type of RNA, called transfer RNA or “tRNA”. One end of each tRNA recognizes complementary mRNA codons. Each tRNA carries its specific amino acid ( the tRNA’s are “loaded” with their correct amino acids by special enzymes in the cell). Once the tRNA  brings the correct amino acid to the mRNA in the ribosome, the ribosome catalyzes formation of chemical bonds between the amino acids. The ribosome in a sense “reads” the mRNA with tRNA help. As a result of this translation process, an mRNA with a specific sequence of nitrogenous bases will be translated into a chain of amino acids in specific sequence. Once these “polypeptides”/proteins are formed, they will fold into complex shapes, their “functional conformations” and thus begin their cellular functions.

 

Example: transcription and translation

DNA sequence              TAC AAC GGT  CAC                                          DNA

-Transcribed into

Complementary

mRNA sequence               AUG UUG CCA GUG                                          mRNA

-Translated into

specific amino acid

sequence                        methionine-leucine-proline-valine                             protein