Antibiotic Resistance I and II

Question 1

variety pathogen genome

Answer 1

• 10^4 bp - 10^12 bp

- linear chromosomes/plasmid, circular plasmids

Question 2

chromosomal genome

Answer 2

• large (megabases - average 4000 genes)
• double-stranded DNA
• tightly packed structure (nucleoid)
• Typically, but not always, haploid
• circular or linear
• can encode for certain antibiotic resistance & virulence factors
• usually within pathogenicity islands

Question 3

plasmid genome

Answer 3

• also called episome
• autonomous, self-replicating, extrachromosomal, double-stranded DNA
• tend to be small (kilobases)
• usually circular, but can be linear
• genes for antibiotic resistance (R plasmids*) and for virulence factors
• easily transferred from host to host
• Found in most species of Gram-positive and Gram-negative bacteria.
• A single cell can harbor more than one plasmid.

Question 4

cojugative plasmids

Answer 4

• (including F and R plasmids)
• can autonomously transfer themselves from one host to another; therefore passing antibiotic resistance to other individuals within the species or even to individuals in other species or genera.

Question 5

Non-conjugative plasmids

Answer 5

• cannot transfer themselves, but they can be transferred by a conjugative plasmid.
• Most plasmids used for genetic engineering are non-conjugative.

Question 6

BACTERIAL VIRUS GENOME

Bacteriophage, phage

Answer 6

-RNA or DNA
-double- or single-stranded
-linear or circular
-3 to 300 kilobases (small)
-can carry virulence factors
e.g., Diptheria Beta-toxin
Cholera toxin*

Question 7

lysogenic infection

Answer 7

• After infection, it might not replicate (called a lysogenic or latent infection).
• The lysogenic phage genome is called a prophage.
• Prophages can circularize and remain autonomous (like plasmids) or integrate into the host chromosome.

Question 8

lytic infection

Answer 8

• the phage genome replicates
• proteins are synthesized and assembled into capsids
• the replicated genomes are packaged within those capsids
• the host cell lyses
• the progeny escape into the environment to reiterate the process.

Question 9

transposition

Answer 9

-relies on the ability of these transposable elements to synthesize their own specific recombination enzyme.

Question 10

transposable elements

Answer 10

-are genetic units that mediate their own transfer from one location in a genome to another location within the same genome or from one genome to another (e.g., from the bacterial chromosome to a conjugal plasmid).
-OCCURS WITHIN THE SAME CELL ONLY
-major carrier of antibiotic resistance genes
evolution of R plasmids

Question 11

Four major types of transposable elements exist:

Answer 11

i) insertion sequence (IS) elements ;
ii) transposons;
iii) transposable prophages;
iv) conjugal transposon.

Question 12

insertion sequences

Answer 12

• Insertion sequence elements are segments of DNA of about 1000 bp.
• IS elements only carry genes for transposition, including its own site-specific recombinase and two distinct nucleotide recognition sequences located at each terminus in inverted order.
• If an IS element transposes into a gene, its insertion will interrupt that gene causing an insertion mutation.
• Transposition occurs infrequently.
• It involves recognition by the recombinase of the terminal inverted repeats and a target area into which a new copy of the IS element will be inserted.
• After transposition into the target area, the original copy remains at its original site.

Question 13

transposons

Answer 13

• Transposons are transposable elements containing genes necessary for transposition and for other functions.
• They are considerably larger than IS elements, because they are hybrids that consist of a central core of genes bounded by two independent IS elements
• The core genes can encode for a variety of functions including resistance to antimicrobial agents and heavy metals.
• conjugation = means of transfer
• = called a transposon if it has a gene for antibiotic resistance between ISs
• must be located within a genome of some kind

Question 14

conjugal transposon

Answer 14

• has genes for conjugation between IS’s
• Not only do these multiply resistant transposons jump from location to location within a given cell, but they also can mediate their own transfer to other cells
• can happen between different species
• found in streptococcal Enterococcus facaelis

Question 15

transposable prophage

Answer 15

• has transposable phage between IS’s
• A bacteriophage that is a transposon. As a phage, it can alternate between lytic growth and lysogeny.
• During lysogeny, it can integrate virtually anywhere in a chromosome. Once integrated, it can transpose itself to another location.

Question 16

R Plasmids

Answer 16

-These plasmids carry genes (often multiple) that confer resistance to antibiotics.
-The genes responsible for resistance usually code for enzymes that inactivate antibiotics or reduce the cell’s permeability to antibiotics.
-In contrast, resistances conferred by chromosomal mutation usually involves the modification of the antibiotic target, e.g. RNA polymerase and streptomycin.
-R plasmids evolve quickly, easily acquiring resistances by fusing to other plasmids or by acquiring transposons
-many Gram-negative bacteria carry R plasmids that carry multiple resistances
-selection for one antibiotic can cause selection for multiple antibiotic resistance because now what’s left is just the antibiotic resistant bacteria
-can transfer between species
-nonpathogenic bacteria that carry R plasmid
often act as reservoir to pathogenic bacteria

Question 17

How do bacterial cells
acquire resistance
that requires multiple changes?

Answer 17

• They do so by acquiring large pieces of DNA from other bacteria or from bacterial viruses (also called bacteriophages).
• The multiple resistance genes already exist together

Question 18

modes of genetic exchange

Answer 18

Transformation
Conjugation
Transduction

Question 19

exogenote

Answer 19

donor DNA

Question 20

endogenote

Answer 20

recipient DNA

Question 21

transformation

Answer 21

-Transformation involves the release of naked DNA into the environment by the lysis of some cells (the donors), followed by the uptake of that DNA by other cells (the recipients).
-Streptococcus pneumoniae, Haemophilus influenzae
& Neisseria gonorrhea use transformation to alter appearance of clinically important surface antigens
-permits evasion of host immune system

Question 22

competence

Answer 22

• the ability to take up DNA from the environ

- often encoded by chromosomal genes

Question 23

transformation chain of events

Answer 23

1) By lysis, trauma, or death, donor cells release naked DNA into the environment. Donor enzymes chop the released DNA into smaller pieces. Since the DNA is naked, it is highly susceptible to environment factors, including detergents that inhibit binding and nucleases that destroy DNA.
2) Pneumococcal cells secrete a protein competence factor that induces cells to synthesize special proteins necessary for competence.
3) A protein located on the cell surface binds double-stranded DNA indiscriminately. It binds any DNA present in the environment. As the DNA passes through the membrane, one strand is degraded.

Question 24

what does the recipient cell need to undergo transformation

Answer 24

• When there is a high enough conc of pheromone (and thus high enough conc of bacteria), they do some population based process such as competence– happens at high conc because there is a high likelihood of having DNA in the environ
• Need proteins on the surface of the recipient to bind DNA, need nucleases that make double stranded DNA = single stranded, then need to protect DNA inside cell from nucleases by using proteins

Question 25

3 fates of the exogenote

Answer 25

i) degradation (there will be no heritable change in the recipient);
ii) circularization (if it can replicate, it will be inherited as a plasmid)- usually doesn’t replicate; or
iii) recombination into the chromosome.

Question 26

conjugation

Answer 26

• Conjugation involves cell-cell contact, therefore it is sometimes called mating.
• The donor cell contains a plasmid (an autonomously replicating, extrachromosomal molecule of circular double-stranded DNA). Because all the genes required for conjugation reside on this plasmid, it is called a conjugal plasmid.
• The recipient does not carry the conjugal plasmid.
• Once the recipient has received the plasmid, it is called the exconjugant.
• Because DNA transfer occurs through cell-cell contact, DNA is never naked and, thus, is not exposed to nucleases or detergents.

Question 27

how to tell between conjugation and transformation in the lab

Answer 27

You can distinguish between conjugation and transformation by throwing in nucleases and detergents because if they still transfer the material = conjugation because no transfer of DNA in the environ.
- Also if you shake it a lot and that stops transfer = conjugation because you are disrupting the mating pairs

Question 28

clinical relevance of conjugation

Answer 28

1) rapid dissemination of drug resistance
via conjugal R plasmids or conjugal transposons
2) mediates inter-species transfer from non-pathogenic Bacteroides to pathogenic Shigella or E. coli or Salmonella or worse
-normal flora acts as resistance reservoir

Question 29

conjugation chain of events

Answer 29

• The donor cell harbors an R plasmid; the recipient cell does not.
• Genes carried by the R plasmid encode a sex pilus (longer and thicker than the common pilus), which facilitates capture of the recipient cell to form a mating pair.
• A conjugation bridge forms between these two cells through which DNA passes. Transfer of DNA occurs through a special form of replication, called transfer replication.
• Replication initiates at the origin of transfer (oriT) located on the R plasmid and proceeds by the rolling circle mechanism.
• First, one of the strands of DNA breaks. Then it is pulled away from its complement through the conjugation bridge.
• The strand left in the donor is replicated to reform the double-stranded R plasmid.
• The transferred strand recircularizes and replicates into a double-stranded R plasmid.
• The cells now detach.
• Both the donor and recipient cells possess a complete copy of the R plasmid. Each can go on to mate with other R- recipients.

Question 30

transduction

Answer 30

-Transduction is form of gene transfer that is mediated by bacterial viruses (bacteriophage).

Question 31

makeup of a bacteriophage

Answer 31

The individual phage or virion consists of a protein capsid enclosing a genomic nucleic acid. The nucleic acid can either be DNA or RNA but not both.

Question 32

clinical relevance of tranduction

Answer 32

• phage can carry virulence factors
  e. g., Beta - toxin produced by Corynebacteria diptheria Beta - phage or cholera toxin produced by Vibrio cholerae CTX phage*

Question 33

phage infection

Answer 33

• A phage infects a sensitive cell by first adsorbing (binding) to a specific receptor on that cell’s surface and then by injecting its genome into the interior of the cell.
• Two types of phage exist: 1) lytic or 2) temperate

Question 34

temperate phage

Answer 34

• Temperate phage (like C. diphtheria ß-phage and V. cholerae CTX phage) may initiate a lytic growth process as described above or alternatively, enter a quiescent form called a prophage.
• The host cell goes about its business as usual except that it harbors a latent prophage that it passes harmlessly on to its descendents. Such a cell is said to be a lysogen (capable of becoming lytic).

Question 35

fate of a prophage

Answer 35

-The prophage of some temperate phage become latent by circularizing as an autonomously replicating plasmid. -The prophage of other temperate phage become latent by physically integrating into the host chromosome.

Question 36

clinical implications of genetic exchange

Answer 36

• Bacteria co-existing in the lower GI-tract, soil, or other environments can exchange DNA encoding diverse antibiotic resistances.
• Antibiotic preparations are often contaminated with DNA, increasing the chance of genetic exchange via transformation between antibiotic-producers & pathogens.

Question 37

bacteriophage

Answer 37

• Bacteriophage or phage reproduce only within bacterial cells: they are obligate parasites.
• They are minimally composed of protein and nucleic acid.

Question 38

lysogen

Answer 38

he host cell goes about its business as usual except that it harbors a latent prophage that it passes harmlessly on to its descendents.
-Such a cell is said to be a lysogen (capable of becoming lytic).