Antibiotic Resistance

Question 1

Mechanisms of antibiotic resistance

Answer 1

Active efflux
Target bypass
Target site modification
Decreased influx
Inactivation of antibiotic
Target protection

Question 2

Active efflux

Answer 2

Pumps facilitate the movement of antibiotic out of cell

Question 3

Active efflux pump families

Answer 3

ABC
MFS
SMR
RND
MATE

Question 4

ABC transporter family

Answer 4

contains both uptake and efflux transport systems. The members of this family are unique in that they use energy derived from ATP hydrolysis.
These pumps transport amino acids, drugs, ions, polysaccharides, proteins, and sugars.
Bacterial ABC transporters usually are made up of six transmembrane segments (TMS) consisting of α-helices, function in the membrane in pairs, either as homodimers or heterodimers, and work in conjunction with cytoplasmic ATPases.
These pumps have fairly specific substrates, and there are very few found in clinically significant bacteria.
One notable ABC pump is found in Vibrio cholerae (VcaM), and is capable of transporting fluoroquinolones and tetracycline

Question 5

MATE transporter family

Answer 5

use a Na+ gradient as the energy source, and efflux cationic dyes, and most efflux fluoroquinolone drugs.
Some MATE pumps have also been shown to efflux some aminoglycosides. Other substrates for these pumps may have unrelated chemical structures.
These pumps are made up of twelve TMS.
Very few of these have been characterized in bacteria, and most are found in gram negative organisms.

Question 7

MFS transporter family

Answer 7

catalyze transport via solute/cation (H+ or Na+) symport or solute/H+ antiport. They are involved in the transport of anions, drugs (e.g. macrolides and tetracycline), metabolites (e.g. bile salts), and sugars. The MFS pumps have the greatest substrate diversity as a group, yet individually tend to be substrate specific.
Examples of this substrate specificity include Acinetobacter baumannii having separate MFS pumps for erythromycin (SmvA) and chloramphenicol (CraA and CmlA), and Escherichia coli having separate MFS pumps for macrolides (MefB), fluoroquinolones (QepA), and trimethoprim (Fsr). There are rare examples of MFS pumps with a slightly broader substrate specificity, such as in the NorA pump in Staphylococcus aureus which transports fluoroquinolones and chloramphenicol (these antimicrobials are the most commonly transported by MFS pumps), or the S. aureus LmrS pump which transports linezolid, erythromycin, chloramphenicol, and trimethoprim. These pumps are made up of twelve or fourteen TMS, and over 1,000 have been sequenced in bacteria. Most MFS pumps have been found on bacterial chromosomes, and nearly 50% of the efflux pumps in E. coli are MFS pumps

Question 8

SMR transporter family

Answer 8

energized by the proton-motive force (H+), are hydrophobic, and efflux mainly lipophilic cations, so may have a very narrow substrate range.
The genes for these pumps have been found in chromosomal DNA and on plasmids and transposable elements. These pumps are made up of four TMS and function as asymmetrical homotetramers.
Drug efflux has only been seen in a few of these pumps, and these most commonly confer resistance to β-lactams and some aminoglycosides. Examples of SMR pumps are seen in Staphylococcus epidermidis (the SMR pump which transports ampicillin, erythromycin and tetracycline) and Escherichia coli (the EmeR pump which transports vancomycin, erythromycin, and tetracycline).

Question 8

RND transporter family

Answer 8

catalyze substrate efflux via a substrate/H+ antiport mechanism, and are found in numerous gram negative bacteria. They are involved in the efflux of antibiotics (all are multi-drug transporters), detergents, dyes, heavy metals, solvents, and many other substrates. Some of these pumps may be drug or drug class specific (Tet pump—tetracycline; Mef pump—macrolides). Many other RND pumps are capable of transporting a wide range of drugs, such as the MexAB-OprM pump in Pseudomonas aeruginosa that confers intrinsic resistance to β-lactams, chloramphenicol, tetracycline, trimethoprim, sulfamethoxazole, and some fluoroquinolones. These pumps are complex multi-component pumps generally made up of twelve TMS and contain two large periplasmic loops between TMS 1 and 2, and TMS 7 and 8. In order to function, these pumps will connect to an OMP and that connection is stabilized by MFPs. Interestingly, these pumps share a high degree of homology among the RND members. The genes for the RND pumps are generally organized as an operon. In many, the gene organization is as follows: the gene for the regulator (which may be transcribed in the opposite direction to the other genes) is adjacent to the MFP gene, which is adjacent to the main pump gene, and then the OMP gene. Probably the most widely studied RND pump is the AcrAB-TolC pump in Escherichia coli, which confers resistance to penicillins, chloramphenicol, macrolides, fluoroquinolones, and tetracycline. The AcrB pump protein contains two binding pockets which allow the binding of substrates of varying size and chemical properties

Question 9

Beta lactamases

Answer 9

Enzymes
Resistance against beta lactams by hydrolysis of amide bond in the beta-lactam ring

Question 10

Clavulanic acid

Answer 10

Beta lactamase inhibitor

Question 11

Specific RNA methyltransferases

Answer 11

Protect the target site from aminoglycoside binding

Question 12

MfpA

Answer 12

blocks gyrase’s ability to twist and untwist DNA.
By binding to gyrase in DNA’s place, MfpA apparently deprives fluoroquinolones of their target; the drugs bind to gyrase-DNA complexes rather than to just the enzyme. MfpA’s inhibition of gyrase function probably slows the bacteria down, but it’s better than being killed by fluoroquinolones.

Question 13

Which efflux pumps pump H+ into the cell

Answer 13

MFS
SMR
RND

Question 14

Which efflux pump uses Na+ into the cell

Answer 14

MATE

Question 15

Which efflux pump requires ATP

Answer 15

ABC

Question 16

Which efflux pump spans both inner and outer membrane

Answer 16

RND

Question 17

Target site alteration

Answer 17

involves alteration of the antibiotic target to reduce binding of the antibiotic.
This can involve mutations in the gene encoding the protein target of the antibiotic molecule or enzymatic alteration of the binding site

Question 18

Target bypass

Answer 18

the function of the antibiotic target is accomplished by a new protein that is not inhibited by the antibiotic, making the original target redundant and the antibiotic ineffective

Question 19

Decreased influx

Answer 19

mediated by changes to membrane structure, for example, the downregulation of porins, which are transmembrane proteins that allow the passive transport of various compounds, such as antibiotics, into the bacterial cell

Question 20

Target protection

Answer 20

generally involves the physical association of a target protection protein with the antibiotic target, thereby relieving it from antibiotic-mediated inhibition

Question 21

Mechanisms of target bypass

Answer 21

The drug target (such as an enzyme) becomes redundant due to acquisition of a gene that encodes an alternative enzyme that fulfils the function of the drug target

The drug target can be replaced by an alternative target that sequesters the drug, thereby allowing the drug target to resume its function

The drug target can be overproduced and thus there is insufficient amount of drug to inhibit the increased available target

Question 22

Mechanisms of drug alteration

Answer 22

Enzymes can hydrolyse the functional group of the drug, thereby destroying its antibacterial activity

Enzymes (such as acetyltransferases, methyltransferases or phosphotransferases) can modify the drug by covalent transfer of various chemical groups to prevent it from binding its target

Question 23

Mechanisms of target protection

Answer 23

Target protection proteins can bind to the drug target and sterically remove the drug from the target

Target protection proteins can bind to the drug target and mediate allosteric dissociation of the drug from its target

Target protection proteins can bind to the drug target and cause conformational changes to allow the target protein to function even in the presence of the drug

Question 25

Modifying aminoglycosides

Answer 25

can be modified by acetyltransferases, phosphotransferases or nucleotidyltransferases, modifying the hydroxyl or amino groups of the drug, which in turn substantially reduces the affinity of the drug to the target

Question 27

Luria-Delbrück fluctuation test

Answer 27

Samples were taken from large culture and were allowed to grow to same density, then exposed to bacteriophage Phage lysed the cells Resistant cells formed colonies The number of colonies fluctuates Explains the random mutation hypothesis

Question 28

Number of codons that can result from a single base change in the tyrosine codon UAU

Answer 28

9

Question 29

Consequence of the fluctuation theory

Answer 29

Mutations are not induced by the presence of an antibiotic, rather pre-existing mutants are selected from the population, favoured and overgrow it

Question 30

Emergence of mutants

Answer 30

Emerge at different rates Decline in the absence of selection pressure

Question 31

Transposons

Answer 31

Move via the cut and paste mechanism For mobilisation of Ds another element is required, called Activator, Ac. It is similar to bacterial IS elements in that it contains only the transposase Inverted repeats form lollipop structures visible in electronmicroscopy when denatured (the double strands are separated by e.g. heat) then allowed to renature slowly

Question 32

Insertion (IS) elements

Answer 32

only carry one gene the product of which is an enzyme required for their mobility: transposase

Question 33

Bacterial transposons

Answer 33

Mostly associated with carrying and transferring antibiotic resistance genes

Question 34

Mottling of grains of maize

Answer 34

discovered a genetic element (termed Dissociation, Ds) that doesn’t only cause mottling but also causes chromosome breakage Her mapping studies also showed that the breakage always occurs at the Ds element She also discovered that the same Ds element didn’t have a constant location but moved to new locations Since then, these elements, now called transposons, have been discovered in every domain of life

Question 35

IS2

Answer 35

Transposons gene Transposase enzyme only

Question 36

Tn5

Answer 36

Express transposon gene Also express antibiotic resistance

Question 37

Inverted repeats

Answer 37

They are in opposite DNA strands in order to preserve the same 5’ to 3’ polarity

Question 38

Mechanism of Tn5 transposition

Answer 38

Tnp binding Synapses Cleavage Target capture Insertion

Question 39

Horizontal gene transfer between bacteria

Answer 39

Transformation Transduction Conjugation

Question 40

Transformation

Answer 40

Recipient cell takes up donor DNA Recombination occurs between donor DNA and recipient DNA

Question 41

Phage transduction- lysogenic pathway

Answer 41

Attachment of virus to host cell Injection of viral DNA Viral DNA is integrated into host DNA Viral DNA is replicated with the host DNA at cell division

Question 42

Lytic cycle

Answer 42

When a bacterial cell is infected with a transducing phage, the lytic cycle may occur. However, during lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes package host DNA accidentally. The result is called a transducing particle. Upon lysis of the cell, transducing particles are released along with normal virions that contain the virus genome. When this lysate is used to infect a population of recipient cells, most of the cells are infected with a normal (lytic) virus. However, a small proportion of the population receives transducing particles that inject the DNA they packaged from the previous host bacterium. Although this DNA cannot replicate, it can recombine with the DNA of the new host.

Question 43

Specialised transduction

Answer 43

When the λ phage lysogenizes a host cell, the phage genome is integrated into the E. coli chromosome at a specific site. This site is next to the cluster of genes that encode the enzymes for galactose utilization. Upon induction, the viral DNA separates from the host DNA by a process that is the reverse of integration. Usually the λ DNA is excised precisely, but occasionally the phage genome is excised incorrectly. Some of the adjacent bacterial genes to one side of the prophage (for example, the galactose operon) are excised along with phage DNA. At the same time, some phage genes are left behind. This transducing particle can subsequently transfer genes for galactose utilization to a recipient cell.

Question 44

Lytic pathway

Answer 44

Lytic events are initiated Phage components are synthesised and virions are assembled Lysis of the host cell and release of new phage virions

Question 45

F plasmid composition

Answer 45

Replication: it encodes a replication initiator protein and multiple replicons (RepFIA, RepFIB, and RepFII ) that are capable of driving autonomous replication of the plasmid Partitioning the sopABC genes encode a type I ATPase partitioning system. Partitioning ensure that daughter cells each will inherit copies of the plasmid Toxin-antitoxin (TA) modules : post-segregational killing (PSK) indicating that cells that have lost the plasmid after cell division die because of the activation of a toxin. The underlying molecular mechanism involves toxin-antitoxin (TA) pairs in which the toxin is not active in plasmid-bearing cells and controlled by the activity of a neutralizing antitoxin DNA transfer region: DNA transfer genes represent the largest (approximately 30 kbp) part of the backbone. These are required for transferring the plasmid from one bacterium to another Leading region (yellow): Genes with known functions for the establishment of the plasmid in a new host are indicated. Cargo genes: which harness their host bacteria with antibiotic resistance and virulence genes, causing increasingly severe problems for the treatment of infectious diseases. IS sequences and transposons.

Question 46

Conjugation

Answer 46

The pilus retracts Fell pairs are stabilised. F plasmid nicked in one strand Transfer of one strand from F+ cell to F- cell F plasmid simultaneously replicated in F+ cell Synthesis of the complementary strand begins in the recipient cell

Question 47

Hfr cells

Answer 47

F plasmid is integrated onto the chromosome

Question 48

Nosocomial infection

Answer 48

(acquired in hospital or other healthcare settings) Procedures to prevent spread in clinical settings Reserving last line antibiotics until absolutely necessary International regulation —- 50 kg ciprofloxacin / day dumped into the river in Hyderabad! Who knows where else this has happened? Resistances to other substances (and other genes) are spread on plasmids – these can also encode antibiotic resistance.