LECTURE - Resistance to Antimicrobials

Question 1

5 main mechanisms by which bacteria become resistant to antibiotics

Answer 1

• limiting access of antibiotic to organism
• enzymatic inactivation of the antibiotic
• active efflux of antibiotic from bacterial cell
• modification or protection of antibiotic target
• failure to activate antibiotic

Question 2

how do organisms limit access of antibiotics to its target?

Answer 2

GRAM NEG :

• some (like vancomycin) are too big to get through porin channels
• single mutations in porins can alter their channels = more resistant

GRAM POS:
- thicker cell wall in some vancomycin-intermediate resistant S. aureus ‘mop up’ vancomycin making it less available to proper targets

Reduced uptake at the cytoplasmic membrane

• not common
• for ex: aminoglycoside antibiotics use a transporter in the bacterial cytoplasmic membrane to enter the cell; alteration of this transporter could prevent entry of the aminoglycoside (possibly during growth in anaerobic conditions)

Question 3

gram neg with beta lactamase

Answer 3

get more “bang for their buck” bc enzyme is concentrated in periplasmic space

Question 4

gram positives beta lactamase

Answer 4

they excrete beta-lactamase into their extracellular environment where they get diluted

Question 5

how can we prevail against beta lactamases?

Answer 5

• by adding clavulanic acid or sulbactam (B-lactam analogs that inactivate bacterial enzymes)
• BUT increased production of beta-lactamase by a bacterium can tip the scales in bacterium’s favour again

Question 6

aminoglycosides inactivation

Answer 6

• when organisms covalently attach acetyl, phosphoryl, or adenyl groups

“-rylation..”

Question 7

other examples of enzymatic inactivation of the antibiotic

Answer 7

• streptogramin acetyltransferase (vat and sat genes) found in staph and enterococci
• possible role for tetX gene in oxidative inactivation of tetracycline

Question 8

one of the most common mechanisms of resistance

Answer 8

active efflux of the antibiotic

- ATP is used by a cytoplasmic pump to expel the antibiotic

Question 9

five classes of efflux pumps

Answer 9

• MATE: multi-drug and toxic compound extrusion
• MFS: major facilitator superfamily
• SMR: staphylococcal multi-resistance
• RND: resistance modulation division
• ABC: ATP binding cassette

Question 10

how do organisms modify/protect antibiotic targets in B lactams

Answer 10

• alternative PBP (transpeptidase enzyme) that doesn’t bind penicillin; no binding = no effect on peptidoglycan synthesis; organisms grow normally
• big problem in gram pos like mecA gene in Staph aureus encoding PBP2’

Question 11

resistance to macrolides, lincosamides, ketolides, etc, by modification or protection of antibiotic target

Answer 11

• rRNA methylase: enzymes encoded by ermA, B, F, and G = can covalently add a methyl group to a specific adenine residue on the 23S rRNA = rendering the 50S subunit immune to these five antibiotic types
• known as MLSKO(B) group = common in Bacteroides species and some gram pos cocci

Question 12

resistance to quinolone and rifampin via modification or protection of target

Answer 12

mutation in DNA gyrase B subunit or RNA polymerase, respectively, renders the enzymes immune to binding by the antibiotics

Question 13

resistance to trimethoprim and sulfonamides via modification or protection of target

Answer 13

mutation in enzymes of the folic acid pathway that are affected by these antibiotics = allows enzymes to still do wok but have a lower affinity for antibiotic than for their intended substrate

Question 14

resistance to metronidazole

Answer 14

• mutation in expression levels of flavodoxin = fail to convert metronidazole into its active form so it has no effect on the bacterium
• Isoniazid for treating TB also has to be activated, by a mycobacterial catalase, and mutation in catalase can cause resistance

Question 15

ways resistance genes are acquired by bacteria: (5)

Answer 15

• mutation
• plasmids
• transposons
• integrons
• conjugative transposons

Question 16

cross-resistance

Answer 16

when one resistance mechanism works for multiple types of antibiotics
(ex: methylase-enzyme modified coded for by ergm genes)

• methylase-modified ribosome is refractory toward macrolides, lincosamides, streptogramins, etc.
• efflux pumps can also work on several antibiotics, rendering an organism resistant to them all

Question 17

antibiotic tolerance

Answer 17

• organism not resistant to antibiotic per se, but avoids its killing potential by another means
  ex: all in the peptidoglycan synthesis inhibitors
• UTIs = E. coli loses its peptidoglycan but doesn’t lyse because of the hypertonic environment of the kidney or bladder
• down-regulation of autolysis enzymes leads to slower (or stoppage of) growth and avoidance of killing B-lactams

Question 18

what is acquired through mutation in bacteria

Answer 18

• not the gene itself, but the altered function of the gene product that can be acquired through mutation
• PBPs = renders them insensitive to B lactams
• DNA gyrase = confers resistance to quinolone and fluoroquinolone antibiotics
• enzymes of the folic acid pathway = resistance to sulfonamides and trimethoprim

Question 19

DNA segments capable of inserting into chromosomal or plasmid DNA independent of homologous recombination

Answer 19

transposons

Question 20

transposons have …

Answer 20

• insertion sequences and genes that code for transposase enzymes = incorporation of transposon randomly into target DNA
• complex transposons typically have genes for antibiotic resistance(s) too

Question 21

integrons

Answer 21

• *basically transposons but have 3 added features**
• att (attachment site)
• gene = integrase which facilitates incorporation of closed circular DNA into the att site
• promoter (P) will allow expression of integrated, promoterless (single open reading frame) genes

** can accumulate many antibiotic resistance genes but his mechanism and then transfer them among organisms, usually by inserting themselves into conjugating pr mobilizable plasmids first) **

Question 22

the Ambler classification

Answer 22

• B-lactamases

- based on AA sequences of the enzymes

Question 23

the Bush-Jacoby-Medeiros groups

Answer 23

• B lactamases

- based on enzyme function

Question 24

resistance to glycopeptide antibiotics

Answer 24

• modification or protection of the target antibiotics
• replaces the D-Ala-D-Ala target with a D-Ala-D-lactate
• vanH converts pyruvate to D-lactate
• vanA and vanB leads to formation of D-Ala-D-lactate; vancomycin cannot bind D-Ala-D-lactate

Question 25

resistance to tetracycline

Answer 25

• modification or protection of the antibiotic target
• ribosome protection
• products of tetO, tetM, and tetQ can bind to 30S ribosome site to protect ribosome from tetracycline binding

Question 26

how can antibiotic resistance genes be regulated by bacteria?

Answer 26

• repression
• attentuation
• activation

Question 27

repression (regulation of resistance genes)

Answer 27

• tetR in resistance to tetracycline, allows for expression of efflux pump (TetB) when they’re needed (i.e. only when tetracycline is present)

blaZ in resistance to penicillin, allows for expression of B-lactamase in MRS only when a B-lactam is present

Question 28

attenuation (regulation of resistance genes)

Answer 28

• ermG in resistance to the MLSKO* group of antibiotics, allows for expression of methylase enzyme when they’re needed (only when an MLSKO antibiotic is present)

Question 29

MLSKO antibiotic

Answer 29

macrolide, lincosamide, streptogramin, ketolide, oxazolidinone

Question 30

activation (regulation of resistance genes)

Answer 30

• two-component, sensor-activator regulatory systems in the cytoplasmic membrane of bacteria, (VanS detects vancomycin in Enterococcus = phosphorylates transcriptional activator, VanR, and triggers transcription of vanH, vanA,and vanX to alter target for vancomycin)
• Amp C B-lactamase can be activated in E.coli
• insertion elements (ISs) = have their own promoters that activate otherwise silent antibiotic-resistant genes (ermF in Bacteroides)
• mutation of promoter = increase ability to activate resistance gen and make organism less susceptible to antibiotics (ampC in Enterobacter)

Question 31

mobile genetic elements (MGEs)

Answer 31

• plasmids
• transposons
• integrons
• conjugative transposons

Question 32

plasmid

Answer 32

Conjugative

• self-transmissable
• have gene (tra) which encode the proteins needed for transfer

Mobilizable plasmids
- have mob genes which allow them to tag along with conjugating plasmids that provide other necessary transfer functions = smaller !

Question 33

transposons

Answer 33

DNa segments capable of insertin into a chromosomal or plasimd DNa independent of homologous recombination