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Biofilms & Microbial communities > Antibiotic resistance > Flashcards

Antibiotic resistance Flashcards

1
Q

what is intrinsic resistance?

A

independent of antibiotic selective pressure and horizontal gene transfer; result of inherent structural or functional characteristics.

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2
Q

what is acquired resistance?

A

mutations in drug targets or transfer of resistance genes through phage-mediated transduction and mobile plasmids.

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3
Q

what is horizontal gene transfer?

A

the acquisition by an organism of genetic information by transfer, for example via the agency of a virus, from an organism that is not its parent and is typically a member of a different species.

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4
Q

why is horizontal gene transfer more frequent in biofilms?

A

HGT is promoted in biofilms
Because cells are close together it facilitate the transfer process (more easily than planktonic)

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5
Q

what ways can horizontal gene transfer occur?

A
  • Transformation: lysed bacterium release DNA
  • Conjugation: occurs via membrane to membrane or via appendages
  • Transduction: via phages
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6
Q

what is intrinsic transformation?

give evidence for this

A

Competent cells take up foreign DNA across their cell membrane and incorporate it into their own genome by genetic recombination.

virulent but killed Streptococcus pneumonia cells added to a living culture of nonvirulent S. pneumoniae causes some cells to become virulent

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7
Q

how did Vitkovitch (2004) demonstrate intrinsic transformation?

A
  • Vitkovitch (2004) demonstrated that biofilm grown Streptococcus mutans was transformed to erythromycin resistance either by the addition of naked DNA or heat killed donor cells carrying the antibiotic resistance genotype.
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8
Q

how muhc higher were the rates of transformation in Vitkovitch’s experiment on intrinsic transformation between biofilms and planktonic cells?

A
  • The rates of transformation were 10 to 600 times greater than those observed in cells in planktonic culture.
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9
Q

describe the new mechanism that uses vesicles in transformation

A

Membrane vesicles: released from the cell surface by many Gram-negative, and some Gram-positive, bacteria and can contain proteins, polysaccharides and importantly for microbial adaptation, DNA.

Carry resistant determinants like β-lactams, and enzymes such as protease, endopeptidases, etc., OMVs give survival advantage to bacteria due to antibiotic resistance traits in biofilms, thereby protecting from antibiotic carnage

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10
Q

what bacterial strain has been shown to release extracellular DNA (eDNA) via membrane vesicles into the developing biofilm ?
what does it provide?

A

Streptococcus mutans
provides therefore an important source for genetic material via this novel mechanism.

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11
Q

how does conjugation occur in gram negative bacteria?

A

via fimbria and pili

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12
Q

Plasmid transfer in the biofilm system was observed to be ____ higher than in planktonic culture. (Dunny et al. 1995).

A

Plasmid transfer in the biofilm system was observed to be 100 times higher than in planktonic culture. (Dunny et al. 1995).

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13
Q

give an example of an experiment involving intergeneric conjugation

A

dual biofilm of Bacillus subtilus carrying a tetracycline resistant gene construct and a sensitive Staphylococcus species.
After 6 and 24 hours, Staphylococcus isolates resistant to tetracycline were recovered and were shown to be carrying the identical tetracycline resistant gene originally borne by the Bacillus (Roberts et al. 1999).

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14
Q

prevention of access to antibiotic target occurs due to:

A

1) reduced permeability of the cell envelope
2) increased efflux activity (accept it goes in but immediately pump out)
3) mutation in antibiotic target
4) enzymatic modification or inactivation of the drug (hydrolysis or transfer of a chemical group)
5) ability to form biofilms greatly enhance antibiotic resistance traits

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15
Q

biofilms have a high level of resistance to two things, what are they ?

A

– Antibiotics
– Biocides

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16
Q

give an example of physical resistance to antimicrobials

A

– Exopolysaccharide production (slime) by biofilms “shields” susceptible cells e.g. to aggressive oxidant biocides

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17
Q

what are the five major types of multidrug efflux transporters

A
  1. resistance-nodulation-cell division (RND; Gram-negative bacteria)
  2. major facilitator superfamily (MF or MFS)
  3. small multidrug resistance (SMR)
  4. multidrug and toxic compound extrusion (MATE, formerly DME)
  5. ATP-binding cassette (ABC).

  • First four groups also known as secondary transporters, use the pre-stored energy of chemical gradients across the membrane
  • ABC transporters directly coupled with energy generation
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18
Q

what are the three H+ drug antiporter groups?

A
  1. resistance-nodulation-cell division (RND; Gram-negative bacteria)
  2. major facilitator superfamily (MF or MFS)
  3. small multidrug resistance (SMR)
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19
Q

give a type of Na+ drug antiporter?

A

multidrug and toxic compound extrusion (MATE, formerly DME)

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20
Q

give a type of ATP hydrolysis-linked drug transporters

A
  1. ATP-binding cassette (ABC).
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21
Q

what type of efflux pump is only found in gram negative bacteria?

A

Acr part of the RND family

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22
Q

what type of antibiotics can get into a biofilm?

(electrochemical status)

A

neutral or charged

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23
Q

whats different about the P. aeroginosa biofilms when they are deficient in Rhamnolipids?

A
  • mutants deficient in rhamnolipid synthesis **do not maintain the noncolonized channels surrounding macrocolonies. **
  • rhamnolipids are not required for the formation of macrocolonies and channels but participate in the maintenance of channels once formed.
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24
Q

how do surfactants maintain the noncolonised channels?

A
  • surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces.
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25
Q

when does rhamolipids synthesis begin?

A
  • high cell density planktonic growth induces the synthesis of quorum-sensing-dependent rhamnolipid production, (in the later stages of biofilm development)
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26
Q

describe how we can map the diffusion into a biofilm

give an example of the tracer used

A

add flourescent stain (eg Rhodamine B) to a biofilm and wait for the marker to appear in the colonies then track it through until it reached the middle of the colony

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27
Q

what was the effective diffusion coefficient of rhodamine B in the biofilm compared to water

A

Data indicate a value for effective diffusion coefficient of rhodamine in the biofilm approx. 15% of its value in pure water.

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28
Q

how fast can antibiotic-sized tracer can access the centre of a large, dense staphylococcal cell cluster

A

within 300 s – 5 minutes.

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29
Q

how does diffusion of solutes in biofilms compare to that in water?

A
  • Biofilms are mostly water and solutes the size of most biocides and antibiotics can diffuse in the biofilm.
  • They do not move as fast as they would in pure water because the cells, EPS, and other constituents of the biofilm hinder their mobility.
  • But measurements of diffusion coefficients suggest that these solutes will typically diffuse at rates approximately 20 to 50% of their rate in water.
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30
Q

what two toxin antitoxin (TAS) systems have antisense RNA as there antitoxin?

A

types 1 and 3

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31
Q

what is the mechanism for toxin neutralisation of type 1 TAS system?

A

antitoxin blocks mRNA of toxin

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32
Q

what is the mechanism for toxin neutralisation of type 2 TAS system?

A

direct protein-protein interactions

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33
Q

what is the mechanism for toxin neutralisation of type 3 TAS system?

A

direct RNA-protein interaction

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34
Q

what is the mechanism for toxin neutralisation of type 4 TAS system?

A

blockage of toxins effect on cellular target

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35
Q

what is the mechanism for toxin neutralisation of type 5 TAS system?

A

RNAasa of antitoxin degrades mRNA of toxin

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36
Q

what is the mechanism for toxin neutralisation of type 6 TAS system?

A

degredation of toxin by ClpXP serine protease

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37
Q

give two examples of cationic antibiotics and how they contact bacteria

A

tobramycin and gentimycin binds to the negatively charged LPS on the outer membrane

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38
Q

what does the tolA gene product do

A
  • tolA gene product affects LPS structure, resulting in decreased aminoglycoside affinity for the outer membrane
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39
Q

what are dormant persister cells
?

A

Persister cells, those cells tolerant to antibiotics, usually comprise about 1% in the stationary state and in biofilms (1, 2). These persister cells arise due to a state of dormancy, defined here as a state in which cells are metabolically inactive

  • All pathogens produce a small subpopulation of dormant persister cells that are highly tolerant to killing by antibiotics.
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40
Q

what is the dormant phenotype characterised by ?

A
  • Isolation of persisters produced a transcriptome which suggests a dormant phenotype characterized by **downregulation of energy-producing and biosynthetic functions. **
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41
Q

how do RelA and MazF toxins cause dormancy

A

cleaving mRNA

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42
Q

give two examples of how toxins help in persister formation in e.coli

A
  • HipA toxin inhibits translation by phosphorylating elongation factor Ef-Tu (chronic infections have higher production)
  • TisB toxin forms a membrane pore, decrease in pmf (proton motive force) and ATP – making membrane leaky costing energy
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43
Q

what are the three major pathways of persister formation in E.coli K12

A
  • Obg/HokB pathway
  • polyphosphate/Lon/mRNA interferase pathway
  • TisB pathway
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44
Q

how do type 1 toxins HokB and TisB induce persister formation

A

Type I toxins HokB and TisB induce persister formation by abolishing proton-motive force (PMF) as membrane-associated peptides,

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45
Q

how do mRNA endonuclease type 2 toxins induce persister formation

A

10 mRNA endonuclease type II toxins interfere with ribosomal translation.

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46
Q

when a cell has damage to its DNA, it induces the SOS system
what does RecA do in response?

A

RecA upregulates tisB which integrates in the the membrane dissipating proton motive forcce

47
Q

which proteins produce and remove nitric oxide in bacteria

A
  • Produced by nitrite reductase, nirS during anaerobic respiration
  • Removed by nitric oxide reductase, norB
48
Q

in the laboratory what molecule is used as a nitric oxide donor

A

Used donor sodium nitroprusside (SNP)

49
Q

what happens to a biofilm when nitric oxide is added?

A

As increased conc of nitric oxide donor the biofilm ratio decreases. NO is dissipating the biofilm.
You can add NO scavengers and you recover biofilm formation

50
Q

what is the effect on a biofilm when SNP is added along with tobramycin

A

When you add tobramycin the wild type is resistant , with the SNP you have an additive effect, virtually eliminating any bacteria on the surface
NO is helping dissipate the biofilm and making it more sentive to antimicrobial agents

51
Q

what is MRSA?

A

Methicillin resistance Staphylococcus aureus

* Gram-positive Staphylococcus aureus

Last standby = vancomycin BUT intermediate and full resistance strains are now appearing – new therapy

52
Q

what are the effect of adding SNP to MRSA biofilm?

A

SNP does make MRSA much more susceptible to antimicrobials, particularly vancomycin

(similar level of biofilm yet the proportion of nonviable cells increases when you have optimum concentration of the nitric oxide donor (500nM)
more effective on immature biofilms

53
Q

Why susceptibility of MRSA and other biofilms to presence of antimicrobials in presence of Nitric Oxide?

A

Nitric oxide activation of Phosphodiesterase (EAL or HD-GYP) reduces cyclic-di-gmp

54
Q

Give an example of a MATE (Na+) efflux pump

can you name any drugs it pumps

A

NorM

aminoglycosides
fluoroquinolones
cationic drugs

55
Q

which family of efflux pumps can work on aminoglycosides, fluoroquinolones and cationic drugs?

A

MATE family (Na+)

NorM

56
Q

give an example of an MFS (H+) efflux pump

can you name any drugs it works on

A

QacA

acriflavine, benzalkonium, cetrimide, chlorhexidine, pentamidine

57
Q

what family of drugs work on acriflavine, benzalkonium, cetrimide, chlorhexidine, pentamidine?

A

MFS (H+)

QacA

58
Q

what family of efflux pumps works on acriflavine, benzalkonium, and cetrimide?

A

SMR (H+) family

QacC

58
Q

Give an example of an SMR (H+) efflux pump?

can you name any drugs it works on

A

QacC

acriflavine, benzalkonium, cetrimide

59
Q

give an example of an RND (H+) efflux pump

include the subunits

A

AcrB

2 AcrA and 2 TolC subunits

60
Q

give an example of an ABC superfamily efflux pump

A

LmrA

61
Q

what kind of ion does the MATE family use for efflux?

A

sodium

62
Q

what kind of ion does the MFS family use for efflux?

A

hydrogen

63
Q

what kind of ion does the SMR family use for efflux?

A

hydrogen

64
Q

what kind of ion does the RND family use for efflux?

A

hydrogen

65
Q

what does teh ABC superfamily use to drive efflux?

A

ATP hydolysis

66
Q

Acr efflux are only found in what type of bacteria?
why?

A

Acr only found in gram-negative
so you can trasnport across the inner and outer membrane

67
Q

what is the DVLO theory

A

What happens when colloidal material comes close to a surface. Initially van de walls forces which pull close. Then double layer repulsive force which pushes them as they get closer. Such you have this net force. Secondary minimum where cell can come close to a surface but you need energy to get past.

68
Q

what is the secondary minimum?

A

Net energy given by sum of the double layer repulsion and van der Waals attractive forces that the particles experience as they approach

69
Q

how do bacteria have sterin stabilisation to aid attachment?

A

When you have a steric stabilization (in bacteria this is enabled by the flagella, fimbrie, pilli). The appendages break through the repulsive barrier, anchoring them to the surface so they can pull them selves closer

70
Q

what electrochemical factors impact attachment?

A

DVLO; PMF; sigma factors
EPS and lectin formation
Adhesion to substratum
Bioelectric effect

71
Q

describe how aerobes are attracted to the biofilm

A

As biofilm forms the metabolism is working so you get microaerophiles initially colonising which then reduces the oxygen redox potential which allows the anaerobes to come into the biofilm – this can be by passive attraction or chemotaxis.
also general oxygen/redox gradients in the biofilm

72
Q

what four colloid scientists developed the DVLO theory?

A

Derjaguin, Landau, Verwey and Overbeek

73
Q

what features may be heterogenous in a biofilm?

A

O2/ redox
EPS and products,
pH,
electrical

74
Q

what is the phenotype of a biofilm with a type 4 pilli (pilA) mutation?

A

you get a flat biofilm (because you lose the ability to form stacks)

75
Q

give a major function of type 4 pilli that contributes to biofilm structure

A

twitching motility, causes migration, cells will migrate towards each other forming the microcolonies/stacks

76
Q
A
76
Q

give three examples of surface attachment defective mutants

A

(i) P. aeruginosa flagellar-mediated motility – initial attachment
(ii) polar-localized type IV pili – twitching motility, 3D
(iii) global virulence regulator GacA – 3D

77
Q

what is biofilm structure affected by a colonic acid mutant?

A

it is has very little EPS and is flat

78
Q

what is ndvB

A

an important facotr for antibiotic resistance in p.aeruginosa
* ndvB, is required for the synthesis of periplasmic anionic glucans - highly glycerol-phosphorylated beta-(1—>3)-glucans, which bind aminoglycosides
* may prevent antibiotics from reaching their sites of action by sequestration in the periplasm

  • Also affect 8 ethanol oxidation genes involved in tobramycin resistance -
79
Q

what is the global stress response sigma factor?

A

rna polymerase alternate factor so it can only bind to rpos sensitive genes

80
Q

what is rpos

A

The gene rpoS (RNA polymerase, sigma S) encodes the sigma factor sigma-38
a central regulator of the general stress response

81
Q

why is the stress response seen in stationary phase planktonic cells similar to what you see in biofilms?

A

because both have a slow growth rate
(also rpos)

82
Q

what differences are there in a p.aeruginosa biofilm with a LasL mutant?

A

structural gene for N-(3-oxydodecanoyl)-L-homoserine lactone synthetase, causes **loss of EPS production and defects in biofilm formation **

83
Q

what AI1 does p.aeruginosa produce in the CF lung?

A

** N-butyryl-L-acyl HSL
and
N-(3-oxydodecanoyl)-L-homoserine lactone**

84
Q

what environmnetal stresses does the stress reponse protect the cell from?

A
  • Cells are protected from the detrimental effects of heat shock, cold shock, changes in pH and many chemical agents.
85
Q
A
86
Q

when RpoS is induced by high cell density, what factors does the generat stress response produce?
(give 2)

A

trehalose (an osmoprotectant)
and
catalase.

87
Q

genes for synthesis of what two structures are suppressed during biofilm maturation?

A

pili and flagella

88
Q

what are the problems with doing microarray assays on cells in a biofilm

a problem of averaging

A

Analysis averages gene expression in biofilm
– which are heterogeneous groups of cells exhibiting different activities
– certain subpopulations in the biofilm may have substantially different patterns of gene expression than did the homogeneous planktonic population or most of the metabolically active cells in the biofilm.

89
Q

how do cationic antibiotics access the cell?

A

bind to the negatively charged LPS of the outer membrane with subsequent transport into cell

90
Q

give two examples of cationic antibiotics

A

tobramycin and gentimycin

91
Q

what does the tolA gene product do to contribute to antibiotic resistance?

A
  • tolA gene product affects LPS structure, resulting in decreased aminoglycoside affinity for the outer membrane
92
Q

give some features of the stress response tobramycin induces

A

activation of dnaK and groES and 2 probable efflux systems (a probable non-RND drug efflux system and a P-type ATPase).

93
Q

what happens to persister cells when the antibiotic concentration drops?

A

Once an antibiotic concentration drops, surviving persisters re-establish the population, causing a relapsing chronic infection.

94
Q

in what areas of the human body are persister cell especially significant?

why

A

Persisters are especially significant when the pathogen is shielded from the immune system by biofilms, or in sites where the immune components are limited - **in the nervous system, the stomach, or inside macrophages. **

95
Q

the persister transcriptome is characterised by what

A

by downregulation of energy-producing and biosynthetic functions.

96
Q

what toxin-antitoxin modules are important in persister dormation

A
  • RelE, MazF toxins cause dormancy by cleaving mRNA
  • HipA toxin inhibits translation by phosphorylating elongation factor Ef-Tu (chronic infections have higher production)
  • TisB toxin forms a membrane pore, decrease in pmf (proton motive force) and ATP - causes reversible dormancy
97
Q

how does HipA help in persister formation

A
  • HipA toxin inhibits translation by phosphorylating elongation factor Ef-Tu

a serine/threonine kinase that is capable of auto-phosphorylation and the phosphorylation of EF-Tu

98
Q

how does the toxin-antitoxin system work to help persisters form

A

in the absence of stress the biofilms have a toxin-antitoxin system at work
as soon as there are antibiotics or stress factors, the antitoxin disappears leaving the toxin to inhibit cell growth/translation/replication
resulting in persister cells

99
Q

what toxin-antitoxin systems are under (p)ppGpp control for persister formation

A

Obg/HokB pathway
and
polyphosphate/Lon/mRNA interferase pathway

100
Q

how is the tisB pathway activated (for persister formation)?

A

activated upon strong SOS induction.

101
Q

following what stimulus does (p)ppGpp accumulate?

A

low nitric oxide or nucelic acids

102
Q

how does (p)ppGpp deplete antitoxin

A

when the cell runs out of NO or nucleotides it produces pppGpp. this stimulates the phosphorylation of PPK which phosphorylates Lon
Lon is able to bind the ribosome replication machinery and shut off transcription of the antitoxin

103
Q

give an example of an alarmone

A

pppGpp

104
Q

what is the antitoxin to the HokB toxin

A

sokB

105
Q

what is the antitoxin to the tisB toxin

A

istR

106
Q

what removes NO?

A

nitric oxide reductase, norB

107
Q
A
108
Q

what produces NO?

A

nitrite reductase, nirS

109
Q

what is the last standby for MRSA treatment?

A

vancomycin

BUT intermediate and full resistance strains are now

110
Q

how does RpoS impact levels of cyclic-di-GMP

A

RpoS is able to bind to DNA and affect gene expression - it can reduce the transcription of cyclases therefore reducing the production of cyclic-di-gmp

111
Q

how does MarA contribute to antibiotic resistance

A

marRAB locus (MarA transcription factor) up-regulates AcrAB-TolC efflux pump and down-regulates OmpF porin influx (intermediate clinical resistance)

112
Q
A

Biofilms & Microbial communities (11 decks)

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