Antibiotic resistance Flashcards
(114 cards)
1
Q
what is intrinsic resistance?
A
independent of antibiotic selective pressure and horizontal gene transfer; result of inherent structural or functional characteristics.
2
Q
what is acquired resistance?
A
mutations in drug targets or transfer of resistance genes through phage-mediated transduction and mobile plasmids.
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.
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)
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
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
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.
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.
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
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.
11
Q
how does conjugation occur in gram negative bacteria?
A
via fimbria and pili
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).
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).
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
15
Q
biofilms have a high level of resistance to two things, what are they ?
A
– Antibiotics
– Biocides
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
17
Q
what are the five major types of multidrug efflux transporters
A
- resistance-nodulation-cell division (RND; Gram-negative bacteria)
- major facilitator superfamily (MF or MFS)
- small multidrug resistance (SMR)
- multidrug and toxic compound extrusion (MATE, formerly DME)
- 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
18
Q
what are the three H+ drug antiporter groups?
A
- resistance-nodulation-cell division (RND; Gram-negative bacteria)
- major facilitator superfamily (MF or MFS)
- small multidrug resistance (SMR)
19
Q
give a type of Na+ drug antiporter?
A
multidrug and toxic compound extrusion (MATE, formerly DME)
20
Q
give a type of ATP hydrolysis-linked drug transporters
A
- ATP-binding cassette (ABC).
21
Q
what type of efflux pump is only found in gram negative bacteria?
A
Acr part of the RND family
22
Q
what type of antibiotics can get into a biofilm?
(electrochemical status)
A
neutral or charged
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.
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.
25
when does rhamolipids synthesis begin?
* high cell density planktonic growth induces the synthesis of quorum-sensing-dependent rhamnolipid production, (in the later stages of biofilm development)
26
describe how we can map the diffusion into a biofilm
## Footnote
give an example of the tracer used
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
27
what was the effective diffusion coefficient of rhodamine B in the biofilm compared to water
Data indicate a value for effective diffusion coefficient of rhodamine in the biofilm approx. 15% of its value in pure water.
28
how fast can antibiotic-sized tracer can access the centre of a large, dense staphylococcal cell cluster
within 300 s – 5 minutes.
29
how does diffusion of solutes in biofilms compare to that in water?
* 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.
30
what two toxin antitoxin (TAS) systems have antisense RNA as there antitoxin?
types 1 and 3
31
what is the mechanism for toxin neutralisation of type 1 TAS system?
antitoxin blocks mRNA of toxin
32
what is the mechanism for toxin neutralisation of type 2 TAS system?
direct protein-protein interactions
33
what is the mechanism for toxin neutralisation of type 3 TAS system?
direct RNA-protein interaction
34
what is the mechanism for toxin neutralisation of type 4 TAS system?
blockage of toxins effect on cellular target
35
what is the mechanism for toxin neutralisation of type 5 TAS system?
RNAasa of antitoxin degrades mRNA of toxin
36
what is the mechanism for toxin neutralisation of type 6 TAS system?
degredation of toxin by ClpXP serine protease
37
give two examples of cationic antibiotics and how they contact bacteria
tobramycin and gentimycin binds to the negatively charged LPS on the outer membrane
38
what does the tolA gene product do
* tolA gene product affects LPS structure, resulting in decreased aminoglycoside affinity for the outer membrane
39
what are dormant persister cells
?
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
## Footnote
* All pathogens produce a small subpopulation of dormant persister cells that are highly tolerant to killing by antibiotics.
40
what is the dormant phenotype characterised by ?
* Isolation of persisters produced a transcriptome which suggests a dormant phenotype characterized by **downregulation of energy-producing and biosynthetic functions. **
41
how do RelA and MazF toxins cause dormancy
cleaving mRNA
42
give two examples of how toxins help in persister formation in e.coli
* 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
43
what are the three major pathways of persister formation in E.coli K12
* Obg/HokB pathway
* polyphosphate/Lon/mRNA interferase pathway
* TisB pathway
44
how do type 1 toxins HokB and TisB induce persister formation
Type I toxins HokB and TisB induce persister formation by **abolishing proton-motive force (PMF) as membrane-associated peptides,**
45
how do mRNA endonuclease type 2 toxins induce persister formation
10 mRNA endonuclease type II toxins **interfere with ribosomal translation.**
46
when a cell has damage to its DNA, it induces the SOS system
what does RecA do in response?
RecA upregulates tisB which integrates in the the membrane dissipating proton motive forcce
47
which proteins produce and remove nitric oxide in bacteria
* Produced by nitrite reductase, nirS during anaerobic respiration
* Removed by nitric oxide reductase, norB
48
in the laboratory what molecule is used as a nitric oxide donor
Used donor sodium nitroprusside (SNP)
49
what happens to a biofilm when nitric oxide is added?
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
what is the effect on a biofilm when SNP is added along with tobramycin
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
what is MRSA?
Methicillin resistance Staphylococcus aureus
| * Gram-positive Staphylococcus aureus
## Footnote
Last standby = vancomycin BUT intermediate and full resistance strains are now appearing – new therapy
52
what are the effect of adding SNP to MRSA biofilm?
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
Why susceptibility of MRSA and other biofilms to presence of antimicrobials in presence of Nitric Oxide?
Nitric oxide activation of Phosphodiesterase (EAL or HD-GYP) reduces cyclic-di-gmp
54
Give an example of a MATE (Na+) efflux pump
| can you name any drugs it pumps
NorM
aminoglycosides
fluoroquinolones
cationic drugs
55
which family of efflux pumps can work on aminoglycosides, fluoroquinolones and cationic drugs?
MATE family (Na+)
| NorM
56
give an example of an MFS (H+) efflux pump
| can you name any drugs it works on
QacA
acriflavine, benzalkonium, cetrimide, chlorhexidine, pentamidine
57
what family of drugs work on acriflavine, benzalkonium, cetrimide, chlorhexidine, pentamidine?
MFS (H+)
| QacA
58
what family of efflux pumps works on acriflavine, benzalkonium, and cetrimide?
SMR (H+) family
| QacC
58
Give an example of an SMR (H+) efflux pump?
| can you name any drugs it works on
QacC
acriflavine, benzalkonium, cetrimide
59
give an example of an RND (H+) efflux pump
| include the subunits
AcrB
2 AcrA and 2 TolC subunits
60
give an example of an ABC superfamily efflux pump
LmrA
61
what kind of ion does the MATE family use for efflux?
sodium
62
what kind of ion does the MFS family use for efflux?
hydrogen
63
what kind of ion does the SMR family use for efflux?
hydrogen
64
what kind of ion does the RND family use for efflux?
hydrogen
65
what does teh ABC superfamily use to drive efflux?
ATP hydolysis
66
Acr efflux are only found in what type of bacteria?
why?
Acr only found in gram-negative
so you can trasnport across the inner and outer membrane
67
what is the DVLO theory
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
what is the secondary minimum?
Net energy given by sum of the double layer repulsion and van der Waals attractive forces that the particles experience as they approach
69
how do bacteria have sterin stabilisation to aid attachment?
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
what electrochemical factors impact attachment?
DVLO; PMF; sigma factors
EPS and lectin formation
Adhesion to substratum
Bioelectric effect
71
describe how aerobes are attracted to the biofilm
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
what four colloid scientists developed the DVLO theory?
Derjaguin, Landau, Verwey and Overbeek
73
what features may be heterogenous in a biofilm?
O2/ redox
EPS and products,
pH,
electrical
74
what is the phenotype of a biofilm with a type 4 pilli (pilA) mutation?
you get a flat biofilm (because you lose the ability to form stacks)
75
give a major function of type 4 pilli that contributes to biofilm structure
twitching motility, causes migration, cells will migrate towards each other forming the microcolonies/stacks
76
76
give three examples of surface attachment defective mutants
(i) P. aeruginosa flagellar-mediated motility – initial attachment
(ii) polar-localized type IV pili – twitching motility, 3D
(iii) global virulence regulator GacA – 3D
77
what is biofilm structure affected by a colonic acid mutant?
it is has very little EPS and is flat
78
what is ndvB
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
## Footnote
* Also affect 8 ethanol oxidation genes involved in tobramycin resistance -
79
what is the global stress response sigma factor?
rna polymerase alternate factor so it can only bind to rpos sensitive genes
80
what is rpos
The gene rpoS (RNA polymerase, sigma S) encodes the sigma factor sigma-38
a central regulator of the general stress response
81
why is the stress response seen in stationary phase planktonic cells similar to what you see in biofilms?
because both have a slow growth rate
(also rpos)
82
what differences are there in a p.aeruginosa biofilm with a LasL mutant?
structural gene for N-(3-oxydodecanoyl)-L-homoserine lactone synthetase, causes **loss of EPS production and defects in biofilm formation **
83
what AI1 does p.aeruginosa produce in the CF lung?
** N-butyryl-L-acyl HSL
and
N-(3-oxydodecanoyl)-L-homoserine lactone**
84
what environmnetal stresses does the stress reponse protect the cell from?
* Cells are protected from the detrimental effects of heat shock, cold shock, changes in pH and many chemical agents.
85
86
when RpoS is induced by high cell density, what factors does the generat stress response produce?
(give 2)
trehalose (an osmoprotectant)
and
catalase.
87
genes for synthesis of what two structures are suppressed during biofilm maturation?
pili and flagella
88
what are the problems with doing microarray assays on cells in a biofilm
## Footnote
a problem of averaging
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
how do cationic antibiotics access the cell?
bind to the negatively charged LPS of the outer membrane with subsequent transport into cell
90
give two examples of cationic antibiotics
tobramycin and gentimycin
91
what does the tolA gene product do to contribute to antibiotic resistance?
* tolA gene product affects LPS structure, resulting in decreased aminoglycoside affinity for the outer membrane
92
give some features of the stress response tobramycin induces
activation of dnaK and groES and 2 probable efflux systems (a probable non-RND drug efflux system and a P-type ATPase).
93
what happens to persister cells when the antibiotic concentration drops?
Once an antibiotic concentration drops, surviving persisters re-establish the population, causing a relapsing chronic infection.
94
in what areas of the human body are persister cell especially significant?
## Footnote
why
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
the persister transcriptome is characterised by what
by downregulation of energy-producing and biosynthetic functions.
96
what toxin-antitoxin modules are important in persister dormation
* 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
how does HipA help in persister formation
* HipA toxin inhibits translation by phosphorylating elongation factor Ef-Tu
## Footnote
a serine/threonine kinase that is capable of auto-phosphorylation and the phosphorylation of EF-Tu
98
how does the toxin-antitoxin system work to help persisters form
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
what toxin-antitoxin systems are under (p)ppGpp control for persister formation
Obg/HokB pathway
and
polyphosphate/Lon/mRNA interferase pathway
100
how is the tisB pathway activated (for persister formation)?
activated upon strong SOS induction.
101
following what stimulus does (p)ppGpp accumulate?
low nitric oxide or nucelic acids
102
how does (p)ppGpp deplete antitoxin
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
give an example of an alarmone
pppGpp
104
what is the antitoxin to the HokB toxin
sokB
105
what is the antitoxin to the tisB toxin
istR
106
what removes NO?
nitric oxide reductase, norB
107
108
what produces NO?
nitrite reductase, nirS
109
what is the last standby for MRSA treatment?
vancomycin
## Footnote
BUT intermediate and full resistance strains are now
110
how does RpoS impact levels of cyclic-di-GMP
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
how does MarA contribute to antibiotic resistance
marRAB locus (MarA transcription factor) up-regulates AcrAB-TolC efflux pump and down-regulates OmpF porin influx (intermediate clinical resistance)
112
