week 4 Flashcards
1
Q
Biomaterial associated infection (BAI) –
A
mostly by staphylococcus epidermidid and S. aureus
2
Q
Race for the surface
A
the sooner the biomaterial gets coated by own cells, the lower the chance of bacteria colonizing the surface
3
Q
biofilms
A
- Quorum sensing – small molecules by bacteria can be recognized
- Difficult to phagocytose
- Not effectively reached by all antibiotics
- In dormant state less susceptible to antibiotics
- Persisters
- A persisting inflammatory stimulus
4
Q
Mouse soft tissue BAI model
A
- Implantation
- Challenge
- Eplantation
- Biopsy for quantitative culture (BM segment sonicated, homogenized tissue), histology, cytokines, mRNA
5
Q
Localization of s epidermitidis
A
not on surface but in deep tissue. S epidermidis in tissue resist antibiotics. Stay in immune cells, are not killing.
6
Q
In human catheter infections
A
high positive culture levels in skin, also in areas not in direct contact with biomaterial. 44% positive, mostly s epidermidis and e faecalis
7
Q
Anti LTA staining
A
colors gram positive bacteria, test to esclude chance of contamination
8
Q
S aureus in bone tissue
A
- Intracellular; long term osteomyelitis patient
- Canaliculi – can even deform to fit structure. Not only surface but also in bone holes
9
Q
Biomaterial associated infection (BAI)
A
- Mostly caused by staphylococci
- Biofilm
- Peri implant tissue colonization
10
Q
Foreign body response –
A
from proinflammatory to anti inflammatory. In specific order immune cells accumulate, orchestrated by cytokines
* mononuclear leukocytes
* neutrophils
* macrophages
* foreign body giant cells (fused macrophages)
11
Q
regulation foreign body response hindrance
A
by pram positive bacterial cell wall. strong inducer inflammation. Depends on combination of physical and chemical combination of material and bacteria, can lead to pro-inflammatory or anti inflammatory response.
12
Q
SEpvp (coating)
A
too strong pro inflammatory result, protracted inflammatory phase (longer), strong delay in foreign body response
13
Q
PApvp (coating)
A
giant cells visible after 14 days, but cultures grow and get incapsulated, dangerous with even systemic dissemination in murine models, sepsis. Localized in cluster like cells
14
Q
low IFN
A
Spreading
Systemic disease
Sepsis
15
Q
Orchestrate local immunity
A
develop materials which host proper response, resulting in infection free healing
16
Q
Release systems antimicrobial coating
A
release antimicrobials
contact killing
anti adhesive
combinations
17
Q
high IFN
A
Strong pro-inflammatory reaction
Protracted inflammatory phase
Strong delay in Foreign body response
18
Q
Considerations for release
A
- Railord to expected pathogen
- Release profile tailored to desired protected area and required concentrations
- Initial burst release, sustained release for sufficient period
- Reservoir of antimicrobial sufficient to realise the above
- Coating sufficiently attached
- Active in vivo situation
- Biocompatible
- Mechanism of release; diffusion, controlled release, biodegradable (but what if antimicrobial is released and coating not yet degraded), triggered
19
Q
Novel antimicrobial approaches
A
- BALI – biofilm alliance
- Superactive; supramolecular biomaterials with antimicrobial and regenerative activity
- PRINT-AID – development of 3d printed devices with antimicrobial properties to prevent biomaterial infections
20
Q
Synthetic antimicrobial and antibiofilm peptides (SAAP)
A
- Derived from human cathelicidin LL37
- OP145 is synthetic derived from P60 with impreoved helicity and amphipathicity; antimicrobial and antibiofilm. Plasma inhibits action of OP145
21
Q
PLEX OP 145 controlled release coating
A
Combination lipids and polymers makes it stable, OP145 10%. In first 10 days 60% released, after that more slowly – burst first. Later improved activity SAAPS in presence of plasma due to optimization without resistance, prevents biofilm formation.
22
Q
Novel ll37 derived SAAPs
A
broad spectrum, active in PBS and plasma AANVULLEN
23
Q
Novel antimicrobial SuperActive (regenerative capacity biomaterial)
A
prevent infection and let tissue regenerate. Base polymer and stacking parts (SAAP). Materials can be dipcoated, effect was not complete elimination of the pathogens
Supramolecular UPy based materials
* Simple mix and match
* Retained activity SAAP148
* Incorporation into solid materials
24
Q
3d printing antimicrobial devices
A
Antimicrobial agent and biomaterial can be made to fit the patient. Control of drug loading and release by adjusting layers. Advantage 10-100x higher concentration, minimal systemic effects. Disadvantage; not enough drug biomaterial options due to heat and sollluability, mixed on stage, antimicrobial resistance.
PRINT-AID Solutions:
Local administration of gentamicin
sulphate (GS) in an 3D-printed PCL
coating
Tailored polymer-SAAP release system by
Droplet-on-demand printing
25
Droplet on demand coat Ti bone plate SAAP148
active against MDR bacteria and biofilm formation. Droplet size controlled by pressure and nozzle opening. Release from one layer almost completed in first day, cumulative release only 13%
26
Coxiella burnettii
* Obligatory intracellular = replicate inside cells
* Enter via receptor mediated endocytosis
* Come into Coxiella containing vacuole CCV
27
CCV coxiella containing vacuole
* Small cell variants (SCVs) – metabolic inactive, resistant and extracellular
* After invasion large cell variants (LCV) – metabolic active, intracellular, acidification triggers transcription
28
what does c burnetti infect
macrophages via actin-dependent phagocytosis
29
Alfavbeta3
* Normally Involved in apoptotic cells via phagocytosis
* C burnetti inhibits inflammation – silent infection
* Adhesion of Coxiella is unknown protein
30
Host cell modulation
* Binding receptor, uptake of c burnetti
* Nasant CCV (coxiella containing vacuole) is formed, phagosome and lysosome interact (no fusion), vacuole becomes larger and bacteria replicate in this low pH
31
Mature CCV
* Heavily loaded with bacteria – large will transform to small
* Retains capacity to fuse and expand
* Now also anti apoptotic markers
32
Coxiella has secretion apparatus
* Needle like structure, injects effector proteins into host cells
* Can manipulate host cells to not combat bacteria anymore
33
Defect in organelle traficing (Dot) intracellular multiplication (Icm)
* Secretion system
* Encoded on islands with relatively low CG contents
* Eukaryotic protein motifs
* Effector protein secreted
* Polymorphic regions between pathotypes
34
How does it work (Dot and Icm)
* Regulate transcription
* Translocation efficiency
* Protein stability
* Expressed after 8h and require low pH
* prmAB important for regulation transcription
35
functional assays used to test function prmAB
made a knock out of prmAB and placed cells in medium, no difference. Inside cells huge difference
36
how dows prmAB work
* pmrA box like a fur box? To test first see if it binds to promotor region – fusion prm and lux (fluorescent). Knock out no translation so it binds there
* regulates transcription
(low pH stimulus)
37
Protein domains point to function
* Anti apoptosis
* Ubiquitylation
* Lipid metabolism
* Membrane trafficking
38
cell wall synthesis
penicillins, cephalosporins, bacitracin, vancomycin
39
Nucleic acid replication
quinolones, rifampin
40
Protein synthesis
chloramphenicol, erythromycin, tetracyclins, streptomycin
41
Plasma membrane
polymyxin B
42
Synthesis essential metabolites
sulfonamide, trimethoprim
43
Types of antibiotic resistance
* Intrinsic resistance – vooral gram negatief difficult, has to go through pore in outer membrane or dissolve. Efflux pump is present and PBPs (penicillin binding protein)
* Target site changes – AB targets protein, comes from gene. Mutation where AB cannot bind anymore, or if other compound binds enzyme.
* Direct interactions with AB
44
PBPs
binds penicillin, those are peptidoglycan synthases and get inactivated by binding penicillin. PBP gets inactivated – destruction cell wall
45
Multi drug resistance (MDR) efflux pumps gram negative bacteria; resistance nodulation division (RND) expression factors
Baseline of expression of proteins of the pump; Acr expression controlled by TetR repressor, can be overruled by AraC family transcription factor. araC is repressed by multi antibiotic resistance (MAR) protein MarR
46
Antibiotics effect on pump
usually bind to MarR protein, derepression, expression pump system
47
Highly resistant bacteria due to which mutation
TetR repressor mutation
48
MRSA expression PBP2a
* Horizontal gene transfer from s epidermidis
* PBP has different structure, but peptidoglycan synthesis not affected
49
Rifampicin resistance
RNA polymerase binding. High frequency rifampicin resistance, s epidermis. Not gradient loss of effect rifampicin, not functional at all anymore
50
Target site mutation rpoB
RNA polymerase of m tuberculosis causing rifampicin resistance
51
tetM
target site protection against tetracyclin. Normally binding to site in ribosome where translation normally starts, not with tetM, binding but function not affected
52
steric hindrance
binding without destroying
53
inactivation by hydrolysis
beta lactam and beta lactamases. Only carbapenem left
54
counteracting beta lactamase –
clavulanic acid. Blocks beta lactamases even in resistant bacteria (by inhibiting enzyme that gets produced by bacteria). In itself it is a beta lactam drug, not antibacterial so needs to be given together with AB
* with amoxicilline – augmentin
* with ticarcillin – co-ticarclav
* however, carbapenemases are still not susceptible to beta lactamase blockers
55
beta lactamases
* ampC – inactivates 2/3 gen cephalosporin, Enterobacter and serratia, chromosomal inducible or constitutive
* ESBL – also 2/3 gen cephalosporins, plasmid localized (can spread)
* Inactivation by clavulanic acid variable
56
Resistance to beta lactam
* Efflux
* Direct interaction; ESBL, carbapenemases
* Target site changes (MRSA)
57
Intrinsic resistance AB
* Prevent influx
* Efflux
58
Target site changes AB
* Mutation
* Protection
59
Direct interactions AB
hydrolysis (enzymatic breakdown)
steric hindrance
60
Antimicrobial peptides
amino acid chains with at least 2 positive charges. Amphipathic, broad spectrum gram pos, neg, fungi, parasite, cancer, virus, counteract sepsis, low MIC, synergy with AB and AMP
61
Human antimicrobial peptides
epithelial, endothelial, leukocytes, platelets
* Rapid; stored and secreted, intracellular killing
* Inducible
* After tissue damage
* Inflammation
* Bacterial products
62
Classes of antimicrobial peptides
* Beta stranded; protegin, alpha/beta defensins
* Alpha helical; ll37, cecropin
63
Membrane disruption types after threshold passed
* barrel stave
* bending backward
* carpet (micel) like soap like structure
64
effects of thrombin on microbes -
thrombin activates thrombocidins (TCs), granules containing alpha granules get released
65
membrane potential measure
DiSC3(5) assay. Higher fluorescence less potetntial, more disturbed
66
TC84 actions
* Membrane depolarization
* Membrane permeabilization
* Membrane invagination
* Cytoplasmic content leakage, membrane disruption
* Unmixes membrane
* Colocalizes with fluid domains
* Colocalizes with membrane invaginations
67
Model of mode of action of TC19/AMP
* Less organized, unmixing due to presence cationic peptides that form patches with membrane that is more liquid
Mechanism of killing by Amp
* Interaction membrane; disruption protein motive force, permeation, disruption, membrane passage (positively charged)
* Interaction targets; nucleic acids, septum, heat shock proteins. The ones that don’t disrupt the membranes (negatively charged)
68
Honey antimicrobial factors
* Osmotic
* Hydrogen peroxide due to glucose oxidase
* Acidity
* Methylglyoxal (manuka)
* Bee defensin-1
69
Biofilm formation
bacteria form extracellular matrix anker on surface, microcolonies, 3d structure
70
Pellicle
protein layer on surface teeth. Bacteria can bind those proteins, other bacteria can bind those bacteria.
71
Ecological succession
change in species in ecological community. Succession occurs when we disturb colonized or after initial colonization. Competitive, more complex over time
72
Biofilm
* Healthy – in slightly higher CO2 concentration, streptococci etc
* Biofilms become thicker – oxygen lower and anaerobic bacteria can grow
* Periodontitis – accumulation dysbiotic bacteria (gum disease)
73
Why buccal cells divide so fast
Buccal cells also get colonized with bacteria, both inside and outside. Possible start for dental plaque. Important in recolonization, early colonizers and in fact early proliferators
74
Oral biofilm from ecological perspective
* Daily removal
* Many environmental changes hourly
* Mostly healthy, yet caries and gingivitis most common worldwide
* Spatio temporal heterogeneity
75
Multi kingdom biofilms
* More complex in vivo
* Fungi, archaea and amoeba
76
Aerobic vs anaerobic biofilms bacteria
* C albicans alters bacterial microbiome, as shown by sequencing; with candida anaerobes, without aerobes
* Antimycin A inhibits oxygen removal, aerobes can grow
77
Caries;
stress (sugar intake and acid production), environmental shift to low pH, ecological shift (strep and a naeslundii to mutants streptococci and lactobacilli), disease enamel and caries
78
Fluoride
prevents caries by preventing low pH to affect teeth, only reduces symptoms
79
Gingivitis
redness of gums, bleed easily. Untreated can lead to periodontitis (bone absorption due to inflammation) and can lead to teeth loss
oral hygiene drops, gingival inflammation, more leakage GCF, protein and iron, more bacteria
80
Root surface bacteria on border enamel and teeth
live of off proteins that are available in high concentrations there
81
Oral microbiome
total of all micro-organisms in a specific location.
82
Holobiont
host/microbiota coevolution. assemblage of a host and the many other species living in or around it, which together form a discrete ecological unit through symbiosis
83
Why rather target tick itself instead of lyme
there are other tick born diseases (1/3 other tick born diseases, 1/5 lyme).
84
Tick saliva
proteins that inhibit coagulation, inhibits inflammatory/immune response.
85
Salp15
in tick saliva inhibits DC and T cells proliferation
86
Tick immunity
less susceptible to tick born diseases by investing animals with ticks multiple times, possibility for vaccine?
87
Cycle of borrelia
Blood enters gut tick (outer surface protein A OspA associated), migrates hemolymph to saliva (OspC expression, which is bound by Selp15 which functions as invisibility cloak), transmitted to host unrecognisable
88
Tick vaccine by targeting salivary proteins
* Identify tick proteins to use; by feeding (not at all, 24h, fully) dissecting, making cDNA libraries of salivary glands, grouping. Most is happening in early tick feeding phase (24h), might be better to target this with vaccine. Narrowing down proteins; expression 5x higher vs uninfected, significant, remove biological replicates, Q RT PCR validate compounds
* Infected ticks injected with RNAi knocking down those transcripts, mice infected, tick weight after feeding stayed the same. Also unfortunately no differences in loads of bacteria in heart and ear compared to normal tick
89
Tick vaccine by targeting salivary gland antigens
* Focusing on tick salivary gland antigens; feeding uninfected ticks, cDNA library, next gen seq, feeding induced tick proteins discovered. Narrowing down; high log2, significantly upregulated etc. 20 validated.
* With antigen 11 present; higher levels of borrelia after infection, phagocytosis reduction
* Without antigen 11 (p19) ticks could feed perfectly normal, borrelia levels significantly lower (culture protected)
90
what provides protection tick borne encephalitis
antigen 22 and antigen 16 provide moderate protection in lethal model against tick borne encephalitis. Combining provides more protection
91
Species complexity higher in Europe
B afzelii, garinii, burgdorferi and bavariensis, need vaccine that targets all 4
92
ospA (expessed by borrelia) downregulation
borrelia migration from gut to salivary gland and host
93
mechanism of ospA based vaccine
antibody goes into tick, binds borrelia in tick, prevents borrelia transfer from gut to saliva
94
first generation LB vaccine (ospA)
* lymerix; whole ospA used, only burgdorferi targeting, 76% efficacy
* imulyme; whole ospA used, phase 3, only burgdorferi, 92% efficacy
* hypothetical vaccine induced adverse effects, mimicry with hLFA1 T cell epitope. Has been disproven, but still discontinued due to lack of demand leading to poor sales. Not licenced
95
second generation ospA vaccine
* VLA15; combination of 3 proteins
* Broad protection to 4 genospecies representing 6 serotypes
* Phase 3 started
96
Experimental LB chimeric ospA
truncated stabilized c terminal fragment to investigate protection by multivalent ospA. Surface shaping approach – multiple epitopes on one protein
* CHECK STEP 1
* Homology modelling/structural scaffold = combining 2 proteins and see if they fold in the way you predicted
* Surface partitioning – see if you can make or keep the patches where Ab can bind
* Patch layout – devide surface in different patches and take patches that bind to create different layouts by differently linking the parts together
* Serotype distribution – serotypes bind different patches, algorithm decides what is the best combination to protect against different serotypes, not always covered all serotypes by one protein. using different disulfide bonds to stabilize
* Immunized mice; ELISA, flow cytometry, bactericidal assay (bind and neutralize borrelia in vivo possible?), collect final sera, organs, serology, another ELISA
* V3 and V5 combination chosen for creation of fusion protein
97
V3-V5 and L2 single antigen ospA vaccine, produced as one protein
* Could induce immune response against all serotypes
* Tested for serotype 1 and 2 especially because those are most prevalent in NL and ENG. Provided 100% protection against tick challenge
98
Destinguish between different malaria species
using microscope based on morphology
99
Genetic variation between plasmodium species measurements
PCR
100
Plasmodium malariae
difficult to see under microscope, when PCR was done, it revealed another species; plasmodium knowlesi.
101
Plasmodium knowlesi
zoonosis
102
Why plasmodium falciparum causes more serious anemia
Multiply inside red blood cell, ruptures. Is worse in falciparum, might be because of the quicker development, higher parasitaemia, blocking blood vessels
103
Temperature chart different plasmodium species
* Vivax/ovale - Ring stage gives fever, rupture also causes fever
* Malariae – slower development.
* Falciparum – patterns more spiked, more quick so quicker development
104
Why quicker development, higher parasitaemia in falciparum why
because preverance of parasite to type of blood cell (infects all), whereas malariae invades old, vivax/ovale invades young, hemoglobin is used, change in surface (knob formation)
105
Overview pathogenesis of malaira
* Change surface
* Lysis
* Use of food source – hemoglobin
* Can all lead to tissue damage
106
Why certain groups are more vulnerable
* Children- no immunity
* Non-immune (travelers)
* Pregnant – harmful for patient and child. Effect depends on age, gravidity, endemicity, HIV status. Parasite can target placenta specifically (not found in non pregnant), also immunostatus decreases
107
difference pregnancy associated and placental malaria
Pregnancy associated malaria – not involve placenta
Placental malaria – infection including placenta
108
Immunity against malaria
only partial, due to different variants, takes many rounds of infections to gain immunity
109
Consequences malaria during pregnancy
* Growth restriction
* Anemia
* Preterm delivery
* abortion
110
microbiome
aggregate of all microbioata (microorganisms in specific environment). Mostly commensial/symbiotic
111
role gut microbiota
* digest fiber
* produce viamins
* synthesize amino acids
* produce short chain fatty acids
* interaction with immune
* protect against pathogens
112
habitats within human gut
lumen mucin and food particles.
113
Challenges studying microbiome
* Hard to culture
* Ecology and community composition important
* Genomic markers important for identification
114
Solutions to challenges studying microbiome
* DNA based
* Metagenomic seq
* DNA probe bases microscopy
115
SSU rRNA vs metagenomics
specific gene used, meta is also for functionality. Gives amount in percentage
116
Recovering genomes from metagenomes –
extract DNA, shotgun metagenomics gives reads. Reads can be assembled into contigs. Map reads to contigs, table to display sequence characteristics and combine both to bin contigs to reconstruct genomes (MAG – metabolic assembled genome)
117
MAGs
give insight into unsequenced organisms. Applicable to every dataset with little bias, incomplete and contamination tho
118
Proximity ligation sequencing (Hi-C)
crosslinker on top of bacteria, connect to different materials and put in different organisms
119
Culturomics
cheap and allows for follow up, but culturing bias and work intensive
120
Combining isolates and metagenomics
combining isoates and metagenomics can help understand strain dynamics and in person evolution
121
Obtain DNA of microbiome
* metagenomics
* Hi-C
* Culturomics
122
What influences our own microbiome
* Relatively stable over time, strain composition even more stable
* Animal based diet increased harmful bacteria, effects of vegan diet were very small
123
Diseases and microbiome
* Dysbiosis
* Single causative agent – C difficile, EHEC etc
124
C difficile
* Can be resistant to AB, makes it worse
* Better to give fecal matter transplantations (after AB)
125
Streptococcus suis
* Pigs
* zoonotic
* Gram positive
* Capsule polysaccharides – multiple serotypes
* Risk of disease increases after viral infection
* Mainly serotype 2 in humans
* meningitis
126
Disease s suis
* Meningitis – irreversible hearing loss in 60%
* Sepsis with high mortality – skin bleeding
* Endocarditis, peritonitis, arthritis
127
Risk factors s suis
* Raw pork consumption
* Exposure to pigs
* Male sex
* Pig related occupation
128
Core genome
pool of genes shared by all members of bacterial species
129
Pangenome
core and accessory genome
130
Accessory genome (dispensable)
pool of genes present in some species
131
Metagenome
gene repertoire of mixed microbial population
132
Whole genome seq with illumina
contigs produced using overlap sequencing reads.
133
Serotype differences s suis
Loss and acquisition of genes may lead to increased virulence and zoonotic potential by polysaccharide capsule switch. However, humans only get infected with 2, known by serotype antibodies measurements
Reinfection s suis is possible
134
Restriction modification system functions
* Restriction function - endonuclease
* Methylation function
* S unit can vary with phase variation mechanism, different phases means different DNA motifs recognized, different gene expression
135
Knocking out xerD in s suis
prevents recombination as seen in FAM assay. Cannot adapt to certain conditions
Order genes s suis - does not affect growth, but does affect pathogenicity
136
Pathogenicity island –
adapt to circumstances, obtained by horizontal gene transfer
137
ICE
Streptococci contain Integrative and Conjugative Elements (ICE) in their accessory genome. Combine features of phages, transposons and plasmids. Integration in genome of ICEs
138
The tragedy of the commons
benefit for individual is enormous, but burden to all
139
Cause of AMR pathogens
Socio economic (availability of AB), environmental (more infections) and ecological changes (use of AB, settings animals)
140
Colistin
last resort AB, but used in large volumes in food animals
141
Colistin resistance
create surface defect, effect against gram negative
142
Anthropogenic and environmental factors associated with high incidence of mcr1 carriage in humans
link between agriculture and mcr1
Mcr1 transposon jumps over time, loss of insertion sequence
143
After banning using colistin in animals
drop in prevalence mcr and resistant e coli
144
PCI
Photochemical
internalization of
gentamicin
mediated cytosolic release of gentamicin
