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Medical Microbiology > Day 7: Skin and Biomaterial associated infection, Respiratory tract infections, Microbial genomics > Flashcards

Day 7: Skin and Biomaterial associated infection, Respiratory tract infections, Microbial genomics Flashcards

HC 17, 18, 19

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1
Q

HC17: Biofilm formation steps

A
  • Adherence
  • Propagation
  • make ‘mushrooms’
  • detachment
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2
Q

Biofilm first steps

A
  • Adhesins bind to surface biomaterial
  • Biofilm formation through exopolysaccharide production
  • Cell death of some bacteria > DNA released > make chains and sticking
    > Sticking through protein contact factors
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3
Q

Proteins in S. aureus biofilm for adherence and accumulation

A
  • Aap protein: accumulation associated protein
  • MSCRAMM protein: microbial surface components recognizing matrix molecules
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4
Q

Accumulation S. aureus through proteins

A
  • Aap/SasG B domain mediated accumulation
  • MSCRAMM mediaed accumulation
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5
Q

Dispersion / detachment of S. aureus in biofilm

A

Phenol-soluble modulins (PSMs)
> strongly amphipathic alpha-helical peptides: can break interactions by replacing them
> activated through quorum sensing
> Biofilm structuring and dispersal
» other virulence functions: toxins for neutrophils: chemokine and toxin function: attract them and kill them

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6
Q

Character of bacteria within biofilms

A
  • Difficult to phagocytose
  • expressing different gene sets than planktonic bacteria
  • Regulate gene expression by quorum sensing
  • Production extracellular polysaccharides increasing biofilm or biofilm dispersing molecules
  • Not effectively reached by all antibiotics
  • In dormant state: persisters and therefore less susceptible to antibiotics
  • a persisting inflammatory stimulus
    » continuous release of structures of biofilm which constantly activate immunity: not good
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7
Q

Biofilms are not the only reason of persisters and BAI, why>

A

Prostheses can get infected upon replacement
> bacteria are also more in the tissue

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8
Q

BAI and infection of tissue: early and late response

A

First: proinflammatory response: PMNs (Polymorphonuclear neutrophils)
Later: macrophages, foreign body giant cells and mononuclear leukocytes, anti-inflammatory

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9
Q

PMNs upon recognition

A

Make cytokines in periphery
> fibroblast activation and proliferation: fibrosis
> PMNs clear most bacteria and macrophages do the rest

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10
Q

Cell wall as inducer of inflammation and with biomaterial

A

In gram-positives
> Lipoteichoic acid (LTA)
> Peptidoglycan
» influence foreign body response
» pro-inflammatory or anti-inflammatory
» bacteria+foreign biomaterial: uncontrolled response

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11
Q

When the local immunity is compromised because mixture of biomaterial and bactieria which derange immune responses, what can happen?

A

Intracellular survival of bacteria within the tissue cells
> persist in immune cells
> clusters in macrophages

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12
Q

S. aureus and S. epidermidis has two niches in BAI:

A
  • Biofilms
  • Tissue
    » just remove the implant with the biofilms is not enough
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13
Q

Cell wall synthesis directed antibiotics

A

Penicillins, Ampicillin (beta lactam antibiotics)

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14
Q

Protein synthesis directed antibiotics

A

Streptomycin, tetracycline, kanamycin

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15
Q

Types of antibiotics

A
  • Cell wall synthesis directed
  • Protein synthesis directed
  • Plasma membrane directed
  • Nucleic acid replication and transcription directed
  • Synthesis essential metabolites directed (in enzymatic metabolic pathways, folic acid)
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16
Q

Antibiotics selectivity

A

Should be directed against specific targets of bacteria, not against human cell components

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17
Q

Antibiotic should act …

A

Rapidly

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18
Q

Disadvantage broad spectrum antibiotics

A

Also affecting the microbiome
> used when uncertain about causing agent

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19
Q

Antibiotics should not cause …

A

allergic response

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20
Q

Pharmocokinetics should match the infection. What is meant?

A

Effective concentration at the site of infection : the route through GI tract etc dilutes the antibiotic

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21
Q

Bacteriostatic vs bactericidal antibiotics

A
  • Bacteriostatic like tetracycline: halt CFU (colony count): stop proliferation
  • Bacteriocidal like penicillin: decrease CFU (colony count): kill the bacteria
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22
Q

Resistance enzyme ESBL

A

Expanded Spectrum Beta Lactamase
> breaks down beta lactam antibiotics

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23
Q

MRSA

A

Multi-resistant Staphylococcus aureus

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24
Q

Resistance enzyme Aminoglycosidases

A

Break down aminoglycosides like kanamycin

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25
Q

Active antibiotic resistance

A
  • Enzymes digesting or modifying antibiotics: beta-lactamase, ESBLs, carbapenemases, aminoglycosidases
  • Active excretion of antibiotics, membrane protein pumps: mainly at gram negative bacteria
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26
Q

Passive antibiotic resistance

A
  • MRSA for example
  • Changes in target so that antibiotics will not bind
  • Alternative PBPs (penicillin binding protein: peptidoglycan synthase)
  • Point mutations
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27
Q

Why do resistant bacteria quickly rule a population?

A

Selective pressure on resistant bacteria
> dependent on target

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28
Q

Resistance transfer to plasmid

A

Transposon combined with plasmid

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29
Q

Horizontal gene transfer of resistance genes

A
  • Conjugative plasmids: sex pili
    > tubes in which plasmids are transmitted
    > from the origin of replication to replicate the plasmid
    > also part of the plasmid can be transmitted (not entire thing)
  • Conjugative transposons: transposase > jumping genes
  • Natural competence: uptake foreign naked DNA (transformation)
  • Bacteriophages: transduction
    > some pack host DNA instead of viral DNA
    > DNA for resistance gene > transfer
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30
Q

Persisters

A
  • Subpopulation of bacteria which can survive many stress situations
  • Heterogenous population contains some persisters
  • Just like the ica locus for biofilm formation but this for stress conditions
    > more tolerant to antibiotics
    > no change in MIC though, so not antibiotic resistant
    > also in biofilms: limitations are nutrients and oxygen
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31
Q

Stress conditions causing growth arrest

A
  • Nutritional deficiency
  • Low membrane potential
  • Intracellular ATP drop
  • Toxin-antitoxin system: become sensitive to own toxins
  • SOS response
  • General stress response
  • Growth arrest itself
    or experimentally
  • high level antibiotic
  • starvation
    » persisters survive the stress conditions
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32
Q

HC18: Upper airway infections

A
  • Mostly viral
  • Sinusitis
  • Pharyngitis: by Group A streptococcus
  • Middle ear infections: otitis media: Haemophilus influencae, Streptococcus pneumoniae
  • Acute epiglottitis: Haemophilus influenzae
  • Diphteria: Corynebacterium diphtheriae
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33
Q

Sinusitis

A

Mostly viral
> Streptococcus pneumoniae, Moraxella catharralis and Haemophilus influenzae also
> excess mucus in sinuses
> inflamed sinus lining

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34
Q

Pharyningitis

A
  • Viral mostly
  • Sometimes Group A streptococcus (S. pyogenes)
    > gram positive cocci in chains
    > normally goes away
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35
Q

GAS infections

A
  • Purulent: abcesses, otitis, sinusitis, mediastinitis
  • Non-purulent: immune complexes, acute rheuma, poststreptococcus glomerulofritis (damage kidneys even when infection controlled)
  • Toxins: Scarlatina / scarlet fever (roodvonk), streptococcal toxic shock syndrome (septic), necrotising fasciitis (flesh eating bacteria)
36
Q

GAS exotoxins, invasins, adhesins

A
  • ExotoxinsL SPE A, B and C
    >Secreted Pyrogenic Exotoxins, superantigens
  • Invasins: streptolysins, streptokinases, proteases
  • Adhesins: M protein, lipoteichoic acid (LTA)
37
Q

GAS molecular mimicry

A

Alpha helical domains M-protein and N-acetyl-beta-D-glucosamine carbohydrate antigen
> resemble myosin
> attack of own muscle tissue by immune system

38
Q

GAS cross-reactivity of antibodies

A

Against own endothelium
> coagulates at heart valve > bacterial endocarditis
> induced by cross-reactive antibodies

39
Q

Otitis media

A
  • Young children affected
  • Often recurrent
  • Tube of Eustachius is more open > connection middle ear and reservoir bacteria
  • possible decrease in countries with S. pneumoniae vaccination
  • protruding the tympanic membrane
40
Q

Haemophilus influenzae

A

Gram negative rod bacterium
> before vaccination introductions
» main cause bacterial meningitis and other invasive infection in children < 5 y/o
» vaccine for HiB induced decline: Capsular polysaccharide antibodies against H. influenzae B

41
Q

Lower airway infections

A
  • Acute bronchitis
    > mostly viral
    > others
  • Acute exacerbation of chronic pulmonary disease (COPD)
    > H. influenzae (NT): not typable, HiB vaccine does not protect
    > others
  • Acute bronchiolitis
    > mostly viral (RSV)
  • Pneumonia
    > Streptococcus pneumoniae and others
    > Legionella pneumophila
  • Tuberculosis
    > Mycobacterium tuberculosis
42
Q

Legionella can survive at …

A

high temperatures

43
Q

Defenses in respiratory tract

A
  • Large respiratory tract has barrier
    > Cilia and epithelium: mechanical barrier
    > goblet cells
  • Small respiratory tract
    > immune cells present
    > antimicrobial peptides made
  • Alveoli
    > two important cell types
    > lining: type 1 cells
    > Alveolar Type 2 cells: progenitor / stem function
    > border capillaries
44
Q

Whooping cough

A
  • Lower airway infection
    > coughing inhibits respiratory function
    > hypoxia, brain damage
    > permanent lung damage
    > stops of breathing, can be terminal for babies
    > highly contagious, in national vaccine program
    > used to be whole cell vaccine, but increased cases because mismatch with circulating strains > later an acellular vaccine
45
Q

Cauding agent Whooping cough and character

A

Bordetella pertussis
> gram negative coccoid rod
> receptors for ciliated epithelium
> pertussis toxin: AB-toxin
> adenylate cyclase toxin: in host cell cAMP is too high > deregulation and cell death
> tracheal cytotoxine: peptidoglycan fragment killing tracheal epithelial cells
» specific form of LPS

46
Q

AB toxins

A

Two components
> one enzyme for transport

47
Q

Bordetella pertussis route of infection

A
  • Inhalation aerosols
  • Bacteria adhere to ciliated epithelial cells
  • Make products
  • Toxin production
  • Damage mucosal cells and act on neurons
  • Paraoxysmal cough
    > or
  • adhere to phagocytes
  • ingested
  • intracellular phase (unknown what happens)
48
Q

ACase effects

A

Inhibit phagocyte migration and oxidative burst

49
Q

Pertussis toxin

A
  • Subunits 1-5
  • S1: enzyme
  • S2-5: binding and transporting part of toxin
  • Ca2+ and calmodulin dependent activation of invaded bacterial ACase
    > also inhibition of Gi
    » rise cAMP levels
50
Q

Pertussis vaccination

A
  • Rise of incidence nowadays due to antivaccination campaigns
  • Acellular vaccines:
    >monovalent: inactivated pertussis toxin PT
    > 3-valent: PT, filamenteus hemagglutinine (FHA) and pertactin (PRN)
    > 5-valent: with also fimbriae agglutinogens 2+3
  • Ineffectivity due to antigenic changes
51
Q

Diphteria

A

Lower airway infection
> Corynebacterium diphtheria
> gram positive rod
> in western countries effective vaccination with toxoid
- toxin producing strains cause airway obstruction

52
Q

Pathogenesis Diphteria

A
  • Adhesion to epithelium using pili
  • Diphteria toxin destroys epithelial cells and PMN
  • Ulcerative, cover with exudate > pseudomembrane
  • Strong inflammation, swelling and choking
53
Q

Diphteria toxin

A

Intact toxin: A and B subunits
> fragment B links toxin to cell and toxin enters cell (toxin cleaved by protease) (receptor-mediated endocytosis)
> A binds NAD
> Catalysis binding NAD to EF-2
> ADP ribose bound to EF-2
> Protein synthesis ceases (elongation polypeptide chain)

54
Q

Vaccine diphteria

A

Toxoid: non-toxic derivative of toxin
Antibodies elicitated will recognize the toxin and neutralize it

55
Q

Pneumonia as old man’s friend

A

Slow death

56
Q

Causative agents pneumonia

A
  • Streptococcus pneumoniae
  • Mycoplasma pneumoniae
  • Legionella pneumophila
57
Q

Pneumonia character

A
  • Inflammation alveoli
  • Abnormal liquid levels in alveoli
  • No proper gas exchange
58
Q

Streptococcus pneumoniae and vaccine

A
  • Pneumococcus
  • Gram positive cocci
  • Capsule with 90 serotypes
  • Pneumonia, sepsis, otitis media, meningitis
  • 13-valent vaccines against polysaccharide capsule > conjugated to protein
59
Q

Route S. pneumoniae

A
  • Aerosol
  • To lungs
  • Escape phagocytosis
  • Inflammation damages lung, damage to respiratory endothelial cells > bacteremia
  • colonization of nasopharynx via adhesins and sIgA proteases > bacteremia and meningitis
60
Q

Why is the airway epithelium not nice for bacteria

A
  • Mucocilliary ladder
  • Phagocytes
  • Antimicrobial peptides
61
Q

Pneumococcus and competence

A

Competence for natural transformation
> can take up naked foreign DNA after producing quorum sensing peptide: Competence stimulating peptide (CSP)

62
Q

Autolysis of pneumococcus and fratricide

A

Lysis of peptidoglycan continuous when synthesis of it is stopped
> end logarithmic phase
> murein hydrolases break it down
> exponential growth when cell wall peptidoglycan synthesis and degradation is balanced
> Some cells induce autolysis of others
» when competence programmed
» competent induced cells lyse the competence deficient cell of some strain
» Microbial fratricide
» for DNA release
» increase efficiency of lateral gene transfer!

63
Q

Which cells in pneumococcus colony become the CSP+ cells which do not undergo autolysis

A

The centered cells> most quorum sensing

64
Q

CibABC

A

CibAB (bacteriocin) + CibC immunity protein
> in fratricide of pneumococcus

65
Q

Benefits fratricide of pneumococcus

A
  • DNA source
  • Nutrients
  • Release virulence factors: pneumolysin
  • Altering the host: avoiding pathogenic population level
    > some immunity created: controlled population
    > limited population better than that high population which kills the population itself eventually
  • Neutralizing antimicrobial peptides which were in lysed cells
66
Q

Tuberculosis

A
  • High prevalence worldwide, not in Europe
  • High lethality
  • Unknown if gram positive or negative: complicated cell wall cauding resistance
  • Mycobacterium tuberculosis needs Ziehl-Neelsen staining
  • When entered > direct colonization macrophages or formation vulcanizing region first (encapsulated)
67
Q

HC19: WGS for vaccine development

A
  • Dry lab: Screened genomes for proteins present which are expressed at cell surface or secreted
  • Wet lab: cloned and tested all protein hits in mice for ability to elicit antibody response
68
Q

Predicting protein structures has become easier:

A

First Cryo-EM after purification, now Alphafold

69
Q

WGS for bacteria workflow

A
  • Bacterial culturing
    > isolate bacteria from: clinical specimens, environment, healthy individuals, laboratory samples
  • DNA isolation
    > extract DNA from cell, purify DNA and remove RNA, proteins and cell wall and sugars
  • Sequence library preparation
    > DNA shearing to desired size
    > Ligate adapters and barcodes
  • Whole-genome sequencing
    > determine base sequence of DNA molecules
70
Q

Illumina Short Read Sequencing

A

PCR based
For WGS
- Produce short 200-500 bp reads
- High accuracy: 99.9%
- Very cheap
- Fragmented genomes
- Shotgun reads > assembled into contigs based on overlap

71
Q

Workflow library preparation

A
  • Shear gDNA into small fragments (200-500 bp)
  • Ligate adapters to both 5’ and 3’ ends of these fragments
    > sequencing binding sites: the polymerase will attach here later on
    > index: barcode for the sample
    > flow cell complementary oligos: bind to the flow cell
72
Q

Cluster generation of Illumina sequencing: on the flow cell

A

Flow cell of 8 lanes with complementary adapters
> fragments are hybridized into sequencing flow cell
> clonal amplification of fragments through bridge PCR
» through fluorescence: much strands needed for all reads
» make cluster of same signal by bridge PCR amplification
» reverse and forward primers used

73
Q

Sequencing by synthesis

A
  • Single stranded fragments are sequenced by polymerising the complementary strand with fluorescently labelled bases
    > one nucleotide at a time, all identical in clusters read simultaneously
    > emission waves length and intensity determines sequence
    > make contigs with overlap
74
Q

Problem short read and de novo assembly

A

Repeat rich regions
> how many repeats, where do the short reads fit?

75
Q

Nanopore sequencing

A
  • Long read sequencing
  • Not PCR based
  • Lower accuracy: 95%
  • More expensive
  • Disruption of current is characteristic per nucleotide base
    » different structure and shape: go through membrane with pore: different signals
  • Long reads complete genomes
76
Q

How do the long reads complete the genomes?

A

In de novo assembly: span the repeat rich regions in the fragmented genome contig based on the Illumina sequencing
> bridge the gap

77
Q

Why is it important to assemble complete genomes?

A
  • Complete information of genes of the strain
  • Critical to understand horizontal gene transfer and mobile genetic elements
  • Plasmids can be transmitted as fragments: but which sequences do they contain
78
Q

Tracing gene transfer of resistance determinants

A
  • Assemble complete genomes
  • Annotation
  • Annotation table, resistance gene report, assembly visualisation
79
Q

Data sequencing

A
  • Fastq files
    > Header
    > Sequence
    > +
    > Quality scores in code
80
Q

Quality control is needed before the …

A

De novo assembly

81
Q

De novo assembly tool

A

Unicycler
> create draft assembly using Illumina short reads with gaps
> then close gaps using Nanopore long reads
> because Illumina more accurate

82
Q

Fasta format

A

> header
sequence

83
Q

Annotation tools

A
  • Prokka
  • ABRicate
  • Bandage Image: assembly graph
84
Q

Annotation to …

A

Find the genes and resistance

85
Q

Prokka

A
  • Identify ORFs > long stretches DNA starting with start codon and non-interrupted by stop codon
  • What ORFs look like
    > compare with large database and select best hits
    > annotation tables
    > non specific
86
Q

ABRicate

A

Annotation of specific genes of interest
> general annotation uses large databases and classifies every gene
> Sometimes interest in subset only: antibiotic genes
> small differences have huge differeces in health
> specific databases used

Medical Microbiology (11 decks)

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