Day 7: Skin and Biomaterial associated infection, Respiratory tract infections, Microbial genomics Flashcards
HC 17, 18, 19
HC17: Biofilm formation steps
- Adherence
- Propagation
- make ‘mushrooms’
- detachment
Biofilm first steps
- 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
Proteins in S. aureus biofilm for adherence and accumulation
- Aap protein: accumulation associated protein
- MSCRAMM protein: microbial surface components recognizing matrix molecules
Accumulation S. aureus through proteins
- Aap/SasG B domain mediated accumulation
- MSCRAMM mediaed accumulation
Dispersion / detachment of S. aureus in biofilm
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
Character of bacteria within biofilms
- 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
Biofilms are not the only reason of persisters and BAI, why>
Prostheses can get infected upon replacement
> bacteria are also more in the tissue
BAI and infection of tissue: early and late response
First: proinflammatory response: PMNs (Polymorphonuclear neutrophils)
Later: macrophages, foreign body giant cells and mononuclear leukocytes, anti-inflammatory
PMNs upon recognition
Make cytokines in periphery
> fibroblast activation and proliferation: fibrosis
> PMNs clear most bacteria and macrophages do the rest
Cell wall as inducer of inflammation and with biomaterial
In gram-positives
> Lipoteichoic acid (LTA)
> Peptidoglycan
» influence foreign body response
» pro-inflammatory or anti-inflammatory
» bacteria+foreign biomaterial: uncontrolled response
When the local immunity is compromised because mixture of biomaterial and bactieria which derange immune responses, what can happen?
Intracellular survival of bacteria within the tissue cells
> persist in immune cells
> clusters in macrophages
S. aureus and S. epidermidis has two niches in BAI:
- Biofilms
- Tissue
» just remove the implant with the biofilms is not enough
Cell wall synthesis directed antibiotics
Penicillins, Ampicillin (beta lactam antibiotics)
Protein synthesis directed antibiotics
Streptomycin, tetracycline, kanamycin
Types of antibiotics
- 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)
Antibiotics selectivity
Should be directed against specific targets of bacteria, not against human cell components
Antibiotic should act …
Rapidly
Disadvantage broad spectrum antibiotics
Also affecting the microbiome
> used when uncertain about causing agent
Antibiotics should not cause …
allergic response
Pharmocokinetics should match the infection. What is meant?
Effective concentration at the site of infection : the route through GI tract etc dilutes the antibiotic
Bacteriostatic vs bactericidal antibiotics
- Bacteriostatic like tetracycline: halt CFU (colony count): stop proliferation
- Bacteriocidal like penicillin: decrease CFU (colony count): kill the bacteria
Resistance enzyme ESBL
Expanded Spectrum Beta Lactamase
> breaks down beta lactam antibiotics
MRSA
Multi-resistant Staphylococcus aureus
Resistance enzyme Aminoglycosidases
Break down aminoglycosides like kanamycin
Active antibiotic resistance
- Enzymes digesting or modifying antibiotics: beta-lactamase, ESBLs, carbapenemases, aminoglycosidases
- Active excretion of antibiotics, membrane protein pumps: mainly at gram negative bacteria
Passive antibiotic resistance
- MRSA for example
- Changes in target so that antibiotics will not bind
- Alternative PBPs (penicillin binding protein: peptidoglycan synthase)
- Point mutations
Why do resistant bacteria quickly rule a population?
Selective pressure on resistant bacteria
> dependent on target
Resistance transfer to plasmid
Transposon combined with plasmid
Horizontal gene transfer of resistance genes
- 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
Persisters
- 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
Stress conditions causing growth arrest
- 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
HC18: Upper airway infections
- Mostly viral
- Sinusitis
- Pharyngitis: by Group A streptococcus
- Middle ear infections: otitis media: Haemophilus influencae, Streptococcus pneumoniae
- Acute epiglottitis: Haemophilus influenzae
- Diphteria: Corynebacterium diphtheriae
Sinusitis
Mostly viral
> Streptococcus pneumoniae, Moraxella catharralis and Haemophilus influenzae also
> excess mucus in sinuses
> inflamed sinus lining
Pharyningitis
- Viral mostly
- Sometimes Group A streptococcus (S. pyogenes)
> gram positive cocci in chains
> normally goes away
GAS infections
- 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)
GAS exotoxins, invasins, adhesins
- ExotoxinsL SPE A, B and C
>Secreted Pyrogenic Exotoxins, superantigens - Invasins: streptolysins, streptokinases, proteases
- Adhesins: M protein, lipoteichoic acid (LTA)
GAS molecular mimicry
Alpha helical domains M-protein and N-acetyl-beta-D-glucosamine carbohydrate antigen
> resemble myosin
> attack of own muscle tissue by immune system
GAS cross-reactivity of antibodies
Against own endothelium
> coagulates at heart valve > bacterial endocarditis
> induced by cross-reactive antibodies
Otitis media
- 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
Haemophilus influenzae
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
Lower airway infections
- 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
Legionella can survive at …
high temperatures
Defenses in respiratory tract
- 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
Whooping cough
- 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
Cauding agent Whooping cough and character
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
AB toxins
Two components
> one enzyme for transport
Bordetella pertussis route of infection
- 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)
ACase effects
Inhibit phagocyte migration and oxidative burst
Pertussis toxin
- 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
Pertussis vaccination
- 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
Diphteria
Lower airway infection
> Corynebacterium diphtheria
> gram positive rod
> in western countries effective vaccination with toxoid
- toxin producing strains cause airway obstruction
Pathogenesis Diphteria
- Adhesion to epithelium using pili
- Diphteria toxin destroys epithelial cells and PMN
- Ulcerative, cover with exudate > pseudomembrane
- Strong inflammation, swelling and choking
Diphteria toxin
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)
Vaccine diphteria
Toxoid: non-toxic derivative of toxin
Antibodies elicitated will recognize the toxin and neutralize it
Pneumonia as old man’s friend
Slow death
Causative agents pneumonia
- Streptococcus pneumoniae
- Mycoplasma pneumoniae
- Legionella pneumophila
Pneumonia character
- Inflammation alveoli
- Abnormal liquid levels in alveoli
- No proper gas exchange
Streptococcus pneumoniae and vaccine
- Pneumococcus
- Gram positive cocci
- Capsule with 90 serotypes
- Pneumonia, sepsis, otitis media, meningitis
- 13-valent vaccines against polysaccharide capsule > conjugated to protein
Route S. pneumoniae
- 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
Why is the airway epithelium not nice for bacteria
- Mucocilliary ladder
- Phagocytes
- Antimicrobial peptides
Pneumococcus and competence
Competence for natural transformation
> can take up naked foreign DNA after producing quorum sensing peptide: Competence stimulating peptide (CSP)
Autolysis of pneumococcus and fratricide
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!
Which cells in pneumococcus colony become the CSP+ cells which do not undergo autolysis
The centered cells> most quorum sensing
CibABC
CibAB (bacteriocin) + CibC immunity protein
> in fratricide of pneumococcus
Benefits fratricide of pneumococcus
- 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
Tuberculosis
- 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)
HC19: WGS for vaccine development
- 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
Predicting protein structures has become easier:
First Cryo-EM after purification, now Alphafold
WGS for bacteria workflow
- 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
Illumina Short Read Sequencing
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
Workflow library preparation
- 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
Cluster generation of Illumina sequencing: on the flow cell
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
Sequencing by synthesis
- 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
Problem short read and de novo assembly
Repeat rich regions
> how many repeats, where do the short reads fit?
Nanopore sequencing
- 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
How do the long reads complete the genomes?
In de novo assembly: span the repeat rich regions in the fragmented genome contig based on the Illumina sequencing
> bridge the gap
Why is it important to assemble complete genomes?
- 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
Tracing gene transfer of resistance determinants
- Assemble complete genomes
- Annotation
- Annotation table, resistance gene report, assembly visualisation
Data sequencing
- Fastq files
> Header
> Sequence
> +
> Quality scores in code
Quality control is needed before the …
De novo assembly
De novo assembly tool
Unicycler
> create draft assembly using Illumina short reads with gaps
> then close gaps using Nanopore long reads
> because Illumina more accurate
Fasta format
> header
sequence
Annotation tools
- Prokka
- ABRicate
- Bandage Image: assembly graph
Annotation to …
Find the genes and resistance
Prokka
- 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
ABRicate
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
