10. Applied - pathogens in the gut microflora, Clostridioides difficile

Applied - pathogens in the gut microflora, Clostridioides difficile

Antibiotics

• Something that kills life

• Anti-microbials → only intend to kill pathogen, not host

• Inhibit essential process

  • Big focus on bacterial cell wall synthesis (vancomycin)

  • DNA gyrase inhibitors

  • DNA-directed RNA polymerase

• Target spectrum of bacteria

  • Big and narrow spectrum

CDI

• Clostridium Difficile Infection

• Disease like pneumonia gets treated with broad spectrum antibiotic

  • Also eliminate symbiotic bacteria in gut necessary for digestion

• Mainly occurs after antibiotic treatment → causes diarhea and gut disease

• Produce toxins A & B → mucosal damage

Clostridium classification

• Cause similar disease profiles and use same toxins

• Even though genetically they are different species, they get classified together

• 96% similarity in genome → same species

• Many C. diff are less than 96% similar → should be different species

Diagnostics

• Symptom based

• Patient material based → stool sample via PCR or enzyme amino assay

  • Depends on DNA or protein marker

• Smell of C. diff is distinct → used as diagnosis

C. Diff gold standard

• Selective culture → plate which only grows C. diff and induces colour

• C. diff is anaerobic → diagnostic in anaerobic cabinet at 37C

• Capillary ribotyping = PCR → gel → interpret gel

• NXT gen sequencing is taking over

Typing

• process in which characteristic of specific strain are determined which can guide clinical decisions

  • Epidemiology surveillance

  • Emergence of new virulant types

  • Recognise hospital outbreaks

  • Comparing strain or patients characteristics

  • Study transmission route

• Age can impact complications of C. diff infections

Transmission route C. diff

• Oral-faecal route

• Spores travel to GI tract into colon where they germinate

• Happens under influence of bile acids

• C. diff will secrete toxins

• Spores will form and cycle starts again

• Spores are dormant life form

  • Specialised cells that survive oxygen exposure

Sporulation

• Special differentiated cell type

• Crucial for transmission → survival in normal atmosphere

• Big problem in hospitals

• Characteristics

  • Small volume, little cytosol, densely packed, chrystalised form

  • Multiple outside layers → specialsed cell wall

  • Very resistant to ethanol, antibiotics cant penetrate spores or because they dont divide

• sp0A is essential for sporulation → KO prevents transmission

Toxins

• Generally located in PaLoc (pathogenicity locus)

• Third toxin → CDTLoc (Binary toxin locus)

• Conserved location

• HGT between isolates

• Toxins are crucial for disease

• Virulance depends on

  • stress response

  • Adhesion factors

  • spore formation

Mechanism toxin A/B

• Toxin A and B

• Internalised via receptor-mediated endocytosis

• Cause glycosylation in Rab/Rho GTPases

  • Important for structure of epithelial cells

• Loss of tight junctions → cause hole in epithelial lining

Binary toxins

• Has two toxin components

• CdtA and CdtB

• ADP ribosylating actin

  • actin filaments keep growing and protrude cell membrane

  • Helps C. diff interact and stimulate infection

• Increase adherance, biofilm formation → increases infection severity

• Alter immune response

Pseudomembranous colitis

• Epithelial layer destroyed

• Neutrophil influx causes increase of pseudomembrane yellow plaques

• Still managable with antimicrobials

Toxic megacolon

• Paralysis of colon

• Colon distents

• Can cause death

Epidemics

• Increase number of cases in 2001

• Hypervirulant version

  • Characteristics is increased disease

  • Higher mortality

• Whole genome sequence of new strains and compare to known strains → identify varinat positions → build phylogenetic tree

• Mutation in gyrase making C. diff resistant to fluorquinolones

• When using this antibiotic, deletion of microflora → space for C. diff to colonise cut and spread

Trehalose

• Most C. diff cases are healthcare associated

• Also community associated CDI → dominated by different types of healthcare associated C. diff

• Trehalose food additive introduced in 2001 → in sync with resistant fluorquinolones C. diff

• Trehalose used as carbon source by C. diff

• A lot of non-antibiotic can affect resistance

Lifestock

• C. diff is big problem in lifestock → pathogen in pigs

• Transmission from pig → farmer → hospital

• Commen origin which causes multiple transmission routes

• One health pathogen

  • Look in environment and veterinary region

resistance to metronidazole

• First infection had no resistance

• Later isolated showed resistance

• Whole genome sequence strain → only difference was presence of plasmid

• Introduction of plasmid into lab strain led to metronidazole resistance

• PCR developed to identify trains which carry plasmid

• Some C. diff use heme-binding proteins (blood) to become resistance to metronidazole

Plasmids in C. diff

• 20% of C. diff strain contain plasmids with unknown functions

• Identify components in plasmids that are neccessary to replicate in C. diff

  • use this to engineer C. diff in lab

• Other plasmid affect susceptibility to vancomycin

• Plasmids can carry toxins

Treatment failure CDI

• Severse disease is performing poorer to metronidazole compared to mild disease

• 20% of severe patients dont get cured

• Possible reasons

  • Sub-optimal antibiotic delivery

  • Micro-environment

  • Other microbiota metabolise antibiotic

• C. diff recurrence decreases treatment success with antimicrobials

Disturbed microbiome

• Dysbiosis of microbiome

• No balance, unstable microbioime community

• Good environment for C. diff

• Restore microbiome via faecal microbiota transplantation

Feacal transplant donor requirement

• Age

  • Ageing immune system, increased risk of cancer

• Travelling

  • Possibility of multi-drug resistant MO

• Obesity

  • Microbiota composition linked to obesity

• Transmitted diseases

  • especially STDs, parasites

FMT disadvantages

• Standardised procedure needed to determine ffects

• Short term negative effects

• Longterm effects are unknown

• More research needed, including controlled trials

  • Uniform donor material needed

• Some microbiota can produce carcinogenic toxins

  • pks+ E.coli

• Abundance of these bacteria decreases with FMT in C. diff → can differ in other diseases

• Microbiota can be reservoir for resistance gene