Bacteria & Fungi - PATH2221

What are the features of a eukaryotic cell (comparable to a prokaryotic cell)

Large (10-100um), have a nucleus, linear DNA, diploid, membrane-bound organelles, 80S ribosomes, mitosis/meiosis, cellulose/chitin in cell walls

What are the features of a prokaryotic cell (comparable to eukaryotic cell)

Small (0.1-10um), absent nucleus, circular DNA, haploid, no organelles, 70S ribosomes, binary fission, peptidoglycan cell walls

What branches are contained in the prokaryotes?

Bacteria and Archaea

What domain is within eukaryotes?

Eukaryota

Briefly outline bacterial cell division

Genetic material replicates, FtsZ protein forms X-ring in centre, constricts, additional proteins form septa, two daughter cells formed.

Describe the difference between Gram negative and Gram positive cells

Gram-positive has an outer membrane and thinner peptidoglycan wall whilst Gram-negative only has a thick peptidoglycan wall

What is the outcome of a Gram stain?

gram positive cells stain purple violet (maintained due to outer membrane)

gram negative cells stain pink (due to violet removed by ethyl alcohol)

How can bacteria be beneficial to the human body?

Include bacteria that make up our microbiome - a healthy gut is associated with resistance to diseases, esp non-communicable (Parkinsons, Sclerosis, diabetes etc.), helping food breakdown, synthesise vitamins, regulate immune system

Outline the effects of microbiome-produced acetate on the body

Acts as an immunomodulator to the immune system in the gut (reduce auto-immune and allergy response) and inhibits some pathogens

How does acetate work as an immunomodulator?

Interacts with G protein-coupled receptor and with naive T cells to help them differentiate, decreases pro-inflammatory cytokines

How can probiotics be beneficial to humans?

Live organism that confer a health benefit in high concs, e.g. lactobacillus is antagonistic to pathogenic bacteria by producing hydrogen peroxide and lactic acid, is a colonisation barrier, inhibits pathogen produced toxins and can immunomodulate the host

Give examples of how bacteria can be helpful in the environment (2)

Bioremediation (used to clean up pollution by breaking down compounds); Rhizobia (symbiotic relationship with plants as they produce nitrogen which increases crop yield)

Outline "the bad guys" of bacteria

Bacteria that are not considered part of the normal body flora and will readily cause disease in healthy individuals e.g. salmonella enterica and helicobacteria pylori

Outline how C.difficile is an opportunistic bacteria

Spore-forming gram positive that is source of hospital acquired diarrhoea as it's precursor is often broad-spectrum antibiotics which depletes the gut microbiome

Outline how bacterial vaginosis is a disease caused by opportunistic bacteria

Vaginal microbiome is low pH due to Lactobacillus but decrease can lead to overgrowth of Gardnerella vaginalis anaerobe found in low numbers causing a bad smell

Who is most at risk of opportunistic infection?

The immunocompromised and those with pathologies directly affecting immune response, eg. AIDS patients

What is the difference between an intracellular and extracellular (epicellular) infection?

For epicellular infection bacteria colonise the surface of the host cell and cause infection whereas intracellular infection requires bacteria to attach and invade host cell

What are virulence factors?

Proteins required for a bacterium to cause disease - include adhesions, secretion systems, host-resistance factors

What are the 4 steps of bacteria causing disease?

- Bacterial exposure

- Colonization

- Immune evasion

- Infection

Outline the lifecycle of an intracellular pathogen

- Host cell invasion/phagocytosis

- Phagosome survival

- Phagosome escape

- Host actin polymerisation

- Multinucleated giant cell and formation

- Host cell apoptosis and bacterial spread

How do epicellular infections colonise the host epithelium

Opa and Pili are used by Neisseria to attach and form microcolonies; type IV-pili of Burkholderia is used to microcolony formation and subsequent intracellular invasion

How do bacteria invade non-phagocytic cells?

1) Zipper: Surface exposed adhesion proteins to bind to receptors on host cells then engulfment

2) Trigger: Type 3 secretion systems to inject effector proteins that stimulate membrane ruffling and internalization

How do type 3 secretion systems allow bacteria to cause disease? (virulence factor)

T3SS of Salmonella enterica promotes invasion into epithelial cells by secretion of effector proteins into host cell causing membrane ruffling (intestinal inflammation)

What is a secretion system?

Machinery that spans gram-negative membrane to allow secretion of effector proteins (virulence factor)

How do type 4 secretion systems allow bacteria to cause disease? (virulence factor)

Can translocate DNA and proteins and play a role in spreading plasmid-mediated antimicrobial resistance by use of effector proteins which attenuate host-cell signaling

How do type 6 secretion systems allow bacteria to cause disease? (virulence factor)

Contractable sheathed needle that bacterium use to stab and inject effector proteins into host cells, e.g. catalase which reduces effectiveness of the host's ROS killing bacteria

How is remodelling of the vacuole influenced by virulence factors?

In L.pneuomophilia T4SS injects effectors into cell facilitating remodelling of vacuole so it is decorated in rough ER and host ribosomes allowing high bacterial replication within

Outline how intracellular bacteria can survive and proliferate outside the phagosome?

Burkholderia pseudpmallei uses T3SS to escape phagosome and adapts to available nutrients in cytoplasm. It hijacks the host cell via BimA, polymerising it into actin tails used for locomotion

What is the difference between exotoxins and endotoxins

Endotoxins are heat-stable lipid A portion of the lipopolysaccharide of Gram negative bacteria, whereas exotoxins are heat-liabile proteins released from bacterial cells

How to type I toxins help bacteria to causes disease?

Bind to host cell surface and secrete proteins that cause a massive non-specific inflammatory response, e.g. toxic shock syndrome toxin

How do type II toxins help bacteria to cause disease?

Act on host cell membranes and destroy their integrity (pore-forming or phospholipase enzymes)

How do type III toxins help bacteria to cause disease?

Proteins with A-B structure. A being active and acts on intracellular targets and B binding to a specific cell receptor

How does cholera toxin help bacteria to cause disease?

A compound A-B type III toxin which enters the cell, increases cAMP production leading to huge Cl- ion secretion resulting in diarrhoea

Outline how primary immunodeficiency diseases (PID) can predispose patients to tuberculosis

Chronic granulomatous disease results in NADPH oxidase deficiency in phagocytes which would generate ROS for killing microorganisms. Lack of ROS means TB becomes rampant.

What sample type would be received from spinal cord testing for meningitis

CSF fluid

What sample type would be received testing for bacteraemia

Blood

What sample type would be received testing for gastroenteritis

Faeces and rectal swab from GI tract

What sample type would be received testing for UTI

Urine

What sample type would be received for STI

Swab, rectal swab, throat swab, eye swab from genital tract

What sample type would be received for sore throat

Swab, aspirate from upper respiratory tract

What sample type would be received for pneumonia, bronchitis

Sputum, aspirate from lower respiratory tract

What sample type would be received for skin infection/wound

Swab, tissue, biopsy, pus from skin/tissue

What is the workflow of a clinical bacteriology lab for phenotypic diagnosis

Samples are received then processed (microscopy, biochem, culture on agar plates), following incubation colonies are identified by rapid biochemical tests and ID confirmation, antimicrobial susceptibility testing is then done to confirm best treatment

What is the purpose of culturing specimens?

To determine is pathogen is present, to perform antibiotic susceptibility test, to keep culture collections for further testing

What is the difference between non-selective and selective culture media?

Non-selective such as blood and CHOC agar have nutrients to support bacterial growth but selective supports growth for one type of organism only providing easier identification

What are the different agars used for selective culture media

MAC/CLED has lactose as carbon source and inhibits Gram positive growth (lactose fermenters appear pink/yellow); CNA agar inhibits growth of Gram negative bacteria

What is the subsequent workflow after identifying a Gram positive or negative bacteria

Gram positive cocci: catalase test

Gram negative bacilli: Growth on MacConkey (MAC)

What is the catalase test?

For rapid differentiation between Staphylocci and Streptococci (Gram positive) as bacteria which express catalase will neutralise hydrogen peroxide and form bubbles

What is the coagulase test?

Differentiates S.aureus from Staphylococcus seeing if they can coagulate EDTA-treated plasma (clumping)

What is the result of haemolysis on blood-containing agar?

Most haemolysis = beta hemolysis. Then alpha then Gamma hemolysis = none. Allows distinction between streptococcus species

What is the oxidase test?

Testing for presence of cytochrome oxidase enzyme by staining blue, differentiating Pseudomonas from other Gram-negatives

What are API strips?

Strips of biochemical suspensions which show colour change when incubated overnight, score is input into database and categorized into most likely species type

How are bacterial species confirmed in labs?

Mass spectometry or MALDI-TOF where colonies are smeared, broken down into protein fragments by laser and analysed against a database

What are the advantages of phenotypic diagnostics?

Culturing, antimicrobial susceptibility tests real-time susceptibility, inexpensive, well validated techniques, easy to construct, interpret and is universal

What are the disadvantages of phenotypic diagnositics?

Reliant on samples being viable (time-sensitive), labour intensive and time consuming, cannot culture all bacteria in the lab and cannot be 100% accurate (contamination is also likely)

Outline what genotypic diagnosis of infection involves

Need to know the sequence of the pathogen, samples are extracted by machine, undergo PCR to determine specific gene product presence, internal control is added to insure sample is not inhibitory of PCR conditions

What are the advantages of molecular diagnostics (genotype)

Rapid, doesn't require culture, sensitive and specific, automated, higher throughput, probes can be added quickly to changing sequence types and whole genome sequence can allow us to identify unknown causes of infection

What are the disadvantages of molecular diagnostics?

High burden of validation required for all tests, highly trained personnel needed, cannot store in culture collection, expensive, mutations in sequence can decrease sensitivity, genotype doesn't always equal phenotype, ultra-cold storage needed (not universal)

What are the basic features of moulds?

Multicellular fungi, have hyphae which end in fruiting bodies filled with spores and have septa separations, moulds look fuzzy/fluffy

What are the basic features of yeast?

Unicellular fungi, optimally grow at 37, egg shaped, look like bacterial colonies on agar plate but slightly bigger

What is superficial mycoses?

Fungal infection of skin/hair/nails, e.g. ringworm and athelete's foot. They occur after the dermatophyte breeches the skins outer layer

What is systemic mycoses?

Mostly opportunistic affecting immunocompromised - Include aspergillus (pneumonia & allergy); histoplasmosis (flu-like from bird droppings); murcomycosis (affects immunocomprimised, nasal pathology)

What opportunistic fungi is associated with HIV?

Pneumocystis jirovecii due to associated low CD4 T cell levels. Causes fever, chills, cough, fatigue

Outline the different candida species mycoses

Most common yeast infection but is commensal at many sites until individual is immunocompromised. C.albicans is most common, C.auris is a public health threat

Outline the action of cryptococcus neoformans

Major yeast pathogen that is inhaled and causes pneumonia and meningitis. It's capsule can be visualised with india ink.

What is the laboratory process behind fungal infection diagnosis

Specimen is received, undergo microscopy & culture, visualised under microscope, yeasts can be identified using MALDI-TOF and undergo antimicrobial susceptibility testing

How are fungae visualised?

Stained with potassium hydroxide to clear other sample debris, then tehy are fluorescently stained with Calcafluor white OR periodic acid-schiff stain OR india ink (cryptococcus) or Lactophenol cotton blue

What are the different kinds of parasites?

They all all eukaryotic but protozoa are unicellular (e.g. plasmodium) and metazoa are multicellular (e.g. helminths)

What is zoonosis?

A disease spread from animal to human

What is anthroponosis?

A disease spread from human to human

Give examples of blood-borne parasitic infections

Trypanosomes (chagas disease, african sleeping sickness) and Leishmaniasis (L.donovani, L.major, L.tropicalis)

What is the lifecycle of a blood-born parasitic disease? (Trypanosoma cruzi)

1) The vector bites human

2) Trypanosome cells move through blood and infect tissues to multiply

3) Intracellular amastigotes burst out of cell and re-infect

4) The vector takes a meal out of infected human and epimastigotes multiply in midgut

5) Trypomastigotes in hindgut infect human when vector feeds again

What is the malaria life cycle?

- Spread to humans by blood meal of Anopheles mosquito

- Human liver cells are infected: schizont replicative cycle

- Human blood stages involve replication within schizont and bursting AND gametocytes taken up by vector biting again

- Mosquito stage involves macrogametocyte forming oocyst which ruptures and releases sporozoites

What is the most severe species of malaria causing parasite

P.falciparum

Outline the general lifecycle of a diarrhoeal parasitic infection

Contracted via fecal-oral route. Cysts are the infective stage and can survive in the environment, upon excystation trophozoites are released to feed, replicate and cause disease. Encystation then occurs and cysts are passed in faeces

Give an example of a diarrhoeal parasitic infection

Giardia lamblia: contracted by contaminated food/water; trophozoite have ventral sucking discs and flagella, cysts can withstand harsh environmental conditions

What are other causes of diarrhoeal infection

E.histolytica and Cryptosporidium are self-limiting parasitic whilst helminth infection presents with similar symptoms

What are the 3 main classes of pathogenic helminths?

Nematodes (roundworms)

Cestodes (tapeworms)

Trematodes (flukes)

Describe the differences between the helminth shapes

Nematodes: elongated, cylindrical, unsegmented

Cestodes: Tape-like segmented

Trematodes: Lead-life unsegmented

Describe the differences between the helminth sexes

Nematodes & Cestodes have separate sexes whilst trematodes do not

Describe the differences between the helminth heads

Nematodes: no suckers, no hooks, well-developed

Cestodes: suckers, with hooks

Trematodes: suckers, no hooks

Describe the differences between alimentary canal for helminths

Nematodes: Present & complete with anus

Cestodes: absent

Trematodes: Present & incomplete with no anus

Describe the differences between body cavity for helminths

Nematodes: Present

Cestodes & Trematodes: adsent

How doe nematodes work?

Can live in the intestines to grow and lay eggs and attach to intestinal wall

How do cestodes infect?

Tapeworms that can growth to multiple metres and are ingested as eggs, can enter brain/muscle/heart

How do trematodes infect?

Transmitted via larvae in seafood, cause lung & liver disease as well as non-specific during migration

Describe bacterial antimicrobial susceptibility testing

Conducted on Mueller hinton agar & blood to determine if susceptible, resistant or intermediate to antiBs.

What are the three tests of bacterial antimicrobial susceptibility testing? (Phenotypic screening)

- Kirby-Bauer disc diffusion

- E-strip

- Microbroth dillution

Describe kirby-bauer disc diffusion

Will show sensitivity or resistance to a known antibiotic concentration by measuring zones of inhibition around filter strip with antibiotic

Describe the E-strip method of screening for resistance

A plastic strip with a gradient of antibiotic concentration is placed on an agar plate and zone of elliptical zone of inhibition forms - MIC can be read

Describe the microbroth dilution method for screening for resistance

Bacteria are exposed to serial dilutions of antibiotic in liquid media

What is the MIC?

Minimum inhibitory concentration: lowest antibiotic concentration that inhibits bacterial growth

What are some non-phenotypic tests for susceptibility?

Vitek platform

Molecular tests for genetic determinants of resistance = GeneXpert

How are antifungals tested for resistance?

Only for non-fastidious fungi (Candida & Cryptococcus & Aspergillus).

Microbroth dilution plate is set up and colour indicates growth (pink)

What are the 2 different types of antibiotics and how do they work?

Bacteriostatic: inhibit ability of bacteria to replicate & grow

Bactericidal: kill bacteria

What is antimicrobial resistance?

Ability of microbes to grow in the presence of a drug that would normally kill or limit its growth

What are the two ways in which microbes become resistant?

Acquired

Intrinsic

What is acquired resistance?

Uptake of genetic elements from other bacteria by horizontal gene transfer, DNA uptake or conjugation

What is intrinsic resistance?

Resistance to antibiotics that occurs due to the presence of enzymes, pores, pumps that exist within the genome of the bacterium

What are ways to circumvent resistance?

Testing for drugs that will kill bacteria

Completing courses

Combination therapy