BM329 Block D

Lecture 1 - Clostridium difficile

Endospore Forming Bacteria

Bacillus consists of aerobic and facultative anaerobic spore formers and includes:

• B. anthracis

• B. cereus (rice)

• B. mycoides

• B. thuringiensis

• B. subtilis

Clostridium consists of strict anaerobic spore formers and includes:

• C. difficile

• C. botulinum

• C. perfringens

• C. speticum

• C. tertinum

• C. tetani

Clostridium difficile

C. diff is the most commonly diagnosed bacterial cause of hospital acquired infectious diarrhoea in resource rich countries. It is an anaerobic spore forming motile gram positive rod which is between 0.5-1 micro metre in diameter and 3-16 micro metres long.

It produces irregular which colonies on blood agar and is difficult to culture.

Endospore forming lifecycle

Spores are survival structures intended to survive environmental changes such as starvation, drying, heat, chemicals and radiation due to their thick outercoat. The sporular phase is a dormant stage but is useful for dispersal via air, wind, water and the gut.

The sporulation phase is only entered when a key nutrient becomes limiting, there is conserved regulation of this process in Clostridium and Bacillus. Spores form from an asymmetric septum - septa usually form at midcell however in sporulation they form at the quarter point.

In C. diff Spo0A which is a response regulator drives the sporulation regulatory network and directly regulates dozens of genes.

Spo0A induces the expression of the first sporulation specific sigma factor (sigma H) which forms a positive feed forward loop with Spo0A leading to more expression (?). Spo0A drives the sequential action of four compartment specific alternative sigma factors F, E, G and K.

Germination of C. difficile spores and vegetative growth occurs only in the lower GI tract due to low oxygen levels (strict anaerobe). Primary bile acids such as taurocholate induce spore germination of the spore into an actively replicating vegetative cell. Addition of taurocholate to cycloserine cefoxitin fructose agar (CCFA), which is a specific agar, increases the recovery of colonies from spores indicating this increases the production of spores. This is controlled by cspBAC locus, CspC binds taurocholate and activates SleC which is a lytic enzyme essential for germination as it degrades proteoglycan spore cortex. On agar plates they must be grown in an anaerobic jar.

Virulence Factors

The ability of C. difficile to cause colitis depends on a range of virulence factors such as toxins which are encoded by the pathogenicity locus, adherence and mobility factors. Toxins which primarily target intestinal epithelial cells are produced in response to limited nutrient availability. Toxin endocytosis and activation in cytosol of epithelial cells causes necrosis which leads to the loss of intestinal membrane integrity which leads to water loss and diarrhoea, host exposure to intestinal microorganisms which leads to other infections due to the loss of the first barrier defence and activation of the host inflammatory response.

Pathogenicity

The pathogenicity locus encodes 5 proteins including 2 of the 3 toxins:

• TcdA (ToxA)

• TcdB (ToxB)

• TcdR

• TcdC

• TcdE

TcdA and TcdB contain several domains: Rho and Rac glucosyl transferase domains at the N-terminus which mediate toxicity by glycosylating and inactivating host Rho and Rac GTPases in the cytosol of targeted cells as well as breaking down tight junctions, cytoskeleton and epithelial integrity, cysteine protease domain which autocatalytically cleaves CDT in the cytosol of eukaryotic cells in association with myoinistitol, hydrophobic protein sequences which are involved in host membrane insertion, and combined repetitive oligopeptide repeat domains (CROP) which are thought to bind to cell surface receptors of epithelial cells before endocytosis and internalisation of TcdA and TcdB.

TcdR is an alternative sigma factor which facilitates the binding of RNA polymerase to the promoters of tcdA and tcdB as well as its own promoter, this means that if there is no TcdR there are no toxins produced. TcdC is expressed in the growth of C. difficile and is thought to act as an anti-sigma factor and thus supresses the expression of tcdA and tcdB transcription. TcdE is a holin like protein which is thought to facilitate the secretion of TcdA and TcdB which lack conventional secretion signal sequences.

Binary Toxin

Some hypervirulent strains of C. diff also express binary toxin or C. diff transferase (CDT). The role of this in virulence is unproven but is associated with higher mortality in patients. CDT is composed of two proteins: CdtA which is an ADP-ribosyl transferase that ribosylates actin in eukaryotic cells interfering with actin polymerisation and cytoskeletal structure and CdtB which forms pores in acidified endosomes and facilitates the transfer of CdtA to the cytosol so it can elicit its effect. For the toxin to work the two components must be cleaved by??

Non-Toxin Virulence Factors

Genes for the regulation of motility and adherence are important for colonisation efficiency and virulence of C. difficile. A lack of flagella is linked to impaired adherence to intestinal epithelium, mutants which lack flagella exhibit dysregulated toxin expression and virulence in vivo.

Flagellum expression is regulated by the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). High levels of this repress flagellum expression and therefore motility, repress synthesis of TcdA and TcdB, and induce expression of pili which interact with intestinal epithelium and contribute to aggregation and biofilm formation. c-di-GMP is a key signal that switches C. diff between a motile, toxin-producing state and an adherent biofilm producing state. It acts as a bacterial hormone which enable survival of the C. diff cells in the colon.

There are many proteins which contribute to adherence and have a role in biofilm formation such as:

• adhesion fibronectin binding protein A

• cell wall proteins such as Cwp66

• S-layer protein A and protease Cwp84

• Spo0A

The extracellular matrix of the biofilm is composed of proteins, polysaccharides and free DNA from dead cells. Together these proteins insulate vegetative cells from oxidative stress, antibodies and antibiotics as well as creating a protected niche for sporulation.

Pathogenesis

Key features of C. diff pathogenesis include impaired tight junctions, cell rounding up and cell death. TcdB activates the calcium influx which causes disintegration of the actin cytoskeleton. TcdA inhibits stimulated mucin exocytosis which means there is no mucus so no protective barrier.

There is also release of inflammatory cytokines (IL-8, IL6, TNFalpha and leptin) which recruits PMNs and the production of reactive oxygen radicals. The toxins increase the release of substance P which is a peptide that enhances fluid loss from the intestines and decreases the exocytosis of protective mucus barrier.

The Gut Microbiome

The human gut microorganisms produce enzymes and amino acids. Human gut microorganisms are believed to be responsible for maturing of the gastrointestinal tract, they may also play a role in obesity. There is great density and diversity if microbes in the gut microbiome.

There is marked inter-individual variation so particular organisms predominate in particular anatomical locations.

Lactobacillus and Streptococcus predominate in the stomach whereas Bacteroides and Clostridium predominate in the distal gut.

Infection

C. difficile is present in the intestine of 2-3% of asymptomatic adults but this may increase to around 30% in hospital environments however this is predominantly in biofilms so there aren’t any pressing issues. This number increases to 60-70% of neonates.

It is a commensal pathogen and normally does not cause any disease. Neonates may not have the necessary adhesion to allow the toxin to bind. The elderly often either enter hospital carrying the pathogen spreading it further or they acquire it in the hospital.

Antibiotics disturb the normal flora creating a niche where C. difficile can proliferate.

Microbiome Mediated Defences

Intact microbiota converts primary bile acids into secondary bile acids, several derivatives of which inhibit the growth of C. diff through detergent induced toxicity to vegetative bacilli.

Commensal bacteria express sialidases which cleave sugars attached to epithelial cells and release sialic acid into intestinal lumen. Commensal bacterial species convert sialic acid into short-chain fatty acids such as succinate. Commensal bacterial populations consume these metabolites as energy sources.

Antibiotics disrupt the microbiota which depletes primary bile acid converters enabling C. diff sporulation and growth. Antibiotics can also deplete competing sialic acid and succinate consumers which liberates an energy source for C. diff as it stops the conversion of primary bile acids to secondary bile acids causing a build up of primary bile acids.

Antibiotic-Associated Diarrhoea

In asymptomatic carriers there is a good IgG and IgA response to C. diff toxins. If there is a poor IgG and IgA response then there is production of toxins, activation of macrophages and upregulation of inflammatory mediators. This results in clinical disease.

C. difficile associated disease and pseudomembranous colitis (PMC) can arise from infection. PMC has significant mortality and is associated with abdominal pain, bloating, watery diarrhoea with a distinct smell, blood in the faeces, perforated colon and toxic megacolon which is where the diarrhoea stops however the pain continues??

Epidemiology

C. diff colonises infection of around 5% of healthy individuals. Antibiotic exposure is associated with overgrowth of the pathogen.

Exogenous infection occurs from spores which are found in infected hospitals. There are highly virulent strains found in USA and Europe.

Sensible use of antibiotics would include no antibiotics meaning no altered gut microbiome, no C. diff growth, no spores and no transmission.

C. diff Diagnosis

This is diagnoses by detection of cytotoxin or enterotoxin in faces. The detection of toxins in faeces is done by ELISA. Diagnosis also involves the detection of glutamate dehydrogenase in faeces.

Culturing must be done anaerobically on cycloserine or egg yolk agar which will produce white/grey colonies. PCR may be done for toxin related genes. A colonoscopy is often done when PMC is suspected and a sigmoidoscopy will be done for severe cases. Genomics may also be used.

Treatment

Treatment with implicated antibiotics has been discontinued but metronidazole or vancomycin may be used in severe cases. Fidoxomicin is used in very severe cases.

Relapses often occur as antibiotics cannot kill spores and multiple course may be necessary. Alternative treatments may include chloestyramine, bacirtcin-fusidic acid, IV immunoglobulin, faecal enema and faecal transplants.

Thorough cleaning will need done to prevent reinfection.

Prevention

Bathroom hygiene is important such as closing the lid when flushing. Infected people should be in isolation with a dedicated toilet and PPE. High touch surfaces should be regularly cleaned with bleach disinfectant. Wards and units may be closed in infected hospitals. All items in a patients room should be thoroughly cleaned after discharge. Hand washing protocol should include soap and alcohol.

Lecture 2 - Shigella and Salmonella

Enteric Bacteria

This is a group of facultative aerobes which are gram negative, non sporulating rods. They are either non-motile or motile by petritichous flagella.

They are distinguished from other gammaproteobacteria by biochemical tests as they are oxidase negative and catalase positive. E. coli, Shigella and Salmonella are mixed acid fermenters which produce acetic, lactic and succinic acids from glucose.

Shigella vs Salmonella

These two species can be distinguished based on various biochemical tests such as the fact that Salmonella produce H2S and shigella do not. Salmonella are peritrichously flagellated whereas Shigella have lost the flagellum biosynthesis genes.

Salmonella can infect a range of animals and is considered a zoonotic bacteria whereas Shigella infects only primates.

Salmonella

S. bongeri is mainly present in cold blooded animals especially reptiles. S. enterica is mainly present in warm blooded animals and the environment with the S. enterica subspecies enterica being responsible for the majority of disease seen in humans. Non-typhoidal serovars result in gastroenteritis and typhoidal serovars result in typhoidal fever. There are over 2500 serotypes which are based upon three major antigens: O, H and Vi. The Vi capsule is only seen in S. typhi. H1 and H2 are flagellar antigens.

Shigella

This is classically separated into 4 species and further into genomic analysis into three clusters. Shigella arose from ancestral E. coli several times independently, this involves the acquisition of key virulence factors such as Shiga toxin as well as gene loss such as those for flagellar biosynthesis and lactose fermentation.

The three main disease causing shigella groups are S. flexneri which is the most frequently isolated species and accounts for 60% of cases in the developing world, S. sonnei causes 77% of cases in the developed world as opposed to 15% of cases in the developing world, and S. dysenteriae which is usually the cause of epidemics of dysentery particularly in confined populations (eg refugee camps)

Salmonella and Shigella Virulence Factors

Key virulence factors include endotoxins, exotoxins, secretion systems (TTSS), flagella and pilli, and biofilm formation.

Many virulence factors are acquired via horizontal gene transfer such as pathogenicity islands (PAIs), plasmids and prophages. This means there is variation from strain to strain. In S. enterica all strains have SP-1 and SP-2. In Shigella virulence plasmids pINV A or B along with several PAI mediate virulence.

Entry into Host Cells

Both species are facultative intracellular pathogens, the bacteria induce their internalisation into non-professional phagocytes which subverts host cell functions such as actin cytoskeleton rearrangements (membrane ruffling). Entry also involves trigger mechanisms (used by both Salmonella and Shigella) and protection from some aspects of the host immune systems, access to nutrients, and spread and dissemination. Bacterium can also enter host cells via the Zipper mechanism, this is used in pathogens like listeria but Salmonella can sometimes use this mechanism instead of the trigger mechanism.

Shigella invades a host cell ruffle produced as a result of TTSS, it interacts with the host cell surface and injects its invasin proteins via this system which choreograph a ;oca; actin-rich membrane ruffle at the host cell. The ruffle engulfs the bacterium and eventually disassembles internalising the bacterium.

Type III Secretion

This system delivers bacterial proteins known as effectors into the host cytosol.

This system injects proteins directly from the bacterial cytoplasm into the host cytoplasm, the proteins in the systems are related to flagellar assembly proteins.

TTSS delivers bacterial effectors into the host cytosol, effectors trigger host cytoskeleton reorganisation which engulfs the bacterium. The effects in each bacteria are similar but the mechanisms may differ.

Sip are Salmonella TTSS proteins. SipBC is a translocon pore, SipC also nucleates actin polymerisation. SopE is the exchange factor which activates Cdc42 and Rac. SopB is phosphatidylinositol.

Ipa are Shigella TTSS proteins. IpaBC is the translocon pore with IpaC also nucleating actin polymerisation. VirA destabilises microtubules.

Intracellular Pathogens

Phagolysosomes are involved in oxidative stress, pH, antimicrobial peptides. Salmonella survive in a vacuole whereas Shigella escape into the cytosol.

Iron acquisition is a challenge for intracellular pathogens as is motility and transmission.

Survival in the Host Cell

Successful pathogens must avoid killing by the phagolysosome. Salmonella does this by modifying the phagosome and blocking the fusion with the lysosome. Shigella instead rupture the phagosome and escape into the cytosol.

S. typhi invades intestinal cells and prevents phagolysosome fusion, it then exists the intestinal cell and invades awaiting macrophages. The bacterium can survive inside the macrophage which then shuttles the pathogen to lymph nodes and circulation.

Salmonella Containing Vacuole

Salmonella both adapt to the intravacuole environment and modify the vacuole preventing maturation, recruiting host proteins to the vacuole membrane and bacterial proteins such as SPI2 pathogenicity islands are also involved.

SCVs migrate to the periphery of the Golgi network, this localisation is required for replication. This is potentially involved in interception of lipids and proteins for nutrients.

Shigella in the Host Cell

Shigella escapes from the phagosome and into the cytoplasm. IpaB and IpaC interact with the vesicle membrane and appear to destabilise it, host factors are also required. Motility in the cytoplasm required for the cell to cell spread. Bacterial protein IcsA is expressed on bacteria surface at the pole and nucleates actin polymerisation. This involves a complicated complex of host proteins being recruited.

Actin Based Motility

The outer membrane protein IcsA is located at the bacterial pole and binds and activates N-WASP. N-WASP then activates the Arp2/3 complex leading to actin polymerisation. Other bacteria such as listeria can also use host actin for motility through different mechanisms.

Exotoxins

Enterotoxins are exotoxins whose sight of action is in the small intestine which leads to fluid accumulation and diarrhoea. Shiga toxin is encoded on a prophage and is also present in some species of E. coli. Shigella enterotoxins include ShET1(PAI SHI-1) and ShET2 (pINV). The Salmonella enterotoxin is known as Stn??

Shiga toxin attaches to ganglioside Gb3 and then enters the cell, cleaving 28S rRNA in eukaryotic ribosomes to stop translation.

Endotoxin and Vi Antigen

Salmonella LPS stimulates an inflammatory response which is a key feature of gastrointestinal salmonella infections. S. typhi LPS does not trigger this response as it has gained a PAI (SPI7) with a capsule biosynthesis locus and the Vi antigen which masks the LPS.

Salmonella in the Host Gut

Tetrathionate metabolism in Salmonella gives it a selective advantage in the host gut. Hydrogen sulphide is produced by the microbiota, this is detoxified to thiosulphate by intestinal epithelial cells, Salmonella then induce inflammation leading neutrophils being recruited and producing ROS which oxidise thiosulphate to tetrthionate. Genes on SPI2 allow Salmonella to use tetrathionate as an alternative electron acceptor during inflammatory response which gives an advantage over normal microbiota which cannot use tetrathionate.

Treatment

Many other bacteria cause diseases of the digestive system such as helicobacter and campylobacter. These bacteria have very similar disease profiles but differential types of faeces. The complications can differ from bacteria such as in E. coli and Shigella HUS can occur damaging the kidneys. The key to treatment is keeping hydrated.

These infections are not large issues in the developed world with a low mortality rate provided that hydration is kept and all treatment is followed. In the developing world there is less access to medical care, clean water or safe foods which results in higher mortality particularly in children.

Disease Transmission

Transmission occurs via the oral faecal route. This occurs via ingestion of contaminated water or food, commonly leafy greens. Infection can also result from poor hygiene.

Salmonella can also be transmitted via contact with reptiles. Shigella has a very low infectious dose of less than 10 cells.

Asymptomatic S. typhi

Some individuals with S. typhi fail to successfully clear the infection which leads to chronic carriage of the bacterium in the gallbladder. This results in intermittent shedding and spread of disease however the carrier will usually be asymptomatic.

typhoid mary

Typhoid Fever Treatment

Untreated typhoid fever has a mortality rate of 10-30%. Antibiotic therapy will reduce this to around 1%. Where resistance is uncommon the treatment choice is a fluoroquinolone which target DNA gyrase such as ciprofloxacin. Otherwise a a third generation cephalosporin which is a class of Beta lactam antibiotic (target??)such as ceftriaxone or cefotaxime is the first choice.

Treatment of Diarrhoeal Diseases

Rehydration therapy involves replacing fluids and salts by oral rehydration supplements typically containing salts NaCl and KCl. Antibiotic therapy is not necessary in most cases but may be used when the disease is severe and the risk of infection spreading is high. In Salmonella the first choice is ceftriaxone whereas in Shigella it is trimethoprim-sulfamethoxazole (target folate metabolism) or ciprofloxacin. Antibiotic resistance rates are increasing so its vital to check prescribing recommendations.

The key to control and prevention is food hygiene and vaccination of hosts.

Lecture 3 - Picornavirus and Rotavirus

Picornaviridae

This is a family of RNA viruses which has 5 main genera:

• Rhinovirus

• Enterovirus

• Apthovirus

• Heparnavirus

• Cardiovirus

There are at least 72 enteroviruses including Polivirus, coxsackieviruses and echoviruses. There are at least 100 serotypes of rhinoviruses which are the major cause of common colds. Aphthoviruses cause foot and mouth disease. Heparnaviruses include Hepatitis A virus which is a waterborne virus.

Picornaviruses have an icosohedral capsid which is 30nm in diameter. It is a naked virus so is hard to kill and enables easy spread. There are 4 virion polypeptides (VP1-4) which assemble into protomer subunits. 5 subunits then assemble into pentamers and 12 pentamers then associate to form the procapsid (provirion). Pleconaril is an antiviral which blocks the receptor binding to the canyon inhibiting virion function .

The genome of Picornavirus is mRNA, they have a single strand positive sense RNA genome which has a small Vpg protein attached to the 5’ end of the genome. They also have a poly A tail at the 3’ end of their genome. The naked genome is infectious.

Picornavirus Replication

The whole genome is translated into one polyprotein which is acted upon by host cell proteases leading to the polyprotein being cleaved into functional units including more proteases. The genome is also a template for more positive strands which can either undergo direct translation or packaging into a new genome.

For replication to occur a receptor on the membrane of a host cell must be bound leading to VP4 being released allowing the genome to be injected through the membrane, this genome can then bind directly to the ribosome.

Poliovirus Assembly

Structural proteins VP0, VP1 and VP3 are cleaved from the polyprotein. These units then assemble into protomer subunits, 5 subunits then assemble into pentamers and 12 pentamers associate to form the procapsid. The gen ome is the inserted and VP2 and VP4 are generated by cleavage of VP0.

Viruses Associated with Gastroenteritis

• Orthoreoviruses

• Rotaviruses

• Calciviruses (noroviruses) - Norwalk virus, Sapporo virus

• Astrovirus

• Enteric adenoviruses

• Others - Parvoviruses, coronaviruses, pestiviruses, toroviruses

Reoviridae

This family consists of several reovirus genera which infect invertebrates and plants. Coltivirus which causes an acute febrile illness known as Colorado tick fever virus. Orbivirus causes a variety of encephalitis conditions such as Blue tongue virus.

Orthoreovirus causes mild upper respiratory tract illness, GI tract illness and biliary atresia.

Rotavirus causes GI tract illnesses and potentially respiratory tract illness.

Reovirus was first recognised in 1959 and were classified incorrectly as echoviruses. They are respiratory enteric orphan viruses. This is a diverse group which infects invertebrates, vertebrates and plants that consists of over 150 species.

Rotaviruses share many structural, replicative and pathogenic features such as icosahedral morphology. They are non enveloped but do have a double capsid. Activation requires proteolytic cleavage of the outer capsid.

The outer capsid is composed of structural proteins, the nucleocapsid core includes RNA dependent RNA polymerase. Reoviruses resemble enveloped viruses with glycoproteins acting as viral attachment proteins, they briefly acquire and then lose an envelope during assembly. They also possess fusion protein activity that promotes the direct penetration of the cell membrane.

Reovirus core proteins include 5’methyl guanosine mRNA capping enzyme and RNA dependent RNA polymerase. The capsid proteins are sigma 1 in reoviruses and VP4 for rotaviruses which extend like spike proteins and sigma 3 in reoviruses or VP7 in rotaviruses which act as viral attachment proteins. The genome is segmented meaning each segment of RNA encodes a different protein.

The genome for reoviruses is composed of 10 dsRNA segments and for rotaviruses consist of 11 dsRNA segments. The segments fall into three distinct categories (λ, μ, σ). 30% of the virion RNA is small single stranded oligonucleotides.

Reovirus Replication

Firstly the virus must be ingested and then the outer capsid must be removed and the ISVP produced. The ISVP proteins at vertices then bind sialic acid containing glycoproteins. The ISVP then penetrates the membrane, the core is then released into the cytoplasm and transcription can be initiated by core enzymes.

The transcription of dsRNA occurs in early and late phases. The negative strand is used as a template by virion core enzymes. The genome has a 5’ methyl guanosine capped, 3’ poly adenylated positive strand mRNA which leaves the core for translation. The positive strand RNA is then copied in new cores to make dsRNA, the new cores then assemble into virions or generate more positive RNA. Orthoreovirus has outer capsid proteins which associate with the core and the virion leaves by cell lysis. Rotavirus has an assembly process which resembles enveloped viruses. For replication the virus must go via the intestine as if it doesnt no infection will occur as the outer capsid cannot be broken down.

Pathogenesis

They are common causative agents of infantile diarrhoea. The virions of these viruses are relatively stable with VP7 and VP4 being used for serotyping. Replication occurs at the columnar epithelial cells of the small intestine. Infection will prevent the adsorption of water. Immunity will require IgA in the gut lumen.

Epidemiology

Infections are seen worldwide with 95% being infected by age 5. These viruses are spread via the faecal oral route and are the most common cause serious diarrhoea in children and can cause gastroenteritis. Symptoms resemble those of other viral diarrhoeas. There will be large quantities of virus in the stool allowing direct detection, there is no specific antiviral therapy.