Gastrointestinal infections - causative agents

requirements for msot pathogens to cause disease (6)

• Enter body

• Colonise host

• Evade host defences

• Multiply and disseminate

• Cause damage to host

• Pathogens produce virulence factors that allow them to infect and damage the host

common modes of entry (4)

faeces

fingers

foods

fluids

methods to overcome acidity of stomach (4)

• Acid resistant

• Present in high numbers

• Protected by food

• Encounter a higher than usual pH in stomach -> eg. antacid use will increase pH

non-invasive bacteria list (4)

• Vibrio cholerae

• Enterotoxigenic E. coli

• Enteropathogenic E. coli

• Enterohaemorrhagic E. coli

invasive bacteria list (4)

• Shigella spp.

• Salmonella spp.

• Yersinia enterocolitica

• Campylobacter

• Aeromonas spp.

viruses list (3)

• rotavirus

• norovirus

• adenovirus

parasites list (3)

• entamoeba histolytica

• giardia lamblia

• cryptosporidium

vibrio cholerae - origin

Environmental organism -> free-living inhabitant of coastal waters and as human intestinal pathogen

vibrio cholerae - method of spread

contaminated food and water contaminated with human faecal waste -> asymptomatic human carriers  (excretors) are major reservoir

Long history characterised by epidemics and pandemics → predominantly in LMIC countries but not present in Aus

vibrio cholerae - symptoms

Causes massive, profuse, watery diarrhoea -> death within hours from dehydration (esp. children) if not treated with oral rehydration salts

"rice water" stools -> cholera cots

vibrio cholerae - serogroups (2)

Two different serogroups causes majority of disease and outbreaks -> serogroup O1 and serogroup O139 depending on categorisation of LPS antigen

Serogroup O1 = subdivided into 2 biotypes (classical and El Tor) → cause of epidemic in Haiti in 2010 following earthquake (El Tor biotype)

No cross protection between O1 and O139 serotypes

O139 now only causing sporadic cases

cholera infection cycle (4)

1. Ingestion of vibrio cholerae in large numbers -> high infectious dose

2. Enters stomach -> sensitive to stomach acid so large dose needed to cause disease unless patient Is achlorhydric or taking antacids

3. Colonisation of small intestine depends on adhesin, toxin production and flagella motility (virulence factors)

   • Flagella to burrow into mucous layer

   • Mucinase to hydrolyse gut mucous

4. Massive loss of fluid and electrolytes without damage to enterocytes -> no blood or white blood cells in stool

vibrio cholerae virulence factors - list (2)

adhesin = toxin co-regualted pius (Tcp pilus)

AB5 choldera toxin

vibrio cholerae virulence factors - Tcp pilus

Pili needed for successful intestinal colonisation of brush border villi

allow bacteria to bind to each other and form micro-colonies and then bind to enterocytes via unknown mechanism

vibrio cholerae virulence factors - cholera toxin subunits

One A catalytic subunit -> toxigenic unit

Five B binding subunits -> binding unit (pentameric)

vibrio cholerae virulence factors - cholera toxin steps to diarrhoea (6)

1. Binds to GM1 gangliosides -> cell surface receptors on intestinal cells

2. A subunit internalised via endocytosis and cleaved into A1 and A2 subunits

3. A1 subunit causes ADP ribosylation of a GTPase -> permanently locked into "on" form

4. Increased levels of adenylate cyclase activity and increased levels of cAMP in cell

5. Inhibition of sodium absorption by villi and increased chloride secretion by crypt cells

6. Increase in [NaCl] in lumen causes osmotic secretion of water -> fluid loss of up to 1L per hour

vibrio cholera - lab diagnosis (2)

1. Grow on thiosulfate citrate vile salts sucrose (TCBS) sugar -> selective indicator medium

   • V. cholerae form yellow colonies -> other Vibrio's form green colonies

2. Follow up with biochemical and serological identification to fully characterise

   • Is not diagnostic -> is indicative only

vibrio parahaemolyticus summary (4)

Natural inhabitant of marine waters -> associated with consumption of contaminated raw or undercooked shellfish

Notifiable infection in Victoria

IP of ~12-24 hours

Produces explosive watery diarrhoea with nausea, vomiting and abdominal cramps

vibrio parahaemolyticus - mechanism of pathogenesis

Mechanisms of pathogenesis not well understood by pathogenic strains have combination of toxins and adhesins

Eg. thermostable direct hemolysin (TDH) toxin which promotes chloride secretion by epithelial cells and also lyse RBCs

E. coli - groups (3)

ETEC = enterotoxigenic
EPEC = enteropathogenic
EHEC = enterohemorrhagic

ETEC - epidemiology summary (3)

Important case of diarrhoea in infants in low and middle income countries -> leading cause of diarrhoea for travellers returning from vacation in these areas

Occasional outbreaks associated with contaminated food in developed countries

Infectious dose for adults ~10^8 cells -> potentially lower for children/ elderly

ETEC - transmission and symptoms

Transmission via faecal contamination of food or water -> human or animal faeces are main sources

Causes mild, self-limiting, watery diarrhoea and abdominal cramping -> no blood, but occasional fever and vomiting

ETEC - pathogenesis

Similar pathogenesis to cholera

Produces pili for colonisation of gut epithelium -> colonisation factor antigen (CFA) = fimbrial adhesin

Produces heat labile (LT) and heat stable (ST) toxins to cause damage

Does not invade intestinal cell

ETEC - heat labile toxin

ETEC heat labile toxin and cholera toxin = identical structure and identical function -> change in cell is permanent

AB5 toxins are cytotonic in action -> pathology results from change in cell's internal biochemistry

ETEC - heat stable toxin

ETEC heat stable toxins have a different structure but similar function to cholera toxin but change in cell is not permanent

EPEC - main demographic affected

Most cases occur in children and neonates -> less than 2 years and esp. less than 6 months

Adults may be infected if ID is high

Outbreaks in paediatric wards, child care centres, adults eating food from buffet

Leading cause of infantile diarrhoea in developing countries → eg. Brazil Mexico, South Africa

EPEC - transmission and symptoms

Produces acute, profuse, protracted watery diarrhoea with vomiting and mild fever

Contracted by drinking contaminated water or food

EPEC - virulence factors (3)

Produces bundle forming pili (Bfp) -> fimbrial adhesin

• Encoded by bfp gene cluster found on EAF plasmid

• Mediates loose attachment to enterocytes similar to Tcp of V. cholerae

Produces attaching and effacing (A.E) lesions for intimate attachment

type III secretion system

T3SS - function

needle and syringe mechanism for translocation of virulence proteins from microbe to host cell cytoplasm

wide spread amongst gram negative animals and plant pathogens

EPEC - pathogenesis (5)

1. Bfp mediates loose adhesion

2. T3SS secretes Tir (receptor) into enterocyte

3. EPEC's outer membrane protein (Intimin) mediates intimate adherence to host cell by binding to deployed Tir

4. Other translocated T3SS effector proteins activate actin polymerisation -> rearrangement of host cytoskeletal structure and pedestal formation characteristic of A/E lesions

Mechanism of diarrhoea not via toxins but potentially via malabsorption caused by flattening brush border villi and A/E formation which then increases intestinal permeability

EHEC - symptoms

Produces a spectrum of disease ranging from abdominal cramps and watery diarrhoea to blood diarrhoea (dysentery/ Haemorrhagic colitis)

Can cause haemolytic uraemic syndrome (HUS) in small % of infected ppl, esp. children

EHEC - reservoirs

Zoonotic pathogen -> transmitted form unaffected animals to humans via raw or undercooked meat, raw or unpasteurised milk, vegetables contaminated with faecal matter

Can be associated with petting zoos

Main reservoir = cattle but also sheep, goats, deer and occasionally other mammals and birds

More typically seen in countries with industrialised agriculture -> spread through densely-farmed animals

EHEC - pathogenesis of major serotype

attaching and effacing pathogen like EPEC-> no invasion but produces toxin

Colonising (adhesin) antigens are unknown

Tir and other effector proteins injected via T3SS -> Tir interacts with Intimin (afimbrial adhesin) -> actin rearrangement in host cell cytoplasm to form pedestal like EPEC

EHEC - major serotype

O157:H7 -> non-sorbitol fermenting

EHEC - toxins

Produces Shiga toxins (Stx1 and Stx2) -> AB5 toxins

Cytotoxic toxin -> identical structure to cholera toxin but different function

EHEC - pathogenesis for bloody diarrhoea (4)

1. Toxin binds to Gb3 receptor on endothelial cells of underlying vasculature → toxins thought to pass between enterocytes to underlying endothelial cells

2. A subunit = an N-glycosidase -> inactivates 60s ribosomal RNA by cleaving N-glycosidic bond in 28S to stop protein synthesis

3. Damage to endothelial cells in vasculature of intestine -> bloody diarrhoea

In small percentage of severe cases, can damage small blood vessels predominantly in renal glomeruli -> haemolytic uremic syndrome (HUS)

EHEC - pathogenesis

Mechanisms of diarrhoea not known but possibly similar to EPEC which also produces A/ E lesions

Blood diarrhoea results from Shiga toxins

enteroaggregative E. coli (EAEC) (3)

Watery, persistent diarrhoea

Affects infants less than 6 months

2011 outbreak associated with specific strain that had gained Shiga toxin from EHEC -> virulence factors from both EAEC and EHEC

ETEC - lab diagnosis (2)

Molecular methods

PCR for gene for heat labile toxin

PCR for gene for heat stable toxin

EPEC - lab diagnosis (2)

Molecular methods:

PCR for bfpA gene

PCR for eae gene -> encodes intimin

EHEC - lab diagnosis via culture (2)

Growth on sorbitol MacConkey agar (SMAC) for O157:H7 -> sorbitol non-fermenter

Special chromogenic agars

EHEC - lab diagnosis via serological tests (2)

Enzyme-immuno-assay for Shiga toxins -> STX1 and STX 2

Identification of cell and flagella antigens -> eg. O157: H7

EHEC - lab diagnosis via molecular methods (2)

PCR for eae gene -> encodes intimin

PCR for stx1 and stx genes

Who uses T3SS (4)

EPEC and EHEC → to inject Tir which binds bacterial Intimin protein

Shigella → deliver Ipas to induce uptake by epithelial cells

Salmonella → encodes 2 - one for invasion, one for intracellular survival

Yersinia → deliver proteins for disruption of innate immune system

shigella - summary (4)

non motile

causes dysentery

low infectious dose and commonly spreads from person to person

human only pathogen → no animal reservoir except occasionally in higher primates

shigella - virulence plasmid encodes… (3)

T3SS

invasion plasmid antigens (Ipas) → translocated into host cell via T3SS to induce membrane ruffling

outer membrane protein - intracellular spread (IcsA) → used to recruit host cell actin and facilitate cell to cell spread

shigella - AB5 toxin similarities to EHEC, ETEC and cholera toxin

Shiga toxin of Shigella dysenteriae subtype 1 and EHEC = same structure, cytotoxic and acts on endothelial cells to inhibit protein synthesis

Cholera toxin and LT toxin of ETEC = cytotonic and acts in intestine to increase cAMP and secretory diarrhoea -> still same structure as Shiga toxin of shigella dysenteriae subtype 1 and EHEC

shigella - infection cycle (7)

1. Shigella approaches intestinal cells via unknown mechanism (non-motile) and invade M cells by unknown mechanism requiring Ipa proteins

2. Bacteria released into lamina propria and engulfed by macrophages → Shigella induces apoptosis of macrophages

3. Engulfment triggers inflammation by stimulating IL-1/ IL-8 cytokine and HXA3 release -> neutrophil migration into area

4. Bacteria that escape macrophage can invade adjacent enterocytes via basal surface by inducing uptake by cell via T3SS -> induces membrane ruffles and uptake via filopodia

5. Bacteria lyse phagosome using 2 Ipa proteins and replicate in cell cytoplasm

6. IcsA protein at one pole of cell recruits and induces rapid polymerisation of actin -> formation of actin tails to propel bacteria through cytoplasm of cell and into neighbouring cells via filopodia

7. Death of adjacent enterocytes forms ulcers in gut -> blood, neutrophils, and bacteria shed in faeces

shigella - possible method to enter basal lamina (2)

Intestinal epithelial cells are resistant to invasion by Shigella on  their luminal surface -> shigella needs to invade via basal surface

Could be via macropinocytosis

Some bacteria access basal surface via leaky tight junctions between cells induced by the presence of bacteria -> possible method

shigella infection cycle - implications of neutrophil recruitment and migration to gut lumen

Neutrophils migrate through mucosal tissues causing separation of tight junctions -> gut becomes leaky and more bacteria invade

shigella dysenteriae subtype 1 - pathogenesis

produces Shiga toxin -> binds to Gb3 receptor on endothelial cells of underlying blood vessels

Inhibits protein synthesis by endothelial cells -> cell death and damage to vasculature of intestine

shigella species - list (4)

typed serologically by O antigens

1. Shigella dysenteriae -> primarily in low and middle income countries

   • Subtype 1 produces Shiga toxin -> same toxin as EHEC

2. Shigella flexneri -> often a STI

3. Shigella boydii

4. Shigella sonnei -> main isolate in industrialised countries

   • Causes mildest Shigella infection -> watery diarrhoea

shigella - lab diagnosis (culture and biochemical tests)

lactose non-fermenter → distinguishable from E. coli which is typically lactose fermenting

serotyping of O antigens

no H (flagellar) antigens → non-motile

no H2S production unlike Salmonella

shigella - treatment

Disease usually self-limiting

antibiotics only required if disease is severe (blood diarrhoea) to reduce duration of illness and period of infectivity to others

Shigella - acid resistant phenotype

Has adaptive acid resistant phenotype which is repressed in stomach but then expressed in small intestine as pH increases

very acid stable

salmonella - summary (4)

Normal flora of many animals inc. chickens, cattle, reptiles

Ingesting in contaminated food or water

Acid labile -> need high infectious dose

Bacteria typically need to multiple in food before ingestion -> food handling implications

salmonella - human pathogens grouping

most grouped in Salmonella enterica subspecies → serotyped by O and H antigens

Salmonella - human pathogens examples (4)

Typhimurium and Enteritidis = cause of human gastroenteritis

Typhi and Paratyphi = human only pathogens that cause systemic disease, not gastroenteritis

Many can cross host species

pathogenicity islands - summary (3)

large area of bacterial chromosome or part of plasmid dedicated to encoding virulence genes

acquired by horizontal gene transfer

contain discrete genetic units

salmonella - pathogenicity island types (2)

Salmonella Pathogenicity Island 1 (SPI-1) = 46kb

Salmonella Pathogenicity Island 2 (SPI-2) = 41kb

both found in bacterial chromosome and encode for T3SS

salmonella - pathogenicity island 1 (2)

Encodes T3SS

Encodes Salmonella Invasion proteins (Sip proteins)

salmonella - pathogenicity island 2 (2)

Encodes T3SS

Encodes Salmonella survival antigen (Ssa proteins)

Salmonella invasion proteins (Sip proteins) - function (3)

Induce membrane ruffling and diarrhoea by cellular mediators

Induce cellular mediators and mobilise intracellular calcium ions

Used to invade M cells and enterocytes

Salmonella survival antigen (Ssa proteins) - function

required for survival of bacterium inside vacuole of macrophages

Eg. inactivate elements of innate immune response

salmonella - infection cycle (5)

1. Cellular mediators induce bacterial uptake and electrolyte accumulation in lumen and inflammatory exudate -> diarrhoea

2. Bacteria move to basal membrane in membrane bound vesicles, escape at basal surface and can leave the gut cells unharmed

   • May be engulfed by macrophages -> SPI-2 Ssa proteins for survival inside vacuole of macrophages

3. Bacteria may be captured in gut lumen by surveilling dendritic cells

4. Transport of bacteria to mesenteric lymph node within leukocytes

5. Bacteria may escape into blood stream to cause transient bacteraemia -> systemic disease symptoms are uncommon

salmonella - treatment

usually self-limiting

treatment with antibiotics generally not used as can prolong excretion of bacteria in faeces

salmonella - lab diagnosis (3)

Lactose non-fermenter -> yellow colonies on MAC and DCA

Growth on DCA: colonies may appear black due to H2S production -> not required

Confirm identity via biochemical tests and slide agglutinations for O and H antigens → strain comparisons using phage-typing or WGS

yersinia enterocolitica - summary (5)

Highly related to yersinia pestis -> the plague

Invasive pathogen -> infects mesenteric lymph nodes

Symptoms may be mistaken for appendicitis

Food borne →associated with cattle and can grow at lower temps so can survive in unpasteurised milk and undercooked meat

Complications with yersinia enterocolitica infection include post-infectious reactive arthritis

Y. enterocolitica - virulence factors list (2)

invasin

large 90kb plasmid

Y. enterocolitica - invasin

outer membrane adhesin protein

Contains an RGD tri-peptide that mimics natural ligand for host beta-1 integrins -> binds to host cell surface

Y. enterocolitica - plasmid encodes… (2)

T3SS → translocates YOP proteins

Various Yersinia outer proteins (YOP proteins) -> translocated into host cell

• Induce apoptosis -> Yop P

• Anti-inflammatory -> Yop M and P

• Prevent phagocytosis and killing by macrophages -> YOP E, T, O, H

Y. enterocolitica - infection cycle (4)

1. Invasion of M cells

2. Uptake by macrophage followed by apoptosis and release of bacteria

   • Pore formation in macrophage membrane

   • Inhibition of phagocytosis

   • Inhibition of TNF production

3. Local and system dissemination

4. Invasion of epithelial cell

Y. enterocolitica - lab diagnosis (5)

lactose non-fermenter

urease positive

oxidase negative

Can grow at low temperatures (psychrotroph) -> plates incubated at 25°C

Grow on selective media (CIN agar) -> bull's eye colonies

campylobacter - summary (5)

Microaerophilic

Curved gram negative rods -> look like gull wings

Large reservoir in animals -> poultry, some domestic animals

Low infectious dose

Long incubation period -> 2-4 days

campylobacter - pathogenesis

not well understood but involves both tissue invasion and cytolethal distending toxin which affects intestines

Can produce some bloody diarrhoea

May cause immune-mediate polyneuropathy -> Guillain Barre Syndrome

campylobacter - ecology

Invasion and toxin production in humans:

Co-associated with free-living amoebae and possibly forms biofilms in water supply -> contamination of milk/ meat

Colonisation but no disease in chickens -> faecal oral transfer between chickens -> inadequate cooking of chicken

campylobacter - lab diagnosis

CAMP medium = highly selective enriched medium containing blood, pyruvate, vitamin B6, and specific antibiotics -> vancomycin, polymyxin, trimethoprim

Incubate at 42°C for 48 hours in microaerophilic conditions -> special gas mix

aeromonas spp. - summary (4)

Common cause of gastroenteritis

Gram-negative motile rods

Found in aquatic environments but also in plants and soils → infections occur due to consumption of contaminated water or food

Endemic to Australia -> disease mostly in young children, young adults and individuals over 50

aeromonas spp. - pathogenesis

Pathogenesis not well understood but increasingly recognised as invasive

Pathogenic strains often make exotoxins

Eg. pore-forming toxins

aeromonas spp. symptoms (3)

watery diarrhoea

abdominal pain

associated with inflammatory bowel disease

aeromonas spp. - most common species for human infections

Three species responsible for most human infections

A. veronii most common in Aus for causing gastro

aeromonas spp. lab diagnosis (5)

Grows well on selective media (CIN agar) -> bull's eye colonies like Yersinia

Facultative anaerobe -> grows well at 25-40°C

Lactose-fermentation is variable

Sequencing of housekeeping genes or whole genome can be used for identification

Likely under-diagnosed by standard biochemical test platforms

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rotavirus - infection and demographic (4)

Infants = most at risk for severe disease

Seasonal virus = predominately winter in US and Aus -> year round in tropics

Transmission by faecal oral route

Asymptomatic excretors play role in spread of virus -> excretion can occur for weeks before onsent of symptoms and for days following resolution

rotavirus - ID and IP

Requires very low ID

IP of ~2 days

rotavirus - structure (3)

Non-enveloped, icosahedral virus with multi-layered protein coat -> very hard in environment and can withstand acid in stomach

Capsid encloses double stranded RNA genome -> segmented genome like orthomyxoviruses inc. influenza

Genome segregation results in several strains -> mainly Group A strain in humans

rotavirus - replication (3)

Entry into cells via enhanced proteolysis of outer capsid protein spikes by trypsin (enzyme)

Viral replication occurs in cell cytoplasm

Brings own RNA-dependent RNA polymerase -> makes mRNA form viral RNA genome

roatvirus - pathogenesis (5)

1. Ingested virus infects mature cells at tips of villi -> immature cells in crypt not infected

2. Infected cells lyse and release virus into lumen -> virus spreads to infect large number of adjacent cells

3. Lysis of infected cells results in blunting of villi -> remaining immature cells have reduced absorptive capacity for sugar, water, salts

4. Virus produces NSP4 which is released from virus infected cells -> enterotoxin effect on enterocytes

   • Stimulates chloride and water secretion into lumen of gut -> fluid accumulation in lumen -> secretory diarrhoea and dehydration

5. Cease of virus replication allows crypt cells to repopulate villi and restore proper villi structure and function

rotavirus - lab diagnosis (2)

Antigen detection assays -> EIA and latex agglutination

Electron microscopy -> need high virus titre or use virus specific antibody to agglutinate virions together

rotavirus - vaccine

Oral live attenuated vaccine = RotaTeq

Induces protective response without inducing disease -> results in decrease winter diarrhoea

Included in childhood vax schedule from 12 wks

Current vaccine has very low risk of intussusception in vaxxed infants 1-7 weeks after 1st dose -> 2 additional cases over non-vaxxed rates per 100,000 vaxxed per year

norovirus - structure

Non-enveloped ssRNA virus -> genome has single segment

norovirus - transmission (4)

Responsible for explosive outbreaks of disease -> seasonal peak in winter

Nursing homes, cruise ships, hospital wards, close contacts -> highly stable in environment

Faecal-oral transmission via contaminated food and water and aerosols from vomiting -> surface contamination

IP of 1-2 days

norovirus - symptoms and treatment (3)

Asymptomatic infection common and viral shedding can occur 2-3 weeks after recovery from illness

Diarrhoea and vomiting generally mild and self-limiting

Induces only short-term immunity -> re -infections 1-2 years later ca occur

norovirus - pathogenesis

1. Virus binds to histo-blood group antigens on cells on gut epithelial cells -> primary host receptors yet to be identified

2. Blunting of villi but intestinal epithelium remains intact

3. Mild inflammatory cell infiltration into lamina propria

4. Diarrhoea associated with transient malabsorption -> intestinal fluid produced during acute illness but not because of enterotoxin

norovirus - understanding of pathogenesis

Mechanisms for induction of diarrhoea and vomiting not well understood

Very difficult to culture

No animal models of human norovirus available

norovirus - resistance to infection

individuals who don’t secrete FUT2 enzyme have low or absent susceptibility to many strains

inactivate FUT2 disrupts synthesis of specific histo-blood group antigens on gut mucosal surfaces → prevent virus from attaching to cell surface

norovirus - lab diagnosis (4)

RT-PCR to look for viral RNA in faeces

Electron microscopy to locate virion in faeces

EIA to look for norovirus antigen in faeces

Rapid antigen tests

norovirus - vaccine

no vaccine

adenovirus - structure

Non-enveloped, double stranded DNA

adenovirus - infection and symptoms (4)

Common cause of GE, respirator tract infections and eye infections

Most commonly affects infants and young children

Causes watery diarrhoea, vomiting, abdominal cramps

Asymptomatic infections common -> virus can be shed for months after infection and virus stable in environment

adenovirus - pathogenesis (2)

Infects epithelial cells and replicates resulting cell death and lysis

Virions produced also promote inflammation