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Learning Outcomes

Gastrointestinal Infectious Disease

Lewis Bingle

Recognise and describe: major GI pathogens (UK and worldwide) Understand and describe: modes of transmission; GI symptoms; current diagnostics; pathogenicity mechanisms

In the UK
~ 20 % population affected / year Mostly mild and self-limiting Under reported If reported 50 % samples do not lead to diagnosis
Non-infectious causes? Unknown microbial cause?

Haiti: cholera epidemic

The Gastrointestinal Tract

From mouth (10 m tube) anus Different physical / chemical conditions along the length Almost all disease causing microbes ingested via mouth Initial interactions with epithelium

Host Defence Mechanisms

Stomach pH < 4 Immunity: phagocytes, immunoglobulins in gut lining Digestive enzymes Peristalsis (gut motility) - flushing Normal (commensal) microbial flora (bacteria, protozoa) competitive exclusion

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Normal Microbial Inhabitants

Stomach: small numbers of lactic acid bacilli, H. pylori Small intestine: approx. 106 organisms / ml
Mainly G+ve

G.I. Symptoms
Incubation period Diarrhoea (dysentery) Vomiting Abdominal pain Nausea Fever Ref. table 10.1: Ford Medical Microbiology

Large intestine (colon): 1011-1012 organisms / g faeces (50 %)

Complex community, mainly G+ve

Bacterial G.I. Pathogens: Campylobacter

Bacterial G.I. Pathogens: Gram negatives

C. jejuni: commonest bacterial cause of infectious GI disease in UK Characteristic S-shape Zoonosis not normally transmitted person-person Often associated with poultry Make sure you cook your chicken!

Bacterial G.I. Pathogens: Helicobacter pylori

Lives in the (highly acidic) stomach > 50% of the world's population carry H. pylori (most asymptomatic) Person-person transmission Gram-negative helical rod Motile: 4-6 polar flagella Urease enzyme neutralises the highly acidic environment of the stomach Causes gastritis; gastric ulcer; gastric cancer

Relationship between H. pylori and C. jejuni

Hp and Cj are morphologically, physiologically, genetically and phylogenetically closely related Hp originally called Campylobacter pylori However, biological niches, persistence in the environment, transmission and pathogenicity are different

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Most scientists and doctors did not believe that any bacteria could live in the stomach (low pH) Barry Marshall and Robin Warren identified spiral bacteria associated with gastritis and in 1982 cultured H. pylori Hypothesis: bacterial cause of peptic ulcer and gastric cancer 2005 Nobel Prize in Physiology or Medicine awarded to Marshall and Warren "for their discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease"

Bacterial G.I. Pathogens: Salmonella

Most common bacterial cause of GI disease after Campylobacter Most common species in UK are S. enteriditis and S. typhimurium Infection via consumption of undercooked / contaminated food or person-person S. typhi / S. paratyphi cause severe systemic disease septicaemia / high fever Carrier state possible (week or months)

Mary Mallon (1869 1938)

1st person in the USA identified as healthy carrier of typhoid fever (S. typhi) Infected at least 53 people, 3 died Quarantined (imprisoned) twice by public health authorities Died in quarantine

Bacterial G.I. Pathogens: E. coli

Many E. coli strains are commensal (e.g K12) Some strains are pathogenic, including:
Enteropathogenic E. coli = EPEC Enterohaemolytic (Vero toxigenic) E. coli = EHEC (VTEC) e.g. O157:H7 Enteroinvasive E. coli = EIEC Enteroaggregative E. coli = EAEC

Most E. coli infections are mild and self-limiting EHEC / VTEC is not!
Bloody diarrhoea HUS kidney failure

EHEC is a zoonosis (commensal in cattle)

hamburger disease

A Recent UK EHEC Outbreak

Bacterial G.I. Pathogens: Shigella spp.

Closely related to E. coli (indistinguishable?) 4 species cause classical bacillary dysentery:
Diarrhoea (blood + mucus) Fever Abdominal cramps

S. sonnei is a significant cause of UK diarrhoeal illness causes mildest shigellosis S. dysenteriae most severe (developing world) Human pathogens not found in animals Low infective dose helps transmission

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Bacterial G.I. Pathogens: Yersinia spp

Y. enterocolitica is the main GI pathogen (only some strains) Symptoms similar to appendicitis Normal gut flora of many food animals infection from undercooked meat Infection may be prolonged, with chronic complications

Bacterial G.I. Pathogens: Vibrio spp

Found in freshwater and seawater Most important pathogen is V. cholerae: cholera
35 million cholera cases / year 100000 120000 deaths due to cholera every year Important cause of pandemics

Ingestion of contaminated water or food Illness ranges from mild GI upset to profuse watery diarrhoea (rice water stools) Can cause rapid death due to dehydration Carrier state possible direct person- person transmission rare

Bacterial G.I. Pathogens: Clostridium spp

Bacterial G.I. Pathogens: Gram positives

C. perfringens and C. difficile: anaerobic members of normal gut flora Also C. botulinum: soil bacterium botox Toxin producers Resistant spores (high cooking temperatures will kill) Cp associated with consumption of poorly cooked food Cd associated with disturbance of normal bowel flora
e.g. broad spectrum antibiotics antibiotic-associated diarrhea (AAD)

Cd is the foremost cause of nosocomial diarrhoea

Bacterial G.I. Pathogens: Bacillus spp

Widespread in the environment Spore-forming (resistant to temperature) Produce heat-resistant toxins B. cereus: Chinese restaurant syndrome
Rice boiled, stored & reheated 2 toxins: emetic (projectile vomiting), diarrheal symptoms Unpleasant but rarely serious

Bacterial G.I. Pathogens: Staphylococcus aureus

Carried on skin and in nose of humans & other animals Food contamination due to
Carriage by food animal Carriage by food handler / cook Transfer from raw to cooked food

B. anthracis = B. cereus + pXO1 (toxin), pX02 (capsule) Anthrax Plasmids conferring virulence!

Produces heat-stable toxins Intoxication rapid onset of vomiting

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Pathogenicity Mechanisms: Adhering to the Epithelium

Best described in some strains of pathogenic E. coli Adhesins (surface antigens) allow attachment to gut epithelium Facilitates gut colonisation Allows localised attack (e.g. via toxin secretion)

Pathogenicity Mechanisms: Invasion

Most (but not all) major bacterial GI pathogens are capable of epithelial invasion Invasion functions plasmid-encoded Usually involves modification of cell wall lipopolysaccharide Food poisoning pathogens (e.g Campylobacters) invade gut epithelium superficially
Inflammation Increase in gut motility (diarrhoea) Occasional bacteraemia

Pathogenicity Mechanisms: Invasion

Some strains of E. coli / Shigella destroy superficial colonic epithelium
Dysentary / bloody diarrhoea with mucus Damage & invasion normally superficial

Pathogenicity Mechanisms: Toxin Production

Infection vs Intoxication
produced in situ vs eaten as preformed toxin speed of onset quicker for intoxication

Enteric fever (e.g typhoid) bacteria invade deeply via lymphoid system (Peyers Patches)
Bacteraemia

Toxins often plasmid or phage-encoded: mobile genes! Mostly polypeptides

Pathogenicity Mechanisms: 3 Toxin Groups

Neurotoxins
e.g. Staph. aureus (toxin B) ; B. cereus (emetic toxin) Often consumed preformed (intoxication)

Pathogenicity Mechanisms: Genomics

Chromosomal Genome Pathogenicity islands (PAIs)
Present in pathogen, absent in commensal relative

Enterotoxins
E.g V. cholerae; Shigella dysenteriae; B. cereus Direct effect on gut epithelium Activation of adenylate cyclase high *cAMP] Epithelial cell membrane proteins affected Water transferred from blood gut Catastrophic fluid loss

Cytotoxins
e.g. Shiga toxin (S. dysenteriae type 1; EHEC / VTEC; C. perfringens) Destroy intestinal cells Inflammatory diarrhoea

Inactive Prophage Mobile Genome (Mobilome) Transmissible (conjugative) plasmids Phage

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Viral G.I. Pathogens

Usually mild gastroenteritis Short-lived (1-2 days) Transmission person-person / aerosols from vomiting Food-borne outbreaks
Infected food handler Food contamination by sewage

Viral G.I. Pathogens

Norovirus
winter vomiting disease affects all age groups Low infective dose Aerosol spread Good survival in environment

Rotavirus
affects young (<2) children

Detection originally by EM, now mainly PCR-based

Rotavirus

Protozoan G.I. Pathogens

Most important: Cryptosporidium parvum crypto
self-limiting diarrhoea, life threatening for immunocompromised patients

Isolation Media for GI Pathogens

Isolation of pathogenic bacteria from faeces is extremely challenging: 1011 bacteria / g 95 % obligate anaerobes Many pathogens closely related to normal commensal flora Initial goal of culture may be enrichment of target organism relative to other flora Second medium for ID See Ford Medical Microbiology table 10.2

Giardia intestinalis
persistent diarrhoea

Entamoeba histolytica
amoebic dysentry

All Survive well in environment

Giardia, Entamoeba form cysts

Often contaminate surface waters

Crypto: immunofluorescence of oocysts from stool sample

Identification from faecal samples: Culture-based Methods

Xylose lysine desoxycholate agar (XLD) Salmonella & Shigella spp appear as red colonies Most Salmonella spp colonies also have black centre Some non-pathogenic bacteria have same appearance Differentiation via urease test: Salmonella and Shigella are urease negative

Identification from faecal samples: Culture-based Methods

Sorbitol MacConkey agar Used for selective isolation of E. coli O157 (EHEC) O157 does not ferment sorbitol pale colonies Other E. coli do ferment sorbitol red colonies Confirmation via serology / biochemistry Toxin genes confirmed by Ref. Lab.

XLD plate showing typical S. typhimurium growth

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Identification from faecal samples: Non-culture methods: IMS

Immunomagnetic separation = selective enrichment Used in detection of enteric pathogens Specific antibodies (e.g. anti-Salmonella) coupled to beads (magnetic)
Beads mixed with faecal suspension bind Salmonella Beads washed & cultured

Identification from faecal samples: Non-culture methods: molecular bacteriology

DNA probes for detection of some species (e.g. pathogenic E. coli) PCR-based analysis Sensitive but expensive Demands technical expertise Specificity maybe a drawback in the diagnostic lab Problems could be partially overcome by global analysis of clinical gDNA
use of microarray technologies High-throughput sequencing / metagenomics

Specificity maybe a drawback in the diagnostic lab

Typing
Differentiation of apparently identical organisms Useful for epidemiology* / research Serotyping: antisera against surface antigens
Polysaccharide O-antigens Flagellar H-antigens e.g. Kauffmann-White scheme for Salmonellae

CLONDIAG's ArrayTube

Phage typing
Differential phage susceptibility

PCR ribotyping
Variation in rRNA

Read Ford section 10.5

*Study of disease origin & spread

Originally developed by HPA / VLA Microarray processing with standard lab equipment Results in 1 day 1 hour hands-on time Custom arrays to identify : Antibiotic resistance profiles Presence of pathogenic strains Presence of pathogenicity genes

High Throughput Sequencing

Modern high-throughput sequencing technologies have already dramatically reduced the cost of genome sequencing High-throughput sequencing technologies obey Moores lawexponential increase in performance / cost Community profiling e.g. gut microbiome Metagenomics Single-cell genomics
Pallen MJ, Loman NJ & Penn CW (2010) High-throughput sequencing and clinical microbiology: progress, opportunities and challenges. Curr. Opin. Microbiol. 13: 17

Bacterial Toxin Detection

Problematic: other components of faeces interfere Routine testing in suspected C. difficile infection Organ / tissue culture labour-intensive and time-consuming Superceded by enzyme immunoassay Specific anti-toxin antibodies conjugated to enzyme detection system

Please read for next week

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The Reference Laboratory

Specialised laboratories with capability to give definitive ID of potential pathogens Develop and evaluate new methods Data collection Samples sent in limited circumstances:
To confirm initial ID To clarify inconclusive ID To provide full characterisation (i.e. To perform difficult testing methods
http://www.hpa.org.uk/ProductsServices/InfectiousDiseases/LaboratoriesAndReferenceFacilities

Therapy
For most common GI pathogens, no specific treatment other than rehydration/ electrolyte balance Simple pain relief (cramps) Antipyretics (fever) Antibiotic treatment is usually contraindicated and may make matters worse! Antibiotic treatment indicated for enteric fevers; severe cases of salmonellosis and shigellosis