The normal gastrointestinal flora

Gram-negative facultative anaerobic rods

Miklos Fuzi

The normal human gastrointestinal flora

• Comprises of 108 – 1010 microbes/gram feces• Not just commensal but symbiotic relationship with host

- fermentation of unused energy substrates

- training of the immune system

- preventing the growth of harmful, pathogenic microbes

- producing vitamins

- trophic effects

- preventing allergy

• Can become harmful- cause infections (if immune system is compromised)

- increase cancer risk

The normal human gastrointestinal flora

• Acquisition and composition of the normal flora:- following birth infants quickly acquire Escherichia

coli and streptococci which generate an anaerobic

environment

- in breast-fed infants a bifidobacterium flora

establishes itself

- in formula fed infants and in older children on solid

diet other anaerobes:

bacteroides (30%!), clostridia, fusobactria, peptococci

and additional enterobacteriaceae colonize the gut

- additional microbes: candida species, protoozoa

The impact of the intestinal flora

on obesity

An obesity-associated gut microbiome with

increased capacity for energy harvest.

Nature 2006, 444, 1027-31

„…colonization of germ-free mice with ‛obese

microbiota’ results in a significantly greater

increase in total body fat than colonization

with a ‛lean microbiota’…”

Ingestion of lactobacillus strain regulates emotional

behavior and central GABA receptor expression in a

mouse via the vagus nerve

Proc. Natl. Acad. Sci. USA 2011, 108, 16050-55

„ …alterations in central GABA receptor expression are

implicated in in the pathogenesis of anxiety…”

„ …L. rhamnosus reduced stress-induced corticosterone

and anxiety- and depression-related behavior…”

„… effects were not found in vagotomized mice…”

Gram-negative facultativeanaerobic rods : Enterobacteriaceae• Habitat: gastrointestinal tract of humans and

animalsplantssoil

• Grow on eosin methylene blue agar• No spore formation• Usually motile• Metabolism : - sugars are fermented

- nitrate degraded to nitrites- catalase: variable- oxidase: negative

Enterobacteriaceae: most important genera

• Escherichia: - The most abundant facultative anaerobic member of the human

gastrointestinal normal flora

- Produces vitamin K

- Some types are enteric pathogens

• Klebsiella: ankylosing spondilitis (HLA-B27); Crohn disease?• Enterobacter• Proteus: rheumatoid arthritis (amino acid sequence homology

between the urease of P. mirabilis and the joint cartilage collagen)

• Citrobacter• Serratia

2006. 03. 06.

Enterobacteriaceae:most important features

- E. coli: production of indole from tryptophan

- Klebsiella: degradation of urea- Enterobacter- Proteus: motile on solid media- Citrobacter- Serratia: production of pigment; capable

of colonizing the respiratory tract

2006. 03. 06.

Identification of Enterobacteriaceae – first level

Kle

bsie

llapne

umoniae

Kle

bsie

llaox

ytoca

Ente

robacte

rcloacae

Ente

robacte

rae

rogene

s

Serratia

Pantoea

agglomerans

Hafnia

Esch

erich

iacoli

Citrob

acter

koseri

Citrob

acter

freund

il

Salm

onella

Edw

ardsie

lla

Providencia

stuartil

Proteus

rettge

ri

Proteus

morganil

Proteus

vulgaris

Proteum

s m

irabilis

Proteus

penne

ri

Motility - - + + + d d + + + + + + + + + + +

H2S - - - - - - - - - d + + - - - + + +

Urease + + d - d d - - d d - - d + + + + +

Indole - + - - - d - + + - - + + + + + - -

Lysin-decar-boxilase

+ + - + + - + + - - + + - - - - - -

Arginine-dihydrol.

- - + - - - - d d d d - - - - - - -

Ornithindecarbox.

- - + + + - + d + - + + - - + - + -

Citrate + + + + + d - - + d + - + + - d d -

VogesProskauer

+ + + + + d d - - - - - - - - - d -

Pigment - - - - d d - - - - - - - - - - - -

Ferment:

- Adonit + + d + d - - - + - - - - + - - - -

- Inositol + + d + d d - - - - d - + + - - - -

- Lactose + + + + d d - + d d - - - - - - - -

-Mannitol + + + + + + + + + + + - - + - - - -

Saccaharose + + + + + d - d d d - - d d - + - +

Polymyxin-B S S S S R S S S S S S S R R R R R R

2006. 03. 06.

Identification of proteus and providencia spp.

Proteus Providencia

mira

bilis

vulgaris

Morga

niissp.m

organii

morga

nii ssp.sib

onii

rettge

ri

inconstans

panne

ri

myxofa

ciens

stuartii

biocsoport

4 stuartii

biocsoport

5 stuartii

biocsoport

6 rustigianii

heim

bach

ae

Urease + + + + + - + + + - - - -

Indole - + + + + + - - + + + + -

H2S + + - - - - d - - - - - -

Methyl red + + + + + + + + + + + d +

Voges-Proskauer d - - - - - - + - - - - -

Gelatine + + - - - - d + - - - - -

Phenylalanine-deaminase

+ + + + + + + + + + + + +

Omithine-decarboxylase

+ - + + - - - - - - - - -

Prod. of gas fromglucose

+ + + + d d d + - - - d -

Fermentation

- Glucose + + + + + + + + + + + + +

- Adonit - - - - + + - - - - + - +

- Inositol - - - - + - - - - + + - d

- Maltose - + - - - - + + - - - - d

- Mannitol - - - - + - - - - - - - -

- Mannose - - + + + + - - + + + + +

- Rhamnose - - - - d - - - - - - - +

- Trehalose + d - + - - d + + + + - -

- Xylose + + - - - - + - - - - - -

2006. 03. 06.

Eosin-methylene blue agar

Selective and differentiating medium:- Eosin and methylene blue inhibit the growth of

Gram-positive bacteria - Anionactive detergent (eg. Na-laurylsulphate)

inhibit the swarming of proteae- Contains lactose. Bacteria degrading lactose

produce acid that precipitates eosin that will be stained by methylene blue → the colonies of lactose positive bacteria are blue those of the lactose negatives are pink or grayish

- Advantage: acid will be formed just under colonies

2006. 03. 06.

E. coliE. coli

Eo

Br

VA

Klebsiella

SSI Bi VA

DC EM Br

Proteus growing on eosin methylene blue

agar

Klebsiella growing on blood agar plate

2006. 03. 06.

Proteus

Serratia marcescens growing on

blood agar plate

2006. 03. 06.

Enterobacteriaceae extraintestinal infections

Most frequent nosocomial infections- Surgical wound infection (late onset)- Urogenital infection /often catheter associated/- Pneumonia /ventilation/- Meningitis- Sepsis (klebsiella in neonatal int. care units)

Most frequent community-acquired infections- Urogenital infections /E. coli/- Pneumonia /klebsiella/- Otitis externa /infection of the external auditory

canal: E. coli, proteus/

Uropathogenic E. coli

• Causes 90 per cent of community-acquired urethral and bladder infections

• Source of infection: normal GI flora• Infection is more frequent in women• Infection can be ascending resulting in

pyelonephritis, prostatitis

• Uropathogenic strains harbour special virulence factors

Uropathogenic E. coli

• Characteristics of uropathogenic strains:- have „P” fimbriae that specifically bind to

P blood group antigens which are present

also on the surface of uroepithelial cells

- produce haemolysins which damage also

epithelial cells

- capable of forming „colonies” inside human

cells (hiding from immune response)

2006. 03. 06.

Enterobacteriaceae: Antibioticresistance

Most frequently used agents:- cephalosporins- carbapenems- aminoglycosides- fluoroquinolones

Most important mechanisms of resistance- Production of β-lactamase- Efflux systems- Alteration of membrane proteins- Production of modifying enzymes- Mutations in target molecules

β-lactamase enzymes

• Large family of enzymes• Many bacteria produces β-lactamases including free-living

species

• Grouping: on the basis of structure or efficacy• Some members of the family enterobacteriaceae

constitutively produce „simple” β-lactamases the genes of

which are usually located on the chromosome

• As a consequence of extensive use of antibiotics a number highly effective variants of the original β-lactamases evolved

in recent decades

β-lactamase enzymes

The most important types of β-lactamases:

- extended-spectrum β-lactamases (ESBLs): klebsiella, E. coli

- genes located on plasmids (easily transmit)

- confers resistance against penicillins and most

cephalosporins

- metallo-β-lactamases (MBLs) and other carbapenemases:

klebsiella, pseudomonas

- genes are located on plasmids or on the chromosome

- confer resistance against all β-lactam antibiotics

including carbapenems

Significance of enteritis

• Estimated to cause 1.6 – 2.1 million deathsannually all over the world (prior to theintroduction of rehydration with glucose-electrolyte solution at the beginning of the1980s the figure could have been twice ashigh )

• Deaths occur mostly in children• Geographical distribution of cases:

Most affected areas: „black Africa”, India, Southeast Asia

Conditions promoting the development of enteric disease

• Lack of safe drinking water (a problem for 30-35% of humanity)

• Lack of appropriate sanitation (affects about 50% of humanity)

• Lack of appropriate food higyene• Lack of appropriate cleaning facilities• High density of population, crowded

residential area

The consequences of recurrent enteric

infections in childhood

• Growth retardation due to malabsorption (average: 8.2 cm until 7 years of age; later the child can ca tch up some growth retardation if infections seize)

• An intellectual retardation of about 10 IQ points ( The brain and synapses develop primarily during the fir st two years of life)

• A weakened immune system („Fitness cost” associated with a predisposition to infections)

• Infection with particular pathogens negatively impa ct development even without enteric symptoms (enteroaggregative E. coli, cryptosporidium)

Genetic susceptibility to infection by enteropathogenic bacteria

• Salmonella: some IL, HLA, IFNGR genes, TNFA

• Helicobacter pylori: some IL genes, IFNGR1, TNFA

• Vibrio cholerae O1: blood group 0• Clostridium difficile: IL -8• Enteroaggregative E. coli: IL -8

E. coli causing enteritis

• Strains of E. coli can acquire pathogenicity factorscarried on plasmids or other mobile genetic elements and become enteropathogenic

• Types of E.coli causing enteritis:- Enteropathogenic E. coli (EPEC)- Enterotoxin producing E. coli (ETEC)- Enteroinvasive E. coli (EIEC)- Enterohaemorrhagic E. coli (EHEC)- Enteroaggregative E. coli (EAEC)

• Types can not always be distinguished: pathogenicityfactors can vary across groups

• Pathogenicity factors are linked to particular O serotyp esin all groups

Enteropathogenic (dyspepsia) E. coli (EPEC)

• Causes disease primarily in children less than 1 ye ar old

• Capable of adhering to the epithelial cells of the small intestine by its pathogenicity factor: „intimi ne”

• Symptoms: can be serious or mild• Pathogenicity is related to certain O serotypes• Earlier caused outbreaks in day care centres fro

young children• Therapy: fluid replacement

when symptoms are serious antibiotics

Enterotoxin producing E. coli (ETEC)

• The causative agent of „travellers’ diarrhea” preva lent in developing countries

• symptoms: serious „watery diarrhea”; affecting the small intestine

• Pathogenicity factors: usually located on plasmids• Serotypes: diverse• Identification: demonstration of pathogenicity gene s with

PCR → underdiagnosed- adhesion factors (colonization factors) –

usually on fimbriae; types sepcific for particular animal species exist

- toxins: LT (heat labile), ST (heat stable) • Therapy: antibiotics, fluid replacement

Enteroinvasive E. coli (EIEC)

• Symptoms are same as those of shigellosis (bloody diarrhea - dysentery)

• EIEC strains carry the same virulence plasmid and virulence genes as shigellae

• EIEC strains are often lactose negative and non-motile

• EIEC O antigens are also related to those of shigellas

• Transmission: food, contact (not as efficient as wi th shigellas)

• Most common serogroup: O124

Enterohaemorrhagic E. coli (EHEC)

• Symptoms: -serious bloody enteritis, affecting primarily the colon

-haemolytic uremic syndrome (HUS): haemolytic anaemia with acute renal failure

• Transmission: food (beef, milk), contact• Pathogenesis:

- adhesion factors- toxins: „Shiga-like” (SLT) – damaging capillaries; transmitted by phages; demonstration: PCR

• Most important serotype: O157 – strains usually sorbitol negative

• Therapy: antibiotics, fluid replacement

Enteroaggregative E. coli (EAEC)

• Attachment to epithelial cells is associated with t he aggregation of the bacteria

• Symptoms: serious watery diarrhea chronic diarrhea

• Pathogenicity factors: pili, fimbriae • The fimbriae aggregate human collagen, fibronectin,

laminin• Identification: demonstration of pathogenicity

factors by PCR → underdiagnosed

Yersinia enterocolitica

• Motile, lactose negative coccobacilli• Many serotypes: mainly the O3 and O9 strains are

widespread in Europe• Symptoms: diarrhea, swelling of lymphatic glands, ile itis

terminalis• Reservoir: animal, human• Transmission: contact, food• Pathogenicity: adhesion proteins• Secondary „autoimmune” sequel: arthritis• Diagnosis: culture of Y. enterocolitica on selective me dia• Therapy: antibiotics

Vibrionaceae family

• Most important group: Vibrio genus• Curved Gram -negative rods, motile, oxidase positive• Natural habitat: saline-, sweet waters• Strains can be grouped on the basis of salt

requirement:halophilic, non-halophilic strains

• Vibrios are usually susceptible to acidic environme nt but tolerate alkaline pH well (characteristic used for isolation)

Vibrio cholerae

• Most important species in the genus Vibrio• On the basis of O antigen more than 100 serogroups

are recognized; all carry the same flagella antigen• Large epidemics are caused by serogroups O1 and

O139 • The O1 strains have two biotypes:

- classic- El Tor

• Both groups are subdivided according to thestructure of the O antigen for serotypes: Ogawa, Inaba, Hikojima

Vibrio cholerae

• Natural habitat: sea (Banghlades), lives in copepods (crustacean); dormant form survives for months in the sediment of estuaries

• Transmission: water, food (many bacteria are required for infection)

• Epidemics: often pandemics (prevalent in India, black Afrika, South -America)

Cholera (1)

• Incubation: from a few hours to a couple of days de pending on the number of infecting bacteria

• Symptoms: serious watery diarrhea, desiccation occu rs within hours often causing hypovolaemic shock

• Pathogenesis: - the pathogen attaches to the epithelial cells of t he small

intestine with its adhesins.- produces cholera toxin (coded for by a phage

inserted in the chromosome) – similar to the heat la bile toxin of the ETEC strains. Toxin’s pathomechanism: activation of adenylate cyc lase,

cAMP accumulated within cells → escape of ions, water

Cholera (2)

• Diagnosis: isolation of the pathogenMedium: TCBS – tiosulphate-citrate-bile-

saccharoseEnrichment: alkaline peptone water

• Therapy: quick fluid and salt replacementantibiotics (tetracycline)

• Vaccine: of limited value

Vibrio colonies on TCBS agar

Other vibrios

• Halophile vibrios – require salt for growth• Important species:

V. parahaemolyticusV. vulnificus

• Transmission: not sufficiently heat treated sea foo d swimming in sea water

• Symptoms: usually not serious• Skin/wound infection does occur

Campylobacter genus• Gram-negative curved rods; motile • Require microaerophilic atmosphere• Oxidase positive• Relevant species: C. jejuni, C. coli, C. fetus, C. lari• Habitat: animals• Transmission: contact, food• Symptoms: variable (can be serious)• Culture: on selective medium in microaerophilic

atmosphere at 42 C• Therapy: macrolide, fluoroquinolone antibiotics• Secondary autoimmune sequel: Guillain-Barre

syndrome

Campylobacter - Gram stain

Colonies of campylobacter on selective medium

Recorded cases of campylobacterosis in the United

Kingdom

Annual number of cases in Hungary: about 6000

Helicobacter pylori

• Gram-negative curved rods, sometimes assume non-culturable coccoid forms

• Microaerophilic• Motile• Oxidase and catalase positives• Strongly urease positives – ensures survival in acidic

gastric environment

• Strains harbouring the „Cag pathogenicity island” are the most virulent (type IV secretion system;

CagA protein)

Helicobacter pylori

• About 50% of humanity carries H. pylori• Carriage is symptomless in about 80% of cases• Conditions caused by H. pylori

- gastritis

- ulcer

- gastric carcinoma

- MALT (mucosa-associated lymphoid tissue)

lymphoma

Helicobacter pylori

• Culture: sample to be taken into transport mediumIsolation: on selective medium (similar to

campylobacter agar), at 37 C microaerophilicatmosphere

• Diagnosis: urease breath test (UBT) – labeled urea swall owed; exhaled labeled carbondioxide detected

detection of antigen in feces (ELISA, latex)

detection of antibody from urine or blood(ELISA)

isolation of pathogen from gastric biopsysample – when determination of antibioticresistance is necessary

demonstration of H. pylori DNA in gastricbiopsy sample by PCR

Helicobacter pylori

Therapy: - cephalosporins- macrolides

Resistence is not rare!

Proton pump inhibitors

Extended, repeated treatment

Helicobacter pylori

Helicobacter pylori: pathogenicity

Helicobacter pylori on gastric

epithelial cells

Thank you for your attention