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