the normal gastrointestinal flora gram-negative facultative ......2014/01/09 · yersinia...
TRANSCRIPT
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The normal gastrointestinal flora
Gram-negative facultative anaerobic rods
Miklos Fuzi
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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
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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
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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’…”
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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…”
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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
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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
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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
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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
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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 + + - - - - + - - - - - -
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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
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2006. 03. 06.
E. coliE. coli
Eo
Br
VA
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Klebsiella
SSI Bi VA
DC EM Br
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Proteus growing on eosin methylene blue
agar
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Klebsiella growing on blood agar plate
2006. 03. 06.
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Proteus
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Serratia marcescens growing on
blood agar plate
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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/
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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
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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)
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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)
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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
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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
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Vibrio colonies on TCBS agar
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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
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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
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Campylobacter - Gram stain
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Colonies of campylobacter on selective medium
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Recorded cases of campylobacterosis in the United
Kingdom
Annual number of cases in Hungary: about 6000
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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)
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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
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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
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Helicobacter pylori
Therapy: - cephalosporins- macrolides
Resistence is not rare!
Proton pump inhibitors
Extended, repeated treatment
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Helicobacter pylori
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Helicobacter pylori: pathogenicity
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Helicobacter pylori on gastric
epithelial cells
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Thank you for your attention