Gram-negative facultative anaerobic rods

The normal gastrointestinal flora
Gram-negative facultative anaerobic
rods
Miklos Fuzi

The normal human
gastrointestinal flora
• Comprises of 10 8 – 10 10 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 facultative
anaerobic rods: Enterobacteriaceae
• Habitat: gastrointestinal tract of humans and
animals
plants
soil
• 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

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.

Klebsiella
pneumoniae
Klebsiella
oxytoca
Enterobacter
cloacae
Identification of Enterobacteriaceae – first level
Enterobacter
aerogenes
Serratia
Pantoea
agglomerans
Hafnia
Escherichia
coli
Motility - - + + + d d + + + + + + + + + + +
H 2S - - - - - - - - - d + + - - - + + +
Urease + + d - d d - - d d - - d + + + + +
Indole - + - - - d - + + - - + + + + + - -
Lysin-decar
Lysin decar-
boxilase
Arginine-
dihydrol. dihydrol
Ornithin
decarbox. decarbox
Citrate
Voges
Proskauer
Citrobacter
koseri
+ + - + + - + + - - + + - - - - - -
- - + - - - - d d d d - - - - - - -
- - + + + - + d + - + + - - + - + -
+ + + + + d - - + d + - + + - d d -
+ + + + + d d - - - - - - - - - d -
Pigment - - - - d d - - - - - - - - - - - -
Ferment: Ferment
- Adonit + + d + d - - - + - - - - + - - - -
- Inositol + + d + d d - - - - d - + + - - - -
- Lactose + + + + d d - + d d - - - - - - - -
-Mannitol Mannitol + + + + + + + + + + + - - + - - - -
Saccaharose + + + + + d - d d d - - d d - + - +
Polymyxin-B Polymyxin S S S S R S S S S S S S R R R R R R
2006. 03. 06.
Citrobacter
freundil
Salmonella
Edwardsiella
Providencia
stuartil
Proteus
rettgeri
Proteus
morganil
Proteus
vulgaris
Proteums
mirabilis
Proteus
penneri

mirabilis
vulgaris
Morganii
ssp.morganii
Identification of proteus and providencia spp.
Proteus Providencia
morganii
ssp.sibonii
rettgeri
inconstans
Urease + + + + + - + + + - - - -
Indole - + + + + + - - + + + + -
2006. 03. 06.
panneri
myxofaciens
stuartii
biocsoport
4
stuartii
biocsoport
5
stuartii
biocsoport
6
H 2S + + - - - - d - - - - - -
Methyl red + + + + + + + + + + + d +
Voges-Proskauer
Voges Proskauer d - - - - - - + - - - - -
Gelatine + + - - - - d + - - - - -
Phenylalanine-
deaminase
Omithine- Omithine
decarboxylase
Prod. Prod.
of gas from
glucose
Fermentation
+ + + + + + + + + + + + +
+ - + + - - - - - - - - -
+ + + + d d d + - - - d -
- Glucose + + + + + + + + + + + + +
- Adonit - - - - + + - - - - + - +
- Inositol - - - - + - - - - + + - d
- Maltose - + - - - - + + - - - - d
- Mannitol - - - - + - - - - - - - -
- Mannose - - + + + + - - + + + + +
- Rhamnose - - - - d - - - - - - - +
- Trehalose + d - + - - d + + + + - -
- Xylose + + - - - - + - - - - - -
rustigianii
heimbachae

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.

Eo
E. coli
E. coli
Br
2006. 03. 06.
VA

SSI Bi VA
Klebsiella
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

Enterobacteriaceae extraintestinal
infections
Most frequent nosocomial infections
- Surgical wound infection
- Urogenital infection /often catheter associated/
- pneumonia /ventilation/
- Meningitis
- Sepsis
Most frequent community-acquired infections
- Urogenital infections /E. coli/
- Pneumonia /klebsiella/
- Otitis externa /infection of the external auditory
canal/
2006. 03. 06.

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)

Enterobacteriaceae: Antibiotic
resistance
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
2006. 03. 06.

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 extended 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): 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 deaths
annually all over the world (prior to the
introduction of rehydration with glucoseelectrolyte
solution at the beginning of the
1980s the figure could have been twice as
high)
• 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 catch
up some growth retardation if infections seize)
• An intellectual retardation of about 10 IQ points (The
brain and synapses develop primarily during the first
two years of life)
• A weakened immune system („Fitness cost”
associated with a predisposition to infections)
• Infection with particular pathogens negatively impact
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 factors
carried 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: pathogenicity
factors can vary across groups
• Pathogenicity factors are linked to particular O serotypes
in all groups

Enteropathogenic (dyspepsia) E. coli (EPEC)
• Causes disease primarily in children less than 1 year
old
• Capable of adhering to the epithelial cells of the
small intestine by its pathogenicity factor: „intimine”
• 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” prevalent in
developing countries
• symptoms: serious „watery diarrhea”; affecting the small
intestine
• Pathogenicity factors: usually located on plasmids
• Serotypes: diverse
• Identification: demonstration of pathogenicity genes 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 nonmotile
• EIEC O antigens are also related to those of
shigellas
• Transmission: food, contact (not as efficient as with
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 the
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, ileitis
terminalis
• Reservoir: animal, human
• Transmission: contact, food
• Pathogenicity: adhesion proteins
• Secondary „autoimmune” sequel: arthritis
• Diagnosis: culture of Y. enterocolitica on selective media
• 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 environment
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 the
structure 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 depending
on the number of infecting bacteria
• Symptoms: serious watery diarrhea, desiccation occurs
within hours often causing hypovolaemic shock
• Pathogenesis:
- the pathogen attaches to the epithelial cells of the small
intestine with its adhesins.
- produces cholera toxin (coded for by a phage
inserted in the chromosome) – similar to the heat labile
toxin of the ETEC strains.
Toxin’s pathomechanism: activation of adenylate cyclase,
cAMP accumulated within cells → escape of ions, water

Cholera (2)
• Diagnosis: isolation of the pathogen
Medium: TCBS – tiosulphate-citrate-bilesaccharose
Enrichment: alkaline peptone water
• Therapy: quick fluid and salt replacement
antibiotics (tetracycline)
• Vaccine: of limited value

Vibrio colonies on TCBS agar

Other vibrios
• Halophile vibrios – require salt for growth
• Important species:
V. parahaemolyticus
V. vulnificus
• Transmission: not sufficiently heat treated sea food
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 medium
Isolation: on selective medium (similar to
campylobacter agar), at 37 C microaerophilic
atmosphere
• Diagnosis: urease breath test (UBT) – labelled urea swallowed;
exhaled labelled carbondioxide detected
detection of antigen in feces (ELISA,
latex)
detection of antibody from urine or blood
(ELISA)
isolation of pathogen from gastric biopsy
sample – when determination of antibiotic
resistance is necessary
demonstration of H. pylori DNA in gastric
biopsy sample by PCR

Therapy:
Helicobacter pylori
- 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