Enterobacteriaceae are a family of Gram-negative bacteria commonly found in the intestines. They include both pathogenic and non-pathogenic species. The document discusses the classification, characteristics, virulence factors and diseases caused by some important genera within Enterobacteriaceae, including Escherichia coli and Shigella. E. coli in particular produces toxins like heat-labile and heat-stable enterotoxins that can cause diarrhea. Laboratory tests are used to differentiate between lactose-fermenting and non-fermenting species on media like MacConkey agar.

1. ENTEROBACTERIACEAE
basics
Chapter I
Dr.T.V.Rao MD
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.V.Rao MD 1

2. Bacteria
Gram
positive
Cocci Bacilli
Gram
negative
Cocci Rods
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.V.Rao MD 2

3. 4/16/2012 Dr.T
.V.Rao MD 3
Enterobacteriaceae
• Commonly present in large intestine
• Non sporing , Non Acid fast, Gram –
bacilli.
• A complex family of organisms,
• Some are non pathogenic
• A few are highly Pathogenic,
• Some commensals turn out to be
pathogenic. as in UTI after catheterization.

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.V.Rao MD 4
Characters of
Enterobacteriaceae
• All Enterobacteriaceae
– Gram-negative rods
– Ferment glucose with acid production
– Reduce nitrates into nitrites
– Oxidase negative
• Facultative anaerobic
• Motile except Shigella and Klebsiella
• Non-capsulated except Klebsiella
• Non-fastidious
• Grow on bile containing media (MacConkey agar)

5. Basic Tests ……
• Basic characters
• Catalase +
• Oxidase -
• Reduce nitrates,
• All are Gram negative and non spore
forming.
• Wide diversity / Antigenic heterogeneity,
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6. Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonella, Shigella
Proteus, Yersinia
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There are several selective and differential media used to
isolate distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In addition to Triple Sugar Iron (TSI) agar

7. Differentiation between LF & NLF by Growth on MacConkey
agar
MacConkey Agar
Contains
Bile salts Crystal violet Lactose Neutral red
 MacConkey agar is selective & differential medium for Enterobacteriaceae
Inhibit growth of G+ve bacteria
Cause of selectivity
Cause of differential
pH indicator
Acidic: Pink
Lactose non feremnters
colorless colonies
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Lactose feremnters
Pink colon
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.V.Rao MD 8
Basic Classification
• I Lactose fermenters E.coli, Escherichia
Klebsiella,
• II Late Lactose fermenters, Shigella
sonnei,
• III Non Lactose fermenters, Salmonella
Shigella,
Commonly tested with MacConkey medium
Many are commensals Lactose fermenters
Also called as coli forms , Enteric Bacilli

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.V.Rao MD 9
Enterobacteriaceae
Taxonomical
• Tribe I Escherichia
Genus – 1 Escherichia,
2 Edwardsville
3 Citrobacter
4 Salmonella
5 Shigella

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.V.Rao MD 10
Enterobacteriaceae.
• Tribe II; Klebsiella
Genus 1. Klebsiella
2.Enterobacter,
3.Hafnia
4 Serratia
Tribe III ; Proteeae
Genus
1,Proteus
2,Morganella
3 . Providencia

11. Classification of Enterobacteriaceae
Contd )
• Tribe IV;
Erwinieae
Genus 1.
Erwinia
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.V.Rao MD 11

12. Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
• Method:
– MacConkey agar is inoculated with tested organism
using streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
• Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g.
Shigella)
Flame & Cool
1 2
3
4
5
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Flame & Cool
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.V.Rao MD
Flame & Cool
12

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.V.Rao MD 13
Highly Pathogenic
Enterobacteriaceae
• Salmonella
• Shigella
• All are Lactose – non fermenters,
• Produce colorless colonies on
MacConkey medium
• LF also called as Para colons,

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.V.Rao MD 14
Escherichia coli
• Named by Escherichia
• Wide group of bacteria on basis of
Bio typing and Serotyping
Produce infections in Humans and
Animals
Detection of E.coli in water indicates
pollution and contamination.

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.V.Rao MD 15

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.V.Rao MD 16
E.coli
• Morphology Gram - ve Straight rods,
• 1-3 X 0.4 -0.7 microns,
• Appear in singles or in pairs,
• Motile by peritrichate flagella.
• Very few strains non motile
• Not spore forming, Non acid fast.

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.V.Rao MD 17
E.coli
Cultural characters
• Aerobic / Facultative Anaerobic
• Grows between 10 – 40 c optimal at 37 c
• Grown in simple medium
• Produce Large grayish ,Thick white , moist
smooth opaque colonies
• May contain capsule.
• On MacConkey medium Produce Bright
pink Lactose fermenters.

18. E.coli on Blood Agar
• Many
pathogenic
strains are
haemolytic
on blood
agar.
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.V.Rao MD 19
E.coli Biochemical
Characters,
• Glucose,Lactose,Mannitol,Maltose
fermented. with A/G
Indole +
Methyl Red +
Voges Proskauer –
Citrate –
Urease not produced.
I,M,Vi,C tests.

20. E.coli
Antigenic Structure
• Somatic 0 170
• Capsular K 100
• Flagella H 75
• Virulence factors
Surface Antigens Toxins
O Endotoxic activity
K protects against the phagocytosis
Fimbriae promote virulence ( important in UTI )
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.V.Rao MD 21
Virulence Factors
• Two types of virulence factors in
E.coli
• Surface antigens and Toxins
• The somatic lipopolysaccharide
surface O antigen has endotoxic
activity and protects from
phagocytosis and bactericidal effects
of complement .

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Fimbriae
• Fimbriae also promote virulence
• Present in large numbers causing
mannose sensitive Haemagglutination
• Colonisation factor antigens is
enterotoxigenic E.coli
• E.coli produce two kinds of Exotoxins
hemolysins and enterotoxins

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.V.Rao MD 23
Toxins
and E.coli
• E.coli produce Exotoxins
• Hemolysins, Enterotoxins causes
Diarrheas,
• Important toxins produces.
• Heat labile HL Heat stable HS
Vero toxins VT Like Shigella
toxins

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.V.Rao MD 24
Toxins
• Enterotoxins – produced by
enterotoxigenic strains of E. coli
(ETEC). Causes a movement of
water and ions from the tissues to
the bowel resulting in watery
diarrhea. There are two types of
enterotoxin:

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.V.Rao MD 25
Toxins
• LT – is heat labile and binds to
specific Gm1 gangliosides on the
epithelial cells of the small
intestine where it ADP-ribosylates
Gs which stimulates adenylate
cyclase to increase production of
cAMP.

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.V.Rao MD 26
Toxins
• Increased cAMP alters the
activity of sodium and chloride
transporters producing an ion
imbalance that results in fluid
transport into the bowel.

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.V.Rao MD 27
Toxins in E.coli
• Produce Enterotoxin L T and S T
• Labile toxin 1956 De experiments in
Rabbit ileal loop causes outpouring of
fluids
• E.coli Labile toxin like Cholera toxin
• L T contains component A and B
• A = Active B= Binding
• B causes Binding with Gm I Ganglioside
receptor on Intestinal epithelial cells

28. E. coli toxins
• Both enterotoxins
are composed of
five beta subunits
(for binding) and
1 alpha subunit
(has the toxic
enzymatic
activity).
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29. 4/16/2012 Dr.T
.V.Rao MD 29
Toxins E.coli
Labile toxin
• Component A Activated to A1 and A2
• A1 Activates adenyl cyclase in the
enterocytes to form cyclic adenosine
5 monophosphate
• Causes to increase outflow of water
and electrolytes in the gut lumen
causes Diarrhea

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.V.Rao MD 30
Toxins of E.coli Stable Toxin
• ST A and ST B
• ST A Acts by activation of Cyclic
guano sine monophosphate.( C GMP )
• Causes fluid accumulation in Intestine.
• E.coli ( Some ) produce
Verocytotoxin causes cytotoxicity
to Vero cells.
• Acts like Shigella dysentery toxin

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.V.Rao MD 31
E. coli infections
• Neonatal meningitis – is the leading cause of
neonatal meningitis and septicemia with a high
mortality rate.
– Usually caused by strains with the K1 capsular antigen.
• Gastroenteritis – there are several distinct types of E.
coli that are involved in different types of
gastroenteritis:
– enterotoxigenic E. coli (ETEC),
– enteroinvasive E. coli (EIEC),
– enteropathogenic E. coli (EPEC) ,
– enteroaggregative E. coli (EAEC), and
– enterohemorrhagic E. coli (EHEC).

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.V.Rao MD 32
Classification of E.coli
1.Enteropathogenic
2.Enterotoxigenic
3.Enteroinvasive
EPEC
ETEC
EIEC
4.Enterohemorrhagic EHEC
5.Enteroaggresive EAEC

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.V.Rao MD 33
Enteropathogenic E.coli
• Causes diarrheal disease in children,
• EPEC O26/O11
• Produce Verocytotoxin
• Infantile enteritis, Involves upper part of Intestine
• Brush border of the intestine is lost
• Intimacin – EPEC adhesion factor.
• Frequent in summer months
• Poor hygiene predisposes.
• Out breaks in Institutions

34. Enteropathogenic E.coli
r.
34
• Causes diarrheal disease in children,
• EPEC O26/O11
• Produce Verocytotoxin
• Infantile enteritis, Involves upper part of
Intestine
• Brush border of the intestine is lost
• Intimacin – EPEC adhesion facto
• Frequent in summer months
• Poor hygiene predisposes.
• Out breaks in Institutions
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.V.Rao MD

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.V.Rao MD 35
EPEC
• EPEC are identified by serotyping by
their O and B antigens
• Diagnosis is difficult during sporadics
• Routine culture is done for isolation
• The EPEC fails to ferment sorbitol
• Causes the disruption of brush border

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.V.Rao MD 36
Laboratory Diagnosis
EPEC
• Confirm with Polyvalent
sera
• Test Sero groups with
polyvalent and monovalent
sera.
• HEp2 – adherence.

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.V.Rao MD 37
Enterotoxigenic E.coli
• Common and causes endemics in
developing countries in all age
groups
• May be mild watery diarrhoea to
fatal conditions
• Fimbrial colonization factor
antigens

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.V.Rao MD 38
Enterotoxigenic E.coli
• Produce Heat stable /Heat labile toxins
• Adheres to epithelium of small intestine.
• Present with Nausea, Vomiting and Lose stool
• H L like cholera toxin
• Causes accumulation of fluids
• Adhesive factors
Fimbriae specific receptor in the intestinal
epithelium CFA
Mortality in children < 5 years

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.V.Rao MD 39
ETEC
• Causes travelers diarrhea
• Water contaminated with Human and Animal
feces predisposes.
• Laboratory Diagnosis
Demonstration of Enterotoxin LT and ST
Tissue culture tests,
ELISA
Passive agglutination tests.
Animal experiments in Rabbit ileal loop test.

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.V.Rao MD 40
Treatment and Prophylaxis in Travelers
diarrhea
• Doxycycline,
• Trimethoprim,
• Norfloxacillin
• Fluroquinolones
• Avoid contaminated food,
• Safe protected water ,prefer bottled
water,
• Hot foods, Hot Drinks,
• Boiled milk

41. 4/16/2012 Dr.T
.V.Rao MD 41
Treatment and Prophylaxis in Travelers
diarrhea
• Doxycycline,
• Trimethoprim,
• Norfloxacillin
• Fluroquinolones
• Avoid contaminated food,
• Safe protected water ,prefer bottled water,
• Hot foods, Hot Drinks,
• Boiled milk

42. 4/16/2012 Dr.T
.V.Rao MD 42
Enteroinvasive E.coli
• Resembles Shigella in many respects
• Non lactose fermenter and non motile
• They invade the intestinal epithelium
• Penetrate HeLa cells in tissue culture
• May produce mild diarrhoea to frank
dysentery
• Sereny test

43. Entero invasive E.coli
• Some are non motile
strains,
• Atypical resembles
like Shigella.
• Clinically mild
diarrhea
• Sereny test positive
animal Rabbit.
• ELISA
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.V.Rao MD 43

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.V.Rao MD 44
Enterohemorrhagic E.coli
• Produce Verocytotoxin or shiga like toxin
• Mild diarrhea - can be fatal hemorrhagic
colitis. and uremic syndrome.
• Present in Human and Animal feces.
• Hemorrhagic complication with O157 in
Japan and USA.
• Salads vegetables, Radish Proper cooking

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.V.Rao MD 45
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46. Enterohemorrhagic E.coli
• The primary target for VT appear to be
vascular endothelial cells
• This may contribute to HUS with
characteristic renal lesion is capillary micro
angiopathy
• The typical EHEC serotype is 0 157 ; H7
• The disease may manifest as food
poisoning
• Occurs due to contamination of feces of
humans or animals
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.V.Rao MD 46

47. 4/16/2012 Dr.T
.V.Rao MD 47
Enterohemorrhagic E.coli can
cause HUS
• HUS develops when the toxin from E. coli
bacteria, known as Shiga-like toxin (SLT)
enters the circulation by binding to
special receptors. These Shiga-toxin
receptors, known as Gb3 receptors , are
probably heterogeneously distributed in the
major body organs allowing disparate
thrombotic (blood clotting) impacts in different
HUS victims, although the greatest receptor
concentration appears to be in the kidneys,
especially in children.

48. 4/16/2012 Dr.T
.V.Rao MD 48
EHEC ( E.coli )
• Culture
• DNA detection methods.
• Cytotoxic effects on Vero
cells.
• Detection with monovalent
sera O157/H7

49. 4/16/2012 Dr.T
.V.Rao MD 49
Enteroaggresive E.coli
• They appear aggregated in s stacked
brick formation Hep-2 cell
• They produce persistent diarrheal
• They produce weight heat stable
enterotoxin called as low molecular
heat stable enterotoxin

50. Enteroaggresive E.coli EAEC
• Stool
Culture
methods
• Detection of
Enterotoxin
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51. Treatment – E.coli Gastrointestina
disease
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.V.Rao MD 51
• Fluid replacement
• Antibiotics
– not used usually unless
systemic infections
prevails
–e.g. hemolytic-uremia
syndrome

52. E. coli
– Clinical
significance
• Is the leading
cause of urinary
tract infections
which can lead
to acute cystitis
(bladder
infection) and
pyelonephritis
(kidney
infection).
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53. Ascending urinary tract infection
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54. 4/16/2012 Dr.T
.V.Rao MD 54
Urinary Tract Infections
• E.coli produce urinary tract infection.
• Majority of UTI s are produce by E.coli.
• Instrumentation, Prostatic enlargement,
Urinary caliculi,Pregnancy,
• Asymptomatic Bacteriuria in pregnant
women,
• Pyelonephritis,

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.V.Rao MD 55
Facts on UTI
• Women suffer more than males Short urethra
Pregnancy, Sexual intercourse /Honey moon
cystitis.
• Other factors
Urethral obstruction,
Urinary stones
Congenital malformation's
Neurological disorders,
Catheterization , Cystoscopy
Usually cystitis is produced from fecal strains
entering urethra

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.V.Rao MD 56
Culturing for E.coli
• Mid stream sample/semi quantitative
culturing (Kass et al ) >_ 1.00,000/ml of
urine. ( significant Bacteriuria )
• Urine should not be kept in wards for > 2
hours and to be preserved at 4 c
• Culture by standard loop method.
• Fixed volume cultured on MacConkey
agar Lactose fermenters I M Vi C
• Antibiotic sensitivity tested.

57. 4/16/2012 Dr.T
.V.Rao MD 57
Other Bacteria and UTI
• Majority of UTI are caused by E.coli
•Other agents which cause UTI,
1 Staphylococcus
2.Proteus.
3.Pseudomonas,
4.Klebsilella spp,Citrobacter,
5.Enteococcus.

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.V.Rao MD 58
Other infection with E.coli
• Pyogenic infections.
• Intraabdominal infections
• Peritonitis. Abscess.
• Septicemias
• Produce Drug resistant
infections.

59. 4/ . 59

60. 4/16/2012 Dr.T
.V.Rao MD 60
Klebsiella, Enterobacter, Serratia & Hafnia sp
• Usually found in intestinal tract
• Wide variety of infections, primarily pneumonia,
wound, and UTI
• General characteristics:
– Some species are non-motile
– Simmons citrate positive
– H2S negative
– Phenylalanine deaminase negative
– Some weakly urease positive
– MR negative; VP positive

61. 4/16/2012 Dr.T
.V.Rao MD 61
Klebsiella species
• Usually found in GI tract
• Four major species
• K. pneumoniae is mostly commonly isolated
species
– Possesses a polysaccharide capsule, which
protects against phagocytosis and
antibiotics AND makes the colonies moist
and mucoid
– Has a distinctive “yeasty” odor
– Frequent cause of nosocomial pneumonia

62. Klebsiella
• Klebsiella is a genus of non-motile, Gram-
negative, oxidase-negative, rod-shaped
bacteria with a prominent polysaccharide-
based capsule. It is named after the
German microbiologist Edwin Kleb's
(1834–1913). Frequent human pathogens,
Klebsiella organisms can lead to a wide
range of disease states, notably
pneumonia, urinary tract infections,
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63. Klebsiella
• The genus was originally divided into 3
main species based on biochemical
reactions. Today, 7 species with
demonstrated similarities in DNA
homology are known. These are (1)
Klebsiella pneumoniae, (2) Klebsiella
ozaenae, (3) Klebsiella rhinoscleromatis,
(4) Klebsiella oxytoca, (5) Klebsiella
planticola, (6) Klebsiella terrigena, and (7)
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64. Klebsiella
• K pneumoniae is the most medically important species of the gro
K oxytoca and K rhinoscleromatis have also been demonstrate
human clinical specimens. In recent years, klebsiellae have becom
important pathogens in nosocomial
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65. 4/16/2012 Dr.T
.V.Rao MD 65
Klebsiella
• Klebsiella
– NF of GI tract, but potential pathogen in other
areas
– TSI A/A + gas
– LIA K/K
– Urea +
– Citrate +
– MR-, VP+
– Motility -
– Has both O and K antigens

66. 4/16/2012 Dr.T
.V.Rao MD 66
Klebsiella species
• Usually found in GI tract
• Four major species
• K. pneumoniae is mostly commonly isolated
species
– Possesses a polysaccharide capsule, which
protects against phagocytosis and
antibiotics AND makes the colonies moist
and mucoid
– Has a distinctive “yeasty” odor
– Frequent cause of nosocomial pneumonia

67. Klebsiella species (cont’d)
– Significant biochemical reactions
• Lactose positive
• Most are urease positive
• Non-motile
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.V.Rao MD 67

68. 4/16/2012 Dr.T
.V.Rao MD 68
Klebsiella
– Virulence factors
• Capsule
• Adhesions
• Iron capturing ability
– Clinical significance
• Causes pneumonia, mostly in
immunocompromised hosts.
– Permanent lung damage is a frequent occurrence (rare
in other types of bacterial pneumonia)
• A major cause of nosocomial infections such as
septicemia and meningitis

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.V.Rao MD 69
Enterobacter species
• Comprised of 12 species; E. cloacae and
E. aerogenes are most common
• Isolated from wounds, urine, blood and
CSF
• Major characteristics
– Colonies resemble Klebsiella
– Motile
– MR negative; VP positive

70. 4/16/2012 Dr.T
.V.Rao MD 70
Serratia species
• Seven species, but S. marcescens is the
only one clinically important
• Frequently found in nosocomial
infections of urinary or respiratory
tracts
• Implicated in bacteremic outbreaks in
nurseries, cardiac surgery, and burn
units
• Fairly resistant to antibiotics

71. Serratia species (cont’d)
• Major characteristics
– Ferments lactose slowly
– Produce characteristic pink pigment,
especially when cultures are left at room
temperature
S. marscens on
nutrient agar →
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.V.Rao MD 71

72. 4/16/2012 Dr.T
.V.Rao MD 72
Hafnia species
• Hafnia alvei is only species
• Has been isolated from many
anatomical sites in humans and the
environment
• Occasionally isolated from stools
• Delayed citrate reaction is major
characteristic

73. 4/16/2012 Dr.T
.V.Rao MD 73
Other Enterobacteriaceae
• Proteus, Providencia, and Morganella
– Are all part of the NF of the GI tract (except
Providencia).
– All motile, with Proteus swarming
– PA +
– Lysine deamination + (LIA R/A)
– Urea + for most, strongly + for Proteus
– TSI variable (know the reactions for each in
the lab!)
– Indole – only P. mirabilis is -

74. 4/16/2012 Dr.T
.V.Rao MD 74
Proteus, Morganella &
Providencia species
• All are normal intestinal
flora
• Opportunistic pathogens
• Deaminate phenylalanine
• All are lactose negative

75. 4/16/2012 Dr.T
.V.Rao MD 75
Proteus species
• P. mirabilis and P. vulgaris are widely recognized
human pathogens
• Isolated from urine, wounds, and ear and
bacteremic infections
• Both produce swarming colonies on non-selective
media and have a distinctive “burned chocolate”
odor
• Both are strongly urease positive
• Both are phenylalanine deaminase positive

76. Proteus species (cont’d)
• A exhibits characteristic “swarming”
• B shows urease positive on right
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.V.Rao MD 76

77. 4/16/2012 Dr.T
.V.Rao MD 77
Morganella species
• Morganella morganii is only
species
• Documented cause of UTI
• Isolated from other anatomical
sites
• Urease positive
• Phenylalanine deaminase positive

78. 4/16/2012 Dr.T
.V.Rao MD 78
Providencia species
• Providencia rettgeri is pathogen of
urinary tract and has caused nosocomial
outbreaks
• Providenicia stuartii can cause
nosocomial outbreaks in burn units and
has been isolated from urine
• Both are phenylalanine deaminase
positive

79. 4/16/2012 Dr.T
.V.Rao MD 79
Citrobacter species
• Citrobacter freundii associated
with nosocomial infections (UTI,
pneumonias, and intraabdominal
abscesses)
• Ferments lactose and hydrolyzes
urea slowly
• Resembles Salmonella sp.

80. 4/16/2012 Dr.T
.V.Rao MD 80
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