This document discusses Enterobacteriaceae, a family of Gram-negative bacteria that includes many common pathogens. It provides details on their characteristics, identification, classification based on lactose fermentation, and important genera such as Escherichia coli. Reasons why E. coli is commonly used for gene cloning are described, including its genetic simplicity, rapid growth, safety, extensive prior study, and ability to host foreign DNA. Identification of Enterobacteriaceae involves examining biochemical reactions and growth on selective media like MacConkey agar.

1. Faculty of Medical Laboratory Sciences
Department of Microbiology
Medical Microbiology course
Dr. Mahadi Hassan Mamoud
PhD Medical Microbiology

2. Enterobacteriaceae
• Commonly present in large intestine.
• Nonsporing , Non Acid fast,Gram – bacilli.
• A complex familyof organisms.
• Some are non pathogenic.
• A few are highly Pathogenic.
• Some commensals turn out to be pathogenic.as in UTI
after catheterization.

3. 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)

4. Enterobacteriaceae: Genetic Properties
• Chromosomal DNA has 39-59% guanine-plus-
cytosine (G+C) content
• Escherichia coli is the type genus and species
of the Enterobacteriaceae
• Species of Enterobacteriaceae more closely related by
evolutionary distance to Escherichia coli than to
organisms of other families (Pseudomonadaceae,
Aeromonadaceae)

5. Identification of Enterobacteriaceae
Biochemical reactions
• Oxidase test
• All members of Enterobacteriaceae are oxidase negative
• Pseudomonas is oxidase positive
• O/F test
• All members of Enterobacteriaceae are O+/F+
• Pseudomonas is O+/F-
• Nitrate reductase
• All members of Enterobacteriaceae are nitrate reductase positive
• Pseudomonas is nitrate reductase negative

6. Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, Shigella
Proteus, Yersinia
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 is selective & differential medium for Enterobacteriaceae
MacConkey Agar
Contains
Bile salts Crystal violet Lactose Neutral red
Inhibit growth of G+ve bacteria
Cause of selectivity
Cause of differential
pH indicator
Acidic: Pink
Lactose feremnters
Pink colonies
Lactose non feremnters
colorless colonies

8. Classification of Enterobacteriaceae according to lactose fermentation (growth on
MacConkey Agar)
Enterobacteriaceae
Lactose Fermenters Lactose Non-Fermenters
Escherichia coli
Klebsiella spp
Enterobacter spp
Citrobacter spp
Salmonella spp
Schigella spp
Proteus spp
Yersinina spp
Pink colonies
Colorless colonies
Acid
Neutral red
No acid

9. 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.

10. Classification
• Domain: Bacteria
• Kingdom: Bacteria
• Phylum: Proteobacteria
• Class: Gamma Proteobacteria
• Order:Enterobacteriales
• Family: Enterobacteriaceae
• Genus: Escherichia
• Species: Escherichia coli (E.coli)

11. 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.

12. 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.

13. • O antigen
– Somatic (on LPS)
– 171 antigens
• H antigen
– Flagella
– 56 antigens
• K antigen
– Capsule and or
fimbrial antigen
– 80 antigens O18ac:H7:K1
18th O antigen 1st K antigen
7th H antigen
Antigenic structure

14. E.coli Biochemical Characters
IMViCtests.
Indole +ve
MethylRed +ve
Voges Proskauer – ve
Citrate –ve
Urease –ve
KIA yellowbutt
yellowslant
Acid&Gas
NoH2S
Set ofSugar Glucose A&G
Lactose A&G
Mannitol A&G
Maltose A&G
Motility Motile

15. Pathogenesis
• Adhesion of the microorganisms to the intestinal
epithelial cells
• Production of enterotoxins Heat labile (LT) and
Heat stable (ST)
• Verotoxin production of cytopathic effects in green
monkey kideny cells
• Invasion of epithelial cells

16. • Heat labile toxin
• like choleragen
• Adenyl cyclase activated
• cyclic AMP
• secretion water/ions
• Heat stable toxin
• Guanylate cyclase activated
• cyclic GMP
• uptake water/ions

17. Urinary tract infection
Is the leading cause of urinary tract infections which can lead to acute
cystitis (bladder infection) and pyelonephritis (kidney infection).
E. coli is the most common cause of urinary tract infection.
Community- vs. hospital-acquired UT infection
Symptoms:
urinary frequency,
dysuria,
hematuria, and
pyuria.
Can result in bacteremia and sepsis.
clinical diseases

18. Other infection with E.coli
• Neonatal Meningitis. particularly K1 strains
• Pyogenic infections.
• Intraabdominal infections
• Peritonitis. Abscess.
• Septicemias
• Produce Drug resistant infections.

19. • Certain strains of E. coli
• ingested in sufficient quantities by host.
• enteritis, enterocolitis, and colitis
• pathogenic strains of E. coli which cause
disease in the intestine are obligate
pathogens

20. six distinct "pathotypes" of E. coli (which cause disease in intestine)
• Enteropathogenic E. coli (EPEC).
• Enterotoxigenic E. coli (ETEC).
• Shiga toxin-producing E. coli (STEC)/enterohemorrhagicE. coli (EHEC),
Verocytotoxin-producing E. coli (VTEC).
• Enteroinvasive E. coli (EIEC).
• Enteroaggregative E. coli (EAEC) (EAggEC)
• diffuselyadherentE. coli (DAE)

21. Enteropathogenic E. coli
• EPEC is main cause of infant diarrhea
• Breast-feeding diminishes the incidence of EPEC infection.
• Rapid person-to-person spread may occur.
• After infection with EPEC there is loss of microvilli.
• Symptoms : Fever, Diarrhea (NO Blood), Vomiting, Nausea.
• Confirm with Polyvalent and monovalent sera.

22. Enterotoxigenic E. coli
• ETEC is a major cause of endemic diarrhea in children
during the first 3 years of life.
• ETEC is the most common agent of traveler's diarrhea.
• There is production enterotoxin
• Produce Heat stable /Heat labile toxins.
• Present with Nausea, Vomiting and Lose stool

23. Enterotoxigenic E.coli
•Laboratory Diagnosis
Demonstration of Enterotoxin LT and ST
Tissue culture tests.
ELISA.
Passive agglutination tests.
Animal experiments inRabbit ileal looptest.
Con.

24. Enteroinvasive E. coli EIEC
a relativelyuncommoncauseof diarrhea.
Some are non motilestrains.
 EIEC sharesmany genetic andclinical featureswith
Shigella.
The diseaseresembles the shigellosis
 The difference
EIEC produces disease only at a large inoculum(108 to
1010 CFU8), with onset generally occurringafter an
incubationperiod of 1 to 3 days.

25. Enteroinvasive E. coli EIEC
Sereny test:
• Instillationof a suspension of freshly isolated EIEC or Shigella
into theeyes of guineapigs leads to mucopurulent
conjunctivitis and severe keratitis.
• Mice can also be used.
CellPenetrationin HeLa or HEP-2 cells.
Plasmid detection:
VMA ELISA: The plasmid codes for outer membraneantigens
called the virulence markerantigens(VMA) which can be
detected by the ELISA (VMA ELISA) test.
Con.

26. Enterohemorrhagic E.coli
Produce Verocytotoxin or shiga like toxin
• Named so because it was first detected by its cytotoxic effecton
Vero cells, a cellline derived from African green monkeykidney
cells.
• It is also known as SLT = shiga liketoxin because it is similar to
the shigella dysenteriae type 1 toxin in its physical, antigenic
and biological properties
Mild diarrhea - can be fatalhemorrhagic colitis. and uremic
syndrome.
Presentin Human and Animal feces.
Hemorrhagic complication withO157 in Japan and USA.

27. Lab. Diagnosis
o Culture
o DNA detection methods
o Cytotoxic effects on Vero cells.
o Detection with monovalent sera O157/H7
Enterohemorrhagic E.coli Con.

28. Dr.T.V.Rao MD 28

29. • Haemolytic anaemia
• Acute renal failure
• Thrombocytopenia
The verotoxin enters the blood stream
EnterohemorrhagicE.colican cause Haemolytic
Uremic Syndrome HUS
Enterohemorrhagic E.coli

30. Enteroaggregative and Diffusely Adherent E.
Coli EAEC and DAEC
 primarily in developing countries and in young children.
 These strains can cause traveler's diarrhea.
 A largeinoculum is required for infection.
 In vitro, the organisms exhibit a diffuse or "stacked-brick"
adherence pattern.
 Can cause Diarrhea Detect by Culture methods
 Brick-like aggregates on cell surfaces
 Mucus biofilm inhibits fluid absorption
 Diarrhea
 Detection of Enterotoxin

31. Culturing for E.coli
• Mid stream sample/semiquantitativeculturing (Kasset
al) >_ 1.00,000/ml of urine. ( significantBacteriuria)
• Urine shouldnot be kept in wardsfor > 2 hours andto
be preserved at 4 c
• Culture by standardloopmethod.
• Fixedvolume cultured onMacConkeyagar Lactose
fermenters I M Vi C
• Antibioticsensitivitytested.

32. Escherichiacolias a Genetic tool.
• The study of Escherichia coli and its plasmids and
bacteriophages has provided a vast body of genetical
information, much of it relevant to the whole of biology.
This was true even before the development of the new
techniques, for cloning and analysing DNA, that have
revolutionized biological research during the past
decade.. Much of the background of knowledge
necessary for the cloning and expression of genetically
engineered information, as well as the techniques
themselves, came fromwork with thisorganism.

33. Why E.coliis preferred
• E. colicells only have about4,400 genes whereas the
human genome project has determined that humans
containapproximately30,000 genes.
• Also, bacteria,includingE. coli, live their entire lifetimein
a haploidstate, with nosecondalleleto mask the effects
of mutationsduring proteinengineering experiments.

34. specimens
• Urine
• Stool.
• Blood
• CSF
Lab Diagnosis

35. • Microscopy
• make distinction is between inflammatory and noninflammatory disease
• Culture
• Identification ofserotypes/pathotypes
 Usuallyperformed atreference lab
 TheE.coliisolate treated ,standardized andallowedto react with antisera (knownantibodies) in
antigenantibodyreactions
 anddesignated
 Specialtests to detect LT and ST are notavailablein mostclinicallaboratories
 Serological characters:
 Somatic(O) antigen.
 Flagellar (H) antigen.
 Manystrainshave capsular(K) antigen.

36. Lab Diagnosis
• Grossly bloody or mucoidstool suggests aninflammatory
process.
• A test for fecal leukocytes (preparation of a thin smear of stool
on a glass slide, addition of a drop of methyleneblue,and
examinationof the wet mount)can suggestinflammatory
disease in patientspresentingwithdiarrhea,
• A testfor fecal lactoferrin, whichis a marker of fecal leukocytes,
is more sensitive and is available in latex agglutinationand
enzyme-linkedimmunosorbentassay formats.

38. 1/24/2020
(Gram stained film of E. coli)

39. 1/24/2020
(E. coli on MacConkey’s agar)

40. 1/24/2020
(Lactose fermenters and non lactose fermenters on
MacConkey’s agar)

41. Reasons E. coli is used for Gene Cloning
• The microorganism Escherichia coli has a long
history of use in the biotechnology industry
and is still the microorganism of choice for
most gene cloning experiments. Although E.
coli is known to the general population for
the infectious nature of one particular strain
(0157:H7) few people are aware of how
versatile and useful E. coli is to genetic
research. There are several reasons E. coli
became so widely used and is still a common
host for recombinant DNA.

42.  Genetic Simplicity
• Bacteria make useful tools for genetic research because
of their relatively small genome size compared to
eukaryotes. E. coli cells only have about 4,400 genes
whereas the human genome project has determined that
humans contain approximately 30,000 genes. Also,
bacteria, including E. coli, live their entire lifetime in a
haploid state, with no second allele to mask the effects of
mutations during protein engineering experiments.

43.  Growth Rate
• Bacteria typically grow much faster than more complex
organisms. E. coli grows rapidly at a rate of one generation
per twenty minutes under typical growth conditions. This
allows for preparation of log-phase (mid-way to maximum
density) cultures overnight and genetic experimental
results in mere hours instead of several days, months or
years. Faster growth also means better production rates
when cultures are used in scaled up fermentation
processes.

44.  Safety
• E. coli is naturally found in the intestinal tracts of humans
and animals where it helps provide nutrients (vitamins K
and B12) to its host. There are many different strains of E.
coli that may produce toxins or cause varying levels of
infection if ingested or allowed to invade other parts of
the body. Despite the bad reputation of one particularly
toxic strain (O157:H7), E. coli are generally relatively
inocuous if handled with reasonable hygiene.

45.  Conjugation and the Genome Sequence
• The E. coli genome was the first to be completely
sequenced. Genetic mapping in E. coli was made
possible by the discovery of conjugation. E. coli is
the most highly studied microorganism and an
advanced knowledge of its protein expression
mechanisms makes it simpler to use for
experiments where expression of foreign proteins
and selection of recombinants is essential.

46.  Ability to Host Foreign DNA
• Most gene cloning techniques were developed
using this bacterium and are still more successful
or effective in E. coli than in other microorganisms.
E. coli is readily transformed with plasmids and
other vectors, easily undergoes transduction, and
preparation of competent cells (cells that will take
up foreign DNA) is not complicated.
Transformations with other microorganisms are
often less successful.