This document provides information on laboratory diagnosis of infections caused by the family Enterobacteriaceae. It discusses the collection and testing of various specimen types, including stool, urine, and blood. A variety of culture media and biochemical tests are described to isolate and identify bacterial pathogens like E. coli, Salmonella, and Shigella from clinical samples. Identification methods include antigenic structure identification using agglutination tests and phage typing for certain Salmonella serotypes. The document provides details on laboratory diagnosis of common infections like urinary tract infections and enteric diseases including typhoid fever.

1. Laboratory diagnosis of infections
produced by germs of the family
Enterobateriaceae

2. Family Enterobacteriaceae
- Clinical significance -
• Intestinal and extraintestinal infections
• Highly pathogenic species of the genera:
– Yersinia
– Salmonella
– Shigella
• Facultatively pathogenic species of the genera:
– Escherichia coli
– Klebsiella
– Enterobacter
– Proteus
– Serratia
– Citrobacter

3. Family Enterobacteriaceae
- Common characters -
• Gram negative bacilli, nonsporulating, non-fastidious
• Glucose-fermenters;
• Lactose is only fermented by some genera – good
differential criterion
• Oxidase-negative
• Catalase-positive
• Habitat:
– soil, plants, human & animal intestines, mucous membranes;
– Particular situation: Salmonella typhi (causative agent of typhoid
fever) – only present in humans (diseased / asymptomatic
carriers)

4. Family Enterobacteriaceae
- Collection of specimens -
• Extraintestinal infections:
– urine, respiratory/digestive samples, wound secretions, blood,
CSF, etc)
• Intestinal infections:
– Faeces: collection close to onset / depending on pathogenesis of
infection
– Transport media: Stuart, Cary-Blair, Amies

5. Collection of urine
When?:
- in the morning (first miction)
How?:
- clean uro-genital area
- eliminate first flow
- collect middle flow in
sterile container
Send to lab immediately or store
at 2-8°C

6. Collection of stool (faeces)
• Disposable stool collection containers (simple / with
transportation medium Carry Blair: non-nutritive medium
which prevents overgrowth of Enterobacteriaceae but
preserves viable enteric pathogens (Salmonella,
Shigella, etc)

7. Family Enterobacteriaceae
- Isolation (inoculation of culture media) -
• Extraintestinal specimens from normally sterile sites:
– Blood agar
• Extraintestinal specimens with moderate bacterial load
(e.g. pus, sputum):
– Blood agar + MacConkey
• Highly contaminated specimens (faeces):
– MacConkey (low selectivity)
– ADCL, Hektoen agar, XLD agar (medium selectivity)
– High selective media e.g. S-S (for Salmonella and Shigella),
Wilson-Blair (for Salmonella)

8. e.g. Hektoen agar
(developed at the Hektoen Institute in Chicago)
• indicators of:
– lactose fermentation
– H2S production;
• inhibitors (bile salts)
to prevent the growth
of Gram positive
bacteria
Lactose H2S
Salmonella (-); alkaline
reaction: blue-
green colonies
(+);
black
centre
colonies
Shigella (-); alkaline
reaction: blue-
green colonies
(-)
E.coli and
others
(+); acid
reaction:
yellow-orange
colonies
(-)

9. Left: lactose (+) = yellow-orange
Right: lactose (-) = blue-green, H2S (+) = black
centre
E.coli / others (definitely not
Salmonella, not Shigella)
• Salmonella (not Shigella
because of H2S production –
black centre colonies)

10. Family Enterobacteriaceae
- Identification -
• Biochemical tests:
– TSI (triple sugar iron) agar
– MIU (motility, indol, urea) agar
– Simmons agar (use of citrate as unique carbon source)
– PAD (phenylalanine deaminase) test
– Fermentation of sugars
• Antigenic structure-based identification:
– Agglutination with antisera

11. Family Enterobacteriaceae
Biochemical tests = testing for enzyme systems
• characterization of bacterial isolate by testing for
characteristic enzyme systems
• Method: re-inoculation of isolated colony (primary
culture) into a series tubes with culture media containing
specific substrates and chemical indicators
• Principle: detection of
– pH changes produced by utilization of substrates /
– colour / other changes produced by specific by-
products

12. Family Enterobacteriaceae
Biochemical tests (continued)
TSI (triple sugar iron) agar – assessment of bacterial
capacity to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S

13. TSI agar:
sucrose, lactose, glucose + mehyl red + ferrous
sulfate
IF:
• only glucose fermented →acid production in the butt of tube →
yellow, but insufficient acid to affect the methyl red in the slant
• either sucrose or lactose fermented → sufficient fermentation
products → both the butt and the slant yellow
• gas during fermentation → gas bubbles/cracking of agar
• no fermentation → slant and butt remain red
• If bacterium forms H2S, this chemical will react with the iron to form
ferrous sulfide = black precipitate in the butt (black butt)

14. TSI agar (continued)
• R = red = no fermentation
(obligate aerobe)
• Y = yellow = some
fermentation (facultative
anaerobe)
• YG = fermentation + gas
• ”+” = Black = H2S

15. Family Enterobacteriaceae
Identification – biochemical tests
• API (Analytical Profile Index)

16. Escherichia coli
• Gram negative, short bacilli, rounded ends,
nonsporulating, motile (peritrichous cilia)
• Normal microbial flora of human and animal intestines;
involved in vitamin synthesis and balance of intestinal
microbiota
• Facultatively pathogenic

17. Escherichia coli
- Clinical significance -
• Enteral infections (5 groups):
– EPEC (enteropathogenic E.coli)
– ETEC (enterotoxigenic E.coli)
– EIEC (enteroinvasive E.coli)
– EHEC (enterohemorrhagic E.coli) – produce verotoxins* (bloody
severe diarrhoea resembling dysentery!)
– EAEC (enteroadherent E.coli)
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

18. *Verotoxins (Shiga-like toxins)
• toxins produced by certain strains of E.coli (EHEC)
which disrupt the function of the ribosome
• Action similar to the toxin produced by Shigella
disenteriae strains (Sh.shiga) – see below
• causes the hemolytic uremic syndrome
• Term “verotoxin” related to the effect on “vero” cell
cultures (lineages of kidney epithelial cells extracted from
African green monkeys; name: abbreviation from “verda
reno” = green kidney in Esperanto)

19. E.coli – Enteral infections
Specimen: stool
• Culture media: MacConkey:
pH indicator = neutral red (red
in acid medium; colourless in
basic medium):
• RED colonies (lactose-
positive), round, shiny, 2-3 mm

20. E.coli colonies on blood agar

21. E.coli colonies on MacConkey:
lactose positive (red) colonies

22. E.coli on medium containing lactose and bile
salts: lactose positive (red) colonies

23. E.coli on Hektoen agar: yellow-orange
colonies (lactose acidification)

24. E.coli – Enteral infections
Identification: biochemical tests

25. E.coli – Enteral infections
Identification on antigenic structure
• Slide agglutination with Ab
against O and B antigens:
– 5 lactose-positive colonies –
pick up with loop and emulsify
with polyvalent anti-EPEC
serum
– If positive test (agglutination)
continue with monovalent
antisera (standard set)
• Similar procedure for EIEC,
ETEC, EHEC (antisera for
antigens O and H)

27. Escherichia coli
- Clinical significance -
• Enteral infections (5 groups):
– EPEC (enteropathogenic E.coli)
– ETEC (enterotoxigenic E.coli)
– EIEC (enteroinvasive E.coli)
– EHEC (enterohemorrhagic E.coli) – produce verotoxins (bloody
severe diarrhoea resembling disenteria!)
– EAEC (enteroadherent E.coli)
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

28. E.coli – Urinary tract infections (UTI)
• Collection of urine for bacterial culture (see above)
• Gram stained smear from urine sediment (after
centrifugation): high no of PMNs + Gram negative bacilli
• Quantitative urine culture:
– A. Dilutions technique
– B. Calibrated loop technique

29. E.coli – Gram stained smear

30. E.coli – Urinary tract infections (continued)
Quantitative urine culture
• Colony counts: method to determine number of viable
bacterial cells in urine specimen
• 1 colony = 1 viable bacterial cell (colony-forming unit =
CFU) → after inoculation: division by binary fission i.e. 1
cell to 2; 2 to 4; 4 to 8; 8 to 16....and so on (at least a
million cells must be present in order to be seen as a
colony with the naked eye!)
• Results reported as: number of bacterial cells per mL of
urine

31. Quantitative urine culture (continued)
A. The dilutions technique:
• 2 dilutions (1/100 and 1/1000) in sterile saline solution
• Inoculate 0.1 mL of each dilution onto blood agar and
MacConkey (lactose containing medium)
• Spread inoculum to obtain isolated colonies (L-shaped
loop); Incubate overnight at 37°C
• Calculate the no of germs / mL urine i.e. multiply:
– No of colonies (CFU) x
– dilution factor (100 or 1000) x
– 10 (we inoculated 0.1 mL of each dilution)

32. Quantitative urine culture (continued)
B. The calibrated loop technique: (nondiluted sample)
• 5 mm loop → 0.01 mL urine (dilution: 1/100) →
MacConkey
• 2.5 mm loop → 0.001 mL urine (dilution: 1/1000) →
blood agar
• Incubate overnight at 37°C
• Calculation: (for each plate)
• No. of colonies (CFU) x dilution = no of germs / mL
• Calculate the arithmetic mean between the 2 counts
(on the 2 plates)

33. Quantitative urine culture (continued)
• Interpretation of results:
• Under 10,000 germs/mL = nonsignificant bacteriuria
(probably contamination from lower urethra)
• 10,000–100,000 germs/mL = nonconclusive; repeat test
• Over 100,000 germs/mL = UTI
• Next steps: identification of causative agent i.e.
– colonial characters, biochemical tests
– Antimicrobial susceptibility testing

34. Genus Salmonella
• The most complex of all Enterobacteriaceae
• over 2400 serotypes – Classification: Kauffmann-White
scheme based on bacterial antigens:
– O (somatic)
– H (flagellar)
– Vi (virulence) – derived from the K (capsular) antigen
• Clinical significance:
– Food poisoning
– Systemic infections (the germs cross the intestinal barrier) –
”enteric fevers”
• Transmission: fecal-oral (via contaminated water, foods)

35. Genus Salmonella
- The Kauffmann-White classification -
• E.g. Based on the “O”
antigens: groups A – I (+
others)
• some examples given in
table →
“O” group A S.paratyphi A
“O” group B S.paratyphi B
S.typhimurium
“O” group C S.paratyphi C
“O” group D S.typhi
S.enteritidis

36. Salmonella typhi
• Prototype agent of ”enteric fevers” (other enteric fevers
caused by Salomnella paratyphi A, B, C – less severe)
• Typhoid fever - fever in plateau (39-40°C), headache,
muscle ache, vomit, diarrhoea /constipation, skin rush
(lenticular maculae; “rose spots”), mental confusion,
hepato-splenomegaly
• Complications: internal bleeding, intestinal perforation +
peritonitis
• Laboratory diagnosis:
– Bacteriology
– Serology

37. Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis -
• Collection of specimens depends on:
– Patient status: diseased / chronic carrier
– Clinical stage (time from onset)
• Secimens:
– Blood for blood culture (best collected during 1st week; percent
of isolation decreases to 25% in the 4th week)
– Bone marrow (in late stages) – lower patient compliance
– Faeces for coproculture (low isolation during 1st week; percent
increases progressively to ~75% in 4th week); also collected
from suspected chronic carriers
– Urine for culture (same isolation curve as for faeces)

38. Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Blood culture:
• inoculated media examined daily for 7-10 days; negative
result only if liquid media remain sterile for 10 days!
• Gram stained smear from turbid tubes: Gram negative
bacilli
• Subculture on agar slant
• Identification based on:
– colonial characters,
– biochemical tests,
– antigenic structure – slide agglutination with anti-O and anti-H
sera (Kauffmann-White classification)

39. Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Coproculture:
• Both in diseased patients and in chronic carrires
• Inoculation in liquid enrichment media (favour
multiplication of salmonellae and inhibit other microbial
flora) e.g.
– Leifson (nutrient broth with acid sodium selenite);
– Muller-Kauffmann (broth with tetrationate and bile)
• Incubate overnight at 37°C
• Reinoculate on selective solid media (nutrients + sugars
+ pH indicator + substances which inhibit other germs)

40. Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Coproculture: (continued)
Colonial characters on selective solid media:
• Wilson-Blair (high selectivity; indicator: brilliant green)
– Black, flat colonies, 1-2 mm, metallic halo
• S-S (selective for Salmonella and Shigella):
– Fine, semitransparent colonies, with black centre (H2S)
• Hektoen enteric agar :
– Fine, green colonies, with black centre (H2S)
• MacConkey (medium selectivity):
– Semitransparent, lactose-negative (colourless) colonies

41. Salmonella on S-S agar: fine, semitransparent
colonies, black centre (H2S)

42. Salmonella on MacConkey agar
• Semitransparent, lactose-
negative (colourless)
colonies

43. Salmonella on agar with lactose and bile salts:
lactose negative (colourless) colonies with black
centre (H2S)

44. Salmonella on Hektoen agar
• black centre
colonies (H2S)

45. Salmonella – Coproculture - continued
• Identification based on:
– colonial characters (see above),
– biochemical tests (API 20E),
– antigenic structure – slide agglutination with anti-O and anti-H
sera (Kauffmann-White classification)
– Phage typing – see next slide

46. (Bacterio)phage typing for Salmonella
• Bacteriophage = virus which specifically attacks bacteria
• Banks of phages developed for Salmonella serotypes
Procedure:
• agar plates flooded with liquid culture of the bacterial
isolate; remove excess liquid; leave culture film to dry;
• inoculate set of phage suspensions onto plate surface;
incubate overnight at 37°C
• Interpretation: phage lysis reactions recorded and
compared to a set of standards (developed by Public
Health England, formerly PHLS, Colindale, England)

47. Phage typing Salmonella enteritidis

48. Salmonella typhi
- Serological diagnosis -
The Widal test:
• Principle: reaction between antibodies in patient serum
and specific antigens of S. typhi → clumping
(agglutination) visible to the naked eye
• easy to perform BUT less reliable than bacteriology:
– cross-reactivity with other Salmonella species,
– the test cannot distinguish between a current infection
and a previous infection or vaccination status
• Still used in low resource areas with high
prevalence of typhoid fever (endemic areas e.g.
India, Pakistan)

49. Genus Shigella
Clinical significance:
• dysentery – diarrhoea with multiple stools with mucus
and blood + general symptoms: dehydration, fever,
abdominal pain + neurologic symptoms (neurotoxin
secreted by Shigella shiga)
Common characters:
• Gram negative bacilli, nonmotile, nonsporulating

50. Genus Shigella - classification
4 serological subgroups (based on biochemical characters
and antigenic structure):
• Subgroup A: Shigella dysenteriae
– Types: Sh. shiga, Sh. Schmitzi, Sh. Large-Sachs
• Subgroup B: Shigella flexneri
• Subgroup C: Shigella boydii
• Subgroup D: Shigella sonnei
_______________
• Shigella shiga: the most severe disease (secretion of
neurotoxin)

51. Kiyoshi Shiga (1871-1957)
• Discovered Shigella dysenteriae during severe
epidemic in 1897: over 90,000 cases
• “shiga” toxin named after him

52. Genus Shigella – Bacteriological diagnosis
• Collection of specimens: faeces (especially portions with
mucus and blood)
• Transport: Cary Blair medium
• Cultivation and isolation: endo-agar, MacConkey, S-S,
Hektoen, XLD
• Colonial characters:
– small, 1-2 mm, transparent, round / irregular contour, convex,
lactose-negative (colourless i.e. colour of the culture medium)
– No H2S production

54. Hektoen agar:
Shigella – colourless colonies;
Salmonella - green colonies with black centre

55. Shigella – identification, continued
• Biochemical characters: API 20E

56. Genus Shigella – Bacteriological diagnosis
- continued -
• Antigenic structure based
identification:
– Agglutination with sets of anti-
sera (polyvalent + monovalent:
subgroups + typing)

57. Genus Klebsiella
• Comensal/Facultatively pathogenic: Colonizes the
respiratory mucosa and the intestine
• In immunosuppressed patients (premature infants,
elderly people) – potential for severe infections
(pneumonia, sepsis, meningitis)
• Hospital acquired infections: surgical wound infections,
urinary infections, sepsis
• Involvement in diarrhoeic diseasae - debated

58. Genus Klebsiella
4 species important for human pathology:
• Klebsiella pneumoniae – comensal of human airways
and intestinal mucosa; facultatively pathogenic
• Klebsiella oxytoca - idem
• Klebsiella ozenae – ozena = chronic inflammatory
infection of the nasal mucosa; mucosal atrophia, crusts
& purulent secretions with unpleasant odour
• Klebsiella rhinoscleromatis – rhinoscleroma = chronic
hypertrophic rhinitis with granulomatous lesions

59. Genus Klebsiella
Common characters:
• Gram negative, short bacilli,
rounded ends, nonsporulating,
arranged in diplo (in pairs) on
the long axis, enacpsulated
• Sometimes bipolar staining
(ends more intensly stained
than middle of rod)

60. Genus Kelbsiella
Isolation:
• Specimens from normally sterile sites (blood, CSF, etc):
– Nutrient broth + reinoculation on blood agar
• Specimens from highly contaminates sites (e.g. faeces):
– Media for enterobacteria: MacConkey, XLD
• Identification:
– Blood agar – large, white-grey colonies, mucous, aspect of
”pouring culture” – in time the colour changes to brown
(”chameleoning” phenomenon)
– MacConkey – large, mucoid colonies, red/pink (lactose positive)
– in time colour changes to yellow (lactose negative) –
“chameleoning” by alkalinisation of the medium

61. Klebsiella – blood agar (non hemolytic
mucoid colonies)

62. Klebsiella: Mucous colonies

63. Klebsiella – pink colonies (Lactose positive)
on MacConkey agar

64. Klebsiella colonies on MacConkey: colour starts to
change - Lactose positive (red/pink) colonies start
to change to lactose negative (yellow) –
alkalinisation

65. Klebsiella – identification – continued
Biochemical characters

66. Genus Proteus
• 4 species:
– P.vulgaris, P.mirabilis, P.penneri, P. myxofaciens
• Common characters:
– Gram negative, short bacilli, rounded ends, high polymorphism,
high motility (peritrichous cili), nonsporulating, nonencapsulated
• Habitat:
– soil, trash, sewage, altered meat, etc. – involved in putrefaction
processes
• Clinical significance:
– comensal of human and animal digestive flora;
– facultatively pathogenic: UTI, otitis, synusitis, meningitis, sepsis
(community or hospital acquired infections)

67. Genus Proteus
• Collection of specimens:
– urine, faeces, pus, sputum, CSF, blood, etc
• Direct microscopy:
– only for naturally sterile specimens e.g. CSF
– PMNs + Gram negative bacilli, noncharacteristic arrangement +
filamentous bacilli (high polymorphism of Proteus spp)
• Isolation and identification:
– Blood agar: swarming phenomenon (concentric growth waves
invading the entire plate after overnight incubation); invades
other bacterial colonies; no isolated colonies
– Selective media: round colonies, same colour as the
medium/transparent, black centre (”cat‘s eye”) – H2S production

69. Proteus
• Swarming
phenomenon on
tryptic soy agar

70. Genera Morganella and Providencia
• Previously classified as species of the genus Proteus
• Morganella morganii (formerly: Proteus morganii)
• Providencia (formerly: Proteus rettgeri)
• Involved in UTI especially in urinary catheterized patients

71. Genera Proteus, Morganella, Providencia

72. Genus Yersinia
3 species important for human pathology:
1. Y. enterocolitica:
– intestinal pathogen (some strains produce an enterotoxin similar
to E.coli; may infect abdominal lymph nodes – apendicitis-like
symptoms)
• Isolation: faeces inoculated on selective media:
– MacConkey – colonies much smaller than of other
enterobacteria
– CIN (cefsulodin, irgasan, novobiocin): overnight incubation at
32°C/48 hours at 25°C: transparent colonies→ (at 48 hours):
larger, pink colonies (increased motility at room temperature)

73. Genus Yersinia
3 species important for human pathology (continued):
2. Y.pseudotuberculosis
• Enteric infection involving also abdominal lymph vessels
and nodes
• Collection of specimens, Isolation and identification
similar with Y.enterocolitica
• Differential diagnosis based on biochemcal tests

74. Genus Yersinia
3 species important for human pathology (continued):
3. Y.pestis – Plague:
– reservoir of germs: rodents (rats)
– interpersonal transmission (human to human)
– Routes of infection:
• Vectors: Flea bites →skin lesions (inflammation,
necrosis, purulent secretion) + swollen lymph
nodes (buboes) = bubonic plague →sepsis
• Airborne: Inhalation →pneumonia = pulmonary
plague

75. Left: Oriental rat flea (vector of Y.pestis)
Right, upper image: Y.pestis infected flea bite
Right, lower image: swollen lymph nodes (buboes)

76. Yersinia on blood agar

77. Yersinia on MacConkey

78. Yersinia – medium with lactose and bile
salts

79. Yersinia on Hektoen agar

80. Yersinia agar

81. Yersinia – identification: API 20E gallery

82. Hektoen agar inoculated with stool sample
• E.coli – red arrow
• Salmonella – blue
arrow
• Proteus – yellow
arrow

83. S-S agar:
A = Klebsiella; B = E.coli; C = Salmonella; D = Proteus;
E = Ps.aeruginosa
• Klebsiella and E.coli –
ferment sugars (red
colonies)
• Salmonella and
Proteus – H2S
production (black
centre)
• Pseudomonas
aeruginosa –
colourless colonies