1. Causetive agents of bacterial intestinal diseases.
Epidemiology, Principles of microbiological diagnosis,
specific therapy.
Tvorko M. S.

2. Enterobacteriaceae
Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Hafnia
Serratia
Proteus
Providencia
Morganella
Yersinia
Erwinia

3. Escherichia coli Characteristics
Discovered in 19th century by Bavarian
pediatrician Theodor Escherich
Gram negative eubacteria
Facultative anaerobe
Usually motile
Universal inhabitant of human (mammalian) colon

4. Escherichia coli
Found in the intestines of humans and animals, this bacterium is usually harmless, but some strains can cause food poisoning and more serious illnesses. Most outbreaks involve contaminated beef that was not cooked thoroughly. The strain known as O157:H7 is considered a potential biological weapon.

5. E. coli Lives in Colon of Healthy People (member of commensal flora)
Includes E. coli

6. Scanning electron micrograph

9. Cultivation.
Colonies of E. coli on meat-peptone agar

10. Colonies of E. coli on Endo's medium

11. Colonies of E. coli on Ploskirev's medium

12. Colonies of E. coli on blood agar

13. Escherichia coli
is highly motile
and will show turbidity throughout the tube.

14. Fermentative properties.
Positive (left) reactions of isolates E. coli in glucose fermentation broth. Note the formation of acid (yellow color) and gas. Observe the bubble in the Durham tube.
“+” -ve test “—” -ve test

15. Indole reaction
A. Salmonella
B. E. coli
is the positive microbe. A B

16. Note the bubble formation. Catalase positive
E. coli can reduce nitrate to nitrite.

17. Antigenic structure.
The antigenic structure of E. coli is characterized by variability and marked individuality. Along with the H- and O-antigens, the presence of other antigens has been shown in some strains, i.e. the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and the thermostable A- and M-antigens.
On the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain For example, one of the most widely spread serotypes is designated 0111 : K58 : H2.

18. Enteropathogenic E. coli (EPEC)
First E. coli pathotype described
UK pediatrician John Bray, 1945
Causes potentially fatal infant diarrhea in developing areas
Contract organism by ingestion of contaminated water, food or fomites

19. EPEC Colonization and Lesion Development
patchy colonization of the small intestine
Generates unique histopathology termed “attaching and effacing” lesion
destroys microvilli
Expresses numerous toxins/effectors that manipulate host cell systems to serve the pathogen
EPEC, like other pathogenic E. coli strains, is a master cell biologist

20. Toxin production.
a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties
endotoxins
thermolabile neurotropic exotoxins
haemotoxins
pyrogenic substances,
proteinases,
deoxyribonucleases, urease,
phosphatase
hyaluronidase
aminoacid decarboxylases

21. Pathogenic E. coli Virulence Mechanisms
Generalities
Virulence systems frequently encoded on mobile genetic elements
successful combinations reflected in pathotypes
Like other mucosal pathogens, pathogenic E. coli use multi-step strategy to infect host
attachment
evasion of host defenses
multiplication
damage host
Pathogenic E. coli often colonize host niches not normally inhabited by E. coli

22. EPEC A/E Lesion and Pedestal Formation

24. Fig. 1.  Scanning electron micrograph showing microcolonies
of EPEC displaying the characteristic localized adherence
pattern of adherence to HEp-2 cells.
Fig. 2.  High power scanning electron micrograph of EPEC displaying localized adherence to
HEp-2 cells.  Note the elongated microvilli to which the bacteria appear to attach.

25. Enterohaemorrhagic E. coli (EHEC)
First described in 1982
Causes bloody diarrhea (haemorrhagic colitis), non-bloody diarrhea and haemolytic uremic syndrome (HUS)
~5% of EHEC infections result in HUS; predominantly in children <5 years old and elderly
~5% of HUS cases are fatal
Contract organism by ingesting contaminated food
common inhabitant of bovine gut
low infectious dose for humans (<100 organisms)
organism may be resistant to stomach acid

26. EHEC Virulence Factors
O157:H7 serotype dominant in North America, UK, Japan
O26 and O111 serogroups more prominent in other countries
Evolved from LEE containing EPEC serogroup O55
Like EPEC, generates A/E lesion
Colonizes the large intestine
Expanded repertoire of adhesion factors
Encodes toxins/effectors in EPEC LEE
Additional virulence factors (plasmid and chromosomal)
RTX toxin similar to haemolysin
StcE activates host Complement cascade
Stx (Shiga) toxin

27. Stx (Shiga) Toxin Toxin encoded on bacteriophage
>200 E. coli serotypes encode Stx
Shiga toxin E. coli (STEC)
most do not encode LEE (not virulent)
A/B toxin
pentameric B subunits bind holotoxin to host cell surface
A subunit cleaves host ribosomal RNA arresting protein synthesis and cell death (apoptosis)
Stx produced in colon travels through bloodstream to kidney
Damages renal endothelium and induces inflammation that may lead to acute renal failure and death

28. Shiga toxin Distinguishing virulence factor Subunit toxin:
A: acts at ribosomal level, inhibits protein synthesis
B: binds glycolipid receptor in mammalian cells (renal endothelium)
Stx1, Stx2
Stx2 variants: 2c,2d,2e,2f

29. HUS: Stx (Shiga) Toxin Activity

30. Direct and indirect contact transmission

31. EHEC Ecology and Transmission

32. Has HACCP led to a reduction in human incidence?
Excretion
Re-colonization
Environment
Death

33. Direct contact transmission

34. Indirect contact (environmental) transmission

36. B.Enterotoxigenic E coli ( ETEC)
Common cause for travelers diarrhoea, and watery diarrhoea in children.
Colonisation factor facilitates the attachment to the
intestinal epithelium.
Some ETEC produces heat labile exotoxin LT and heat stable or either of the toxins
LT has two sub units A B
Action -Activate Adenylate cyclase
Increase local CAMP
Intense, prolonged hypersecretion of water ,
Lumen of gut fill with water
Hypermobility and diarrhoea results.

37. LT is antigenic and cross reacts with the enterotoxin of Vibrio cholerae.
Some ETEC produces heat stable enterotoxin STa/b
STa activates guanylyl cyclase.
STb activates cyclic nucleotides.
Releases water

38. Enteroinvasive E coli (EIEC)
Produces disease similar to shigellosis.
In adults this has been isolated with Shigella
Commonly affect children in developing countries,
and travelers.
Disease is due to invasion into mucosal cells of the intestine
multiply inside the cells and destruction /inflammation/ulceration
diarrhoea with blood
EIEC are nonlactose fermenter, or late lactose fermenter
and non motile.

39. E. coli Enteroinvasive E. coli member normal gut flora
Agent of bacillary dysentery like disease

40. Normal Colon Dysenteric Colon healthy GI tract diseased GI tract Fever
Severe abdominal pain
Bloody discharge with mucous

41. E. Enteroaggregative E coli (EAEC)
Produce acute/chronic diarrhoea in persons in developing countries.
Sepsis When normal host defense is poor sepsis can happen.
Common in new born babies whose IgM level is low.

46. Treatment of E.coli related diarrhoea
1st Line
Nitrofurantoin
Nalidixic acid
Norfloxacin ABST’ SHOULD BE DONE
Ampicillin
Cotrimoxazole
2nd line
Ciprofloxacin/Ceftriaxone/Cefuroxime
Gentamicin

47. Salmonella Salmonella enterica
one species, ~2000 serovars
Non standard nomenclature
S. enterica serovar Typhimurium
or S. typhimurium
rod-shaped, non-spore-forming Gram-negative bacterium
belongs to the family Enterobacteriaceae
close relative of E. coli
Motile by peritrichous flagella (H antigen).
nonmotile exceptions: S. gallinarum and S. pullorum

48. Salmonella infections in humans
Enteric fever
typhoid and paratyphoid fevers
typhi, paratyphi A, B, C
systemic infection
infects only humans
GI symptoms may not be evident
Salmonella gastroenteritis
non-typhi serovars
zoonosis: predominantly food-borne
can be complicated by septicaemia
more common with some serovars, e.g. S. dublin (15% mortality rate when septicemic in the elderly)
Metastatic disease, e.g. osteomyelitis

49. 10 most frequently isolated Salmonella strains causing human disease
S typhimurium (22.1%)
S enteritidis (26.1%)
S enteritidis heidelberg (4.8%)
Salmonella enteritidis newport (4.3%)
Salmonella hadar (2.7%)
Salmonella enteritidis agona (2.0%)
Salmonella enteritidis montevideo (1.7%)
Salmonella oranienburg (1.6%)
Salmonella muenchen (1.5%)
Salmonella enteritidis thompson (1.5%)

50. Salmonella typhi
Scanning electron micrograph
Gram’s staining

51. Cultivation.
Colonies of S. paratyphi on Ploskirev's medium

53. Colonies of S. typhus on Ploskirev's medium

54. Colonies of Salmonella on Mac-Conkey medium

55. Colonies of Salmonella on CLED medium

56. Colonies of S. typhus on on bismuth-sulphite agar

57. Fermentative properties.

58. Antigenic Structure Kauffmann-White antigenic scheme O antigens
agglutination reactions with specific antisera against Salmonella antigens
O antigens
characteristic sequence of repeating polysaccharide units in LPS.
H antigens
flagellar antigens (protein) and may occur in one of two phase variations.
Vi antigen
a capsular polysaccharide homopolymer of N-acetyl galactosamineuronic acid

60. Toxin production.
S. typhi contains gluco-lipo-protein complexes. The endotoxin is obtained by extracting the bacterial emulsion with trichloracetic acid. This endotoxin is thermostable, surviving a temperature of 120° C for 30 minutes, and is characterized by a highly specific precipitin reaction and pronounced toxic and antigenic properties. Investigations have shown the presence of exotoxic substances in S. typhi which are inactivated by light, air, and heat (80° C), as well as enterotropic toxin phosphatase, and pyrogenic substances.

61. Infectious dose typically about 1,000,000 bacteria
much lower if the stomach pH is raised
much lower if the vehicle for infection is chocolate
protects the bacteria in their passage through the stomach
an infectious dose of about 100 bacteria

62. Clinical Features Enteric Fever
incubation period 10 to 14 days
septicaemic illness
myalgia and headache
fever
splenomegaly
leukopenia
abdominal pain
Rose spots (macular rash on abdomen)
10% fatal
positive blood, urine, and stool cultures
Sequelae: intestinal haemorrhage and perforation

63. Pathogenesis and diseases in man.
The causative agent is primarily located in the intestinal tract. Infection takes place through the mouth (digestive stage).
Salmonellae penetrate into the intestine, during which inflammatory processes develop in the isolated follicles and Peyer's patches (invasive stage).
Salmonellae enter the blood (bacteriemia stage). Here they are partially destroyed by the bactericidal substances contained in the blood, with endotoxin formation.
During bacteraemia typhoid salmonellae invade the patient's body, penetrating into the lymph nodes, spleen, bone marrow, liver, and other organs (parenchymal diffusion stage).

64. During the second week of the disease endotoxins accumulate in Peyer's patches, are absorbed by the blood, and cause intoxication. The general clinical picture of the disease is characterized by status typhosus, disturbances of thermoregulation, activity of the central and vegetative nervous systems, cardiovascular activity, etc.
On the third week of the disease a large number of typhoid bacteria enter the intestine from the bile duct. This results in the development of hyperergia and ulcerative processes. (excretory and allergic stage).
Clinical recovery (recovery stage) does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery, and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, sometimes, for life.

65. Epidemiology carrier states
carrier state may last from many weeks to years with faecal shedding
convalescent carrier
chronic carrier
~3% of persons infected with S. typhi
~0.1% of those infected with non-typhoidal salmonellae
potential for cross-contamination of foods by the infected handler
“Typhoid Mary” Mallone
but more common in textbooks than in real life

66. Typhoid Mary's real name was Mary Mallon.
Irish immigrant who made her living as a cook.
Mallon was the first person found to be a "healthy carrier" of typhoid fever in the United States.
She herself was not sick – but over 30% of the bacteria in her feces were S. typhi
Mallon is attributed with infecting 47 people with typhoid fever, three of whom died.

68. Laboratory Diagnosis
Isolated from stool, blood and urine in enteric fever (blood cultures need to be taken!) Isolation of haemoculture.
Isolated from stool in gastroenteritis
Appears as a non-lactose fermenter
on MacConkey agar or similar selective agar

69. (18-24hrs/every 2nd day for 7days) MacConkey
Blood ( ml)
BHI
Incubate 37 0C
Subculture
(18-24hrs/every 2nd day for 7days)
MacConkey
Stools and urine first should be cultured in enrichment media

70. Laboratory Diagnosis
Biochemical tests and serological tests must be done in parallel
Some other bacteria, e.g. Citrobacter, may have similar serological profiles
The isolated culture is identified by inoculation into a series of differential media and by the agglutination reaction. The latter is performed by the glass-slide method using monoreceptor sera or by the test-tube method using purified specific sera

71. LABORATORY DIAGNOSIS
1. Isolation of the bacteria
2. Demonstration of antibodies
3. Demonstration of circulating antigens
4. Blood picture

72. Isolation of bacteria - Specimens - stools/urine/blood
Blood culture - before giving chloramphenicol
- repeat the culture
% +ve 1st week up to10days
- +ve during relapses
- can culture bone marrow +ve
1-2 days after drug therapy ? good -

73. When blood culturing is done there is diluting of the drug
Stool and urine culture
Stools - during the second and third week
Urine - during the third and the fourth week, less frequently +ve than stools
Repeated culture is necessary when blood culture is -ve

74. Serological test Detection of antibodies to H and O antigens
Widal test
Antibodies appear at the end of the first week
Rises during the 3rd week of the enteric fever
Two specimens are taken at interval of 7 to 10 days

75. Interpretation of Widal test
1. Antibody appears at the end of 1st week/rises during 2nd and 3rd/steady at the 4th week then decline
2. Four fold rise of both H and O antibody in an interval of days (between 1st and the 3rd week is highly significant)

76. Commercial kits commonly used, e.g. API20
Phage typing done for epidemiological purposes
E.g. to find source of outbreak
Certain phage types predominate nationally
S. typhimurium PT4
S. enteritidis DT109

77. Salmonella vaccines
Vaccination of travellers against typhoid recommended, but does not remove need for good hygiene
Three licensed vaccines
Traditional heat-killed
very reactogenic
Vi subunit vaccine
live oral vaccine, S. typhi Ty21A
Salmonellas can act as live attenuated carriers for other antigens
So far only experimental
No vaccines for gastroenteritis

78. Enterocolitis Common manifestation of Salmonella
Common pathogen is S typhimurium
8-48 hrs after ingestion
nausea/ headache/ vomiting and profuse diarrhoea,
low grade fever Resolution 2-3 days.
Inflammatory lesions in bowel are present

79. S. typhimurium in the mouse
causes gastroenteritis in humans
causes typhoid-like disease in mice
infection can be established orally or systemically
used as model of typhoid
primary mechanisms of pathogenesis
invasion of the intestine
survival and growth in macrophages

80. Salmonella in eggs
various Salmonella serovars isolated from the outside of egg shells
S. enteritidis PT4 present inside the egg, in the yolk
vertical transmission
deposition of the organism in the yolk by an infected layer hen prior to shell deposition.

81. Sources of in infection
contaminated water
contaminated dairy products
shellfish
dried or frozen eggs
meat and meat products of infected animals
Household pets

82. Clinical features Gastroenteritis
incubation period depends on dose
symptoms usually begin within 6 to 48 hours
Nausea and Vomiting
Diarrhoea
Abdominal pain
Myalgia and headache
Fever
duration varies, usually 2 to 7 days
seldom fatal, except in elderly or immunocompromised

83. Pathogenesis Gastroenteritis
Pathogenic salmonellae ingested in food survive passage through the gastric acid barrier
invade intestinal mucosa
invasion of epithelial cells stimulates the release of proinflammatory cytokines
induces an inflammatory reaction
causes diarrhoea and may lead to ulceration and destruction of the mucosa

84. Stools remain +ve for sometime after recovery
Bacterimia rare
Blood culture -ve
Stools remain +ve for sometime after recovery
Diagnosis
1.Faeces, vomitus, food stuffs (mainly a single source)
2. Convalescent serum often agglutinates the suspension of casual serotype
Prevention and control
Sanitation
Thorough cooking
Carriers should not handle food
Vaccination not successful long-term.

85. Vibrio cholerae: 1-pure culture; 2- flagellate vibrios

91. Sheep erythrocyte hemolysis Lysis by specific O-1 subgroup phages
Vibrio
Fermentation
within 24 hrs
Sheep erythrocyte hemolysis
Lysis by specific O-1 subgroup phages
Agglutination by O-1 cholera serum
Sensitivity to polymixin B
sacharose
mannose
arabinose
Vibrio cholerae biovar cholerae A – +
Vibrio cholerae biovar El Tor
Vibrio cholerae biovar Proteus
Vibrio cholerae biovar albensis А

97. The golden rules for prevention of food poisoning
1- Choose foods processed for safety.
2- Cook food thoroughly.
3- Eat cooked food immediately.
4- Store cooked food carefully.
5- Reheat cooked food thoroughly.
Do any of you eat poisonous plants? (ask for show of hands). No one would intentionally eat a poisonous plant.
Now, raise your hand if you eat apples, peaches, or apricots (show of hands). Believe it or not, all of these are poisonous plants. What part of them do you avoid eating? The pits or seeds. And does anyone know what poison is the seeds? Cyanide. If you grind up a lot of them, enough cyanide could be produced to cause illness.
People have been poisoned by mistaking hemlock roots for turnips or carrots, mistaking lily bulbs for onions, by eating fruits like ackee before they’re ripe, or by not preparing food properly (pokeweed has to be boiled well before eating).

98. 6- Avoid contact between cooked and raw food.
7- Wash hands repeatedly. using lots of friction
W—warm
A—and
S—soapy
8- Keep all kitchen surfaces meticulously clean. Wash dishes and utensils after contact with raw meat or eggs.

99. 9- protect foods from insects,
rodents and other animals.
10- use safe water.
Keep hot food hot and cold food cold.