1. Gram-Negative Rods

3. General Classification
Based on source or site of infection
1. Enteric tract
2. Respiratory tract
3. Animal sources

4. Source or site of infection
Enteric tract
Primarily within:
Shigella, vibrio, Campylobacter
Both within and outside:
Escherichia, Salmonella
Enteric but outside only:
Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Morganella, Pseudomonas, Bacteroides

5. Source or site of infection (continued)
Respiratory tract:
Haemophilus, Legionella, Bordetella
Animal sources:
Brucella, Francisella, Pasteurella, Yersinia

6. Classification Based on morphology, biochemical traits and genetic (phylogenetic) relationship
Cocobacilli
Vibrionaceae
Pseudomonaseae
Enterobacteriaceae

7. Enterobacteriaceae A heterogenous family
Mostly found in colon of human and other animals
Different pathogenetic mechanisms
Facultative anaerobic
Glucose fermentation
None have cytochrome oxidase
Reduce nitrates to nitrites

8. Most important genuses in Enterobacteriaceae family
Escherichia
Shigella
Salmonella
Klebsiella
Enterobacter
Serratia
Proteus
Yersinia

9. In contrast to Enterobacteriaceae
Pseudomonaceae
Gram negative rods:
Non-fermenting (Strict aerobic)
Not reduce nitrate
Oxidase-positive

10. General structure of cells in Enterobacteriaceae
All have Endotoxin
Some have Exotoxins, mostly called enterotoxins
Three surface antigens:
O antigen: outer polysaccharide portion of the lipopolysaccharide (repeating 3-4 oligosaccharide sugars times). A basis for the serologic typing (about 2000 types of Salmonella and 150 types of E. coli).
H antigen: on the flagellar protein (in E.coli and Salmonella and not in Klebsiella and shigella). Unusual H antigens in Salmonella called phase 1 and phase 2. The organism can reversibly change in antigenicity to evade the immune response.
K polysaccharide antigen: In encapsulated organisms such as Klebsiella. Identified by quellung (capsular swelling) reaction in the presence of specific antisera used for epidemiologic purposes. In S. typhi, it is called Vi (or virulence) antigen.

11. Quellung (capsular swelling) reaction

12. Coliforms E. coli Enterobacter Klebsiella Citrobacter
That part of this family which are normal inhabitants of the colon:
E. coli
Enterobacter
Klebsiella
Citrobacter
So, E.coli is the indicator for
fecal contamination of water supply:

13. Coliforms
Lactose fermentation, Acid and gas production, growth at 44.5 C and typical colony on EMB.
4 colony count per dL in drinking water is indicative of unacceptable fecal contamination.

14. Antibiotic therapy
Must be individually tailored to the antibiotic sensitivity test (Antibiogram).
Penicillin and cephalosporin families. Aminoglycosides (Gentamicin, amikacin, kanamycin, streptomycin …), Chloramphenicol, tetracyclines, quinolones and sulfonamides.

15. Laboratory diagnosis Culture for isolation
Suspected specimens are inoculated onto 2 media: 1. Blood Agar 2. A selective differential medium (MacConkey’s agar or Eosin-methylene blue, EMB agar. The differential ability is based on lactose fermentation as the most important criterion in identification of these organism. Non lactose fermenters form colorless colonies. Selective effect is exerted by bile salts or bacteriostatic dyes.

17. Laboratory diagnosis (continued)
Culture for identification
Screening biochemical tests for a final definitive identification:
Array of 20 or more biochemical tests to identify the species.

18. Serology
Usually in Salmonella, Shigella and E.coli the final detection is by serotyping using agglutination Ag+Ab test.

20. Triple Sugar Iron Agar Almost enough to identify the genus
Indicator : Phenol red
Components:
Iron or Ferrous sulfate
FeSO4 (ferrous sulfate) + Solfate reductase  SH2  Sodium tiosufate
Ferrous sulphide (FeS)
Black FeS indicates the production of SH2
3 sugars: glucose (0.1%), lactose (1%), and sucrose (1%)
Pepton and yeast extract

24. Different observations for TSI
Reactions
Slant
Button
Gas
H2S
Representative genera
Acid + -
Escherichia, Enterobacter, Klebsiella
Alkaline
Shigella, Serratia
Alkalin
Salmonella, Proteus
Pseudomonas
Kligler Iron Agar can be used as alternative with two sugers: glucose & lactose

25. Lysine iron Agar (LIA) The indicator: bromocresol purple.
Components: Lysine, Glucose, FeS
Bacteria that anaerobically decarboxylate lysine  alkaline reaction (purple colour) in butt.
Not decarboxylation  alkaline slant and an acid butt.
Deamination the lysine  red slant and acid butt.
Hydrogen sulphide  blackening in
the medium

26. Lysine iron Agar (LIA)

28. Urea Agar Indicator: Phenol red Component : Urea
If the bacterium Produces urease:
Urea (NH2)2CO is hydrolyzed to NH3 and CO light orange changes to reddish purple (in Proteus and K pneumoniae

29. Amonium citrate (Simmons Citrate)
Indicator: Bromothymol blue
If the bacterium can utilizes ammonium dihydrogen phosphate (a salt of ammonium) and sodium citrate as sole source of nitrogen and carbon, the indicator turns to blue at alkalin pH due to releasing ammonia.

30. Motility SIM medium: SH2, Indole, Motility Proteus: Swarms
Differentiation between Enterobacter cloacae (motile) from Klebsiella pneumoniae (Non motile)

31. Indole (in SIM)
Tryptophan + Tryptophanase  Deamination  Intermediate products: Indole ….
Detection: 5 drops Kovac’s reagent (contains para-dimethyl-amino-benzaldehyde = PDAB) is added  Benzyl pyrol  Red ring

32. MR-VP 1. Methyl Red MR-VP medium Components: Glucose, Phosphate
After 48 hours  Methyl-red (1 droplet to 1 ml of medium) :
If pH < 4.4  Mixed acidic fermentation (Red color).
If pH ≥ 5  Butylen glycol fermentation (Yellow color).

33. MR-VP 2. Voges Proskauer Butylen glycol fermentation
MR-VP medium: After hours  Alcoholic alpha naftool (15 droplets to 1 ml medium) + KOH 40% (10 droplets) (may come along cratin)  min:
Red (If Acetoin “Acetyl methyl carbynol” exists.)
No color change (If no Acetoin exist)

34. API 20E system
A plastic strips consist of 20 small wells containing dehydrated media components
The bacterium is suspended in sterile saline and added to each well.
The strip is incubated for hours.
The colour reactions are noted as either positive or negative.
The test results can be entered into a computer programme to identify the bacterium.

35. API 20E system

37. Escherichia coli Diseases 1. Diarrhea or dysentery 2. UTI
3. Sepsis (The most common cause among negative rods)
4. Neonatal meningititis (One of the 2 important agents. The other is the group B streptococci due to colonization of vagina by these organisms in about 25% of pregnant women ).
5. Nosocomial infection

38. Neonatal meningititis
UTI
Sepsis
Neonatal meningititis

39. Escherichia coli

41. Virolence factors: Pili Capsule Endotoxin
Two exotoxins (enterotoxins).

42. Pathogenesis
E. coli attaches to the surface of jejunum and ileum by Pili  Bacteria synthesize enterotoxins (exotoxins determined by plasmids)  Diarrhea
The toxins are strikingly cell-specific. Cells of colon are lack of receptors for the toxins.

43. E. coli pili (fimbriae)
mannose
galactose
glycolipids
glycoproteins

44. Most important sub species of pathogenic E. coli
Enteropathogenic E. coli (EPEC)
Enterotoxigenic E. coli (ETEC)
Enteroinvasive E. coli (EIEC)
Enterohemorrhagic E. coli (EHEC)

45. Enteropathogenic E. coli (EPEC)
destruction of surface microvilli
Gut lumen
fever
diarrhea
vomiting
nausea
non-bloody stools
Diarrhea is self-limited and short duration (1-3 days)

46. Enterotoxigenic E. coli
Travellers diarrhea
Diarrhea like cholera but milder
Diarrhea is self-limited and short duration (1-3 days)

47. Enterotoxigenic E. coli (ETEC)
Heat labile toxin (LT)
like choleragen
Activation of Adenylate cyclase
Cyclic AMP concentration
Secretion water/ions (potassium and chloride)
Heat stable toxin (ST)
Activation of Guanylate cyclase
Cyclic GMP concentration
Uptake water/ions (Sodium and Chloride)

48. Enterohemorrhagic E. coli (EHEC)
Produce verotoxin which works like Shiga toxin
Hemorrhagic
bloody, copious diarrhea
few leukocytes
Hemolytic-uremic syndrome
thrombocytopenia (low platelets)
hemolytic anemia
kidney failure

49. Enterohemorrhagic E. coli
Usually O157:H7
Flagella
Transmission electron
micrograph

50. Enteroinvasive E. coli (EIEC)
Very similar to shigella species
(in biochemical and morphological traits)
Invades to epithelial mucosal cells
Cause enteric inflammation
Non lactose fermentative
Non motile

51. E. coli Transmission By:
Meat products or sewage-contaminated vegetables

52. UTI The most common agent for UTI and nosocomial UTI.
(Cystitis, pyelonephritis): fever, chills, flank pain
Occurs primarily in women

53. Systemic infection Capsule and endotoxin play a prominent role
Capsular polysaccharide interferes with phagocytosis (Serotype having K1 causes neonatal meningitis).
LPS during sepsis causes fever, hypotension and disseminated intravascular coagulation.

54. Treatment Antibiogram for most infections
A combination of ampicillin and gentamicin in neonatal meningitis
Rehydration for diarrhea

55. Prevention No passive or active immunization
Prompt withdraw of catheters and intravenous lines
Caution regarding uncooked food and unpurified water while traveling.

56. Klebsiella Klebsiella ozaenae
- Ozena (the atrophy of nose with bad smelling)
K. rhinoscleromatis
- Rhinoscleroma (A granulomatosis in nose and pharynx)
Klebsiella penomoniea
- 5% in upper respiratory and GI systems
- Nosocomial infection
Klebsiella oxytoca

62. Lab detection
Large mocoidal colonies.
Lac pos.
Not motile

63. Proteus P. mirabilis UTI (Alkalic environment in urinary tracts)
due to urease  kidney stone
P. vulgaris
Nosocomial infection

66. Shigella Species: S. dysenteriae S. sonnei S. flexeneri S. bouedi
There are more than 40 shigella serotypes.
Important properties
Non lactose fermenting
Distiguishable from Salmonella by: no gas, no H2S, nonmotile.
Having an enterotoxin called Shiga toxin

68. Shigellosis Only a human disease
Transmitted from person to person by asymptomatic carriers (oral-fecal transmission)
4 F’s – fingers, flies, food, feces
Food-born outbreaks outnumber water-born outbreaks by 2 to 1.
In mental hospitals and day-care nurseries
Children <7 accounts for half of shigella positive stool culture

69. Shigella disentery type 1 (Shiga bacillus): - Labile Exotoxin
(effective both on intestine and CNS works like verotoxin in E.coli)

70. Pathogenesis Exclusively in gastrointestinal tract
Bloody diarrhea (dysentery): Invading the mucosa of the distal ileum and colon.
Local inflammation accompanied by ulceration occurs
The organisms rarely penetrate the wall or enter the bloodstream unlike salmonellae.

72. Invasion to epithelial cells (M cells)  spreading to next cells  microabcess formation in the wall of colon and distal ileum  necrosis of mocusal layer  ulcer  bleeding  psudomemberane.

74. Clinical findings Incubation period: 1-2 days
Symptoms: Fever, abdominal cramps, followed by diarrhea (watery at first but later contains blood and mucus).
Mild or severe disease depending:
- The species of Shigella
- The age of the patient

75. Most invasive species S. dysenteriae causes the most severe disease
S. sonnei causes mild disease but more frequent

76. Shiga toxin neurotoxic effects
Shiga toxin CNS abnormalities can include
lethargy
disorientation
paralysis
coma
Neurotoxicity occurs most often in children and the elderly and is often fatal.

77. Clinical findings
Resolves in 2-3 days but antibiotic can shorten the course.
Serum antibodies appear after recovery but are not protective.
Most patients spread bacteria only short time after recovery but few people stay chronic carriers.
Most shigellosis cases are in children less than 10 years.

78. Treatment In 50% cases self recovery is in 2-5 days.
The main treatment: Fluid and electrolyte replacement.
No antibiotic in mild cases
Antibiogram test: Trimethoprim - sulfamethoxazole or Ampiciln.

79. Prevention
Interruption of fecal-oral transmission by proper sewage disposal, chlorination of water and personal hygiene.
No vaccine
Antibiotic prophylactic is not recommended.

80. Lab diagnosis Samples: feces, rectal swabs
Culture and biochemical tests: Non-lactose fermenter
Serology tests:
Slide agglutination to detect its species

81. Lab diagnosis Invasive enteric infections:
Shigella, Salmonella or Campylobacter
Toxin-producing organism:
V. cholerae, E. coli, Clostridium perfringens or certain viruses or Entamoeba histolytica
Methylene blue stain of a fecal sample to determine whether PMNs are present.

86. Salmonella Important properties Not lactose - fermentive
Produce H2S, Gas, motile

90. Salmonella nomenclature
Is complicated.
Currently there are two recognized species:
S. enterica and S. bongori
S. enterica has 5 main subspecies.
S. enterica enterica is relevant with human infections (specific or zoonotic).

91. Naming the salmonella S. enterica enterica: S. typhi
S. paratyphi (A, B, C…)
S. typhimurium
S. choleraesuis
S. enteritidis (1500 serotypes)

92. Diseases Enterocolitis (S. typhimurium & S. enteritidis)
Enteric fever (typhoid fever) (S. typhi and S. paratyphi)
Septicemia with metastatic abscesses (S. choleraesuis)

93. Diseases
Enterocolitis (S. typhimurium & S. enteritidis) An invasion of epithelial and subepithelial tissue of small and large intestines. Penetration both through and between the mucosal cells: Inflammation and diarrhea. PMN response limits the infection to the gut and the adjacent mesenteric lymph nodes. The dose of Salmonella required: at least while for Shigella: 103 organisms.

94. Clinical findings of Enterocolitis
Incubation period: 6-48 hours Symptoms: Nausea, Vomiting, Abdominal pain and diarrhea with or without blood The disease is self-limited. Treatment only in very young and very old. S. Typhimurium & S. enteritidis : the most common cause of enterocolitis.

95. S. typhimurium colonies

96. Typhoid or Enteric fever
Typhoid or Enteric fever caused by S. typhi and S. paratyphi (A, B and C).
The illness is slow, with fever and constipation rather than vomiting and diarrhea.
After the first week, bacteremia becomes sustained. High fever, tender abdomen, and enlarged spleen occur.

97. Typhoid (Enteric fevers)
Infection begins in small intestine but few gastrointestinal symptoms occur.
The organisms multiply in the mononuclear phagocytes of peyer’s patches, then spread to the phagocytes of the liver, gallbladder and spleen leading to bacteremia and then fever.

98. Typhoid (continued)
Rose spot (rose-coloured papules) on the abdomen are associated with typhoid fever but occur only rarely.
The disease begins to resolve by the third week but intestinal hemorrage or perforation can occur.
3% of typhoid fever patients become chronic carriers. The carrier rate is higher among women.

100. Septicemia S. choleraesuis: most often cause septicemia. Symptoms:
Fever
Little or no enterocolitis
Focal symptoms: bone, lung, or meninges.

101. Septicemia (S. choleraesuis)
Accounts for only about 5-10% of Salmonella infections and occurs:
More common in patients with chronic disease or children with enterocolitis.
It leads to seeding of many organs commonly: osteomyelitis, pneumonia, and meningitis

103. Typhoid vaccine Two types: Live vaccine
Subunit vaccine (a vi capsular polysaccharide vaccine)

104. Transmission of salmonella
Ingestion of food and water contaminated by human and animal wastes.
S. typhi, transmitted only by humans, but other species have a significant animal reservoir.
Human sources:
1. Temporarily excrete the organism during or shortly after enterocolitis
2. Chronic carriers

105. Transmission
The most frequent animal source is poultry and eggs, but meat products that are inadequately cooked have been implicated as well.
Dogs and other pets including turtles are additional sources.

106. Lab. diagnosis Enterocolitis: isolated from stool
Enteric fever: blood culture during first 2 weeks of illness.
Septicemia: Blood culture

107. Lab. diagnosis MacConkey, EMB, Hekton Enteric agar, XLD, Endo agar
Lactose -, other biochemical tests
Gas and H2S (S. type: no gas)
Serological tests by their O, H and Vi antigens
Serological detection for Ab if culture is negative: Vidal test

108. Treatment Enterocolitis: Self-limited.
Fluid and electrolyte replacement.
Antimicrobial agents are indicated only for neonates or persons with chronic disease who are at risk of septicemia and disseminated abscesses.

109. Treatment Enteric fever and Septicemia: Ampicillin or chloramphenicol
Ampicillin: in patients who are chronic carriers of S. typhi.
Cholecystectomy may be necessary to abolish the chronic carrier state.
Focal absesses should be drained.

110. Prevention Public health and personal hygiene measures.
Proper sewage treatment
A chlorinated water supply
Cultures of stool samples from food handlers
Two vaccine confer protection against S. typhi but no common
Proper cooking of poultry and meat
Pasteurization of milk

111. Genus: Yersinia Species: Yersinia pestis and Yersinia enterocolitica
Small gram negative bacillus, bipolar staining (like a safety pin)
Capsule in freshly isolated organism but lost with passage
Plague or black death
None motile
Disease

112. Virulence factors
Capsule antigen (F-1) which protects against phagocytosis.
Endotoxin
Exotoxins (block beta adrenergic receptors)
Coagulase
Fibrinolysin
Pesticin I (a bacteriocin)
V antigen protein
W antigen protein

113. Pathogenesis Urban cycle
Transmission of the bacteria among urban rats with the rat flea as vector to human.
This cycle predominates during times of poor sanitation, eg. Wartime, when rats proliferate and come in contact with the fleas in the sylvatic cycle.
Sylvatic cycle
Humans are accidental hosts and cases of plague occur as a result of being bitten by a flea that is a part of the sylvatic cycle.

116. Event within the flea
The flea ingests the bacteria while taking a blood meal from a bacteremic rodent.  The blood clots in the flea’s stomach owing to the action of the enzyme coagulase made by the bacteria  The bacteria are trapped in the fibrin and proliferate to large numbers.  The mass of organisms and fibrin block the proventriculus of the flea’s intestinal tract. During its next blood meal the flea regurgitates the organisms into the next animal  Because the proventriculus is blocked, the flea gets no nutrition.  becomes hungrier  loses its natural host selectivity for rodents  more readily bites a human  The bacteria inoculated by bite spread to the regional lymph nodes

117.  become swollen and tender called buboes and this plague is called bubonic plague. The organisms can reach high concentrations in the blood and disseminate to form abscesses in many organs including lungs.  The endotoxin-related symptoms, including disseminated intravascular coagulation and cutaneous hemorrhages, probably were the genesis of the term ‘black death’.

118. Respiratory droplet transmission
Respiratory droplet transmission of the organism from patients with pneumonic plague can occur.

119. Clinical findings
Bubonic plague, is the most frequent form, begins with pain and swelling of the lymph nodes draining the site of the flea bite and systemic symptoms such as high fever, myalgia, and prostration.
The buboes are an early characteristic finding.
Septic shock and pneumonia are the main life-threatening subsequent events.

121. Epidemiology
Endemic in the wild rodents of Europe and Asia for thousands of years.
99% of cases of plague occur in Southeast Asia.
Enzootic (sylvatic) cycle consists of transmission among wild rodent by fleas.
Rodents are relatively resistant to disease.
Humans are accidental hosts and cases of plague occur as a result of being bitten by a flea that is a part of the sylvatic cycle.

122. Pneumonic plague can arise either from inhalation of an aerosol or from septic emboli that reach the lung.
Untreated bubonic plague is fatal in approximately half of the cases.
Untreated pneumonic plague is invariably fatal.

123. Lab. diagnosis
The best procedure: Smear and culture of blood or pus from the bubo.
Giemsa’s or Wayson’s stain reveals the typical safety-pin appearance of the organism better than does Gram’s stain.
Fluorescent-antibody staining can be used to identify the organism in tissues.
A rise in antibody titer to the capsule antigen can be useful.

125. Treatment
The treatment of choice is a combination of streptomycin and tetracycline.
Due to the rapid progression of the disease, treatment should not wait for the results of the bacteriologic culture.
Incision and drainage of the buboes are not usually necessary.

126. Prevention

127. Prevention Controlling the spread of rats in urban areas.
Preventing rats from entering the country by ship or airplane.
Avoiding flea bites
Avoiding contact with dead wild rodents.
A patient with plague must be placed in strict isolation (quarantine) for 72 hours after antibiotic therapy is started.

128. Only close contacts need to receive prophylactic tetracycline.
There is no vaccine for citizens normally. But a killed organism vaccine protecting bubonic but not pneumonic plague was used by USA forces during Vietnam wars.

129. Yersinia enterocolitica
Motile: flagella are present at 22 c.
No capsule
Primarily a zoonotic disease (cattle, deer, pigs, and birds(
Yersiniosis : Incubation 1-2 days.
Acute Y. enterocolitica infections produce severe diarrhea in humans, along with Peyer's patch necrosis, chronic lymphadenopathy, and hepatic or splenic abscesses. Fever and right-sided abdominal pain.

130. Yersinia enterocolitica

131. Lab diagnosis
Culture: Cold enrichment
Culture on blood
Sample: Feces

132. Y. pseudotuberculosis
Motile: flagella are present at 22 c., No capsule. Culture on blood.
Primarily a zoonotic disease (cattle, deer, pigs, and birds).
In animals, can cause tuberculosis-like symptoms, including localized tissue necrosis and granulomas in the spleen, liver, and lymph node.

133. In humans, symptoms of Pseudotuberculosis (Yersinia) are similar to those of infection with Yersinia enterocolitica (fever and right-sided abdominal pain), except that the diarrhea is often absent.