1. PHT313 Lecture 2 2nd Term
Dr. Hesham Radwan

2. Objectives By the end of this lecture the student must be:
A) Identify the genus of Enterobacteriaceae
B) describe the chemical tests for Family
C) Differentiate between different genuses
D) List and match the symptoms, diagnosis and treatment for different sps.
E) Recognize the differences between lactose fermenters and lactose non-fermenters

3. The Gram-negative cell wall is composed of a thin, inner layer of peptidoglycan and an outer membrane consisting of molecules of phospholipids, lipopolysaccharides (LPS), lipoproteins and sutface proteins. The lipopolysaccharide consists of lipid A and O polysaccharide.

4. Enterobacteriaceae Coliforms (lactose fermenters)
Normal inhabitants of GIT of human and animals
Source of noscomial infections
Opportunistic or cause secondary infections of wounds, urinary and respiratory tracts and the circulatory system e.g. E. coli, Klebsiella
E. coli used as biological indicator in water pollution
True pathogens (Lactose non-fermenters)
Salmonella spp., Shigella spp., Yersinia spp.
Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)

5. General characteristics of most Enterobacteriaceae
Gram negative rods
Non-spore forming
Grow in simple media
Ferment glucose and produce acid
Have peritrichous flagella and are motile
Possess a capsule

6. Media for isolation Selective differential media for enteric pathogens
MacConkey agar
EMB agar
SS agar
Selective by incorporation of dyes and bile salts
Differential by incorporation of lactose and/or Fe+3
Fe+3 is incorporated to detect H2S
Classified as lactose fermenters & non-lactose fermenters

7. The Enterotube® II contains 12 different agars enabling the performance of a total of 15 biochemical tests as well as an enclosed inoculating wire.

8. I- Escherichia coli General characteristics: Gram-negative Motile rods
Non-spore forming,Facultative anaerobic, Oxidase -ve
Ferment glucose and lactose
Normal flora of intestine
Opportunistic pathogens
E. coli may be pathogenic inside or outside Intestine
Some strains (Pathogenic) cause various forms of gastroenteritis

9. EMB
Gram Stain
MacConkey agar
Citrate
Methyl Red +ve and VP -ve
Indole

10. Virulence factors Adhesions (Colonization factors)
Pili or fimbriae & nonfimbrial factors
Host defense
Capsule
OMPs are involved in helping the organism to invade by helping in attachment and in initiating endocytosis
Exotoxin production (Enterotoxin)
Heat-Labile (LT) & Heat-Stable Toxin (ST) (ETEC)
Shiga-like toxin (Verotoxin) (EHEC)
Endotoxin (Pyrogen)
Lipid A of LPS causes fever and endotoxic shock

11. Diseases caused by E. coli
Intestinal: Diarrhea (Toxin and/or adhesion)
Enterotoxegenic E. coli (ETEC)
Enteropathogenic E. coli (EPEC)
Enteroinvasive E. coli (EIEC)
Enterohemorrhagic E. coli (EHEC)
Enteroaggregative E. coli (EAEC)
Extraintestinal:
Urinary Tract Infections [UTI] (Pili)
Neonatal meningitis (K1 antigen)
Sepsis (commonly in debilitated hospitalized patients)
Endotoxic shock
Due to lipid A (Pyrogen)
Fever and sudden hypotension
Acquired by ingestion of
contaminated food and water

12. Summary of E. coli gastroenteritis
Symptoms
Diseases
Invasion
Pathogenesis
Site
M.O.
Watery diarrhea, cramps, nausea, low grade fever
Traveler's diarrhea
Infant diarrhea
Non invasive
LT + STcAMP + cGMP  Fluid + electrolyte loss
Small intestine
ETEC
dysentery-like diarrhea, severe inflammation, fever
Dysentery
Invasive
nonfimbrial adhesin (NFA): OMP
Large intestine
EIEC
Watery diarrhea
With mucous
without blood or pus
With fever & vomiting
Infantile diarrhea
Poorly invasive
NFA: intimin EPEC adherence factor
Some reports of shiga-like toxin
EPEC
Hemorrhagic colitis with sever abdominal cramps, watery diarrhea followed by blood, no fever
HUS
Bloody diarrhoea and haemolytic uraemic syndrome
Cytotoxic shiga-like toxin (verotoxin)
EHEC
Vomiting
Abdominal pain
Without inflammation or fever
Aute and persistent diarrhoea in children and adults
Production of enterotoxin that similar to ETEC
EAEC

13. Urinary Tract Infection (UTI)
E. coli is the most common organism causing UTI
Community acquired 90%
Hospital acquired (50%)
UTI is the disease of female (Short urethra)
Fecal E. coli acquires pili to colonize mucosa of UT
Travel up urethra & infect balder (Cystitis)& sometimes move further up to infect kidney (pyelonephritis)
Symptoms:urinary frequency, dysuria, hematuria, pyuria

14. Diagnosis of UTI Specimen MSU (Mid-Stream Urine) Culture
On MacConkey agar
Gram negative, Lactose fermented (Pink colonies)
Viable count
≥100,000 (105) cfu/ml urine
IMViC

15. Neonatal meningitis E. coli is the second most common cause
S. galactaiae (Group B) is the first
Occur during the first month of life
Lab diagnosis
Specimen: CSF
Culture: Pink colonies on MacConkey agar (LF)

16. II- Salmonella General characteristics:
Gram-negative rods belonging to Enterobacteriaceae
Do not ferment lactose and H2S positive
Salmonellae live in the intestinal tracts of animals
Not considered part of normal intestinal flora in human
Always PATHOGENIC to human

17. Classification of Salmonella
Kaufman-White- Le Minor Classification
Based on O and H antigen serotyping
64 O and 114 H variants identified
Classified into 9 groups (A-I) according to O antigen
Each group can be classified into subgroups according to H Ag
Salmonella can be detected by its group O antigen and then by its type specific H antigen
>2500 known serovars

18. Classification of Salmonella
US CDC (Center for Disease Control)
S. enterica
Subdivided into 6 subspecies enterica, salamae, arizonae, diarizonae, indica, houtanae
Of these six subspecies, only subspecies enterica is associated with disease in warm-blooded animals
S. enterica subsp. enterica serovar Typhi or S. Typhi
S. enterica subsp. enterica ser.Typhimurium or S. Typhimurium
S. enterica subsp. enterica ser. Enteritidis or S. Enteritidis
Salmonella bongori (Subspecies V)

19. Species of Salmonella
Two important members of Salmonella causing diseases:
Salmonella causing enteric fever
Salmonella Typhi or Salmonella Paratyphi A,B, and C
These organisms penetrate intestinal mucosa
Detected in blood, urine and stool
Salmonella causing food poisoning
Salmonella Enteritidis and Salmonella Typhimurium
These organisms do not penetrate intestinal mucosa
Detected in stool only

20. Enteric Fever “Typhoid”
Enteric fevers are severe systemic forms of salmonellosis.
Caused by S. typhi whereas a milder form “Paratyphoid” caused by S. paratyphi A, B or C
Mode of Transmission
Via fecal-oral route through fecally-contaminated food or water from either chronic carrier or case
A few individuals continue to harbour Salmonella in their gall-bladders and intermittently excrete organisms in their faces
Temporary excretors: Patients who excrete bacteria for <year
Chronic carrier: Patients who excrete bacteria for > year
Incubation Period
10-14 day during which bacteria multiplies in Peyer’s patches
Pass to blood via lymphatics resulting in bacteriaemia in 1st week
In 2nd week M.O. passes to different organs including peyer’s patches causing ulcers, gall bladder, liver, kidney & rarely menings

21. Symptoms of Enteric Fever
Symptoms begin after an incubation period of 2 weeks
Enteric fevers may be preceded by gastroenteritis, which usually resolves before the onset of systemic disease
The symptoms of enteric fevers are nonspecific and include
Fever, headache, delirium (sustained fever), malaise and tender abdomen
Complications include intestinal hemorrhage and perforation

23. Laboratory diagnosis Direct diagnosis:
Specimen: Blood during 1st week, urine during 2nd week and stool during 3rd week
Isolation of microorganism:
From blood using blood culture
Five to 10 ml of blood is taken during the 1st week of infection,
Add to ml sterile nutrient broth and incubate at 370C for 24 hrs.
Subculture is done on MacConkey's agar which shows colorless colonies in positive case

24. Laboratory diagnosis From stool
By culture on enrichment medium such as selenite F or tetrathionate broth which inhibits the growth of coliform and allow the growth of Salmonella and Shigella
Subculture on MacConkey, SS or DCA agar(Deoxycholate Citrate Agar)
On MacConkey's agar they give colorless colonies
On SS agar they give colorless colonies with black edges due to H2S production
Biochemical Reactions:
The suspected colonies were subjected to biochemical reactions
Oxidase negative, not ferment lactose and sucrose, H2S positive

25. Laboratory diagnosis Indirect: Serological diagnosis (Widal Test):
In the 2nd week of the disease, antibodies against Salmonella are present in the patient's serum and can be detected serologically by Widal test (agglutination test)
Widal test is positive and valid during 2nd week
Serial dilutions of patient's serum are added to an equal volume of common O and specific H antigens
Agglutination of O- antigen and one only of the H-antigens at a titer 1/80 or above is diagnostic

26. Salmonella causing food poisoning
The causative agent : S. typhimurium & S. enteritidis
Mode of infection:
Consumption of contaminated food
Food as cakes, pastries and various milk and egg dishes
Cattle, sheep, hens, ducks and turkeys are often infected and the organism may contaminate meat and meat products
Infective dose 100,000 bacteria
Gastric juice is the an important host defense and decreased acidity is a predisposing factor
Incubation period is hrs and recovery within 4-7 days
It is self limiting disease
Manifestations include
Nausea, vomiting, and abdominal discomfort, non-bloody diarrhea and slight fever

27. I- Shigella General Characteristics Gram negative rods Non motile
Non spore-forming
Non capsulated
Oxidase negative
Ferment glucose with acid only
Non lactose fermentating organism
H2S negative

28. Shigella species Disease: Bacillary dysentery (Shigellosis)
S. sonnei is the most common, followed by S. flexneri
Mode of Transmission
Oral-fecal transmission
<200 bacilli are needed for infection in health individuals
Incubation periods
It varies between 1-3 days
Symptoms
Ranges from asymptomatic to severe bacillary dysentery
Watery diarrhea changing to dysentery with frequent small stools with blood, pus and mucus
Fever, tenesmus and abdominal cramps

29. Stages of shigellosis Early stage:
Ingestion of contaminated food or water
Noninvasive colonization and cell multiplication
Production of the enterotoxin in the small intestine
Watery diarrhea attributed to enterotoxic activity of Shiga toxin (similar to LT of ETEC)
Fever attributed to neurotoxic activity of toxin
Second stage:
Adherence to and tissue invasion of large intestine
Typical symptoms of dysentery
Cytotoxic activity of Shiga toxin increases severity

30. Laboratory identification
Specimen:
Stool (mucous bloody part of stool) or rectal swap
Stool culture
Specimen is inoculated in Selenite broth at 370C for 24 h
Then subculture on MacConkey or SS
On MacConkey agar: they give colorless colonies
On SS agar: they give colorless colonies
Gram stain
Gram negative bacilli, NON MOTILE
Biochemical reactions
Oxidase negative, ferment glucose, non ferment lactose and H2S NEGATIVE

31. COMMENSAL ENTEROBACTERIAE
Opportunistic pathogens and are common cause of nosocomial infections
Klebsiella
Enterobacter
Serratia
Proteus
Morganella
Providencia
Lactose Fermnters
Non-Lactose Fermnters

32. Klebsiella-Enterobacter-Serratia group
General characteristics:
These organisms are very similar
All are motile except Klebsiella
MR negative; VP positive, Lactose Fermenter
Simmons citrate positive
H2S negative
Some weakly urease positive
Phenylalanine deaminase negative
These organisms belonging to Enterobacteriaceae
Frequently in large intestine but also found in soil and water
Usually opportunistic pathogens
Wide variety of infections: primarily pneumonia, wound and UTI

33. Klebsiella pneumoniae
Virulence factors
Polysaccharide capsule
Protects against phagocytosis and antibiotics
Makes the colonies moist and mucoid
Adhesions
Clinical significance
Major cause of nosocomial infections
Nosocomial Pneumonia
Important respiratory tract pathogen outside hospitals
3% of bacterial pneumonia
Bloody sputum (50%) (Thick, Jelly and Red Sputum)
Septicemia, Meningitis and UTI

34. Proteus-Providencia-Morganella
These organisms belonging to Enterobacteriaceae
All are normal intestinal flora but also found in soil and water
Opportunistic pathogens
Non-lactose fermenters
Phenylalanine deaminase and urease positive
Urease positive after 2-6 hrs (urea → NH3+ CO2)
All are highly motile
Proteus sp. swarming on blood agar
Swarming characterized by expanding waves (rings) of organisms over the surface of blood agar

35. Proteus species
P. mirabilis and P. vulgaris are widely recognized human pathogens
Isolated from urine, wounds and ear and bacteremic infections
They found in colon and able to colonize urethra especially in female
P. mirabilis causes urinary tract infections (UTI):
Proteus sp. produces urease which alkalinizes urine →precipitation of calcium and magnesium salts → stone formation →renal epithelium damage
P. vulgaris causes nosocomial infections (pneumonia, bacteremia) and UTIs

36. Laboratory diagnosis Specimen: Culture:
Urine or Stool.
Culture:
On MacConkey agar →non lactose fermenters→ colorless colonies
On SS agar →non lactose fermenters → colorless colonies with black center
On ordinary media, such as nutrient agar, blood agar, show swarming (successive waves on the surface) due to high motility of Proteus
The Weil-Felix test which is an antigen antibody reaction
Biochemical reactions: Urease , phenyldeaminase and H2S positive