Enterobacteriaceae

Family
Enteric Gram Negative
Rods

1
 Enterobacteriaceae
Natural habitat:
• The intestinal tract of humans & animals
• About 32 genera, contains more than 130 spp.
• Of these, about 14 genera are medically important
• Some, e.g. E. coli, are part of the normal flora & incidentally
cause disease,
• Salmonella & Shigella - regular pathogens for humans
Morphology:
• Gram negative, facultative anaerobic bacilli,
• Non-motile or motile by peritrichous flagella;
• Non-sporing, some encapsulate
2
 Enterobacteriaceae
Toxins:
Endotoxins
• O Ags / LPS consist sugars & lipid A.
• Present in the cell wall of GN bacilli, responsible for
many pathological effects during human infection

Exotoxins
• Proteins liberated extracellularly from the intact
bacterium by a few spp, e.g. S. dysenteriae; toxignic
strains of E. coli.

3
 Enterobacteriaceae
Biochemical tests
– Biochemically active & ferment many carbohydrates
a.Glucose fermentation:
– Produce lactic, formic & acetic acids
– Some spp produce H2 & CO2 gas during glucose ferm
b. Lactose fermentation:
– Used to separate into LF/NLF
– Differential & selective media: for separation of group
• MacConkey (contains bile salts to inhibit G +ve)
• LF develop pink coloration in isolated colonies.

4
 Enterobacteriaceae
• Other biochemical tests employed include:
– H2S production
– Indole formation from tryptophan
– Use of citrate as a carbon source

5
 Enterobacteriaceae
Pathogenicity:
• All Enterobactriaceae should be considered potentially
pathogenic.
• Patients with underlying disease, immunosuppression,
mechanical or medical manipulation & other forms of
debilitation are susceptible to infection.
• All Entrobactriaceae release LPS as they die - result in
endotoxin shock in humans.
• The lipid A moiety of LPS is responsible for most of the
symptomatology associated with endotoxin shock.

6
 Enterobacteriaceae
Effects of LPS
1. Fever: occurs within 30 min of exposure to LPS.
2. Hypotension:
– Small doses of LPS cause decrease in BP in 30min
– Large doses are lethal – (cause fatal hypotension).

7
 SALMONELLAE
• Predominantly animal pathogens cause disease in man.
• The natural habitat is animal gut.
• Food stuffs from animal sources are important vehicles
in the transmission of infection.
Classification
• There are 2 spp - S. enterica & S. bongori.
• 4 [S. typhi, S. paratyphi A, B (S. schottmulleri) & C]
cause enteric fever in man, Eg. S enterica serovar Typhi

8
 Salmonellae
• Other spp cause salmonella food poisoning or
enterocolitis - e.g. S. typhimurium and S. enteritidis
• Salmonella typhi and paratyphi A  - strictly human
serovars that may cause  diseases often associated with
invasion of the blood stream

9
 Salmonellae
Morphology:
– Gram-negative bacilli, motile.
Cultural characters:
– Facultative anaerobes; grow on simple media.
– Selective: SS
– On MacConkey - produce pale NLF colonies.
– On Wilson & Blair's bismuth sulphite agar - produce
black metallic colonies due to H2S production.

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 Salmonellae

Salmonella
11
 Salmonellae
Antigenic structure:
– They possess O and H Ags.
– There’s sharing of O Ag b/n S. typhi, S. paratyphi A, B, C
Pathogenesis & Diseases caused by Salmonellae:
• They cause a variety of conditions (salmonellosis).
1. Enteric fever
2. Enterocolitis (food poisoning)
3. Bacteremia and septicemia.

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 S. Typhi: Typhoid fever
I. Typhoid fever (Enteric fever)
– Produced by S. typhi, S. paratyphi A, B & C.
– Source of infection: stool or urine of cases or carriers.
– Infecting dose is about 105 organisms
– The organism enters the body by the oral route in
contaminated food or drinks, e.g. raw vegetables, fruits,
raw shell fish & milk products.
– After ingestion, the organism multiplies, passes through
the lymphatics to the blood stream causing bacteremia
that persist for 1 week.
– It then disseminates to the kidney, excreted in urine.

13
 Typhoid Fever
– To the liver & is excreted in bile to reach the intestine.
– It rarely reach the lungs or meninges.
Clinical features:
– Incubation period: 10 -14 days
– Fever, malaise, headache, constipation & myalgia with
enlargement of the liver & spleen, also rose spots in
light colored skin. The fever is a gradually rising
– Patients become stuporous and may go into coma
– Mortality rate: 10 -15%, but now reduced to <1%.

14
 Typhoid Fever
– After recovery some individuals continue to harbour
salmonella in their tissues (gall bladder or urinary tract)
as convalescent or chronic carriers, which intermittently
excrete it in the stools / urine.
– Such individuals should not work as food handlers.

15
 Typhoid Fever
Diagnosis of enteric fever:
• Diagnosis depends on the stage of the disease by:-
1. Isolation of the organism from blood, urine & stools
2. Detection of Abs in the serum of the patient.
I. During the 1st week:
Isolation from blood:
• The organism is found in the blood during the 1st week.
A blood culture is done by adding 5-10ml of blood to
50-100ml. of bile salt broth which is incubated at 37oC.
Subcultures are made on MacConkey.

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 Typhoid Fever
• NLF colonies are further identified by morphology,
biochemical activities & serologic typing by slide
agglutination with anti salmonella sera.
• The blood culture may have to be repeated to increase
the chance of isolation of the organism.
• The organism disappears from the blood shortly after
chloramphenicol therapy.
II. In the second week onward:
Isolation from stools:
• Organism found in stools throughout the course of illness
but frequent during the 2nd & 3rd weeks of illness.

17
 Typhoid Fever
• Repeated stool exam should be done.
• Stool sample inoculated into test tubes of selenite &
tetrathionate broth
• After incubation of 1-2 days, this is placed on differential
& selective media, e.g. MacConkey, SS agar.
• Any pale NLF colonies are picked & identified by
biochemical reaction patterns and slide agglutination
tests with specific sera.
Isolation from urine:
– The organism appears in urine from 2nd wk onwards

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 Typhoid Fever
– Its appearance may be intermittent, hence isolation
from urine needs repeated examination.
– The urine sample is centrifuged & the deposit is
inoculated on MacConkey.
– Pale colonies are identified as mentioned above.

2. Serological diagnosis:
– serological techniques are used to identify unknown
cultures with known sera & may also be used to
determine antibody titre in pts with unknown illness.

19
 Typhoid Fever
a. Rapid slide agglutination test:
– known antisera & unknown culture are mixed on a slide
& the mixture observed under microscope.
– clumping, when it occurs, can be seen within few mins
b. Tube agglutination test (Widal test):
– Abs to salmonella appear in the serum of the patient
during the second week of illness
– Serial dilutions of the patient's serum (1/20,1/40,1/80
etc) are made in test tubes.

20
 Typhoid Fever
• 5 sets of tubes are made; to each set salmonella
suspension is added
• The tubes are incubated, then examined for presence of
agglutination
• The highest dilution of serum which gives agglutination is
the end titre.

21
 Typhoid Fever
Interpretation of the Widal test:
• For proper interpretations, 2 serum samples separated
by 7-10 days interval should be tested.
• The detection of a "rising titre" in the 2nd serum sample
indicates active enteric fever.
The results are interpreted as follows:
1. Rising titre of O > 1:160 suggests that active infection
present, as anti-O Abs disappear faster than anti-H Abs.
2. High titre of H >1:160 suggests past immunization or
past infection. O suspension is not agglutinated in this
case

22
 Typhoid Fever
3. Recently vaccinated individuals will possess agglutinins
to S. typhi & S. paratyphi.
4. If the test is done during the first week it gives false
negative results as the antibodies start to appear during
the second week & on ward.
5. If the patient received antibiotic treatment early in the
disease, the antibody titre will be suppressed. This is due
to reduction of the antigenic mass.

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 Typhoid Fever
Prevention:
1. Sanitary measures must be taken to prevent
contamination of food and water by the organism.
2. Carriers must not be allowed to work as food handlers.

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 Typhoid Fever
3. Avoiding risky foods and beverages
4. Vaccine for enteric fever.
– Two vaccines are available:
• Contains killed Salmonella typhi bacteria
• Vaccine taken by mouth containing a live but
weakened strain of the bacteria
Treatment:
• The drugs of choice are chloramphenicol, ampicillin &
trimethoprim-sulfamethoxazole.
• Ampicillin, amoxicillin, ciprofloxacin, ceftriaxone,
cefotaxime are also effective
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 Salmonella Enterocolitis
II. Salmonella Enterocolitis:- Food Poisoning
– The most important are S. typhimurium & S.
enteritidis. These organisms are common pathogens
of animals or birds.
Transmission:
– The organism is transmitted from improperly cooked
meat of infected animals, eggs of infected birds or
from food contaminated with rat excreta.
Symptoms:
• The disease is characterized by nausea, vomiting,
abdominal discomfort, diarrhoea & slight fever.

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 Salmonella Enterocolitis
• The condition occurs after multiplication of the organism
in the intestine, i.e. it is an infection and not due to toxin
• The incubation period is 12-18 hrs to allow for such
multiplication.
• Recovery follows within 1week.
Diagnosis:
• Based on the isolation & identification of the organisms
from the stools, vomitus & food remnants.

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 SHIGELLAE
– The natural habitat of shigellae is limited to the intestinal
tracts of humans & other primates, where they cause
bacillary dysentery
– They are closely related to E. coli.
Morphology:
– Slender Gram-negative rods; coccobacillary forms
occur in young culture.
– Non-motile & non-capsulated.
Culture:
– They produce pale non-lactose fermenting colonies
on MacConkey.

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 Shigellae
Species
S. dysenteriae
S. boydii
S. flexneri
S. sonnei
Pathogenesis:
• Shigella spp are highly infective (highly communicable).
About 102 organisms can initiate infection.
• The infective dose is low, particularly in S. dysenteriae =
10 -100 organisms

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 Shigellae
• The infection occurs by the ingestion of contaminated
food or drinks (fecal-oral contamination).
• They invade the mucosal epithelium causing micro-
abscesses in the wall of the large intestine and terminal
ileum leading to necrosis of the mucous membrane,
superficial ulceration, bleeding, and formation of a
‘pseudomembrane’ on the ulcerated area.
• It is limited to the GIT without blood invasion.
• This consists of fibrin, leukocytes, cell debris, necrotic
mucous membrane, and bacteria.
• As the process subsides, granulation tissue fills the
ulcers & scar tissue forms.
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 Shigellae
• The IP is short with an onset of abdominal pain,
diarrhoea, tenesmus & fever.
• Recovery occurs spontaneously.
• Very few patients remain as chronic carriers.
Toxins:
Shigellae produce 2 types of toxins:
1. Endotoxin: upon autolysis, all shigellae release their
toxic LPS. The endotoxin probably contribute to the
irritation of the bowel wall.

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 Shigellae
2. Shigella dysenteriae Exotoxin:
• S. dysentriae type 1 (shiga bacillus) produces a heat -
labile exotoxin that affects both the gut & the CNS.
• The Shiga toxin, also called verotoxin, is produced by
S. dysenteriae
• Acting as a neurotoxin, which causes the extreme
severity & fatal nature of S. dysentriae infections (i.e.
meningitis).
• Acting as enterotoxin, S. dysentriae produces diarrhoea
as does the heat - labile E. coli enterotoxin.
• Shigellae cause diarrhea (bloody), fever, stomach ache

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 Shigellae
• Shigella dysenteriae type 1, causes deadly epidemics
in many developing regions and nations.
• Shigella flexneri causes bacillary dysentery, the
symptoms of which include abdominal pain, diarrhea,
fever, vomiting and blood or mucus in the stool.
• Shigellosis resolves within 5-7 days

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 Shigellae
Diagnosis: Culture:
• Specimen - Fresh stool, mucous flecks, and rectal
swabs. Large number of faecal leukocytes & some red
blood cells are seen microscopically.
• The material are streaked on MacConkey's
• Non-lactose (pale) colonies are identified by morphology,
biochemical reactions & serologically by agglutination
with specific antisera.
• Enrichment culture of faeces in selenite F broth, with
subsequent subculture on to MacConkey agar, may
improve isolation.

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 Shigellae
Prevention:
• No specific prophylaxis is available.
• Proper hygienic measures should be followed.

Treatment:
• Restoration of the fluids & electrolyte balance
• Antibiotics, e.g. ampicillin, tetracyclines, trimethoprim /
sulfamethoxazole, nalidixic acid, fluoroquinolone,
ciprofloxacin, sulphonamides are effective in treatment.

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Gram negative curved rods

 Campylobacter
 Helicobacter
 Vibrio
• Aeromonas
• Plesiomonas

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 HELICOBACTER
• Helicobacter pylori: spiral-shaped, Gram negative rod,
produce urease, has multiple flagella at one pole
• Found closely associated with gastric mucosa & is
associated with gastritis, gastric & duodenal (peptic)
ulcer disease & gastric carcinoma.
Growth characteristics
• Catalase positive,
• Motile

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 Helicobacter
Pathogenesis
• Grow optimally at pH 6 -7 but not at pH of gastric lumen
• Found deep in the mucus near the epithelial surface
with pH 7.4
• Produce protease that modifies the gastric mucus
• Produce urease –yield ammonia which buffers acid
• Motility helps it to find its way to the epithelial surface
• Destruction of the lumen
• Toxins, LPS & ammonia directly damage the cells

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 Helicobacter
Epidemiology
• Present in the gastric mucosa of 20% persons <30 yrs,
but in 40-60% of persons aged 60 years
• Developing countries: prevalence about 80% in adults
• Person-to-person transmission is likely
Diagnosis
• Gastric biopsy - used for histology, used for culture
• Culture – grows in 3-6 days
– Grows optimally at pH 6.0 -7.0 in Skirrow’s medium
• Serum Ab test

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 Helicobacter
• Stool antigen detection

Treatment
Triple therapy with:
1. Metronidazole and either
2. Bismuth subsalicylate or Bismuth subcitrate plus
3. Amoxicillin or tetracycline - for 14 days eradicates H.
pylori in 70-95% of patients.

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 Vibrionaceae family
• Vibrio: Gram negative curved bacilli.
• Vibrios are wide spread in nature, mainly in water
• Only one serovar V. cholerae, is the cause of cholera.
• Non-cholera vibrios of different serovars exist within the
spp
• Other spp: V. parahaemolyticus, V. vulnificus, V.
mimicus
Similarities to Enterobacteriaceae
• Gram-negative
• Facultative anaerobes
• Fermentative bacilli
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 Vibrionaceae
Habitat:
• Water contaminated with patient feces or carriers

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 Vibrio cholerae
Morphology:
• Gram negative comma-shaped bacilli, arranged in pairs
or short chains.
• Actively motile by single polar flagellum, non-sporing,
non-capsulated
Cultural characters:
• Aerobes, grow readily on ordinary media over a wide
temp range (optimum 37oC).
• Growth is inhibited at acid pH (optimal pH for growth:
alkaline pH >8.0).

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 V. cholerae
• Grow on variety of simple media including:
• MacConkey’s agar
• TCBS (Thiosulfate Citrate Bile salts Sucrose) agar
• V. cholerae grow without salt
• Most other vibrios are halophilic
Enrichment medium:
• Alkaline peptone water (pH 8.4) promotes the rapid
growth of V. cholera from mixtures of other bacteria, e.g.
fecal samples.

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 V. cholerae
Selective media:
1.Thiosulphate citrate bile sucrose (TCBS) medium
– TCBS agar, pH 8.6
– V. cholerae produce large yellow sucrose - fermenting
colonies after 18-24 hrs incubation.

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 V. cholerae
Toxins:
– Endotoxins & exotoxins are recognized.
– The enterotoxin stimulates persistent and excessive
secretion of fluid by the intestinal mucosa

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 Cholera
• The word cholera is derived from a Greek term that
means "flow of bile"
• Also called Asiatic or epidemic cholera - is a severe
diarrheal disease caused by the bacterium V. cholerae.
• Cholera is an acute infectious disease characterized by
severe vomiting & watery diarrhoea (rice water stools)
resulting in dehydration & collapse.
• V. cholerae produces cholera toxin
• IP: short 1- 4 days (24 - 48 hours)
• Symptoms begin with the sudden onset of painless
watery diarrhea
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 Cholera
Pathogenesis
• Infection occurs by the oral route through contaminated
food or drinks.
• Source of infection: a case or a carrier who excretes
the organism in the stools.
• The infectious dose is 103 -106 organisms; less if
ingested with food.
• V. cholerae O1 & V. cholerae O139 cause clinical
disease by producing an enterotoxin.
• Infection is restricted to intestine with no blood invasion.
• The organisms attach to the microvilli of the brush
border of epithelial cells - multiply & liberate enterotoxin
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 Cholera
• Enterotoxin blocks the absorption of sodium & chloride
by the microvilli & promote the secretion of chloride &
water
• Enterotoxin results in the excretion of electrolytes such
as chloride & bicarbonate ions along with massive
quantities of water.
• Severe diarrhoea & vomiting may lead to dehydration &
death.
• The loss of potassium ions may result in cardiac
complications and circulatory failure.

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 Cholera
Epidemiology
• The disease is endemic in the Indian subcontinent &
used to occur in world wide epidemics.
• Transmission to humans is by water or food (feco-oral
route).
• Person to person infection is rare
• The natural reservoir of the organism is not known but
could be aquatic environment
• 8 cholera pandemics have occurred
7th pandemic:
• V. cholerae O1
• Began in Asia in 1961
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 Cholera
Clinical manifestations
• Incubation period: 2-3 days
• Abrupt onset of vomiting & life-threatening watery
diarrhea (15-20 liters/day)- fishy odor at beginning.
• As more fluid is lost, feces-streaked stool changes to
rice-water stools:
• Colorless,
• Odorless,
• No protein
• Speckled with mucus

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 Cholera
Mortality
• Untreated cholera frequently results in high (50-60%)
mortality rates
 Death attributable to:
· Hypovolemic shock (due to abnormally low volume
of circulating fluid (plasma) in the body)
· Metabolic acidosis (pH shifts toward acid side due to
loss of bicarbonate buffering capacity)

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 Cholera
Diagnosis of cholera:
A. First case in a non-endemic area
– Specimen for culture: mucous flecks from stools.
1. Stool inoculated on alkaline peptone water
2. Subcultures made from the surface pellicle after 6-8 hrs
on TCBS or alkaline agar.
Colonies are identified by:
• A wet mount which is examined for motility.
• Smears stained show Gram negative comma-shaped
bacilli.

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 Cholera

B. Secondary case during an epidemic

• In an established epidemic, cases can be diagnosed by
microscopic examination of stools for comma-shaped
Gram negative bacilli with characteristic motility, which
can be immobilized by adding specific antisera.

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 Cholera
Treatment
1. IV fluids to correct the fluid & electrolyte balance
2. Antibiotics have a secondary role in treatment.
– Tetracyclines are the most effective, but plasmid
mediated resistance is increasing.
– If resistant to tetracycline– fluoroquinolones
(ciprofloxacin), erythromycin are alternatives

55
 Cholera
Prevention/Prophylaxis:
• Community & personal hygiene to prevent feco-oral
transmission.
• Isolation of cases & proper disposal of sewage.
• Koll's Vaccine which is heat killed suspension of the
organism is given in 2 doses.
• The role of vaccine in protection is questionable as it
stimulates anti-bacterial & not anti-toxic Abs.

56
 Pseudomonadaceae
• Pseudomonads: widely distributed in water, soil,
sewage, animals, plants
• The bacteria favor moist areas, such as sinks, toilets,
inadequately chlorinated swimming pools and hot tubs,
and outdated antiseptic solutions
• The typical Pseudomonas bacterium in nature might be
found in a biofilm, attached to some surface or
substrate
• Pseudomonas is one of the most vigorous, fast-
swimming bacteria by (means of its flagellum) seen in
pond water samples.

57
 Pseudomonas
Species
More than 13 spp are identified, but the common are:
• P. aeruginosa
• Pseudomonas cepacia
• Pseudomonas pseudomallei (now Burkholderia
pseudomallei)
• Pseudomonas mallei (now Burkholderia mallei)

58
 Pseudomonas aeruginosa
Pseudomonas aeruginosa
– An opportunistic pathogen of humans, almost never
infect the uncompromised tissue
– The major human pathogen of the group is the third
most common cause of nosocomial infections after S.
aureus & E. coli.
– Nearly 30% of hospitalized patients show colonization.
– It is isolated from the intestinal tract of about 5% of
healthy people
– In nature might be found in a biofilm

59
 P. aeruginosa
Morphology:
– P. aeruginosa: G-ve, almost all are motile by means of
a single polar flagellum, aerobic bacilli, occurs as single,
in pairs or occasionally short chains.

60
 P. aeruginosa

Gram stain of Pseudomonas aeruginosa cells

61
 P. aeruginosa
Cultural characters:
– Grow on all routine media, wide temp range (5- 42oC);
grow well at 37- 42oC.
– Growth at 42oC helps differentiate from other
pseudomonas spp
– Broth cultures: show uniform turbidity & surface
pellicle, evidence of its aerobic growth requirement.

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 P. aeruginosa

1. P. aeruginosa growth in certrimide agar

2. P. aeruginosa colonies on other agar
3. Soluble blue pigment pyocyanin produced by many strains of P.
aeruginosa
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 P. aeruginosa

Antigenic structure:
– Pili extend from the cell surface & promote attachment
to host epithelial cells.
– PS capsule - responsible for the mucoid colonies seen
in culture.
– LPS - is responsible for many of the endotoxic
properties of the organism.
Pathophysiology
• Most P. aeruginosa produce various toxins & proteases

– Exotoxin A - cause tissue necrosis, lethal for animals

64
– Enzymes: proteases, lipases
 P. aeruginosa
• P. aeruginosa is both invasive and toxigenic.
• The 3 stages of Pseudomonas infections are
– 1. bacterial attachment and colonization,
– 2. local infection, and
– 3. bloodstream dissemination and systemic disease
Pathogenicity:
– P. aeruginosa is a common human saprophyte, it rarely
causes disease in healthy persons
– An important cause of hospital acquired infections.
– Most infections occur in patients with burns &
malignancy.
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 P. aeruginosa
– Most infections with this organism occur in
compromised hosts
Common Infections caused by P. aeruginosa
• In compromising conditions: disrupted physical barriers
to bacterial invasion (e.g, burn injuries, IV lines, urinary
catheters, endotracheal tubes) and
• dysfunctional immune mechanisms, such as those that
occur in neonates, AIDS, neutropenia, complement
deficiency

66
 P. aeruginosa
Common infections
1.UTI:
– Chronic infections associated with indwelling catheter
2. Bacteremia / septicemia (Blood stream infections) –
especially in immunocompromized individuals
3. Skin and soft tissue infections,
– Wound infections, dermatitis, burns, Folliculitis,
infected skin lesions, e.g- pressure sores.
– Muscle, tendons, ligaments, fat.
4. Ear infections: including otitis externa (swimmer's ear) /
malignant external otitis.
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 P. aeruginosa
5. Respiratory infections - LRTIs – severe pneumonia in
hospitalized pts – especially ICU
6. Eye infections secondary to trauma or surgery –
damages the cornea
7. Bone & joint infections
8. CNS infections: causes meningitis & brain abscesses
9. Gastrointestinal infections

68
 P. aeruginosa
Diagnosis:
Specimens: from skin lesions, pus, urine, blood, sputum
1. The pus from the lesions may be greenish in colour
2. Smears stained by gram stain show Gram-ve bacilli
3. Biochemical tests:
– It does not ferment lactose
– Oxidase test is positive.
4. Cultures on nutrient agar show the characteristic
greenish colouration of the medium.

69
 P. aeruginosa
Antibiotic Sensitivity / Resistance
• Resistant to most usual antibiotics - Antibiotic sensitivity
should be done to give the proper antibiotic
• Only few antibiotics are effective against P. aeruginosa,
including fluoroquinolones, gentamicin and imipenem, &
even these antibiotics are not effective against all strains

70
Spirochaetales

Spirochaetales

71
 SPIROCHAETES
• Spirochaetales (Greek – spira = coiled; chaete = hair)
• Heterogenous group of spiral-shaped, motile organisms
• Order Spirochaetales contains 2 families:
• Family: Spirochaetaceae
– Genus: Treponema
– Genus: Borrelia
• Family: Leptospiraceae
– Genus: Leptospira
Habitat:
• Most are commensals in the mouth & genitalia but few
cause human diseases.
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 Spirochaetes
• Better stained with silver or Giemsa stains.

Genera:
• Genus treponema includes few pathogenic members.
Three major genera in the family pathogenic to man are:
1. Treponema causes syphilis, bejel, yaws & pinta.
2. Borrelia causes relapsing fevers & Lyme disease.
3. Leptospira causes leptospirosis (Weil's disease).

73
 Spirochetes morphology

Treponema

Leptospira

Borrelia
Morphology of the 3 genera 74
 Treponema pallidum
Pathogenic Treponema
1. Treponema pallidum ssp pallidum: causes syphilis.
2. Treponema pallidum ssp endemicum – causes bejel
3. Treponema pallidum ssp pertenue: causes yaws
4. Treponema carateum: causes pinta
• Treponemes responsible for these diseases cannot be
distinguished serologically, morphologically.
• They have not been successfully cultivated on
artificial media.
• *Non-pathogenic treponema spp (Reiter’s strain) -
part of the oral flora
75
 Treponema pallidum
Treponema pallidum
• Pathogen of humans only, no animal model
• Extremely fastidious, intracellular pathogen, survive
only briefly outside host.
Morphology:
• Slender, microaerophilic bacterium with spiral coils
regularly spaced 1um apart
• T. pallidum is a fragile spiral bacterium 0.1- 0.25 by 5-
10m in diameter.

76
 Treponema pallidum
• Possess an outer membrane but not LPS in structure
• Its small size makes it invisible on light microscopy
• Treponema spp are not stained with Gram stain, but:
• Seen by dark-ground microscopy in unstained
preparations
• Dark-field microscopy allows a lower limit of resolution
than bright-field microscopy (0.1um vs 0.2um)
• Are actively motile by means of an axial filament

77
 Treponema pallidum
Virulence Factors of T. pallidum
• The Ags of T. pallidum are unknown, but certain
immunologic responses are elicited by infection:
a. Induced Treponemal Abs are capable of immobilizing &
killing the spirochaetes.

78
 Treponema pallidum
Some virulence factors
1. Outer membrane proteins promote adherence
2. Tissue destruction & lesions are primarily result of
host’s immune response (immunopathology).
Resistance
• T. pallidum is a very delicate organism
• Susceptible to heat, drying - inactivated by elevation of
temp to 41-420C/hr – the basis of the ‘fever therapy’
for syphilis.

79
 Treponema pallidum
• In whole blood or plasma stored at 4°C, organisms
remain viable for at least 24 hours, but are killed in 1-3
days at 0-40C
• So that transfusion syphilis can be prevented by storing
blood for at least 4 days.
• Inactivated by contact with O2, distilled water, soap,
arsenicals, mercurials, bismuth, common
antiseptics, antibiotics

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 Syphilis
• A chronic sexually transmitted disease (STD) of blood
vessels and of the perivascular areas.
• Incubation Period: 10-90 days – 21 days (median)
• Infectious dose: About 57 organisms
Epidemiology
• Highest rates in South & SE Asia, sub-Saharan Africa,
S. America and the Caribbean.
• Infectivity is maximum during first 2 years of disease
– primary & secondary stages

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 Syphilis
Pathogenesis of T. pallidum pallidum
Acquired syphilis:
• The manifestations are protean with different stages
occurring over time in untreated disease
Symptoms and stages
• The disease follows 3 stages if left untreated.
1. Primary syphilis:
• The bacteria enter the body through tiny microscopic
abrasion/breaks in the skin & mucous membranes.
• Then, enter the lymphatics; the regional lymph
nodes become involved & bacteremia occurs.
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 Syphilis
• Primary chancre develops at the site of inoculation 2-
10 wks after exposure to infection as a papule on the
genitalia → ulcerates, heals spontaneously in 1 month.
• It is a well defined indurated, hard, painless ulcer mainly
on the genitalia (90%) & extra-genital on lips (5-10%).
• In female, chancre occurs in the cervix, labia.
• Chancres may not be detected in females if on cervix.
• Primary syphilis is highly infectious.
• It heals spontaneously
• Serologic tests often negative when the chancre first
appears.
83
 Syphilis

Hard chancre 84
 Syphilis
2. Secondary syphilis:
• Lesions occur 6-8 weeks after the appearance of the
chancre & are characterized by the manifestations:
• E.g. Skin rash, condylomata lata of vulva, mucous in
the mouth, fever, headache, malaise
• Skin rash is painless, reddish raised bumps that
eventually rupture and ooze a discharge
• The spirochetes may also be transmitted by kissing

85
 Syphilis
• The lesions are rich in spirochetes
• Secondary syphilis is highly infectious.
• T. pallidum is found in large numbers in the lesions of
both primary & secondary stages & highly infectious.
• Lesions heal & the person enters a latent stage - are
no signs or symptoms of the disease, last for several
years to decades.
• Serological tests becomes almost uniformly positive.
• About 25% of patients are cured, 25-30% progress to
the tertiary stage.
• Early syphilis: <2 years – highly infectious
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Generalized rash Palmar / plantar rash 87
 Syphilis

Condylomata lata
88
 Syphilis
Latent stage
• Quiescent stage which follows secondary stage
• Host suppresses the infection enough so that no
lesions are clinically apparent.
• After about 2 years, the syphilis is NOT normally
infectious, except from mother to the fetus.
• First 4 years = early latent
• Subsequent period = late latent
• About 40% of late latent patients progress to late
tertiary syphilitic disease
• Only evidence is positive serologic test for syphilis
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 Syphilis
3. Tertiary syphilis:-
– Late syphilis: >24 months
– After 2-20 years, approximately 30% of untreated
patients progress to the tertiary stage
– Rare because of the widespread availability and use of
antibiotics
Lesions
A. Gummatous Lesions:
• Appearance of gumma in the internal organs (liver,
bones, testes) 5-40 years after initial infection, if not
treated.
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 Syphilis
B. Neurosyphilis
• Neurosyphilis is defined as a CSF WBC count of 20
cells/µL or a reactive CSF VDRL test result.
• The pathogenesis of neurosyphilis is similar to that in
the rest of the body.
• The neurologic presentation is with loss of pain
sensation, loss of peripheral reflexes, impairment of
vibration & position senses.

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 Syphilis
Syphilitic meningitis
• Is the early manifestation - occurring within 6 months
of the primary infection.
• Patients with acute syphilitic meningitis have signs of
meningeal irritation, headache, nausea, vomiting. Fever
is unusual.
C. Other sites
– Skin, Bone, Joints
•Tertiary stage is not infectious

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 Syphilis

Late syphilis - Serpiginous Gummata of Forearm

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 Congenital syphilis (CS)
• A pregnant syphilitic women can transmit T. pallidum to
the fetus through the placenta.
• This may lead to abortion or still birth at term or child
is born live but develops congenital syphilis.
• About half of infected fetuses die shortly before or after birth
• Syphilis – is teratogenic (Teratogen: Any substance or
condition that might disrupt embryonic development and
cause birth defects).

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 Congenital syphilis
Some signs of congenital syphilis
 Early Congenital syphilis:
a. Skin rash: maculopapular rash
b. Mucosal lesion: Mucocutaneous lesions
c. Hepatosplenomegaly
 Later manifestations of congenital syphilis include:
– Hutchinson’s teeth
– Bone & Teeth deformities: “saddle nose” (due to
destruction of the nasal septum).

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 Congenital syphilis

Hutchinson’s teeth
96
 Congenital syphilis

• Perforated palate
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 HIV Infection and Syphilis
• Syphilis and HIV infections commonly coexist.
• Syphilis enhances risk of transmission of HIV
• Clinical course is similar to non-HIV-infected patients.
• Serological tests for syphilis are usually equivalent in
sensitivity in HIV-infected and non-infected persons.

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 Syphilis
Lab Diagnosis:
1. Detection of sphirochaetes in the lesion
• Wet mount is prepared from exudate from the chancre
in primary stages or from the skin eruptions & mucous
patches in secondary stages.
These are examined by:
a. Dark-ground microscope - shows living motile
spirochaetes with characteristic slow cork-screw motility
on microscopic examination

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 Syphilis

Darkfield microscopy of Treponema pallidum 100

 Syphilis
2. Serologic diagnosis (STS):
– Positive within 5-6 weeks after infection
– Strongly positive in secondary phase
– The use of only one type of serologic test is insufficient
for diagnosis

These are either:

• Non-treponemal Ags: positive in 50%
• Treponemal: positive in 90%

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Serological diagnosis for Syphilis 102
 Syphilis
I. Non-Treponemal antigen tests:
A. Rapid plasma reagin (RPR):
– Test is non-specific, may give false positive in other
diseases, e.g. autoimmune disease, measles, leprosy,
malaria
– A positive test should be confirmed by one of the specific
treponema Ag tests.

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 Syphilis
II. Treponemal antigen tests:
– Antigens for these tests are prepared from virulent
treponema.
– The tests are highly specific as they detect anti-
treponemal Abs but they are more complex &
expensive
– They are used primarily as confirmatory tests.

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 Treatment
Therapy for Primary, Secondary & Early Latent Syphilis
• Benzathine penicillin G 2.4 million units IM in single dose
• If penicillin allergic:
– Doxycycline 100mg orally twice daily for 14 days, or
– Tetracycline 500mg orally 4 times daily for 14 days

105
 Treatment
Therapy for Syphilis in Pregnancy
• Treat with penicillin.
• Erythromycin is no longer an acceptable alternative
drug in penicillin-allergic patients.
• Patients who are skin-test-reactive to penicillin should
be desensitized in the hospital and treated with
penicillin.

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 Genus Borrelia
• Borreliae: Many members are commensals
• Pathogenic borrelia cause R. fever & Lyme disease.
• Lice or ticks become infected by feeding on patient or
rodent blood during the bacteraemia stage.
Morphology
• Large spirals with irregular coils spaced 2-4m apart.
• Motility is by both a rotating & a twisting motion.
• Are stained with ordinary stains & are Gram-negative
• Also stained with Giemsa or Wright's stain

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 BORRELIA
Cultural characters:
• They can be cultured in the fluid media containing blood,
serum or tissue.

Antigenic structure:
• Borreliae are antigenically variable
• Borrelia recurrentis show antigenic variation in vivo.

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 Relapsing Fever (RF)
Relapsing Fever (Borreliosis)
• A disease characterized by repeated bouts of fever
alternating with periods of apyrexia.
Relapsing fever is divided in to 2 forms:
1. Epidemic Relapsing fever (LBRF)
• It is caused by B. recurrentis and its variants and
transmitted by body lice – Pediculus humanus corporis
2. Endemic Relapsing fever
• Tick-borne relapsing fever (TBRF) - transmitted by
soft-bodied ticks – ornithodoros genus.
• Man is the only host for B. recurrentis and B. duttoni
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 Relapsing Fever (RF)
Epidemic Relapsing fever
Pathogenesis
• Borrelia multiplies in the blood of the louse & infection is
transmitted by the bite or rubbing crushed lice into bite
wounds.
• After IP of 3-10 days, there is sudden onset of fever
which lasts about 4 days followed by afebrile period of
3-10 days when the pt develops another bout of fever.

110
 Relapsing Fever
• The organism is found in large numbers in the blood
during the febrile stage.
• Abs against Borrelia appear during the febrile stage;
these agglutinate & destroy the organism and the attack
is terminated.
• Antigenic variants of Borrelia emerge, cause the
relapse.
• Has evolved strategies, including antigenic variation, to
evade innate (e.g complement) & adaptive immune
defense
• The louse is infected for life time

111
 Relapsing Fever
Epidemiology
• Currently found in Africa
Transmission
• By the bite of infected body louse or feces of an infected
the arthropod vector.

112
Diagnosis:
• Blood is obtained during the rise in temperature for
smear & animal inoculation.
1. During the febrile stage:
– Blood films stained with Leishman or Giemsa stain
reveal large numbers of loosely coiled spirochetes
among red cells in 70% of patients.
2. During the afebrile stage:
– The organism is scanty in the blood & blood film is –ve

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 Relapsing Fever

Borrelia in blood smear

114
 Relapsing Fever
3. Animal inoculation:
– Injecting white mice IP with 1-2ml patient blood. After 2-
4 days, films from tail blood are stained & examined.
– The animal acts as in vivo enrichment medium.
Risk factors
– Crowding, poor hygiene, and poverty are risk factors for
large outbreaks, such as the epidemics

Mortality
– 30 -70% without antimicrobial treatment but can be
lowered to 2-6% if treated.
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 Relapsing Fever
Treatment:
– Penicillin, tetracycline, erythromycin, chloramphenicol
– Erythromycin or chloramphenicol can be used in the
treatment of pregnant women and children.

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 Endemic Relapsing Fever
• Endemic RF: Caused by at least 15 borrelia species
• Rodents are reservoir for the organism

Some borrelia species causing Endemic RF

• Borrelia duttoni
• Borrelia hermsii
• Borrelia parkeri
• Occurs in Africa, Spain, Saudi Arabia, Asia, and certain
areas in the USA & Canada.

117
 Endemic RF
Pathogenesis
• Soft ticks feed for short periods during the night and then
fall off the host.
• A patient can be infected with Borrelia within minutes
after the tick attaches to the host.
• This is an important distinction from other tick-borne
diseases, such as Lyme disease, in that the ticks feed
for longer periods of time and it can take several hours
before Borrelia burgdorferi cells infect the human host.
Mortality
• The mortality rate of endemic relapsing fever is 30–70%
without treatment & 1% with treatment
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 Endemic RF
Treatment
• Tetracycline

Prevention
• To prevent endemic relapsing fever, it is important to
wear proper clothing that keeps ticks from gaining
access to the skin
• treated with insecticide
• rodents should be controlled for endemic disease

119