ESSAY

1. Organisms causing meningitis it's pathogenicity, lab diagnosis of meningococcal

meningitis, the role of BACTEC in rapid diagnosis of causative agents in bacterial
meningitis
2. Anthracis
3. Clostridia:
4. Causative agents of enteric fever, diagnosis of typhoid fever.

5. Classify mycobacterium, details about pulmonary tuberculosis?

6. Dysentery, causative agents, details about bacillary dysentery.
7. Describe Corynebacterium diphtheriae. add a note on its prophylaxis

8. Name 3 coccobacillus : Bordetella pertussis

Haemophilus influenzae

Chlamydia trachomatis

Discuss in detail about etiology, pathogenesis, and lab diagnosis of Bordetella pertussis

9. Leptospirosis
10. Syphilis
11. Rickettsiaceae
12. Laboratory diagnosis of respiratory tuberculosis

SHORT NOTES
1. el tor vibrio
2. Gonorrhea
3. Non gonococcal urethritis (NGU)
4. x and v factors
5. What is the bile solubility test? Describe its principle
6. Zoonotic infections
7. BCG
8. Food poisoning
9. Unique characteristics of enterococcus
10. Nagler’s reaction
11. What is the disease caused by mycoplasma?
12. Llab diagnosis of typhoid fever:
13. Actinomycosis
14. Nonsporing anaerobic infections
15. Weil’s disease: (hepato- renal hemorrhagic syndrome)
16. Causative agent for relapsing fever:
17. Various mechanisms by which escherichia coli produce diarrhea
18. Mantoux test
19. Name 4 pigments produced by bacteria
20. Milk ring test and its uses
21. Neil mooser reaction
22. Write a note on satellitism
23. Clostridium botulinum
24. Quellung reaction
ESSAY
 1. MENINGITIS
● Meningitis is an inflammation of leptomeninges surrounding the brain and spinal cord with
involvement of the subarachnoid space
● TYPES OF MENINGITIS
○ Acute meningitis
○ Chronic meningitis
● Organisms causing bacterial meningitis
○ ACUTE OR PYOGENIC MENINGITIS
■ Streptococcus pneumonia is the most common cause of pyogenic meningitis
■ Other agents include meningococcus, Streptococcus agalactiae, Listeria, and
Haemophilus influenzae.
■ Neonates-streptococcus agalactiae, gram-negative bacilli such as Escherichia
coli and Klebsiella
■ Elderly - Streptococcus agalactiae and Listeria monocytogenes
○ CHRONIC BACTERIAL MENINGITIS
■ Mycobacterium tuberculosis
■ Borrelia birgdoferi
■ Treponema pallidum
■ Rate bacterial agents such as Nocardia, actinomyces, Tropheryma whipplei,
Leptospira, and brucella
● PATHOGENESIS - bacteria transmitted from person to person through droplets of
respiratory secretion from cases or nasopharyngeal carriers
○ Routes of infection
■ Hematogenous spread - most common route but entry into the subarachnoid
space is gained through the choroid plexus or through other blood vessels of
the brain.
■ Direct spread from an infected site present close to the meninges-otitis
media, mastoiditis, sinusitis, etc.
 ■ Anatomical defects in the central nervous system may occur as a result of
surgery, trauma, congenital defects, which can allow organisms for ready and
easy access to CNS
● CLINICAL MANIFESTATION
○ Important symptoms include fever vomiting intense headache altered consciousness
and occasionally photophobia
○ Signs of meningism
■ Nuchal rigidity
■ Kernig's sign
■ Brudzinski's sign
■ Organism specific finding (eg) purpuric rash as seen in meningococcal
meningitis
● LABORATORY DIAGNOSIS OF MENINGOCOCCAL MENINGITIS
○ Specimen collection and transport
■ First suspected meningococcal meningitis the specimens are nasopharyngeal
swabs pus or scrapings from rashes; should be carried in a transport medium
such as Stuart's medium.
○ Cytological and biochemical analysis

○ Gram staining
■ Gram-negative diplococci capsulated with adjacent sides flattened - Neisseria
meningitides which causes Meningococcal meningitis
● ROLE OF BACTEC IN RAPID DIAGNOSIS OF CAUSATIVE
AGENTS IN BACTERIAL MENINGITIS
○ BACTEC principle is based on fluorometric detection of growth; use an
oxygen-sensitive fluorescent dye present in the medium
○ Mycobacteria Growth Indicator Tube(MGIT) is an automated culture system
available for laboratory diagnosis of Chronic bacterial meningitis caused by
Mycobacterium tuberculosis.
○ MGIT works on the fluorometric principle of detection similar to BACTEC
 2. ANTHRACIS
● Bacillus anthracis is gram-positive, aerobic, non-motile, sporulation bacteria

SPORES:
● Formed under unfavorable conditions
● Highly resistant to physical and chemical agents
● Are formed in soil/culture, not in the animal body

VIRULENCE FACTORS:
(1)CAPSULE-

● made of polyglutamate
● Inhibits phagocytosis
● It is plasmid-borne

(2)TOXIN HAS THREE FRACTIONS

● Edema factor-active fragment

Acts as adenylyl cyclase and increases cAMP level
Cause edema
● Protective factor-binding fragment
Binds to host cell receptors and facilitates entry of other fragments into
host cells
● Lethal factor-causes cell death

CLINICAL MANIFESTATIONS:
ANIMAL ANTHRAX

● Anthrax is a zoonotic disease.

● Herbivores are more commonly affected than carnivores
● Ingestion of spores in the soil causes the disease
● Anthrax presents as fatal septicemia
● Infected animals discharge many bacilli from the mouth, nose, and rectum which
sporulate in soil and may act as a source of infection for man

HUMAN ANTHRAX

● Humans acquire disease via abraded skin

● Inhalation of spores
 ● Ingestion of anthrax infected animal carcasses

Types:

● Cutaneous anthrax-(hide porter’s disease)-malignant pustule is seen.

● Pulmonary anthrax-(wool sorter’s disease)-causes bioterrorism outbreaks commonly
Hemorrhagic pneumonia is seen
● Intestinal anthrax-when undercooked/uncooked meat is consumed

EPIDEMIOLOGY:
● High incidence in Africa, Central, and southern Asia
● Human anthrax
Non-industrial-agricultural exposure to animals
Industrial-from hides, hair, bristles, wools

LABORATORY DIAGNOSIS:
● Specimen collection-
● pus, swab, or tissue from the pustule
● Sputum in pulmonary anthrax
● Blood in septicemia
● CSF in hemorrhagic meningitis
● Gastric aspirate, feces, food in intestinal anthrax
● Ear lobes for dead animals
● Direct demonstration-
● Gram staining
● Mc Fadyean's reaction-Capsule appears as amorphous purple around bacilli when
reacted with Gurr’s polychrome methylene blue for 30sec
● Direct immunofluorescence test
● Ascoli’s thermo precipitation test
● Culture-
● B. anthracis is non-fastidious
● Nutrient agar-Medusa head appearance, irregular, opaque, grayish-white with
frosted glass appearance
● Blood agar-dry wrinkled, non-hemolytic colonies
● Gelatin stab agar-inverted fir tree appearance
● Selective media-Solid medium with penicillin-bacilli in a string of pearl appearance
● PLET medium
Reference: Ananthanarayan and Paniker’s Textbook of Microbiology-11/E-pg no.235-fig 24.4

● Culture smear-
● Gram-staining-bamboo stick appearance of bacilli with non-bulging spores
● Spores-demonstrated by Ashby’s method or acid-fast staining

Reference: Ananthanarayan and Paniker’s Textbook of Microbiology-11/E-pg no.232-fig 24.1

● Molecular diagnosis-PCR with specific primers

PREVENTION:
● Animals died of anthrax should be burnt or buried deep in lime pits
● Decontamination(by autoclaving) of animal products
● Protective clothing and gloves when handling infectious materials
● Immunization-Pasteur’s anthrax vaccine
● Sterne vaccine-live attenuated non-capsulated
● Mazzucchi vaccine

TREATMENT:
● Post-exposure prophylaxis-Adsorbed toxoid vaccine (BioThrax) along with
antimicrobial therapy
 3. CLOSTRIDIA
● Clostridia are obligate anaerobic gram-positive bacilli with bulging spores.
● Infection causing clostridia are
1. C. perfringens
2. C. tetani
3. C. botulinum
4. C. difficile

Clostridium tetani

MORPHOLOGY:
● Obligate anaerobic, gram-positive bacillus with terminal round spore (drumstick
appearance)
● Non-capsulated
● Has peritrichous flagella and exhibits swarming motility

PATHOGENESIS:

● C. Tetani produces two exotoxins – tetanolysin and tetanospasmin

● Tetanolysin is a hemolysin, not involved in the pathogenesis
● Tetanospasmin (tetanus toxin) is a neurotoxin and is responsible for disease
manifestations. It gets toxoided spontaneously or by formaldehyde.
● Mechanism of action:
Tetanus Toxin binds to receptors present on the motor nerve terminal

Causes presynaptic inhibition of glycine and GABA (inhibitory neurotransmitter)

Spastic muscle contraction

MODE OF TRANSMISSION:

● It is non-infectious (no person to person spread)

● Injury (superficial abrasions, puncture wounds, road traffic accidents)
● Surgery is done without proper asepsis
● In neonates following delivery due to unhygienic practices
● Otitis media

CLINICAL MANIFESTATIONS:

● The incubation period is about 6-10 days. Muscles of the face and jaw are
affected 1st(shorter distance for a toxin to reach presynaptic terminals)
● First symptom: increase in masseter tone leading to trismus or lockjaw,
followed by muscle pain and stiffness, back pain, and difficulty in swallowing
● As the disease progresses, painful spasm develops, it can be:
Localized: involves affected limb
Generalized: painful muscle spasm leading to descending spastic paralysis
● Deep tendon reflexes are exaggerated
● The autonomic disturbance is maximal during the 2nd week of severe tetanus,
characterized by:
Low or high BP
Tachycardia
Intestinal stasis
Sweating
Increased tracheal secretions
Acute renal failure
● In neonates, difficulty in feeding is the initial presentation

COMPLICATIONS:

● Risus sardonicus: characterized by abnormal, sustained spasms of facial

muscles that appear to produce grinning
● Opisthotonos position: the abnormal posture of the body occurring due to
generalized spastic contraction of the extensor muscle
LAB DIAGNOSIS:

Specimen:

● Necrotic tissue from the site of injury

Microscopy:

● Gram staining reveals drumstick appearance

● Unreliable (cannot distinguish from other clostridia)

Culture:

● More reliable
● Robertson cooked meat broth: C. tetani being proteolytic turns meat
particles black and produces foul order
● Blood agar with polymyxin B: C. tetani produces swarming growth

Toxigenicity test:

● Performed in vivo mouse inoculation test on specimens like serum or

urine.

PREVENTION:
Active immunization
● Monovalent vaccine: Tetanus Toxoid
● Combined vaccine: DPT (diphtheria pertussis and tetanus), Td (tetanus
and diphtheria), pentavalent vaccine (DPT, hepatitis B and Hib)
TREATMENT:

Passive immunization

● 250 IU of HTIG (Human Tetanus immunoglobulin) or 1500 IU of ATS

(Anti tetanus serum) given as a single IM dose.
● Effect of HTIG last for 30 days and ATS last for 7-10days

Combined immunization:

● Both active and passive immunization

 ● In a non-vaccinated person, 1st dose of TT is given in one arm along with
HTIG or ATS in another arm followed by a complete course of TT vaccine

Antibiotics:

● They play a minor role as they cannot neutralize toxins that are already
released
● They are useful in early infection, before the expression of toxin(<6hrs)
● They prevent further release of toxin
● Metronidazole is a drug of choice given for 7-10 days

Other measures:

● Endotracheal intubation and early tracheostomy are useful to protect the

airway
● Anti-spasmodic to eliminate reflex spasm
● Beta-blockers to control sympathetic hyperactivity
● Entry wound should be cleaned and debrided of necrotic material, to
remove anaerobic foci of infection
● The patient should be isolated in a separate room as any noxious stimulus
can aggravate the spasm

PREVENTION OF TETANUS AFTER INJURY:

Complete course of Not tetanus prone Tetanus prone

vaccination wound wound

Taken within 5 years nothing required Nothing required

Taken within >5to<10 Td 1 dose Td 1 dose

year

Taken >10 years back Td 1 dose Td 1 dose + HTIG

Unknown/incomplete Td complete dose Td complete dose

immunization +HTIG
 4. CAUSATIVE AGENTS OF ENTERIC FEVER

Salmonella is a gram-negative bacterium, belonging to the family Enterobacteriaceae.

TYPHOIDAL SALMONELLA: Includes

1. Salmonella typhi
2. Salmonella paratyphi A, B, C

● Enteric fever includes both typhoid fever and paratyphoid fever.

PATHOGENESIS:

● Infection is acquired by ingestion of contaminated food or water.

● Infective dose : 103 to 106 Bacilli
●
CLINICAL MANIFESTATIONS OF ENTERIC FEVER:

● The incubation period is 7-14 days

● Step ladder pyrexia
● Headache, Malaise, Anorexia
● Abdominal discomfort
● Rashes (Rose spots)
● Complications like intestinal bleeding, intestinal perforation, Meningitis,
Hepatosplenomegaly.

LABORATORY DIAGNOSIS OF TYPHOID FEVER:

Isolation of bacilli from a patient, Demonstration of Antibody in Patient serum.

1. Culture Isolation
● Blood Culture
85-90 % positive in the first week of illness
70% positive in the third or fourth week of illness
percentage positivity decreases thereafter
● Stool and Urine Culture
2. Culture Smear and Motility: Gram-stained smear reveals gram –ve bacteria. They are motile
with peritrichous flagella.
3. Biochemical identification
4. Slide agglutination test
5. Serum Antibody Detection (WIDAL TEST)
6. Antigen Detection: ELISA
7. Molecular Methods: Nested PCR
8. Non Specific Findings: WBC count – Neutropenia is seen in 15-25% of cases.
9. Antimicrobial Susceptibility Testing: Disk diffusion method on Mueller-Hinton Agar or MIC
based method (VITEK)
10. Detection of Carriers:
● Culture: By stool, urine, or bile culture.
● Detection of Vi Antibodies: here even a titer of 1:10 is considered a significant
OTHER ANTIBODY DETECTION TESTS:

● Typhidot test: It uses a dot ELISA format to detect both IgM and IgG separately after 2-3
days of infection.

1. CULTURE ISOLATION :

A)Blood Culture: Ideal Method

i) Inoculation of blood in the medium at dilution of 1:5 = Antibacterial components will

get diluted.
ii) Incubation @ 37℃ . Salmonella grows within 24 hrs.
iii) Subcultures are made and inoculated on blood agar and Mac Conkey agar.
iv) Colony Appearance on Blood Agar: Nonhemolytic moist colonies
v) Colony appearance on Mac Conkey agar: Non-Lactose Fermenting(NLF).
● Bone Marrow specimens: 55-90% sensitive, it is used when blood culture is
negative as the patient is on antibiotics.
● Duodenal Aspirate: if blood and bone marrow culture is –ve.
● Other specimens like rose spots, pus from suppurative lesions, CSF, Sputum,
Autopsy Specimen of the gallbladder, mesenteric lymph nodes where salmonella
can be isolated.
C)Urine Culture: Salmonella are shed in urine irregularly, infrequently. Urine samples are
centrifuged and deposits are inoculated in enrichment /selective media.

BIOCHEMICAL IDENTIFICATION :

S. Typhi is Anaerogenic

i. Indole test –ve

ii. Citrate test –ve
iii. Urease test –ve
iv. Triple Sugar Test: No gas production, production of small speck H2S.

SLIDE AGGLUTINATION TEST : It is done with polyvalent O and Polyvalent H antisera.

They react with isolated bacteria on the slide.

i) +ve agglutination implies isolation belongs to the genus Salmonella.

ii) Serotypes can be identified by specific O antisera
S.typhi agglutinates with O9 antisera
S.Paratyphi A agglutinates with O2 antisera
S.Paratyphi B agglutinates with O4 antisera.

WIDAL TEST: Discovered by Fernand Widal.

PRINCIPLE: Agglutination test, Detects H and O Antibodies in patients of enteric/

typhoid fever.
 Kauffmann-White Antigenic Classification

Salmonella Somatic O antigen Capsulated Vi Flagellar H

antigen antigen

S. Typhi 9,12 +(capsulated) d

S. Paratyphi A 1, 2 ,12 -(non capsulated) a/1,5

S.Paratyphi B 1, 4 ,5, 12 -(noncapsulated) b/1,2

APPARATUS: Two types of tubes

i. Felix Tube –A short round bottom tube for O agglutination.

ii. Dreyer’s Agglutination tube – A narrow tube with a conical bottom for H
agglutination.

ANTIGENS USED :

i) O antigen of S. Typhi (TO)

ii) H antigen of S.Typhi(TH)
iii) H antigen of S.Paratyphi A (AH)
iv) H antigen of S.Paratyphi B (BH).
The Paratyphi O is not employed as it cross-reacts with typhoid O antigen due to their
sharing factor 12.

PROCEDURE :

i. Patient serum is serially diluted from 1/10 to 1/640 in 4 sets of tubes.

ii. To each set of diluted sera four antigens, ( TO, TH, TA, TB) suspension are added.
iii. Incubated in a water bath @ 37℃ overnight.
RESULT:

O antibodies +O antigens = granular / H antibodies + H antigen = Fluffy/ Cotton

Chalk Agglutination./ disc shaped Wooly Agglutination.

● If Agglutination occurs, there is clear supernatant fluid.

● If Agglutination does not occur, Hazy supernatant fluid and button formation due to
deposition of antigen.

INTERPRETATION: The highest dilution of sera at which agglutination occurs in antibody

titer.

i) Significant Titre: Higher titer are significant. In India, a significant titer is

● H agglutination titre more than 200
● O agglutination titre more than 100.

In the above diagram, the TO titer is 1:160 implies it is a significant titer.

The titer is 1: 320 implies it is a significant titer.

● Demonstration of rising in titer by testing sera at a 1-week interval is more meaningful

than a single high titer.
 ● A rise in titer in anamnestic response is transient, usually falling after a week. In true
infection antibody titer increases fourfold after 1 week.

Widal test Result Suggestive of

Rise of TO and TH antibody Enteric fever due to S.typhi

Rise of TO and AH antibody Enteric fever due to S.Paratyphi A

Rise of TO and BH antibody Enteric fever due to S. Paratyphi B

Rise of TO antibody only Recent infection: due to S.Typhi or

Paratyphi A,B.

Rise of TH antibody only Convalescent stage/Anamnestic

response

Rise of all TH, AH, BH Post TAB vaccination.

False +ve result in:

i. Anamnestic response
ii. Bacteria fimbriae in Bacterial Antigen suspension.
iii. Prior Immunization ( TAB vaccine)

False –ve result in Early-stage ( 1st week of illness), Patient on Antibiotics.

TREATMENT :

● DOC : Ceftriaxone for empirical treatment.

● Alternative drug: Azithromycin, Ciprofloxacin
 5. MYCOBACTERIUM
✧ CLASSIFICATION OF MYCOBACTERIUM:

MYCOBACTERIA

M.tuberculosis complex. M.leprae. Non-tuberculous bacteria

(Further classified by Runyon)
❖ Photochromgens
❖ Scotochromgens
❖ Nonphotochromogens
❖ Rapid growers

PULMONARY TUBERCULOSIS :

● It is caused by the bacteria- Mycobacterium tuberculosis complex

M.tuberculosis complex includes
1. M.tuberculosis
2. M.bovis
3. Other members like M. caprae, M. africanum ,M. microti, M.
pinnipedii

MORPHOLOGY:
M.tuberculosis has an antigenic structure.
1. CELL WALL(INSOLUBLE)ANTIGENS:
The cell wall consists of several distinct layers
● Peptidoglycan layer: It maintains the shape and rigidity of the cell
● Arabinogalactan layer: It facilitates the survival of
M.tuberculosis within the macrophages
● Mycolic acid layer: It is the principal constituent, made up of
long-chain fatty acids attached to arabinogalactan. It confers very
low permeability to the cell wall and is responsible for acid fastness
and also reduces the entry of most antibiotics
● Outermost layer: It consists of lipids (mycocerosates and
acylglycerols), glycolipids, and mycosides (phenolic glycolipids)
 ● Proteins (e.g. porins, transport proteins): They are found
throughout the various layers
● Plasma membrane: This layer is present beneath the cell wall, into
which various proteins, phosphatidylinositol mannose, and
lipoarabinomannan {LAM) are inserted. LAM is an important
antigen, helps in attachment to host cells, and is also a target
antigen used for diagnosis.

2. CYTOPLASMIC(SOLUBLE)ANTIGENS:
These include antigen 5, antigen 6, antigen 60; and are used in serodiagnosis of
tuberculosis.

(fig. 63.1) Pg:624 (ref. Essential of Medical Microbiology by Apurva. S Sastry-3/E)

PATHOGENESIS:

❖ Source of infection: (1) human (e.g. cases of pulmonary tuberculosis), (2) bovine source
(e.g. consumption of unpasteurized infected milk).

❖ Mode of Transmission: M. tuberculosis is mainly transmitted by inhalation of droplet

nuclei, generated while coughing, sneezing, or speaking of infected patients.
Other modes are Inoculation and ingestion
 ❖ Risk factors: include low cell-mediated immunity, age(late adulthood and early
adulthood periods are prone), sex(Women at 25-35age, men at an older age are prone)
CLINICAL MANIFESTATIONS
Tuberculosis (TB) is classified as pulmonary and extra-pulmonary forms.
Pulmonary Tuberculosis (PTB)
Pulmonary tuberculosis (PTB) accounts for 80% of all cases of tuberculosis (TB).
It can be further categorized into primary or postprimary (secondary) types

(Table 63. 1) pg 626(ref. Essential of Medical Microbiology by Apurva. S Sastry-3/E)

LABORATORY DIAGNOSIS : Mycobacterium tuberculosis :

DIAGNOSIS OF ACTIVE TUBERCULOSIS
SPECIMEN COLLECTION:
• In pulmonary TB: Sputum (2 specimens-spot and early morning), gastric
aspirate (in children)
• In EPTB: Specimens vary depending on the site involved.
Digestion, decontamination, and concentration of specimen:
• Modified Petroff's method (4% NaOH) or NALC (N-acetyl-L-cysteine) + 2%
NaOH.
 DIRECT MICROSCOPY BY ACID-FAST STAINING:
• Ziehl-Neelsen (ZN) technique-long slender, beaded, less uniformly stained red
color acid-fast bacilli
• Kinyoun's cold acid-fast staining
Fluorescent (auramine) staining-it is more sensitive and Conventional culture
media- take 6-8 weeks
• Solid media, e.g. Lowenstein Jensen (LJ) medium- shows
rough, tough, and buff-colored colonies in 6-8 weeks
• Liquid media- Kirchner's medium and Middlebrook 7H9
medium.
AUTOMATED CULTURE METHODS:-(take 3-4 weeks)
• MGIT system: Detects growth and resistance to antitubercular
drugs (ATDs); with a turnaround time of 2-3 weeks
• BacT/ALERT: Detects growth
• Versa Tek System: Detects growth and resistance to ATDs.
CULTURE IDENTIFICATION:
• MPT 64 antigen detection-
• Biochemical identification- Niacin test (obsolete).
MOLECULAR METHODS:
• PCR detecting IS6110 gene
• CBNAAT (GeneXpert)- for identification and detection of resistance to
rifampicin; has a turnaround time 2 hour
• Line probe assay (e.g. Genotype TB)-for identification and detection of
resistance to 1st and 2nd line ATDs; has a turnaround time 2- 3 days.
DIAGNOSIS OF LATENT TUBERCULOSIS:
•By tuberculin test (e.g. Mantoux test) and interferon-gamma release assay (IGRA).
DRUG SUSCEPTIBILITY TESTING:
PHENOTYPIC METHOD:
❖ MGIT (used for 1st and 2nd line drugs): Resistance is determined by the
growth of TB bacilli in the drug-containing tube as compared to control tube
(drug-free) within 4-21days
❖ Proportion method (used for 1st and 2nd line drugs): An isolate is considered
resistant to a given drug when the growth of 1% or more is observed in the
drug-containing LJ medium compared to the control LJ medium without drug
after 42 days of incubation.

GENOTYPIC METHODS:
❖ GeneXpert (used only for rifampicin): Five probes targeting different wild
sequences of the rpoB gene are used. Turnaround time is <2 hours
❖ Line probe assay: Detects resistance to both 1st and 2nd line drugs in 2- 3 days
of turnaround time.
TREATMENT:
Anti-tubercular drugs (ATDs) can be classified into:
✔ First-line drugs
✔ Second-line drugs
FIRST-LINE DRUGS:
⮚ isoniazid (H)
⮚ Rifampin (R)
⮚ Pyrazinamide (Z)
⮚ Ethambutol (E)
⮚ Streptomycin (S)
SECOND-LINE DRUGS:
⮚ Ethionamide and prothionamide
⮚ Quinolones: levofloxacin, moxifloxacin and ofloxacin .
⮚ Aminoglycosides: kanamycin, capreomycin, and amikacin
⮚ Cycloserine and para-aminosalicylic acid
⮚ Macrolides: clarithromycin
 ⮚ Bedaquiline (approved in 2015)

Pg:632 Treatment regime based on drugs susceptibility testing (ref. Essential of

Medical Microbiology by Apurva. S Sastry-3/E)

RESISTANCE TO ANTITUBERCULAR DRUGS (ATDS)

MONO RESISTANCE
Defined as resistant to one first-line ATD only.
POLY RESISTANCE
Defined as resistant to > 1 first-line ATD except other than both INH and
rifampicin resistance to Antitubercular Drugs(ATDs)
RIFAMPICIN RESISTANCE (RR)
 Defined as rifampicin resistance with or without resistance to other ATDs
(excluding isoniazid).
MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB)
MDR-TB is defined as resistance to isoniazid and rifampicin with or without
resistance to another first-line anti-tubercular drug
EXTENSIVELY DRUG-RESISTANT TUBERCULOSIS (XDR-TB)
They are MDR-TB cases which are also resistant to Fluoroquinolones
(ofloxacin/levofloxacin) and At least one injectable aminoglycosides (kanamycin,
amikacin or capreomycin).

DRUG RESISTANCE GENES FOR M.TUBERCLOSIS:

(table 63. 70 Pg: 634 (ref. Essential of Medical Microbiology by Apurva. S

Sastry-3/E)

BACILLUS CALMETTE GUERIN VACCINE (BCG): Vaccine for the prevention of

tuberculosis
⮚ BCG strain: Live-attenuated M. Bovis was the strain
⮚ Types of vaccine: BCG is available in two forms
• Liquid (fresh) form
 • Lyophilized from (freeze-dried) form
⮚ Administration of BCG
• Dose and strength: 0.1 mL containing 0.1 mg TU
• Alcohol should not be used to wipe the skin
• Site: It is given above the insertion of the left deltoid
• Route: It is administered by intradermal route by using a 26 gauge tuberculin
syringe
⮚ Indications of BCG:
• Direct BCG: BCG is directly given to newborns soon after birth. This strategy
is followed by most of the developing countries including India. If not given at
birth it can be given later, maximum up to 2 years
• Indirect BCG: BCG is given after performing the tuberculin test.
⮚ Contraindications to BCG include:
• HIV-positive child
• Child borne to AFB positive mother
• Child with low immunity
• Generalized eczema
• Pregnancy.
 6.BACILLARY DYSENTERY
Dysentery is characterized by diarrhea with increased blood and mucus, often associated with
fever, abdominal pains, and tenesmus.

CAUSATIVE AGENTS:

● Shigella species
● Campylobacter jejuni
● Enterohemorrhagic E. coli
● Enteroinvasive E. coli
● Vibrio parahaemolyticus

BACILLARY DYSENTERY

● Bacillary Dysentery is characterized by the passage of loose stool mixed with blood and
mucus.
● Causative agent is – Shigella.
● It is classified into four species and based on O antigen they are further classified into
several serotypes.
● Shigella dysenteriae has 15 serotypes.

PATHOGENESIS

● Mode of transmission:

Infection occurs by ingestion through contaminated fingers (most common), food, water and
rarely flies.

● Minimum infective dose:

As low as 10-100 bacilli can initiate the disease, probably because of the ability to survive in
gastric acidity.
 ● Entry via M cell:

● Exotoxins

Some serotypes of Shigella produce enterotoxins:

Shigella enterotoxin and Shiga toxin

● Endotoxins

Induces intestinal inflammation and ulceration.

CLINICAL MANIFESTATIONS

5 phases:

▪ INCUBATION PERIOD
Lasts for 1-4 days
▪ INITIAL PHASE
Watery diarrhea with fever, malaise, anorexia, and vomiting
▪ PHASE OF DYSENTERY
Frequent passage of bloody mucopurulent stools with increased tenesmus and abdominal
cramps.
▪ PHASE OF COMPLICATION
Seen in children less than 5 years of age
⮚ Intestinal complications- toxic megacolon, perforations, and rectal prolapse
⮚ Metabolic complications- hypoglycemia, hyponatremia, and dehydration
⮚ Ekiri Syndrome or toxic encephalopathy- altered consciousness, seizures,
delirium, abnormal posturing, and cerebral edema
 ▪ Postinfectious phase
After months, autoimmune reactions are developed

EPIDEMIOLOGY

▪ Risk factors include overcrowding, poor hygiene, and children less than 5 years
▪ It tends to occur as an epidemic in developing countries such as the Indian Subcontinent
and sub-Saharan Africa
▪ Humans are the natural host and cases are the only source of infection.
▪ Children less than 5 years account for nearly 69% of cases
▪ With improved sanitation, the incidence is decreasing and the drug resistance among
Shigella strains is increasing

LABORATORY DIAGNOSIS

The fresh stool is the ideal specimen.

PATHOLOGY:

▪ Shallow ulcer
▪ Inflamed intervening mucosa

STOOL MACROSCOPIC FEATURE:

▪ Number of motion: >10/day

▪ Amount: small quantity
▪ Colour: bright red
▪ Odorless
▪ Alkaline in nature
▪ Adherent to the container

MICROSCOPIC FEATURE:

▪ Presence of RBCs – discrete or rouleaux

▪ Pus cells- numerous
▪ Macrophages- numerous
Stools can be used to:

● Wet mount preparation

● Culture
● Selective media such as MacConkey Agar
● Culture smear and motility testing
● Serotyping
● Colicin typing

TREATMENT

● Ciprofloxacin is the drug of choice

● Alternative drugs include ceftriaxone, azithromycin, and fifth-generation quinolones
● Oral Rehydration Solution (ORS) should be started for correction of dehydration and
nutrition

PREVENTION

● Handwashing after handling children’s feces and before handling of food is highly
recommended
● Stool decontamination with Sodium hypochlorite
● No vaccine is available

REFERENCE:

Essentials of Medical Microbiology by Apurba S Sastry and Sandhya Bhat – Third Edition
 7. CORYNEBACTERIUM DIPHTHERIAE

Corynebacterium diphtheriae in methylene blue-stained smear

Ref: fig 24.1A pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● The causative agent of diphtheria infecting the throat if not treated properly, it may be
life-threatening

MORPHOLOGY:

● Gram-positive but tend to decolorize easily

● Encapsulated, nonsporing, nonmotile bacillus
● Frequently shows club-shaped swellings
● Has several species called diphtheroids
● Two characteristic features of Corynebacterium diphtheriae
● Chinese letter arrangement: appear as V or L shaped because the bacterial
cells divide and the daughter cells tend to be in acute angle to each other
● Metachromatic granules: when taking up the Loeffler’s methylene stain they
appear as bluish granules at the end of the poles of the bacilli. they are also
called volutin or babes-Ernst granules

Virulence factors(Diphtheriae toxin): Primary virulence factor responsible for the disease
STRUCTURE OF TOXIN:

MECHANISM OF DIPHTHERIA TOXIN:

FACTORS REGULATING TOXIN PRODUCTION:

● Remains toxigenic as long as the phage are present inside the bacilli
● Toxin production depends on iron concentration
● Diphtheria toxin is also produced by C. ulcerus and C. pseudotuberculosis

PATHOGENICITY:

● Incubation period : 3 to 4 days

● Diphtheria is toxemia but never bacteremia
● Bacilli are noninvasive but the toxin produced by them enters the bloodstream causing
the disease

RESPIRATORY DIPHTHERIA:

● The most common form of diphtheria

● Affected areas: nose and larynx
 ● Facial diphtheria:

Pseudomembrane coat is classically seen in diphtheria

Ref: pg no 263 fig.no 24.2A Essentials of medical microbiology by apurba Sastry 1st edition

Bull neck appearance

Ref: pg no 263 fig.no 24.2B essentials of medical microbiology by apurba Sastry 1st edition

● Elicits an inflammatory response

● This leads to the formation of mucosal ulcers lined by leathery greyish white
pseudomembranous coat
● Pseudomembrane extends in severe cases leads to airway obstruction
● Massive tonsillar swelling and neck edema bull neck appearance
SYSTEMIC COMPLICATIONS:

NEUROLOGIC MANIFESTATIONS :

● Polyneuropathy
● Peripheral neuropathy
● Ciliary paralysis

CARDIOLOGICAL MANIFESTATIONS:

● Myocarditis
● Arrhythmias
● Dilated cardiomyopathy

Polyneuropathy and myocarditis are the late toxic manifestations of diphtheria

LABORATORY DIAGNOSIS:

Specimen: throat swab and a portion of pseudomembrane

Direct smear:

Ref: fig 24.1B pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● Gram stain: club-shaped gram-positive bacilli with Chinese letter arrangement

Ref: fig 24.1C pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● Albert stain: green bacilli with bluish-black metachromatic granule

Culture media:

Ref: fig24.4A pg no 264 essentials of medical microbiology by apurba Sastry 1st edition

● Enriched medium: blood agar, chocolate agar, Loeffler’s serum slope

Ref: fig24.4B pg no264 essentials of medical microbiology by apurba Sastry 1st edition

● Selective medium: potassium tellurite agar produce black colonies

IDENTIFICATION:

● Biochemical tests such as hiss serum sugar fermentation test

● Automated identified systems such as MALDI-TOF or VITEK
● Diphtheria toxin identification:
● In vivo: guinea pig inoculation in subcutaneous and intracutaneous region
● In vitro:
 Elek’s gel precipitation test

Ref :fig.no24.5 pg no265 essentials of medical microbiology by apurba Sastry 1st edition

Elek's gel precipitation test

Detection of tox gene by PCR
Cytotoxicity on cell lines
Detection of toxin by ELISA or ICT

EPIDEMIOLOGY:

● Source of infection: by carriers

● Carrier: maybe nasal or throat. Nasal carriers are more dangerous than throat due to
frequent shedding
● Transmissions: by droplets
● Reservoir: humans
● Age: common among children of age 1 to 5. But due to immunization, it has been
changed to school children
● The situation in the world: due to widespread immunization, the cases are drastically
declined. but in recent years around 16648 cases are recorded
● The situation in India: India still has the highest cases of diphtheria ( around 8788) in
2018
PROPHYLAXIS:

Infection control measure: patient kept in an isolated room to prevent droplet infection

Post-exposure prophylaxis: booster dose of diphtheria vaccine + penicillin G +erythromycin (7

to 10 days) is given

VACCINATION:

● Induces antitoxin production in the body

● Can’t prevent the cutaneous diphtheria
● Can’t eliminate the carrier state

TYPES OF VACCINE:

● Single vaccine: Diphtheriae toxoid = diphtheriae toxin + formalin +alum (adjuvant)

● Combined vaccine:

DPT = DT( diphtheria toxoid)+ Pertussis+TT(tetanus toxoid)

DaPT = DT+PT+acellular pertussis(aP)

ADMINISTRATION OF DIPHTHERIA VACCINATION :

Schedule: by national immunization schedule of India 2020

Children:

DOSE TYPE OF DOSE GIVEN AT THE AGE OF

First dose Pentavalent vaccine 6th week
Second dose Pentavalent vaccine 10 th week
Third dose Pentavalent vaccine 14 th week
Fourth dose Booster dose 16 – 24 months
Fifth dose Booster dose 5 yrs
Sixth dose Booster dose 10 yrs
Seventh dose Booster dose 16 yrs
Pregnant women: two doses of Td at the one-month interval

Site: IM at the lateral aspect of the thigh

Storage: kept at 2-8*C

Adult immunization: recommended as cases are increasing across the world

● Adults who have completed primary vaccination: Td booster dose is indicated every 10
years till 65 years
● Adults who have not completed primary vaccination: 3 doses of Td given at 0, 1 month,
and 1 year

ADVERSE REACTION OF DPT ADMINISTRATION:

Mild: fever and local reaction

Severe: associated with neurological complications

Absolute contraindication:

● Hypersensitivity to the previous dose

● Progressive neurological disorder
 8. BORDETELLA PERTUSSIS

ETIOLOGY

● Bordetella is highly fastidious, very small, gram-negative coccobacilli, non-fermenter,

family Alcaligenaceae
● Bordetella pertussis: cause whooping cough in children, a highly contagious
vaccine-preventable bacterial disease
o characterized by a paroxysmal cough ending in a high-pitched inspiratory sound
described as "whoop".
o B. pertussis causes a violent paroxysmal productive cough in children called whooping
cough or pertussis.

PATHOGENESIS

▪ B. pertussis produces a wide array of toxins and biologically active products

▪ Pertussis Toxin (PT) most important virulence factor

▪ It causes ADP ribosylation of G protein, which activates adenylyl cyclase, leading to

increased concentrations of cAMP; responsible for producing a variety of biological
effects, such as:
✔ T cell mitogenicity
✔ Hemagglutination
✔ Adhesion to respiratory ciliated cells
✔ Inhibition of neutrophil oxidative burst monocyte migration, histamine release from mast
cells
✔ Induction of leukocytosis
✔ Enhancement of insulin secretion leading to hypoglycemia

▪ Tracheal cytotoxin

It is a part of cell wall peptidoglycan, which causes damage to the cilia of respiratory
epithelial cells by producing interleukin-1 and nitric oxide intracellularly.

▪ Adenylate cyclase toxin: It activates cyclic AMP, which impairs the host immune
function.

▪ Dermonecrotic toxin: contribute to respiratory mucosal damage.

▪ Endotoxin: has properties similar to those of other gram-negative bacterial LPS.

▪ Adhesins: role in bacterial attachment.

Examples include:

✔ Filamentous hemagglutinin (FHA)

✔ Pertactin, an outer-membrane protein
✔ Fimbriae or pili or agglutinogens
✔ BrkA (Bordetella resistance to killing) protein: mediates the serum resistance and
adhesion.

LAB DIAGNOSIS

• Specimen collection

Nasopharyngeal secretions are the best specimens which may be obtained by-

➔ Nasopharyngeal aspiration (best method)

➔ Perinasal swab
● For culture, alginate swabs are the best followed by dacron swabs.
● Cotton swabs are not satisfactory as fatty acids present in cotton may inhibit the growth
of the bacilli. However charcoal-impregnated cotton swabs (Stuart’s) may be useful.
● It is recommended to collect six swabs, at 1- 2 days intervals to achieve maximum yield.
 ● Transport: Specimens- processed immediately. If the delay is expected, then suitable
charcoal-based medium (Amies) can be used.

DIRECT DETECTION:

● B. Pertussis may be directly detected from nasopharyngeal secretions by direct

immunofluorescence test using fluorescein labeled polyclonal or monoclonal
antibodies.Because of poor sensitivity and specificity, it is not widely used.
● Culture: Nasopharyngeal Culture remains the gold standard method of diagnosis.
● B. pertussis is a strict aerobe, grows best at 35-37°C.
● It is fastidious, requires special complex media for primary isolation, such as-
● Charcoal agar supplemented with 10% horse blood and cephalexin (Regan and Lowe
medium). It is currently the medium of choice.
● Bordet Gengou glycerine-potato-blood agar was a traditional medium used before.
● Colonies are greyish while, convex with a shiny surface appear after 3- 5 days, described
as mercury drops or bisected pearls appearance

Reference: Essentials of medical microbiology by apurba Sastry –2/E–pg 368- fig 3.4
CULTURE SMEAR: Gram-staining of culture reveals small, ovoid coccobacilli (0.5 µm), tend
to arrange in loose clumps, with clear spaces in between giving a thumbprint appearance

Reference: Essentials of medical microbiology by apurba Sastry-2/E-pg:368-fig:3.4c

DETECTION SERUM ANTIBODIES

● Enzyme immunoassays (EIA~) using purified antigens of B. pertussis, such as PT, FHA,
and pertactin are the methods of choice.

MOLECULAR METHODS:

● PCR is being increasingly used in many laboratories replacing the culture, because of
increased sensitivity, specificity, and quicker results.
● The most common targeted genes are 1S481 and the PT promoter region genes.
TYPING OF B. PERTUSSIS

● It is important during outbreak investigation to find out the epidemiological link between
the isolates.
● Serotyping: It is based on two fimbrial antigens ( type 2 and 3) and one
lipooligosaccharide antigen (type l ) of Bordetella pertussis.
● Genotyping: It can be carried out by gene sequencing, and pulsed-field gel
electrophoresis~ (PFGE).
● Others: Lymphocytosis is common among young children but not among adolescents.
 9. LEPTOSPIROSIS

INTRODUCTION:
● Leptospirosis is a Spirochetal infection caused by Leptospira
● Leptospira is a thin, flexible, coiled helical bacteria.
● They have many tightly coiled spirals, with hooked ends.
● 6- 20 μm in length.

CLASSIFICATION:

● Leptospira is broadly classified into:

● L. interrogans
● L. biflexa
● These are further classified into various subgroups based on Liposaccharide antigens.

L. interrogans 26 Serogroups

L. biflexa 32 Serogroups

● SEROGROUPS OF LEPTOSPIRA INTERROGANS:

Australis Grippotyphosa Sarmin

Autumnalis Hebdomadis Sejroe

Ballum Icterohaemorrhagiae Semaranga

Bataviae Javanica Tarassovi

Canicola Leptonema Hurstbridge

Celledoni Lyme Ranarum

Cynopteri Mini Turneria

Djasiman Pomona Manhao

Pyogenes
PATHOGENESIS:

● There are two stages of pathogenesis:

FIRST PHASE (Septicemic phase)

Leptospira enter through the mucosa (Conjunctival /oral)

or abraded skin

L.interrogans enter the bloodstream and spread to various

organs including brain, liver, lung, heart & kidney.

● Vascular damage occurs Spirochetes seen in walls of capillaries, Small & Large
blood vessels.
● Penetration and invasion of tissues due to Active motility and Hyaluronidase
release.

SECOND PHASE: (Immune phase)

Antibodies develop and form

Antigen-Antibody complexes deposited in various organs

Renal colonization occurs where bacilli are adherent to the proximal tubular brush border

Excreted in urine
CLINICAL MANIFESTATION:
● INCUBATION PERIOD: 1 to 30 days.
● MILD ANICTERIC FEBRILE ILLNESS: Occurs in 90 % of patients.
● A biphasic course involving the Septicemic phase and
● Presents with flu-like illness with:
➔ Fever
➔ Chills
➔ Headache
➔ Nausea & Vomiting
➔ Conjunctival suffusion
➔ Myalgia
➔ Abdominal pain
 10. SYPHILIS

● Syphilis is a sexually transmitted disease caused by Treponema pallidum

● These bacteria come under the genus Spirochetes
● Trep- turn (helical); nema- thread-like ; pallidum- pale staining

PATHOGENESIS OF SYPHILIS

1) MODE OF TRANSMISSION
2) SPREAD

3) IP- INCUBATION PERIOD

● 9 to 90 days
● Note- blood is infectious even during incubation and early stages

CLINICAL MANIFESTATIONS

STAGES- 4
1) PRIMARY SYPHILIS
▪ 2 characteristic features are hard chancre and regional lymphadenopathy

NOTE:

If transmitted by

▪ Direct contact 🡪 primary chancre is extragenital, usually on fingers

▪ Blood transfusion 🡪 no chancre

2) SECONDARY SYPHILIS
● Develops after 6-12 weeks after healing of the primary lesion
● Skin and mucous membrane commonly affected
● Generalized lymphadenopathy
Note: condyloma lata vs accuminatum

3) LATENT SYPHILIS
● No lesions but the patients are serologically positive ie) Antigens and Antibodies present in
serum
● So the patient can transmit the infection via blood or in utero
4) LATE/TERTIARY SYPHILIS
● Gumma – destructive granulomatous lesions of skin, bones
● Neurosyphilis- chronic meningitis, general paralysis of insane and tabes dorsalis
● Cardiovascular syphilis- aneurysm of ascending aorta and aortic regurgitation
Note:

- latent + late/tertiary syphilis = Quaternary syphilis

CONGENITAL SYPHILIS

● vertical transmission from mother to baby

● consists of a triad of symptoms

DIAGNOSIS:

● laboratory diagnosis of treponema consists of demonstration, detection of antibodies, PCR

A) MICROSCOPY (common for all spirochetes)

1) DARK GROUND MICROSCOPE:

● Since Treponemes cannot be visualized by light microscope, the dark ground microscope is
used
● Advantages- no stain used so organisms are live and motile
● Motility- i) Flexion- extension motility
ii) Cock screw motility (similar to H. pylori)

Dark background, light spirochetes

Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3A

2) DFA- TP (Direct Fluorescent Antibody Staining for
T. pallidum)
● Stain🡪 fluorescent- labeled monoclonal antibody targetted
against T. pallidum surface antigens
● Dark background, apple green fluorescent colored bacilli

Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3B

3) BRIGHT-FIELD MICROSCOPY
● Since spirochetes are thin, silver impregnation stain is used
● 2 types of stain i) FONTANA stain- yellow background and brown spirochetes
ii) Levaditi stain
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3C

B) IMMUNOLOGICAL TEST/ SEROLOGY

● As microscopy is difficult and culture methods are not available, antibody detection methods
are of paramount importance in the diagnosis of syphilis

I) Non-Treponemal or Standard Test for Syphilis(STS)

● Here nonspecific antibodies (Reagin antibody) are detected
● These antibodies are secreted against Cardiolipin antigen present in Oxheart
● Type- Ig G or rarely Ig M
VDRL test

● This test was named after Venereal Disease Research Laboratory (VDRL), New York, where
the test was developed
● Principle🡪 Slide flocculation
● Procedure
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 760, fig 77.4
Uses- cheaper, preferred as a screening test (high sample load), detect neurosyphilis

RPR test

● Similar to VDRL with the following differences

● Prolonged shelf life, so used for the individual test (low sample load)
● No need for a microscope, results can be read with naked eyes
● More expensive

PROS AND CONS OF NON SPECIFIC OR NON TREPONEMAL TESTS

PROS

● To monitor prognosis
● IOC- investigation of choice
● VLDR- to detect neurosyphilis (CSF antibody)
● 98- 99 % specificity

CONS

● BFP- biologically false-positive reactions 🡪 positive results in nontreponemal tests, with

negative results in treponemal tests, in the absence of syphilis and not caused by technical
faults
● Prozone phenomena
● Low sensitivity

II) TREPONEMAL OR SPECIFIC TESTS:

● Here specific treponemal antibodies are detected in patient serum
● Extract of live or killed T. pallidum is used
● Advantage- High specificity and High sensitivity,
● Used as Confirmatory test
C) CULTURE
● Pathogenic Treponema are NONcultivable so Animal Inoculation is used
● Eg; Rabbits testes 🡪 Nichols Strain

D) PCR/ MOLECULAR METHODS

● To amplify and detect T. pallidum specific genes
● PCR is of paramount importance in the diagnosis of congenital and neurosyphilis

SYPHILIS AND HIV

● HIV 🡪 increases the risk of syphilis because Genital syphilis facilitates the transmission of
HIV through the abraded mucosa
● HIV patients if gets syphilis will rapidly progress to late stages of syphilis and also gets
neurosyphilis
● Also, diagnosis of syphilis in HIV patients is difficult
TREATMENT

● DOC of all stages of syphilis 🡪 Penicillin

● But for patients with penicillin allergy, tetracycline is used
PREVENTION:

● Treatment of cases and contacts (sexual partners)

● Education about safe sex practices
● Prophylactic use of barrier contraceptive methods.

Reference:

Essentials of Microbiology Apurba Sastry 3/E

 11. RICKETTSIACEAE
GENERAL FEATURES
● They are obligate intracellular organisms.
● Cannot grow on artificial media, but can grow on cell line /mice inoculation (except
bartonella).
● Transmitted by arthropod vectors (except Coxiella by inhalation mode.
● Final target site: vascular endothelial cells.

CLASSIFICATION
Order Rickettsiales

Family Rickettsiaceae Family anplasmataceae

Rickettsia Ehrlichia
Orientia Anaplasma
Wolbachia
Neorickettsia

FAMILY RICKETTSIACE
RICKETTSIAL INFECTIONS
1. Typus group.
2. Spotted fever group.

ANTIGENIC STRUCTURE
● OMP antigens – for serodiagnosis .
● LPS antigens - serves basis of Weil Felix test .

PATHOGENESIS

● Transmission: through arthropod vectors.

● Tick and mite through a bite can act as reservoirs to maintain the organism by
transovarial transmission.
● Louse and flea through rubbing or scratching the vector on abraded skin or mucosa.
● Spread: lymphatics then lymph nodes then bloodstream.
 ● Target sites: endothelial cells.
● Intracellular survival: though they get phagocyted they resist lysosomal killing and
inhibit phagolysosomal fusion.
● Multiplies by binary fission.
● Cell to cell spread by actin polymerization.

FEATURES

Epidemic typhus (louse-borne)

● Vector: louse.
● Species:R.prowazekki .
● Clinical manifestations: acute febrile disease accompanied by incubation period 1_2
weeks.
1. Headache
2. Eye discharge
3. Rashes: begins on the trunk then spreads to extremities except for palms and soles
4. Myalgia
5. CNS involvement: confusion, coma
● Risk factors: outbreaks occur in unhygienic conditions.
● Brill Zinsser disease: recrudescent illness and the R.prowazekii remains latent for years.

ENDEMIC TYPHUS (FLEA-BORNE)

● Vector: flea.
● Species: R.typhi
● Reservoir: rodents (Rattus rattus )
● Clinical manifestations: incubation period 1_2 weeks
 1. Similar to epidemic typhus but it is milder and rarely fatal.
2. Rashes on skin involving the trunk more often than the extremities.
● Geographical distribution: worldwide.

ROCKY MOUNTAIN SPOTTED FEVER (TICK-BORNE)

● Vector: Tick.
● Species: R.rickettsi
● Most severe form: associated with high mortality rate.
● Reservoir: ticks as both vector and reservoir.
● Clinical manifestations: incubation period 4_ 14 days.
1. RMS fever _ fatal
2. Rashes on palm and soles
3. Maculopapular and becomes hemorrhagic
4. CNS involvement
● Geographical distribution: America.

INDIAN TICK TYPHUS (TICK-BORNE)

● Clinical manifestations: Eschar at the site of tick bite is more severe in diabetes, alcoholic
patients.
● Vector: Tick.
● Species: R.conori
Geographical distribution: Europe, Asia.

RICKETTSIAL POX (MITE BORNE)

● Vector: mite.
● Species: R.akari
● Clinical manifestations:
1. Vesicular rashes
2. Eschar _ painless black lesions
3. Regional lymphadenopathy
● Geographical distribution: America and Ukraine.

LABORATORY DIAGNOSIS
WEIL FELIX TEST
● It is a heterophile agglutination test; where rickettsial antibodies are detected by using
certain Proteus strains (OX19, OX2, OXK strains) due to the cross-reactivity of alkali
stable LPS antigen.
 ● Procedure: It is a tube agglutination test; serial dilutions of patient serum are treated with
motile strains of P.vulgaris OX19 and OX2 and P.mirabilis OXK.
● Results:
1. In epidemic and endemic typhus _sera agglutinate mainly with OX19 and OX2 are
elevated
2. In tick-borne spotted fever _antibodies to OXK were raised.
3. The test is negative in rickettsial pox Qfever ehrlichiosis and bartonellosis .

False-positive titer
● It May be seen in presence of underlying Proteus infection .hence a fourfold rise of
antibody titer in paired Sera is more meaningful than a single titer.
It may occur due to excess antibodies in patients Sera (prozone phenomenon).this can be
obviated by testing with serial dilutions of the patient's sera.
● Weil Felix test being a non-specific test should always be confirmed by specific tests.

SPECIFIC ANTIBODY DETECTION TESTS

● Indirect immunofluorescence assay
● CFT
● Ig M capture Elisa
● Latex agglutination test

Other methods:
● Cutaneous biopsy – histological examination of sample from lesion.
● Isolation can be done by cell lines since they can’t grow in artificial media
● Neil Mosser reaction: specimens are inoculated in guinea pigs
1. R.rickettsiae: produces scrotal necrosis
2. R.prowazekki: produce only fever without any testicular inflammation
3. R.conori,R.akari:produce positive tunica reaction
● PCR.

TREATMENT OF RICKETTSIOSIS
● Doxycycline is the drug of choice for the most rickettsial illness.
● Chloramphenicol as an alternative.
SCRUB TYPHUS
● Agent :Orientia tsutsugamushi. It differs from rickettsia by both genetically as well as
lacking in LPS in the cell walls.
● Vector: mites the larval stage called chiggers stage of mite are the only stage that feeds on
humans hence scrub typhus is also known as chiggerosis
● Clinical manifestations: triad
1. Eschar
2. Lymphadenopathy
3. Maculopapular rash
● Antigenic diversity: three major types
1. Karo
2. Gillian
3. Kato
● Zoonotic tetrad: mites, rats, shrews, scrub vegetations, wet season.
● Indian scrub typhus is more common in India.
● Diagnosed by Weil Felix test (OXK raised).

EHRLICHIOSIS
● Pathogenic species: Ehrlichia chaffeensis ,Ehrlichia ewingii,Anaplasma
phagocytophilum,Neorickettsia sennestu
● Transmission: ticks except Neorickettsia sennestu_by fish carrying flukes.
● Epidemiology: USA and Asia.
● Clinical manifestations: headache, myalgia, lymphadenopathy, vomiting, diarrhea,
change in mental status.
● Inclusions: morula, elementary and initial bodies.
● Treatment: Doxycycline

Q FEVER
● Species: Coxiella burnetti
● Transmission: contaminated urine feces and milk of sheep and cattle.
● Geographical distribution: India, most part of the world except cold region.
● Clinical manifestations
1. Acute fever: pneumonia, hepatitis, pericarditis
2. Chronic fever: endocarditis
3. No rashes
● Lab diagnosis: isolation followed by IFA, PCR can also be done
● Treatment
1. Acute fever _ Doxycycline and quinolones
2. Chronic fever _ hydroxychloroquine
 ● Prevention _Vaccination, good disposal of cattle wastes, good pasteurization of milk.

BARTONELLOSIS
● B.hensele :
1. Transmission: cats
2. Cat scratch disease: causes regional lymphadenopathy and painless pustule.
● Bacillary angiomatosis: neovascular lesions in HIV infected persons.
● Bacillary peliosis: Angio proliferative disorder involving liver and spleen.
● B.quintana : Causes TRENCH FEVER.
● B.bacilliformis : Oroya fever or carissons disease, Verruca peruana.
Laboratory diagnosis
● Antibody detection_ IFA, PCR.
Treatment
● Cat scratch disease _azithromycin.
● Bacillary angiomatosis _ erythromycin or Doxycycline.
 12. LABORATORY DIAGNOSIS OF RESPIRATORY
TUBERCULOSIS

1. SPECIMEN COLLECTION:
Spot sample(on the same day under supervision)
2 sputum samples
early morning sample(on next day)
gastric aspirate – children(tend to swallow sputum)
- ICU patients (aspiration)
▪ alternatively, 2 spot samples at least one hour apart can be collected.
▪ Early morning sputum specimen: collected on an empty stomach, after rinsing the
mouth
▪ Deep inhalation and cough out from chest during exhalation and spit the sputum
in container
▪ Sputum – 3-5 ml, thick and purulent.

2. DIGESTION, DECONTAMINATION, AND CONCENTRATION:

Sputum and specimens are

i. Digested – to liquefy thick pus cells and homogenization.

ii. Decontaminated – to inhibit normal flora
iii. Concentrated – to increase yield.

METHODS:

A. Modified Petroff’s method- 4% NaOH ; recommended for LJ culture

B. NALC( N-acetyl -L-cysteine) + 2% NaOH: recommended for automated culture

systems.
 3. DIRECT MICROSCOPY BY ACID-FAST STAINING:

A. ZEIHL-NEELSEN (ZN) TECHNIQUE ( HOT METHOD )

Smears are prepared from the thick part of sputum

Smear is stained by acid-fast stain

M.tuberculosis appears slender, beaded, less uniformly stained red color acid-fast bacillus.

PRESUMPTIVE DIAGNOSIS: “acid-fast bacillus resembling M. tuberculosis are seen bt smear

microscopy by ZN stain “.

ADVANTAGES: rapid, easy to perform, cheaper

DISADVANTAGES: less sensitive than culture, cannot determine the viability.

B. KINYOUN’S COLD ACID – FAST STAINING

C. FLUORESCENCE STAINING:

- Primary stain- auramine -phenol (7-10 min )

- Decolourizer – 0.5 % acid alcohol ( 2min )
- Counterstain – 0.1% potassium permanganate ( 30 secs )

Examine under a fluorescent LED microscope

Bacilli – brilliant yellow against a dark background

⮚ Smears are screened faster – The screening method

⮚ More sensitive than ZN staining
A: ZN staining of sputum smear showing long, slender, and beaded red-colored acid-fast bacilli.

B. auramine phenol staining sputum smear – tubercle bacilli appear bright brilliant green against
a dark background.

REFERENCE: Essentials of medical microbiology by apurba Sastry- 3/E-pg no.628-fig 63.3A

and B.

4 . CULTURE METHODS:

A gold standard method of diagnosis of TB.

a. Conventional solid media ( Lowenstein – Jensen medium ):

Inoculated for 6- 8 weeks (long generation time of 10-15 hours )
Colonies: M. tuberculosis produces typical rough, tough, and buff-colored colonies.
b. Conventional liquid media: Kirchner’s medium and Middle brook 7H9 -used.
c. Automated liquid culture:
- Faster turnaround than compared to conventional culture methods
- Middle brook 7H9 medium + growth media + antibiotic mixture ----- used.
BACTEC MGIT ( mycobacteria growth indicator tube )-
- Detects growth of mycobacteria
- Performs drug susceptibility testing against all first-line and second-line
antitubercular drugs.
 CULTURE IDENTIFICATION :

Colonies of LJ media and broth from automated culture bottle

First subjected to acid-fast stain

If stain is positive, further tests are done

i) MPT 64 ANTIGEN – detection by rapid immunochromatographic

test(ICT),antigen-specific for M.tuberculosis complex and negative for
Nontuberculous mycobacteria.
ii) AUTOMATED IDENTIFICATION SYSTEM: such as MALDI-TOF
iii) BIOCHEMICAL TESTS: niacin test, rabbit pathogenicity test.

5. MOLECULAR METHODS:

i) PCR – Nested PCR – detects IS6110 gene.

- Automated Real-time PCR –

1. Catridge -based nucleic acid amplification test (CBNAAT) – eg;

GeneXpert-for identification and detection of rifampicin resistance-95%sensitive and
98%specific-turnaround time 2 hours.

2. Chip-based real-time PCR: eg; Truenat.

ii) Line Probe Assay (LPA):

⮚ involves probe-based detection of amplification DNA in specimen

⮚ used for identification and detection of resistance to 1st and 2nd line ATDs
⮚ Turnaround time 2-3 days.
SHORT NOTES
 1. EL TOR VIBRIO
⮚ It caused the 7th pandemic – originated In Indonesia in 1962
⮚ It produced milder cholera but was associated with more carrier rate than
classical.
⮚ O139 (BENGAL STRAIN )- isolated from Chennai and appears as derivative of
EL Tor, but has a distinct LPS and is capsulated.

PATHOGENESIS:

✔ TOXIN- MEDIATED (endotoxin ).

✔ Transmitted by – contaminated food and water
✔ In the small intestine, vibrio penetrate the mucous layer and reach
epithelial cells
✔ Adheres to the epithelium by type IV fimbria – toxin coregulated pilus.
✔ Gene for cholera toxin – phage coded.

CLINICAL MANIFESTATIONS

Incubation period – 24 to 48 hours

● Watery diarrhea
● Rice water stool
● Vomiting
● Muscle cramps

LABORATORY DIAGNOSIS:

1. SPECIMEN: freshly collected watery stool- for acute cases

Rectal swab – for convalescent patients or carrires.

1. TRANSPORT/HOLDING MEDIA: in 10-20 ml of :

● Venkatraman-ramakrishnan (VR) medium
● Alkaline salt transport medium
● Cary-Blair medium: for salmonella and shigella
● Autoclaved seawater.
 2. DIRECT MICROSCOPY :

Gram staining – of mucus flakes of feces – curved comma-shaped gram-negative rods,

arranged in rows- fish in stream appearance.

Motility testing by hanging method – darting motility.

3. CULTURE: aerobic and grows well in original media – nutrient agar.

Enrichment broth – alkaline peptone water(APW)

Monsur’s taurocholate tellurite peptone water

Selective media :

- TCBS Agar: yellow colonies- sucrose fermenters

- Alkaline bile salt agar (BSA): translucent oil drop colonies
- Monsur’s gelatin taurocholate trypticase tellurite agar (GTTTA): translucent
colonies with a black center and a turbid halo
- Mac Conkey agar: translucent and pale -pink on prolonged incubation – Late
lactose fermenters.

5. CULTURE SMEAR AND MOTILITY TESTING:

Culture smear – short curved gram-negative bacilli

Hanging drop – darting motility.

6 . IDENTIFICATION :

Automated systems – MALDI-TOF or VITEK

Conventional biochemical tests-

a. Catalase and oxidase +

b. ICUT test:

● indole test +
● Citrate test- variable
● Urease test – negative
 ● TSI ( triple sugar iron agar test ) – sucrose fermenter – shows acid
– gas absent – H2S absent.
c . Hemodigestion: on blood agar – greenish clearing around the main inoculum.
d.String test: vibrio mixed with 0.5% sodium deoxycholate on the slide, suspension loses
its turbidity and becomes mucoid – when lifted with string forms string.

7. BIOTYPING: classical and El tor biotypes are differentiated by:

Biotypes of V. cholerae 01 Classical biotype El Tor biotype

β-hemolysis on sheep blood agar Negative Positive
Polymyxin B Susceptible Resistant
Group IV phage susceptibility Susceptible Resistant
El Tor phage V susceptibility Resistant Susceptible
VP(Voges- Proskauer ) test Negative Positive

8. SEROGROUPING: species confirmed by agglutination test on the slide with V. cholerae

polyvalent O antisera.

First tested with O1 antisera

If found negative if agglutinated, then simultaneous serotyping with Ogawa

and Inaba antisera

Then test with O139 antisera

9. ANTIGEN DETECTION: by cholera dipstick assay

10. MOLECULAR METHOD: BioFire FilmArray –(automated multiplexed PCR).

11. ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST): on Mueller Hilton agar by disc

diffusion method.

PREVENTION

GENERAL MEASURES :

● Provision of safe water

● Improved sanitary disposal of feces.
● Proper food sanitation
● Prompt outbreak investigation and taking necessary steps to reduce transmission

CHEMOPROPHYLAXIS:

Tetracycline – drug of choice

VACCINES:

Oral cholera vaccines

a. Killed whole-cell vaccine: whole-cell vaccine

Whole-cell recombinant B subunit vaccine
b. Oral live attenuated vaccines (OCV)
 2. GONORRHEA
It is a sexually transmitted disease.
CAUSATIVE AGENT – Neisseria gonorrhoeae- capsulated, gram-negative, kidney-shaped
diplococcus manifests as cervicitis, urethritis, conjunctivitis
VIRULENCE FACTORS:
1. Pili or fimbriae- helps in adhesion
2. Outer membrane proteins: protein I and protein II

CLINICAL MANIFESTATIONS:

IN MALES –

- Purulent urethral discharge

- Complications - Epididymitis, prostatitis, balanitis
- Water – can perineum – infection spreads to periurethral tissues causing abscess
with sinus formation.

IN FEMALES-

- Mucopurulent cervicitis
- Vulvovaginitis
- Spreads to the endometrium and fallopian tube
- Fitz-Hugh – Curtis syndrome - a complication of peritonitis and perihepatic
inflammation.

IN PREGNANT –

- Premature delivery
- Chorioamnionitis
- Sepsis in infant

LABORATORY DIAGNOSIS:

● SPECIMEN – urethral swab – men, vaginal swab – women

● TRANSPORT MEDIA: Stuart‘s medium
● MICROSCOPY: gram staining – gram-negative intracellular kidney-shaped diplococci
 ● CULTURE: Thayer martin medium
● IDENTIFICATION: gonococci are catalase and oxidase positive. Ferment only glucose.
● MOLECULAR METHOD: PCR
● TREATMENT: drug if choice – 3 rd generation cephalosporins.

PROPHYLAXIS: No vaccine.
 3. NON GONOCOCCAL URETHRITIS (NGU)
● It is a condition of chronic urethritis where gonococci can’t be demonstrated. The cause
could be gonococci but may not be detectable because they persist as L forms or it could
be caused by other microorganisms
● Sometimes: urethritis + conjunctivitis +arthritis 🡪 REITER’S SYNDROME

ONSET: take more than a week

URETHRAL DISCHARGE: mucous to mucopurulent

CAUSE DIAGNOSTIC METHODS TREATMENT

1) Bacterial Chlamydia trachomatis 🡪 culture on McCoy and Doxycycline,

HeLa cell lines AZITHROMY
CIN
Ureaplasma AZITHROMY
urealyticum CIN

Mycoplasma hominis

Gardenerella vaginalis

Acinetobacter lwoffi
2) Viral Herpes

Cytomegalovirus

3) Fungal Candida albicans detection of budding yeast Clotrimazole

in discharge

4) Protozoal Trichomonas vaginalis detection of trophozoite Metronidazole

 4. X and V Factors

● Haemophilus influenzae require accessory growth factors present in blood :

● FACTOR X: could be hemin/protoporphyrin IX / other iron-containing porphyrins
(group of heat-stable compounds)
● required for the synthesis of cytochrome, catalase, and peroxidase (involved in aerobic
respiration)
● This factor is required only during aerobic respiration
● FACTOR V: heat-labile factor present in RBCs, could be NAD or NADP. This is
synthesized by fungi bacteria (by staphylococcus aureus) plant and some animal cells.
● Haemophilus grows well on blood agar with factor V. Thus staph. aureus can also be
added to provide a source
● Ordinary blood agar is not suitable for its growth as V Factor is not freely available More
so, sheep blood contains NADase that destroys factor V.
● However, it is freely available in chocolate agar since at 750C NADase gets inactivated
and RBCs are lysed releasing Factor V.
 5. BILE SOLUBILITY TEST
● A bile solubility test is done to differentiate pneumococcus from other streptococci.
● Pneumococci are soluble in bile.

2 METHODS

TUBE METHOD PLATE METHOD

10% sodium deoxycholate solution + 1ml Loopful of 10% deoxycholate +

Broth culture (neutral Ph) S.Pneumoniae colony on blood agar

Culture clear due to lysis Colony lyses within few minutes

PRINCIPLE:

Based on the presence of autolytic enzyme amidase present in the pneumococcus.

 6. ZOONOTIC INFECTIONS
● A zoonosis is any disease or infection that is naturally transmissible from vertebrates to
humans

● Zoonotic infections associated with bacteria :

❖ TULARAEMIA
⮚ FRANCISELLA TULARENSIS is the causative agent of the infection
⮚ It is primarily a plague-like disease of rodents and other small animals
⮚ Results from
✔ Interactions with biting or blood-sucking insects (ticks and tabanid
flies)
✔ Ingestion of contaminated water or food
✔ Inhalation of infective aerosols
⮚ Clinical manifestations :
✔ Ulceroglandular tularemia: ulcerative lesions at the site of
inoculation with regional lymphadenopathy
✔ Pulmonary, oropharyngeal, oculoglandular, and typhoid like illness
✔ Complication: suppurated lymph node, acute kidney injury,
hepatitis, rhabdomyolysis
✔ Category A agent of bioterrorism
⮚ Lab diagnosis
✔ Culture: needs special media such as BCG agar
✔ Specimen: ulcer scrapings and lymph nodes biopsy
✔ Species identification is done by conventional biochemical tests or
by automated identification systems such as VITEK
⮚ Treatment
✔ Gentamicin is considered the drug of choice
✔ Doxycycline or ciprofloxacin can be given as alternatives

❖ PASTEURELLA INFECTION
⮚ Pasturella species are harbored as normal flora in the oral cavity of dogs
and cats
⮚ Clinical features
✔ The affected area becomes red, swollen, and painful with regional
lymphadenopathy
✔ In serious cases, bacteremia can result, causing osteomyelitis or
endocarditis, or meningitis
⮚ Lab diagnosis
✔ P.multocida is a gram-negative coccobacillus that grows in culture
media
✔ Identification is done by MALDI-TOF or VITEK
 ⮚ TREATMENT
✔ Penicillin G or amoxicillin-clavulanate

❖ CAPNOCYTOPHAGA INFECTION
⮚ Certain species are commensals in the mouth of a dog
⮚ C.canimorsus can cause fulminant septicemia following a dog bite
⮚ Associated risk factors
✔ Patients with anatomic or functional asplenia
✔ Heavy alcohol intake
✔ Liver cirrhosis
⮚ Lab diagnosis
✔ Fusiform or filamentous gram-negative coccobacilli
✔ Capnophilic
✔ Produce orange-colored colonies, take up to 14 days to grow
✔ Identification through MALDI-TOF and VITEK
⮚ Treatment
✔ Β lactam / β lactam inhibitor combinations such as ampicillin
sulbactam

❖ RAT BITE FEVER

⮚ Characterized by septic fever, petechial rashes, and painful polyarthritis
⮚ Caused by streptobacillus moniliformis and spirillum minus
⮚ Transmission
✔ Contact with rodents carrying these bacteria
✔ Consumption of food contaminated with urine and droppings of
rodents carrying bacteria
⮚ Treatment
✔ Penicillin

Reference :
Apurba s Sastry ; 3rd edition
 7. BCG
LIVE ATTENUATED VACCINE (BACTERIAL)
● The live attenuated vaccines lose their ability to induce disease but retain
immunogenicity.
● Attenuation is achieved by passing the organism into a foreign host.

ADVANTAGES
● More potent immunizing agent.
● Multiply inside the host hence antigenic dose would be larger than administered
● Capable of inducing the production of Ig A antibody production.

PRECAUTIONS
● Contraindications:
● should not be administered in immunodeficiency patients such as leukemia, lymphoma.
● Pregnancy.
● When two live vaccines are given, there should be an interval of at least 4 weeks.
● Dosage: given in single dose.
● The risk of gaining the virulence: should be given in an effective because there is always
a risk of gaining the virulence.
● Storage: must be cautiously retained.
REFERENCE
● Essentials of Apurba Shastry 3rd edition.
● Table 31.1 pg no:311; Essentials of Apurba Shastry 3rd edition.
 8. FOOD POISONING

❖ Food poisoning refers to the illness acquired through consumption of food or drinks
contaminated either with microorganisms or their toxins

BACTERIA CAUSING FOOD POISONING:

INCUBATION PERIOD 1 – 6 hrs

● Here the toxins are already present in the contaminated food (preformed toxins) that’s why
the incubation period is less

1) Staph aureus

TOXIN- Enterotoxin (preformed toxin)

● Heat stable toxin, resistant to gastric juice.

● Site of the action 🡪 vagus nerve, vomiting center, intestine
● Serotypes producing enterotoxin 🡪 Serotype A to E, G to P (15)
● Serotype F is responsible for Toxic shock syndrome and doesn’t cause food poisoning
● Detection of enterotoxin: ELISA, PCR, Latex agglutination test

SYMPTOMS

● Nausea, Vomiting
● Occasional diarrhea
● Hypotension
● Dehydration
● However, there is NO FEVER

TREATMENT: Supportive, correcting the electrolyte balance

2) Bacillus cereus
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 395

INCUBATION PERIOD 8- 16 hrs

● Here, diarrhea and abdominal cramps are present

1) Bacillus cereus (diarrheal type)

2) Clostridium perfringens
● After ingestion of contaminated food, the toxin causes severe abdominal cramps and
diarrhea

INCUBATION PERIOD >16 hrs

Vibrio cholera

Symptoms begin 1- 4 days after exposure, characterized by

● Watery diarrhea
● Abdominal cramps
● Fever, chills
● Nausea, vomiting

REFERENCE- Essentials of Microbiology Apurba Sastry 3/E

 9. UNIQUE CHARACTERISTICS OF ENTEROCOCCUS
● Enterococci show the following characteristics that help in their identification:
➔ They are gram-positive oval cocci arranged -in pairs; cocci in a pair are arranged
at an angle to each other (spectacle eyed appearance)
➔ Non-motile cocci (except gallinarum and casseliflavus).
➔ Blood agar: It produces non-hemolytic translucent colonies.
➔ MacConkey agar: It produces minute magenta pink colonies.
➔ Nutrient agar: It grows poorly.
➔ Bile aesculin hydrolysis test is positive
➔ PYR (Pyrrolidonyl-beta-naphthylamide) test is positive.
➔ they can grow in presence of extremes of conditions such as 6.5% NaCl, 40%
bile, pH 9.6, 45 degrees C, and 10 degrees C.
➔ Heat tolerance test: they are relatively heat resistant, can survive 60°C for 30
minutes.

Reference: Essentials of medical microbiology by apurba sastry-2/Epg.no 237.

 10. NAGLER’S REACTION

● Nagler’s or lecithinase reaction is used to detect Clostridium perfringes.

● C. perfringes produces α-toxin which has lecithinase activity.
● C. perfringes is grown on egg yolk or serum agar with antitoxin spread on one half of the
culture plate and the other half without antitoxin.

Reference:jaypeedigital.com

THE HALF OF THE PLATE WHICH HAS NO ANTITOXIN :

Lecithinase which is a phospholipase in the presence of Ca++ and Mg++ splits lecithin in the
medium into phosphorylcholine and triglyceride which is seen as a zone of opacity around
colonies.

THE HALF OF THE PLATE WHICH CONTAIN ANTITOXIN :

This opacity isn’t seen around colonies in antitoxin containing half of the plate, due to
toxin-antitoxin neutralization reaction,i.e., antitoxin neutralizes α toxin and hence it’s lecithinase
activity is lost and hence no opacity.

● This test is also positive for C.bifermentans, C.baratti, C.sordellii.

Reference: Ananthanarayan and Paniker’s Textbook of Microbiology – 11/E-pg no. 245,248 and
Essentials of Medical Microbiology 3/E-pg no. 539,540,541,542.
 11. MYCOPLASMA
● Smallest microbes. Pleuropneumonia-like organisms. (PPLO)
● Lack rigid cell wall
● Highly Pleomorphic.
● Better stained by Giemsa stain.
● Mycoplasma colonies – Fried egg appearance.

DISEASE CAUSED BY MYCOPLASMA :

● Mycoplasma Pneumoniae are strict human pathogen, transmitted by respiratory droplets.

● M.Pneumoniae produces various infections.
They are :
i. UPPER RESPIRATORY TRACT INFECTIONS: Pharyngitis,
Tracheobronchitis, Otitis media.
ii. PNEUMONIA: causes atypical community-acquired interstitial pneumonia.
♦ Also referred to as Eaton’s agent Pneumonia or Primary atypical pneumonia
or Walking Pneumonia.
♦ Onset is gradual with fever, malaise, headache, sore throat.
♦ Characterized by wheezing, dry cough, Peribronchial Pneumonia, Streaks of
interstitial infiltration on chest X-RAY.
iii. EXTRAPULMONARY MANIFESTATION :
♦ Neurologic: Meningoencephalitis, encephalitis.
♦ Dermatologic: Skin rashes, Steven Johnson Syndrome.
♦ Cardiac: Myocarditis, Pericarditis.
♦ Hematologic: Anemia.
● Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum cause urogenital
infections. Non-gonococcal urethritis caused by Ureaplasma urealyticum.
✔ Transmitted by sexual contact.
✔ Causes urethritis, proctitis in males; cervicitis, vaginitis in females
 12. LAB DIAGNOSIS OF TYPHOID FEVER

Laboratory tests Diagnosis

Culture isolation: Blood and Bone marrow ✔ Bacterial colonies on broth.

culture on BHI broth/agar or on BACTEC in
1st week of illness. Incubated @37℃.

Stool culture: in 3-4 weeks of illness.

✔ Enrichment broth such as Selenite F

broth, tetrathionate broth.
✔ Low selective medium: MacConkey
agar. ⮚ Translucent NLF colonies
✔ Highly selective media :
● DCA(deoxycholate citrate ⮚ NLF colonies with black center
agar)
● XLD(Xylose lysine
deoxycholate) ⮚ Pink colonies with a black center.
● Wilson Blair green bismuth
sulfite. ⮚ Jet black colonies with metallic
sheen due to H2S production.

Culture smear and motility Gram-Negative, Motile

Biochemical Identification of S.Typhi

● Indole test o –ve

● Citrate test o –ve
● Urease test o –ve
 ● Triple sugar test o Small speck H2S production.

Widal test: 2-3 weeks of illness. Antibodies O antibodies produce granular chalky
are detected against TO, TH, BH antigens. clumps when O ag. H antibodies produce
cotton wools clumps with H antigen.

▪ InS. Typhi infection: TO and TH

antibodies increase.

Antigen detection (Serum and Urine) By ELISA

Molecular Method PCR detecting flagellin gene

Nonspecific findings Neutropenia, leukocytosis.

 13. ACTINOMYCOSIS:

It is a clinical condition caused by Actinomyces

Actinomyces are

▪ Gram –ve rods.

▪ Non- acid-fast
▪ Normal flora in the oral cavity, GIT, Vagina.

A.israelii is the most common species infecting man.

PATHOGENESIS: Actinomycosis is a chronic suppurative & granulomatous infection

characterized by multiple abscesses, formation of sinuses, discharge containing sulfur granules,
indurated swellings.
Reference : Essentials of Medical microbiology Apurba Sastry 3rd ed pg : 543.fig: 55.4

LAB DIAGNOSIS: The specimen to be collected include pus with sulfur granules, bronchial
washing, cervicovaginal secretion.

TREATMENT :

✔ Less duration of antibiotics usage may cause relapse.

✔ I.V Penicillin G for 4-6 weeks
✔ Followed by Oral Penicillin V for 6-12 months.
✔ Surgical Removal of affected tissues for the extensive lesion.
✔ Penicillin allergy patients are treated with Doxycycline, Ceftriaxone, Clindamycin.

REFERENCE: ESSENTIALS OF MEDICAL MICROBIOLOGY- APURBA SASTRY

3RD ED.
 14. NONSPORING ANAEROBIC INFECTIONS

CLINICAL PRESENTATIONS
i. ANAEROBIC COCCI
● Peptococcus and peptoStreptococcus - normal flora of skin
mouth intestine and vagina. However are recovered from various
clinical infections such as puerperal sepsis, skin and soft skin
infections, and brain abscess
● Veillonella - usually non-pathogenic
ii. GRAM-POSITIVE NONSPORING ANAEROBES
● Bifidobacterium species - beneficial normal flora in the mouth
and gut
● Eubacterium species - commensals in mouth and intestine
● Propionibacterium species - cause acne vulgaris and folliculitis.
Also, cause prosthetic hip joint and prosthetic heart valve
infections
● Lactobacillus species - normal flora of mouth gut and vagina
iii. GRAM-NEGATIVE NON - SPORING AND ANAEROBIC BACILLI
● Bacteroides fragilis
a. Virulence factor include
i. Capsular polysaccharide
ii. Lipopolysaccharide
 iii. Enterotoxin
b. It causes peritonitis and intraabdominal abscess following
bowel injury and pelvic inflammatory disease
c. Antral toxigenic strains can cause diarrhea
● Prevotella
a. Pigmented (eg) P.melaninogenica
i. Has been isolated from lung or liver abscess
mastoiditis pelvic and genitourinary infection and
lesions of intestine and mouth
b. Non-Pigmented (eg) P.buccalid
i. Cause periodontal disease and aspiration
pneumonitis
● Porphyromonas
a. P.gingivalis - periodontal disease
b. P.endodontalis - dental root canal infections
● Fusobacterium
a. F.nucleatum - Normal inhabitant of mouth but can cause
oral infection pleuropulmonary sepsis CNS infection and
septic arthritis
b. F.necrophorum - is an agent of Lumiere’s syndrome
● Leptotrichia buccalis
a. Part of normal oral flora
b. Are implicated in acute necrotizing gingivostomatitis
known as Vincent's angina.
Reference: Essentials of medical microbiology Apurba Sastry 3rd edition
 15. WEIL’S DISEASE
(Hepato- renal hemorrhagic syndrome)

➔ Severe form of icteric illness.

➔ Occurs in 10% of patients.
➔ Biphasic course may not be present.

CLINICAL FINDINGS INCLUDE:

➔ High grade fever

➔ Liver jaundice and raises liver enzymes
➔ Hemorrhages: Pulmonary hemorrhage, Gastrointestinal hemorrhage,
Conjunctival hemorrhage
➔ Raised Serum Urea and Creatinine with Renal failure.

LABORATORY DIAGNOSIS OF WEIL’S DISEASE:

LABORATORY FIRST STAGE SECOND STAGE

DIAGNOSIS (Septicemic phase) (Immune phase)

Specimens Blood and CSF Urine

Serology for Antibody Serum IgM absent Serum IgM present

detection

Antibiotic treatment Susceptible to Refractory to

antibiotics antibiotic treatment

LABORATORY DIAGNOSIS OF LEPTOSPIROSIS:

● Specimens used:
● First Stage: Blood & CSF
● Second Stage: Urine
1. MICROSCOPY:
● Wet Films: Observed under dark ground / Phase contrast microscope. Leptospira
exhibits spinning & translational movements. They are highly motile. Leptospira
interrogans- spirally coiled with hooked ends visualized.

● Staining: Stained by Silver impregnation stain - Fontana stain and modified

Steiner technique.
● Disadvantages: Require technical expertise and be less sensitive. Serum proteins
in blood resemble Leptospires.

2. CULTURE ISOLATION:
● Culture Condition: Incubated at 30o for 4 to 6 weeks. Culture fluid should be
examined under a dark ground microscope for leptospires’ presence.
● Culture Media: EMJH (Ellinghausen, McCullough, Johnson, Harris) medium,
Korthof’s and Fletcher’s medium.

3. SEROLOGY FOR ANTIBODY DETECTION:

Antibody detection tests are classified into:

Genus specific test:

Uses broadly reactive genus-specific antigen derived from a non-pathogenic L.biflexa
Patoc 1 strain.
● ELISA: detects IgM and IgG separately.
● Lepto dipstick array test: detects IgM antibodies.
● Immunochromatographic test: detects IgM and IgG separately.

Serovar-specific test:
● Microscopic agglutination test: Gold standard method and reference test for
Leptospirosis diagnosis. Used to detect the presence of antibodies.
 Patient’s serum mixed with live antigen suspension of leptospirosis endemic in the
locality, in a microtitre plate

Incubated for: 2-4 hrs at 30o

Examine under dark ground microscopy

Check for the presence of Agglutination.

Cross agglutination and absorption test:

● To detect relatedness between strains.

4. MOLECULAR METHODS:
● Polymerase chain reaction: Genes like 32 kDa lipoprotein gene, 16s/23s rRNA,
IS1533 insertion sequence targeted.
● PCR: Not antigen-specific
.
5. NON SPECIFIC FINDINGS:
● Elevated Blood Urea Nitrogen & Serum Creatinine
● Elevated Bilirubin and Liver enzymes in serum.

TREATMENT:
● Mild Leptospirosis:
● Oral doxycycline- 100 mg twice a day for 7 days
● Amoxicillin
● Severe Leptospirosis:
● Penicillin is the drug of choice- 1.5 million units IV, 4 times/day for 7 days.
● Cefotaxime
● Ceftriaxone
 16. CAUSATIVE AGENT FOR RELAPSING FEVER:
● Epidemic relapsing fever is caused by various species of Borrelia, a spirochete.
● Relapsing fever: Recurrent episodes of fever with nonspecific symptoms following
exposure to insect vectors.
● Two types:
Caused by: Vector

EPIDEMIC RF B.recurrentis Louse

ENDEMIC RF B.duttonii, B.hermsii, Lice

B.turicatae

● PATHOGENESIS:
Mode of transmission

EPIDEMIC RF Crushing of louse by scratching.

ENDEMIC RF Bite of infected lice.

Borrelial surface antigen undergoes repeated antigenic variation, hence evading the
immune host system.

● CLINICAL MANIFESTATIONS:
Recurrent febrile episodes - 3 to 5 days with intervening afebrile periods of 4-14
days.
Hemorrhages most likely in Epidemic RF (Epistaxis & blood-tinged sputum)
Neurologic features- seizures, meningitis & psychosis may occur in 10-30% of
cases, these are common in Epidemic RF.

● LABORATORY DIAGNOSIS:
● Specimen: Blood
● Microscopy:
➔ Peripheral thick/thin smear- stained by Wright or Giemsa stain
➔ Dark ground or Phase contrast microscopy- detect motile spirochetes
● Culture:
 ➔ Confirmation is done by isolation of Borrelia from blood in the afebrile
period
● Serology: To detect antibodies
➔ ELISA & IFA
➔ GlpQ assay
● Molecular method: PCR
● TREATMENT:
● Doxycycline and Erythromycin- drug of choice
● Single-dose for Epidemic RF & 7-10 days for Endemic RF.

Reference: Essentials of Medical Microbiology by Apurba S Sastry - 3/E

Reference: Complete Microbiology for MBBS by CP Baveja - 7th edition –pg 739 - Fig 49.1.7
17. VARIOUS MECHANISMS BY WHICH ESCHERICHIA COLI
PRODUCE DIARRHEA.
● E. coli causes diarrhea mainly through Enterotoxins. The various enterotoxins are,

1. LT (HEAT - LIABLE TOXIN ):-

● Mechanism of action: It has 2 peptide fragments A and B.

● Fragment B – It is a binding fragment; binds to GM1 ganglioside receptors present on the
intestinal epithelium following which A fragment is internalized.
● Fragment A – It is the active fragment, causes ADP ribosylation of G protein 🡪 activates
adenylate cyclase 🡪 results in the intracellular accumulation of cyclic AMP 🡪 leads to
increased outflow of water and electrolytes into the gut lumen, with consequent diarrhea.

2. ST (HEAT – STABLE TOXIN ):-

● Mechanism of action: It binds to the guanylate cyclase C 🡪 increased production of

cyclic GMP 🡪 fluid accumulation in the gut lumen 🡪 diarrhea.

3. VEROCYTOTOXIN OR SHIGA TOXIN:-

● Mechanism of action : It has 2 fragments A and B

● Fragment B binds to the globotriosyl ceramide ( gb3) receptor on intestinal epithelium.
● Fragment A is the active fragment . It inhibits protein synthesis by inhibiting 60S
ribosomal subunit.

There are 6 pathotypes of diarrheagenic Escherichia coli,

1. Enteropathogenic E. coli (EPEC)

2. Enterotoxigenic E. coli (ETEC)
3.  Enteroinvasive E. coli (EIEC)
4. Enterohemorrhagic E. coli (EHEC)
5. Enteroaggregative E. coli (EACE)
6. Diffusely adherent E. coli (DAEC)
 ⮚ Enteropathogenic E. coli – causes infantile diarrhea.
Mech. Of action :
● Adhesion of intestinal mucosa, mediated by plasmid coated bundle forming pili,
which form cup-like projections called pedestals.
●  A/E lesions ( attaching and effacing lesions ) are typical lesions produced on the
intestinal epithelium ; which leads to disruption of brush border epithelium causing
increased secretion and watery diarrhea.

⮚ Enteroinvasive E. coli – The epithelial cell invasion is mediated by

plasmid – coded antigen called Virulence marker antigen ( VMA ).

⮚ Enterotoxigenic E. coli – causes Traveler’s diarrhea.

Mech. of action :
● Attachment to intestinal mucosa is mediated by fimbrial protein called CFA (
colonization factor antigen )
● Toxin production – Heat liable toxin (acts by inc. cAMP) and heat stable toxin ( acts
by inc. cGMP).

⮚ Enterohemorrhagic E.coli – acts by secreting shiga toxin ( by inhibiting protein

synthesis by inhibiting 60S ribosome ).
⮚ Enteroaggregative E. coli – Intestinal colonization is mediated by aggregative
adhesion fimbriae I ( regulated by aggR gene ). It also produces EAST 1 toxin
(enteroaggregative heat stable enterotoxin 1 ). 

REFERENCE:

Essentials of Medical Microbiology by Apurba S Sastry and Sandhya Bhat – Third Edition
 19. MANTOUX TEST

⮚ Also known as Tuberculin test/Pirquet test

⮚ Antigens used in tuberculin test:
• OT (Old tuberculin antigen): It is a crude preparation of tubercle bacilli, now
rarely used
• PPD (Purified protein derivative antigen): It is a purified preparation of the
active tuberculoprotein.
⮚ DOSAGE: It is expressed in the tuberculin unit (TU). One TU is equal to 0.01
mL of OT or 0.00002 mg of PPD
⮚ PROCEDURE:
• Mantoux test: It is the most commonly employed method. 0.1 mL of PPD
containing 1 TU is injected intradermally into the flexor surface of the forearm
⮚ Reading: It is taken after 48-72 hours. At the site of inoculation, an induration
surrounded by erythema is produced. If the width of the induration is:
• 2:10 mm: Positive (tuberculin reactors)
• 6-9 mm: Equivocal/doubtful reaction
• <5 mm: Negative reaction.
⮚ INTERPRETATION OF RESULT:
Adults: Positive tuberculin test in adults-only indicates present or past exposure
with tubercle bacilli but does not confirm the presence of active stage of the
disease.
Children: In children, a positive test indicates active infection and is used as a
diagnostic marker
⮚ False-positive: The test becomes positive after
• BCG vaccination (after 8-14 weeks)
• Nontuberculous mycobacteria infection.
⮚ False-negative: The test may become negative in various conditions such as early
or advanced TB, miliary TB, decreased immunity (HIV-infected people)
 19. NAME 4 PIGMENTS PRODUCED BY BACTERIA
Pigments Bacteria Color
Staphyloxanthin Staphylococcus aureus Golden yellow

Zeaxanthin Bacillus Brown

Pyocyanin Pseudomonas aeruginosa Blue-green

Anthraquinone Penicillium oxalium Red

20. MILK RING TEST AND ITS USES

● It is done to detect Brucella antibodies in milk
● Procedure: Sample of whole milk mixed well with a drop of stained Brucella antigen
(Concentrated suspension of killed Bacillus abortus/B.melitensis stained with
hematoxylin)
● Incubation in water bath at 70°C for 40-50 mins
● If antibodies present in milk bacilli agglutinated and rise with cream to form blue ring at
top leaving milk unstained
● Positive : Blue ring at top leaving milk unstained
● Negative: No ring .Milk remains uniformly blue
● Uses: For diagnosis of brucellosis in animals
● Screening test to detect presence of Brucella antibodies in infected cattle

21. NEIL MOOSER REACTION

● Animal pathogenicity testing done in guinea pigs to speciate rickettsial species
● R.typhi and R.prowazekii are similar but can be differentiated by which R.typhi transmit
endemic typhus while later does not
● When male guinea pigs inoculated intraperitoneally with blood from a case of endemic
typhus / culture of R.typhi
● They develop fever & characterised scrotal inflammation
● This is called Neil Mooser / Tunica reaction
● This reaction is negative with R.prowazekii
 22. VIRULENCE FACTORS OF STAPHYLOCOCCUS AUREUS
CELLWALL CELL SURFACE PROTEINS EXTRACELLULAR
ASSOCIATED ENZYMES
Peptidoglycan layer: Clumping factor/Bound coagulase: Coagulase:
● Thicker ● It is a fibrinogen binding adhesion ● Acts with
● Induce ● Responsible for slide coagulase test coagulase reacting
inflammatory ● Fibronectin binding adhesion factor (CRF) in
response ● Collagen binding adhesin plasma
● Endotoxin like ● To bind with &
activities activate
prothrombin
● Forming fibrin
clots that protect
bacteria from
phagocytosis
● This serves as the
basis for the tube
coagulase test
Teichoic acid: Protein A: ● Heat stable
● Made of ribitol ● 42k Da polypeptide encoded by spa thermonuclear
phosphate gene and DNase
polymers ● Anticomplementary,antiphagocytic, ● Staphylokinase/
● Helps in ○ Chemotactic,mitogenic fibrinolysin
adhesion of ● Induce platelet damage ● Breaks fibrin clot,
cocci to ● Inhibit opsonization spread infection
mucosal ● Mediates staphylococcal ● Hyaluronidase
surfaces co-agglutination reaction: ● Breaks connective
● Inhibit ● Protein A bind to the Fc terminal of IgG tissue
complement-m ● Leaving free Fab region that binds with ● Lipase and
ediated its specific antigen phospholipase
opsonization ● Microcapsule: ● Breakdown of
● Has polysaccharide lipids
● Inhibits phagocytosis by neutrophils
●
TOXINS OF STAPHYLOCOCCUS AUREUS
CYTOLYTIC TOXINS ENTEROTOXIN EPIDERMOLYSIS/
EXFOLIATIVE
Hemolysins: ● Responsible for ● Responsible for
● Alpha hemolysin- Staphylococcal food Staphylococcal Scalded
leucocidal, cytotoxic, poisoning Skin Syndrome (SSSS)
dermonecrotic ● A preformed toxin, ● Has 2 proteins ET-A &
● Lyse rabbit cells incubation period 1-6 hrs ET-B
● Beta hemolysins – is a ● Has 15 serotypes ● Localized tender
sphingomyelinase (A-E,G-P) blisters, bullae
● Lyse sheep cells ● Antigenic & is neutralized formation to exfoliation
● Gamma hemolysin by specific antibodies ● Separation of outer
● Delta hemolysin epidermal layer leaving
denuded underlying
skin- Nikolsky's skin
● Often occurs in
newborns (Ritters
disease)& children
● Milder forms-
pemphigus neonatorum,
bullous

Panton Valentine leucocidin Toxic Shock Syndrome Toxin

(PVL) ● TSST-1 & TSST-2
● Has bicomponent S ● Act as superantigen
and F ● Shows fever, hypotension,
● Gamma hemolysin and myalgia, diarrhea,
PVL are called ● Erythematous rash,
synergohymenotropic
toxins
● Leucocidal &
hemolytic
● PV toxin expressed on
MRSA

Ref: Ananthanarayan and Paniker’sTextbook of Microbiology Ed: 11

Essentials of Medical Microbiology by Apurba S Sastry Ed:3
 22. SATELLITISM:
Satellitism of H. influenzae (schematic diagram)

Ref: fig.no33.1 pg no 364 essentials of medical microbiology by apurba Sastry 1st edition

● Seen in the Haemophilus influenzae

● H. influenzae is highly fastidious require factor X and V for their growth
● H. influenzae grows well on blood agar and chocolate agar
● Colonies of H. influenzae can grow adjacent to S. aureus- satellitism

Satellitism of H. influenzae around S.aureus

Ref: fig.no33.2A pg no 364 essentials of medical microbiology by apurba Sastry 1st edition

● When S.aureus is streaked across blood agar plate perpendicular H. influenzae streak
line factor V is released
 ● Thus, it forms larger colonies across the S. aureus streak line which gradually decreases
away from the line
● This property is routinely employed for the isolation of H. influenzae

REFERENCE:

● Essentials of medical microbiology by apurba Sastry and Sandhya Bhat 3rd edition
● Ananth Narayanan and paniker’s textbook of microbiology seventh edition
● Images reference: essentials of medical microbiology by Apurba Sastry and Sandhya
Bhat First edition
 23. CLOSTRIDIUM BOTULINUM

MORPHOLOGY:

● Gram-positive spore forming (oval and subterminal) obligate anaerobes

● Non-capsulated
● Has peritrichous flagella
● Produces powerful toxin called botulinum which causes botulism

PATHOGENESIS:

C.botulinum is non-invasive. The pathogenesis is due to botulinum toxin

SEROTYPE:

● Has 8 serotypes—A, B, C1, C2, D, E, F&G

● A, B, E causes human diseases; A most severe
● All serotypes produce neurotoxin except C2 which produces an enterotoxin

TOXINS:

● BT differs from other toxins as it is produced intracellularly and appears outside

after autolysis of bacterial cell
● Nontoxic protoxin active neurotoxin
Trypsin or
Proteolytic enzymes

MECHANISM OF ACTION OF BT:

● After entry, BT is transported via the blood to peripheral cholinergic nerve

terminals
● The most common nerve terminals are NMJ, postganglionic parasympathetic
nerve endings, and peripheral ganglia. It does not affect CNS
● BT blocks the release of Ach, leading to paralysis
RECOVERY:

● Blocking of Ach release is permanent but action is short-lasting and recovery

occurs in 2-4 months, once new terminal axons sprout

THERAPEUTIC USES:

● BT can be used in the treatment of spasmodic conditions like strabismus,

blepharospasm, and myoclonus

CLINICAL MANIFESTATIONS:

Manifestations appear after an incubation time of 18-36 hrs which include:

● Diplopia, dysphasia, dysarthria

● Flaccid paralysis of voluntary muscles
● Decreased deep tendon reflexes
● Constipation
● Respiratory muscle paralysis may lead to death

TYPES OF BOTULISM:

FOODBORNE BOTULISM:

● Due to consumption of food contaminated with preformed BT

● Sources: homemade improperly canned food or fermented food and beverages
● Most cases are sporadic; outbreaks are rare

WOUND BOTULISM:

● Due to contamination of wounds with C. Botulinum spores

● Presents like foodborne botulism except for the absence of GIT features
● Can be recurrent in injection drug users
INFANT BOTULISM:

● Most common type (75% of total cases)

● Due to ingestion of contaminated food with spores of C. Botulinum
● Floppy child syndrome is seen which includes an inability to suck, swallow,
weakened voice, ptosis, floppy neck, and extreme weakness.
● Managed by supportive care and assisted feeding
● Rarely progresses to generalized flaccidity, respiratory failure, and sudden death

ADULT INTESTINAL BOTULISM:

● Rarely, in patients with suppressed normal flora, the colonized clostridial

spores may germinate producing toxin: as in infant botulism

IATROGENIC BOTULISM:

● Due to injection of an overdose of toxin while used for therapeutic

purpose

INHALATION BOTULISM:

● Aerosolization of spores may occur as in act of bioterrorism

LAB DIAGNOSIS:

ISOLATION OF THE BACILLI:

● Done on blood agar or Robertson’s cooked meat (RCM) broth

● In RCM broth they can be either proteolytic and saccharolytic turning the
meat particles black and pink respectively
● Growth on culture media can be confirmed by gram staining and
biochemical tests or by automated methods such as MALDI-TOF
● Serotyping is done with type-specific antisera
TOXIN DEMONSTRATION (MOUSE BIOASSAY):

● Specimens are injected into mice; dial develops paralysis in 48 hours; which can be
inhibited by prior administration of specific antitoxin.
● The sensitivity of the mouse bioassay varies inversely with the time elapsed between the
onset of symptoms and sample collection.

TREATMENT:

● Meticulous intensive care support is needed (such as mechanical ventilation if respiratory

paralysis develops).
● Botulinum antitoxin: It should be administered immediately on clinical suspicion,
without waiting for laboratory confirmation. Earlier the administration better is the cure
rate because antitoxin can neutralize the unbound free toxin molecules. However, once
toxin binds to nerve ending antitoxin has no role.
● In wound botulism, suspected wounds and abscesses should be cleaned, debrided, and
drained promptly Antibiotics such as penicillin or metronidazole may be useful.

CLOSTRIDIUM CAUSING GAS GANGRENE:

Established agents: C. peirfringens (most common, 60% of the total cases) and C. novyi and C.
septicum, (20- 40%).

• Probable agents: there are less commonly implicated; e.g.- C. histolyticum., C. sporogenes, C.
fallax, C. bifermentans, C. sordellii C. aerofoetidum C. tertium.
 24. QUELLUNG REACTION:
● Also known as Neufeld reaction
● This test was routinely done in the past at the bedside, directly from sputum
samples from pneumonia cases. When the specimen is treated with type-specific
antiserum, along with methylene blue dye: the capsule becomes swollen, sharply
delineated, and refractile
● Currently, serotyping is done by a latex agglutination test using type-specific
antisera.