Medical Microbiology

1. •ALLAH TAALAH KI RAZA
•RASOOL E AKRAM (SAW)
KI KHUSHNOODI
THE ULTIMATE PURPOSE OF QARSHI

2. Mission of Qarshi Organization
Insaniyat ki Bhalai
Ummah ki Sarbulandi
Pakistan ki Taraqqi
3

3. QARSHI VALUES
•NAIK NIYATI
•JUSTICE
•SELF RESPECT
•POSITIVE THINKING
QARSHI CULTURE
• OWNERSHIP
•CLEANLINESS
•COOPETITION
•PRODUCTIVE HARD
WORK

4. Mission Statement of Qarshi University
To be a premier source of knowledge and leadership for the
achievement of marketable education, learning, and research
to produce managers of change in the society based on Islamic
values.

5. Vision
“Distinctive leadership in
quality education, human
resource, research and
development, and corporate
social responsibility”

6. Dr. (NM) Shehla Unmbreen

7. CLASSIFICATION
Kingdom: Bacteria
Phylum: Proteobacteriacea
Class: Betaproteobacteria
Order: Neisseriales
Family: Neisseriaceae
Genus: Neisseria
Species: gonorrhoeae
meningitidis
lactamica,etc

8. INTRODUCTION & HISTORY:
Discovered Neisseria
gonorrhoeae (1879)
Albert stain
Mycobacterium
leprae
Albert Ludwig Sigesmund
Neisser

9. CHARACTERISTICS:
characteristics
Gram
negative/
cocci
Aerobes
Oxidase
positive
diplo
cocc
i

10. CONTAINS TWO IMPORTANT PATHOGENS
🞭 Nesseria meningitidis
🞭 Nesseria gonorrhoeae

11. IMPORTANT DIFFERENCE BETWEEN
N.gonorrhoeae & N. meningitidis
N.meningitidis N. gonrrhoeae
Note:both can be differentiated by biochemical tests using serum sugar
 I have got an antibiotic
resistant plasmid
 Oval shape
 Have pilli
 Ferment Glucose
 I have got a polysaccharide
capsule.
 Obligate aerobe
 Semicircular with flat ends-
diplococci
 Non spore forming
 Move by pilli
 Ferment Maltose & Glucose

13. NEISSERIA MENINGITIDIS
Described and isolated by weichselbaum from spinal fluid of patient,1887
 Neisseria meningitidis (meningococcus) has a prominent
polysaccharide capsule that enhances virulence by its
antiphagocytic action.
 The capsule also is the immunogen in the vaccine that
induces protective antibodies .

14. SEROGROUPS AND SEROTYPES
On the basis of Antigenicity:
 specificity of capsular polysaccharide antigens
divided into 13 serogroups .
 These are A,B,C,D,X,Y,Z,W -135,29-E,H,I,K and L.
 Five serotypes cause most cases of meningitis and meningococcemia:
A, B, C, Y, and W-135.
 Serogroups A,B,C,X,Y,W 135 : most commonly associated with
meningococcal disease
 GroupA: epidemics
 Group C: localised outbreaks
 Group B: both epidemics and outbreaks

15.  🞭 Serotypes:
 🞭 Based on the outermembrane protein serogroups
further divided into serotypes
 🞭 About 20 serotypes have been identified
 This is because the group B polysaccharide is not immunogenic in
humans and therefore is not part of the vaccines that contain the
capsular polysaccharide of the other four groups.
 In 2014, a vaccine against group B meningococci containing factor
H binding protein as the immunogen was approved.

16. EPIDEMIOLOGY
Natural
habitat and
reservoir
human nasopharynx
urogenital tract
anal canal
Nasopharyngeal carriers 5-10% adults asymptomatic
carriers
Modes of infection Direct contact or respiratory droplets from
the nose and throat of infected people
Prevelence of meningitis is highest in meningitis belt of Africa
(frequent epidemics occurred there)
In 1996,largest 150000 cases 15000 deaths reported

17.  Humans are the only natural hosts for meningococci.
 The organisms are transmitted by airborne droplets; they
colonize the membranes of the nasopharynx and become
part of the transient flora of the upper respiratory tract.
Carriers are usually asymptomatic.
 From the nasopharynx, the organism can enter the
bloodstream and spread to specific sites, such as the
meninges or joints, or be disseminated throughout the body
(meningococcemia).
 About 5% of people become chronic carriers and serve as a
source of infection for others.

18.  The carriage rate can be as high as 35% in people who live in
close quarters (e.g., military recruits); this explains the high
frequency of outbreaks of meningitis in the armed forces prior
to theuse of the vaccine.
 The carriage rate is also high in close (family) contacts of
patients.
 Outbreaks of meningococcal disease also have occurred in
college students living in dormitories.
 Two organisms cause more than 80% of cases of bacterial
meningitis in infants older than 2 months of age:
Streptococcus pneumoniae and N. meningitidis.
 Of these organisms, meningococci, especially those in group
A, are most likely to cause epidemics of meningitis.

19.  Group B meningococci cause many cases of meningitis in
developed countries because they are not present in the
capsular polysaccharide vaccine.
 Overall, N. meningitidis ranks second to S. pneumoniae as a
cause of meningitis but is the most common cause in persons
between the ages of 2 and 18 years.

20. Inhalation of contaminated droplets
Adherence of organism to nasopharyngeal mucosa
Local invasion and spread from nasopharynx to meninges through
blood stream (directly along perineural sheath of olfactory nerve,
cribriform plate to subarachnoid space)
In meninges, organsims are internalised into phagocytic cells
They replicate and migrate to subepithelial spaces
Incubation period : 3-4 days
PATHOGENESIS (STEPS)

21. VIRULENCE FACTORS
🞭 Fimbrae (common pili)-
🞭 Lipooligosaccharide:
🞭 Capsule
🞭 Cell membrane proteins
🞭 IgA protease-

22. Meningococci have four important virulence factors:
(1) A polysaccharide capsule that enables the organism to
resist phagocytosis by polymorphonuclear leukocytes (PMNs).
The capsule is the immunogen in several commonly used
vaccines against meningococci.
(2) Endotoxin, which causes fever, shock, and other
pathophysiologic changes (in purified form, endotoxin can
reproduce many of the clinical manifestations of
meningococcemia).
(3) An immunoglobulin A (IgA) protease that helps the
bacteria attach to the membranes of the upper respiratory tract
by cleaving secretory IgA.

23. (4) Factor H binding protein (FHBP) on meningococci, which
binds Factor H, an inhibitor of complement factor C3b.
 The presence of Factor H on the surface of meningococci
reduces the opsonizing activity of C3b and reduces the
amount of membrane attack complex produced.
 FHBP is the immunogen in the vaccine against group B
meningococci.

24.  Resistance to disease correlates with the presence of
antibody to the capsular polysaccharide.
 Most carriers develop protective antibody titers within 2
weeks of colonization. Because immunity is group-specific, it
is possible to have protective antibodies to one group of
organisms yet be susceptible to infection by organisms of the
other groups.
 Complement is an important feature of the host defenses,
because people with complement deficiencies, particularly in
the late-acting complement components (C6–C9), have an
increased incidence of meningococcal bacteremia.
 Patients receiving eculizumab, a terminal complement
inhibitor used in the treatment of paroxysmal nocturnal
hemoglobinuria, have a 1000-fold increase in meningococcal
disease.

25. CLINICAL FEATURES
 Febrile illness : Mild and self limiting
 Pyogenic meningitis : High fever, stiff neck, Kernig’s sign, severe
headache, vomiting, photophobia, chills
 Meningococcemia : acute fever with chills, malaise, prostation,
Waterhouse- frederichsen syndrome, DIC
 Other Syndrome : Pneumonia, arthritis, urethritis,
respiratory tract infection.
The two most important manifestations of disease are
meningococcemia and meningitis. The most severe form of
meningococcemia is the life-threatening Waterhouse–Friderichsen
syndrome, which is characterized by high fever, shock, widespread
purpura, disseminated intravascular coagulation, thrombocytopenia,
and adrenal insufficiency.

26. Hemorrhage in the adrenal glands in
Waterhouse- Fridericksen syndrome
Meningococcal disease is favoured by defieciency of the terminal complement
components (C5-C9)

27. Meningococcemia. Note purpuric lesions on leg caused by
endotoxin-mediated disseminated intravascular coagulation
(DIC).

28.  Bacteremia can result in the seeding of many organs, especially
the meninges.
 The symptoms of meningococcal meningitis are those of typical
bacterial meningitis, namely, fever, headache, stiff neck, and an
increased level of PMNs in the spinal fluid.

29. Laboratory Diagnosis
 The principal laboratory procedures
are smear and culture of blood and
spinal fluid samples. A presumptive
diagnosis of meningococcal meningitis
can be made if gram-negative cocci
are seen in a smear of spinal fluid.
 The organism grows best on chocolate
agar incubated at 37°C in a 5% CO2
atmosphere. A presumptive diagnosis
of Neisseria can be made if oxidase-
positive colonies of gram-negative
diplococci are found

30.  The differentiation between N. meningitidis and N. gonorrhoeae is
made on the basis of sugar fermentation: meningococci ferment
maltose, whereas gonococci do not (both organisms ferment
glucose).
 Immunofluorescence can also be used to identify these species.
 Tests for serum antibodies are not useful for clinical diagnosis.
 However, a procedure that can assist in the rapid diagnosis of
meningococcal meningitis is the latex agglutination test, which
detects capsular polysaccharide in the spinal fluid.
Neisseria meningitidis
growing on chocolate
agar
growing on
sheep blood
agar

31. CULTURAL CHARACTERISTICS
Media used:
🞭 non selective media:
🞭 Blood agar
🞭 Chocolate agar
🞭 Mueller-Hinton starch casein hydrolysate agar
🞭 Selective media
🞭 Modified Thayer-MartinAgar (Larger colonies,
bluish grey in color and may be mucoid)
🞭 Colony characteristics
🞭 Color: Bluish grey
🞭 Shape: Round
🞭 Size:About 1mm
🞭 Surface: Smooth
🞭 Elevation: Convex
🞭 Opacity: Transluscent
🞭 Consistency: Butyrous

32. BIOCHEMICAL TESTS
• Oxidase positive
• Catalase positive
• Ferments glucose and maltose with acid production
• Doesn’t ferment lactose, sucrose and fructose
• Nitrate negative
• Colistin resistant
• Gamma-glutamyl aminopeptidase positive
• DNAase Positive

33. PROPHYLAXIS
a. Chemoprophylaxis :
🞭 Rifampicin
🞭 Minocycline
🞭 Ciprofloxacin
b. Vaccination:
🞭 A vaccine containing capsular polysaccharide of
serotypes A and C : for infants below 2 years
🞭 A quadrivalent vaacine constituted by
polysaccharides of serotypes A,C,Y and W-135 :
for children and adults
🞭 conjugate vaccine:
polysaccharide antigen is conjugated to diptheria
toxoid

34. LABORATORY DIAGNOSIS
1. Specimen:
🞭 CSF
🞭 Blood
2. Examination of CSF:
🞭 Increased Pressure
🞭 Turbid
🞭 The collected CSF is divided into 3 portions (for
microscopy, for biochemical tests and for culture)

35. 🞭 Microscopy:
🞭 Gram stained smear of CSF deposit commonly shows Gram
negative intracellular diplococci.
🞤 White cell count increases to several thousand per cubic mm
with 90-99% PMNs.
🞤 Biochemical tests:
🞤 Glucose is markedly diminished
🞤 CSF protein is markedly raised CSF:
🞤 Culture:
🞤 Inoculated into chocloate agar
🞤 Incubated at 37c in 5-10% Carbondioxide and high humidity
🞤 After 24 hours bacterial colonies appear
🞤 The organism is tested for biochemical and agglutination reaction

36. Normal CSF:
Clear ,
colorless
0-5
lymphocytes
Sterile
150-450 mg /l
protein
2.8-3.9mmol/l
glucose
CSF in viral meningitis
Clear or slightly turbid
10-500 cells mainly
lymphocytes
Stool culture, or
serology +ve
Normal or slightly raised
protein
Normal glucose
CSF in TB
meningitis:
Clear or slightly turbid
10-500 cells,mainly
lymphocytes( polys early)
AFB in Z-N stain
Grow in LJ medium
Moderately raised
protein
Sugar reduced
CSF in bacterial
meningitis:
Turbid
500-20,000
cells,few
lymphocytes
Bacteria in Gram
stain
Markedly raised
protein
Reduced or
absent glucose
CSF IN DIFFERENT
MENINGITIS

37. 3.Blood culture:
🞭 Blood culture is positive in over 40% cases of
meningiococcal meningitis
4.Other Cultures:
🞭 Nasopharyngeal swab
🞭 Skin lesions
🞭 Joint fluid
🞭 Tracheal aspirate
🞭 Urethral discharge
🞭 Serology
🞭 Petechial lesions

38. d) Detection of antigen:
🞭 For Detection of Meningiococcal DNA
🞭Polymerase Chain Reaction (PCR)
🞭 For detection of soluble polysaccharide antigen
🞭 Counter current immunoelectrophoresis (CIEP)
🞭Latex agglutination test

40. Neisseria gonorrhoeae (Gonococcus)
🞭 N. gonorrhoeae causes the sexually
transmitted disease gonorrhoea.
🞭 first described by Neisser in 1879 in
gonorrheal pus.
🞭 resembles meningococci very closely in many
properties.

41. MORPHOLOGY:
Gram negative
oval/spherical cocci
usually found with in the polymorphs
Arranged in pairs (adjacent sides concave)
Kidney shaped
possess pili on their surface

42. CULTURE & CULTURAL CHARACTERISTICS:
🞭 fastidious organisms do not grow on ordinary culture
media.
🞭 aerobic but may grow anaerobically also
🞭 The optimum temperature for growth is 35-36°C &
optimum pH is 7.2-7.6.
🞭 It is essential to provide 5-10% CO2.

43. Media used:
a) Non selective media: Chocolate agar,
Mueller-Hinton agar
Modified New York City
medium
b) Selective media: Thayer Martin medium
with antibiotics (Vancomycin, Colistin &
Nystatin)

44. Colony morphology: Colonies are
small
round
translucent
convex or slightly umbonate
finely granular surface
lobate margins.

45. Biochemical reactions:
1) Oxidase test: Positive
2) Ferments only glucose
but not maltose.

46. PATHOGENICITY:
Source of infection:
1. Asymptomatic carriers
2. Patients
Mode of infection:
1. Venereal infection (sexual contact)
2. Nonvenereal infection

47. Antigenic structure & virulence factors:
1. Pili
2. Lipooligosaccharide: Endotoxic.
3. Outer membrane proteins: 3 types
a) Protein I (por)- it is a porin & helps in adherence.
b) Protein II (opa)- helps in adherence.
c) Protein III (rmp)- it is associated with protein I.
4. IgA1 protease: Splits & inactivates IgA.

48. Antigenic structure & virulence factors

49. Mechanism of pathogenesis:
Gonococci adhere to epithelial cells of urethra or
other mucosal surface through pili
penetrate through the intercellular space
reach the sub epithelial connective tissue &
causes inflammation
Leads to clinical manifestations
Incubation period: 2-8 days.

50. Disease:
A) In men:
The disease starts as an acute urethritis with a
mucopurulent discharge
extends to the prostate, seminal
vesicles & epididymis
In some it may become chronic urethritis leading to
stricture formation
The infection may spread to the periurethral tissues,
causing abscesses & multiple discharging sinuses
(Watercan perineum)

51. B) In women:
The initial infection is urethritis & cervicitis but vaginitis
does not occur in adult female (vulvovaginitis can
occur in prepubertal girls)
The infection may extend to Bartholin’s glands,
endometrium & fallopian tubes causing
Pelvic Inflammatory Disease (PID)
Rarely peritonitis may develop with perihepatic
inflammation (Fitz-Hugh-Curtis syndrome)

52. C) In both the sexes:
Proctitis, pharyngitis,
conjunctivitis, bacteraemia which may lead to
metastatic infection such as arthritis,
endocarditis, meningitis, pyemia & skin rashes.
D) In neonates:
Opthalmia neonatorum (a
nonvenereal gonococcal conjunctivitis in the
newborn) results from direct infection during
passage through birth canal.

53. LABORATORY DIAGNOSIS:
Specimens collected:
A) In men:
a) Acute infection- Urethral discharge
b) Chronic infection-
iii)
i) Morning drop
ii) Discharge collected after prostatic massage
Centrifuged deposit of urine
B) In women:
i) Urethral discharge
ii) Cervical swabs

54. C) In both the sexes: Blood, CSF, synovial fluid,
throat swab, rectal swab & material from skin
rashes.
Transport: If there is delay in processing than the
specimens should be sent in “ Stuart’s medium”.

55. Methods of examination:
A) Direct microscopy:
1. Gram staining:
Smear provides a
presumptive
evidence
of gonorrhea in men.
Gram negative
diplococci are found.
But it is unreliable
in women.

57. 2. Immunofluorescence:

58. B) Culture:
• Media used:
• Colony morphology: Gram’s smear:
• Reveals Gram negative cocci in pairs
with adjacent sides concave.
• Biochemical reactions:

59. C) Serology:
🞭 Complement fixation test,
🞭 Precipitation,
🞭 Passive agglutination,
🞭 Immunofluorescence,
🞭 Radioimmunoassay.(uses whole-cell
lysate,pilus protein and lipopolysaccharide
antigen)

60. TREATMENT:
🞭 Previously Penicillin was drug of choice but
resistance developed rapidly.
🞭 Penicillin resistant is due to production of
penicillinase enzyme & the strains are called
as penicillinase producing Neisseria
gonorrhoeae (PPNG).
🞭 Now Ceftriaxone or Ciprofloxacin plus
Doxycycline or Erythromycin is useful.

61. EPIDEMIOLOGY:
🞭 Gonorrhoea is an exclusively human disease.
🞭 The only source of infection is a human
carrier or less often a patient.
🞭 Asymptomatic carriage in women makes them
a reservoir to spread infection among their
male contact.
🞭 Gonorrhoea is an venereal disease (STD).

62. PROPHYLAXIS:
🞭 Early detection of cases,
🞭 Tracing of contacts,
🞭 Health education,
🞭 General measures,
🞭 Vaccination has no role in prophylaxis.

63. NONGONOCOCCAL (NONSPECIFIC) URETHRITIS
🞭 Urethritis due to causative agents other than
gonococcus.
🞭 Etiology:
a) Bacteria- Chlamydia trachomatis
Mycoplasma urealyticum
Ureaplasma urealyticum
b) Parasites- Trichomonas vaginalis
c) Viruses- Herpes simplex
Cytomegalovirus
d) Fungi- Candida
🞭 NGU can be a part of Reiter’s syndrome- a clinical
condition characterized by urethritis, arthritis &
conjunctivitis.

64. COMMENSAL NEISSERIAE
N. lactamica
N.pharyngis
N. polysaccreae
N. cinerea
N. flavescens
N. mucosa
Commensal
neisseriae

65. On the basis of Laboratory Investigations how will you
differentiate Neisseria gonorrhea from Neisseria
meningitidis
03
25 years old patient was seen in emergency department
of hospital with fever, headache and stiffness of neck.
His Kernig’s sign was positive. There were petechial
rashes on his body.
A. What is the diagnosis?
B. Name the virulence factors of this bacterium and
how they evade human host Immune system?
C. What is the route of transmission of this bacteria?
D. Briefly discuss its pathogenesis.
E. If it is due to Gram negative diplococci, how would
the diagnosis be confirmed in Laboratory?
0.5
02
1.5
03
03

66. A 20 years old boy presents with purulent urethral exudate
and dysuria for last 2 weeks. Gram Smear of pus discharge
shows a large number of Gram negative intracellular
diplococci.
A. What is the diagnosis?
B. Name the bacterium that is responsible for such
symptoms?
C. Name the virulence factors of this bacterium and how
they evade human host Immune system?
D. Briefly discuss its pathogenesis.
E. Enlist Three clinical complications of this infection seen
in women and one in infants.
0.5
1.5
03
03
02

67. A 5 years old child develops high grade fever and headache
for last 4-5 days. He is brought to the pediatric emergency
room. On examination he had neck stiffness. A lumber
puncture is performed. Preliminary report of cerebrospinal
fluid analysis suggests the diagnosis of Acute bacterial
meningitis.
A. Name the possible organism responsible for disease in
him?
B. Give portal of entry of this microbe.
C. How will you proceed in the laboratory to further confirm
the diagnosis and establish the causative organism?
01
01
03

68. A young fashion designer presents to the outdoor patient
department with complaints of painful micturition, with
discharge of yellowish creamy pus. The Gram smear of his
discharge revealed Gram negative diplococci within
polymorph nuclear leukocytes.
A. What is your diagnosis?
B. How will you proceed for further isolation and
identification of this organism?
C. Name the virulence factors of this bacterium and how they
evade human host Immune system?
D. Briefly discuss its pathogenesis.
E. Enlist Three clinical complications of this infection seen in
women and one in infants.
0.5
03
02
2.5
02