1. GRAM NEGATIVE DIPLOCOCCI

2. Layout: Neisseria: Taxonomic classification General features Species
Pathogenic Neisseriae
OtherNeisseriae.
Moraxella species
Oligella species

3. Neisseriae

4. TAXONOMIC CLASSIFICATION:
KINGDOM: Bacteria
PHYLUM: Protobacteria
CLASS: Betaproteobacteria
ORDER: Neisseriales
CLASS: Neisseriaceae
GENUS: Neisseriae

5. GENERAL FEATURES: Gram Negative diplococci
Aerobes and facultative anaerobes.
Non sporing
Non motile
Optimum temperature ºc
Optimum ph=
5-10% CO2 enhances growth.
Fatidious in growth requirements.

6. CONTD…. Oxidase positive. Catalase positive.
Superoxol test: Positive in N.gonorrhoeae and Negative in N.meningitidis & N.lactamica.
Carbohydrates are oxidatively utilized.
Mainly differentiated on the basis of carbohydrate utilization tests: Glucose, maltose, lactose and sucrose.

7. TRANSPORT MEDIA: Stuart’s media
Amies media.
SELECTIVE MEDIA:
Thayer Martin
Modified Thayer Martin
Martin Lewis
Modified New York City medium.

8. - + +/- Species Pigment Growth at 22ºc Oxidation of carbohydrate
Blood/serum required for growth
Growth on selective media
N.gonorrhoeae - +
N.meningitidis
N.lactamica
+/-
N.pharyngis
N.subflava
N.flava
N.perflava
N.sicca
N.Cinerea
N.polysacchariae
M.catarrhalis

9. + - +/- Species Glu Mal Fruc Suc β galactosidase Glut amino peptidase
Prol amino peptidase
Polysaccharase
Tribut esterase
DNase
N.gonorrhoeae + -
N.meningitidis
+/-
N.lactamica
N.Pharyngis
N.Subflava
N.Flava
N.Perflava
N.sicca
N.cinerea
N.polysacchareae
M.catarrhalis

10. Important Human Pathogens
Neisseria gonorrhoeae
Neisseria meningitidis
Other species normally colonize mucosal surfaces of oropharynx, nasopharynx and anogenital mucosal membranes

11. Meningitis,bacteremia, pneumonia,arthritis.
ORGANISMS
DISEASES
N.gonnorrhoeae
Urethritis, cervicitis, salpingitis, PID,proctitis,bacteremia, arthritis,conjuntivitis,skin lesions,pharyngitis.
N.meningitidis
Meningitis,bacteremia, pneumonia,arthritis.
Other Neisseria species
Opportunistic infections

12. NEISSERIA MENINGITIDIS
Synonyms
-Diplococcus intracellularis Meningitidis
-Meningococcus
-Weichselbaum first discovered and isolated in 1887 from the spinal fluid of a patient.
WEICHSELBAUM.

13. -Arranged in pairs(adjacent sides are flat). -Non-motile.
MORPHOLOGY
-Gram Negative cocci
-Oval/Spherical
-Capsulated
µm in size.
-Arranged in pairs(adjacent sides are flat).
-Non-motile.
-Generally intracellular/may be extracellular.
NEISSERIA MENINGITIDIS

14. Cultural characteristics:
Exacting growth requirements.
Blood,serum,ascitic fluid media.
Strict aerobes.
Opt. temperature 35°c-36°c(25-42^c).
Opt. pH
Growth facilitated by 5%-10% CO2.

15. COLONIES THAYER-MARTIN AGAR -Small,1mm in diameter. -Translucent
-Round,convex
-Bluish grey
-Smooth glistening surface
-Entire edges
-Lenticular shape
-Butyrous consistency
-Easily emulsifiable
-Smooth and rough forms
THAYER-MARTIN AGAR

16. -Blood agar-weak haemolysis -Liquid media –Granular Turbidity
Different media
Blood agar
Chocolate agar
Mueller-hinton starch
Casein hydrolysate
agar
Modified Thayer-martin
(Vancomycin
Colistin
Nystatin)

17. -Oxidase test positive -Freshly prepared 1% oxidase reagent
BIOCHEMICAL REACTIONS
-Oxidase test positive
-Freshly prepared 1% oxidase reagent
﴾tetramethyl paraphenylene diamine dihydrochloride) ↓
Culture media
Deep purple colonies

18. KOVAC'S METHOD -Growth in a loop ↓
Rub on a strip of filter paper moistened with oxidase reagent
Deep purple colour

19. -Catalase test positive
-Indole,hydrogen sulphide not produced
-Nitrates not reduced.
-Glucose and maltose are utilized with acid production only(Peptone serum agar slope, RCUT).

20. RESISTANCE
-Delicate
Freeze drying , Microbanking, Liquid nitrogen.
-Highly susceptible
-Heat
-Dessication
-Disinfectants
-Pencillin.
-Alterations in pH

21. -Based on capsular polysaccharide.
CLASSIFICATION
-Based on capsular polysaccharide.
13 serogroups
Group.A
epidemics
Group.B
epidemics+
Localised outbreaks
Group.C

22. Contd….
Serotyping based on class1 OMP and subtyping based on class 2 and 3
Group, type and subtype are phenotypic characteristics.
Types 2, 15 and recently type 4 have been associated with group B meningococcal disease.
The majority of group C meningococci belong to type 2(2a or 2b).

23. ANTIGENICITY AND VIRULENCE
CAPSULE
Antiphagocytic
Opsonizing anti-capsular antibodies are protective.
Meningococcal vaccine
OUTER MEMBRANE PROTEINS
5 classes; class 5 protein(opa and opc) is involved in attachment and invasion of epithelial cells.Class 2 and 3 are major OMPs.

24. LIPOOLIGOSACCHARIDES
Lack repeating O-Ag(Rough LPS).
Lipid A moiety-portion that mediates induction of inflammatory cytokines.
OTHER FACTORS:
IgAase
Pili
Iron is obtained from transferrin.

25. Inflammatory mediators and molecules involved in meningococcal disease:
IL-1
IL-6
IL-8
PAF
PROSTAGLANDINS
TNF
H2O2
SINGLET EXCITED OXYGEN
HYDROXYL RADICAL
CD 62 , ELAM-1
ICAM-1, CD 18.

26. PATHOGENICITY
-Human pathogens –nasopharynx
-Cerebrospinal meningitis
-Meningococcal septicemia
-Rhinitis
-Pharyngitis
-Conjuctivitis

27. Nasopharyngeal acquisition & colonization
transmission
Pneumonia
Bloodstream invasion
Meningitis Meningitis Meningococcemia
Meningococcemia

28. CEREBROSPINAL MENINGITIS
Perineural sheath of olfactory.N
Cribriform plate of ethmoid bone
Subarachnoid space/blood stream
CNS
Suppurative lesion in meninges
Spinal fluid

29. FEATURES Nausea Vomiting Headache Neck stiffness Lethargy Confusion
MENINGITIS

30. OTHER MANIFESTATIONS Arthritis Primary meningococcal pneumonia
Meningococcal pericarditis
Endocarditis
Conjunctivitis

31. COMPLICATIONS Cranial.N palsy Cortical venous thrombophlebitis
Cerebral edema
Children develop subdural effusion
Permanent sequelae include mental retardation,deafness,hemi paresis.

32. Clinical features Chills Malaise Prostration Petechial rashes
Metastatic involvement of joints,ears,eyes,lungs,a drenals.
10% pneumonia.

33. WATER HOUSE FRIDERICHSEN SYNDROME
-Fulminant meningococcemia
-Fatal condition
-Shock
-DIC
-Multisystem failure
-Deficiency of C5-C9
-Pathogenic component-LPS(endotoxin)
-Adrenal haemorrage and shock

34. EPIDEMIOLOGY Transmission-airborne droplets/fomites
Common in children b/w 3mnt-5yrs
Crowded conditions-jails,ships,army camps
MENINGITIS BELT OF AFRICA- Ethiopia-Senegal
,000cases
deaths

36. Indian scenario:
Meningococcal disease is endemic in Delhi and sporadic cases of meningococcal meningitis have been occurring in Delhi in the past.
Isolated cases of meningococcal meningitis during were also reported from several states of India including Haryana, Uttar Pradesh, Rajasthan, Sikkim, Gujarat, Jammu & Kashmir, West Bengal, Chandigarh, Kerala and Orissa.
Serogroup A has been associated with all the repeated outbreaks of meningitis, although serogroup B and C have been detected in a few sporadic cases.

37. LAB DIAGNOSIS:
CSF COLLECTION

38. CSF examination: General appearance: Purulent
Biochemical parameters: Glucose <35mg/100ml ,
proteins mg/100ml.
Cytology: ,000 PMNs/µl.
Centrifuge the CSF sample
Plate out the deposit on BA and CA and incubate at 37ºc in 5-10% CO2 for 24 hours.
Add glucose broth to remaining deposit, incubate overnight and subculture in the same way. Examine the colonies by Gram stain and Oxidase test.

39. CONTD….
Make 2 smears of the centrifuged deposit and stain with Gram stain and methylene blue respectively.
FITC coupled antiserum may be examined for the direct identification of the meningococcal serogroup responsible for infection.
Divide the supernatant into 2 aliquots- one for biochemical reactions and the other for menigococcal polysaccharide antigen detection by latex agglutination, CIEP or coagglutination.
Biochemical reactions
Antibiotic sensitivity testing.
Serogrouping.

40. -Blood culture-incub. For 4-7days
-Nasopharyngeal swab
-Petechial lesions
-Autopsy-lateral ventricle/surface of brain &S.C.
Retrospective evidence-detection of Abs. (Chronic meningococcemia)
Molecular diagnosis-detection-Meningococcal DNA-PCR.
Serological tests: CFT, HA, ELISA.

41. Treatment: Penicillin G or ceftriaxone(Confirmed cases).
Emperical treatment:
Upto 3 months: ampicillin+cefotaxime or ceftriaxone.
3 months to 7 years: cefotaxime or ceftriaxone.
>7 years: ampicillin or penicillin G + cefotaxime or ceftriaxone.
Dexamethasone may be started 15 minutes before starting antibiotics.

42. PREVENTION
-Meningococcal polysaccharide vaccines(univalent and polyvalent vaccines).
ANTIMICROBIAL PROPHYLAXIS
Rifampicin , Ciprofloxacin.

43. Vaccines:
Five serogroups, A, B, C, Y and W135 are responsible for virtually all cases of the disease in humans. Vaccines are currently available against four of the five strains, and a vaccine against the B strain is in development.Menactra, Menomune of Sanofi-Aventis, Mencevax of GlaxoSmithKline and NmVac4-A/C/Y/W-135 (has not been licensed in the US) of JN-International Medical Corporation are the commonly used vaccines. Vaccines offer significant protection from three to five years (plain polysaccharide vaccine Menomune, Mencevax and NmVac-4) to more than eight years (conjugate vaccine Menactra).

44. -1879-Albert.L.S.Neisser NEISSERIA GONORRHOEAE
First described in gonorrhoeal pus.
-1885-Bumm-cultured & proved it’s pathogenicity
-1960-Douglas Kellogg discovered phase variation in gonococci.
-Fred Sparling- showed that small colony gonococci were piliate and virulent large colony gonococci were non piliated and avirulent.

45. MORPHOLOGY Gram negative Diplococcus Adjacent sides concave
Kidney shaped
Exclusively intracellular
Pili-adhesion to mucosal surface

46. CULTURAL CHARACTERISTICS
Aerobes/ facultative anaerobes pH Temp. 35ºc-36ºc 5-10% CO2 Chocolate agar,Mueller-hinton agar,Thayer martin media, Modified TM medium, Modified NYCM.

47. Colonies
-Small
-Round
-Transluscent
-Convex/slightly umbonate
-Granular surface,lobulate margins

48. - Non Pigmented colonies
TYPES OF COLONIES
T1 &T2
-small brown colonies
-piliated
-autoagglutinable
-virulent
T3 ,T4 & T5
- Non Pigmented colonies
-non-piliated
-smooth suspensions
-avirulent

49. BIOHEMICAL FEATURES: Utilize only glucose. Catalase positive.
Oxidase positive.
Superoxol test positive

50. RESISTANCE Delicate Killed-heat,drying,antiseptics.
Dies-1-2hrs outside the body in exudates.
Cultures dies-3-4days.
Survives-slant culture-35˚c-sterile paraffin wax.
Stored-(-70˚c) for yrs.

51. ANTIBIOTIC RESISTANCE:
Chromosomally resistant N. gonorrhoeae(CMRNG).
PPNG(Penicillinase producing N.gonorrhoeae): Mainly 3.2MDa and 4.4MDa plasmids(TEM-1 β lactamase).
Tetracycline resistant N. gonorrhoeae(TTNG):25.2MDa plasmid- tetM gene.

52. ANTIGENIC PROPERTIES
-Pili
-Hair-like structure
-Composed of repeating peptides subunits(pilins).Pilins contain 159 amino acids.
-Cyanogen bromide digestion has recognized 3 regions.Region 2 is receptor binding region and 3 is type specific region.

53. OMPs P1: Principal outer membrane protein. P2:OPA
TRILAMINAR OUTER
MEMBRANE PROTEIN
Protein 1 (por)
-2 variants
-IA &IB-
serowars-A1-24
And B1-32
Protein 2
-OPA
-Clumping
Protein 3
OMPs
P1: Principal outer membrane protein.
P2:OPA
P3: Binds with P1 to form porin channels

54. Contd…. LOS: Damage to cilia sloughing of epithelial cells.
Lacks somatic O antigen.Sialation protects from antibody mediated defence processes.
Peptidoglycan: C6 hydroxyl group is o- acetylated.Induces fever, ciliary damage, epithelial damage, arthritogenic and activates complement.Also prevents lytic action of lysozymes.
IgAse: Cleaves Fc portion of sIgA.
Alpha protein or factor.

55. Designation
Location
Contribution
PilE
major fimbrial protein
initial binding to epithelial cells
P.II (Opa)
outer membrane protein
contributes to invasion
P.I (Por)
outer membrane porin
may prevent phagolysosome formation in neutrophils and/or reduce oxidative burst
LOS
outer membrane lipooligosaccharide 
elicits inflammatory response, triggers release of TNF
P.III (Rmp)
elicits formation of ineffective antibodies that block bactercidal antibodies against P.I and LOS
Tbp1 and Tbp2
outer membrane receptors for transferrin
iron acquisition for growth
Lbp
outer membrane receptor for lactoferrin

57. Adhesion-urethral mucosal surface
Cocci penetrates-intercellular spaces
Subepithelial C.T-3 day
Incubation period -2-8 days

58. IN MAN
Acute urethritis
Mucopurulent discharge
Urethra
Prostate
Seminal vesicles
Epididymis

59. Chronic urethritis
Stricture formation
Periurethral tissues
Abcesses and multiple
discharging sinuses
WATERCAN PERINEUM

60. -Urethra & cervix-affected
IN WOMEN
-Urethra & cervix-affected
-Vaginal mucosa not affected
-stratified squamous epth.
-acidic pH
-Vulvo-vaginitis-prepubertal girls
-Urethra,cervix,bartholin’s glands,F.T
-P.I.D & salphingitis-sterility
-Peritonitis-perihepatic inflammation
(FITZ-HUGH-CURTIS SYNDROME)

61. IN BOTH THE SEXES
-Proctitis
-Conjuctivitis
-Arthritis
-Ulcerative endocarditis
-Rarely meningitis
-Pyemia
-Non-Veneral-GONOCOCCAL OPTHALMIA
-Gonococcal bacteremia-haemorragic papules
Hands,forearm;arthritis-knee,ankles

62. ANORECTAL GONORRHOEA
-Anorectal pain/pruritus
-Tenesmus
-Purulent rectal discharge
-Rectal bleeding
PHARYNGEAL GONORRHOEA
OCULAR GONORRHOEA
-Autoinoculation-from infected site

63. EPIDEMIOLOGY Source-human carrier Mode of infection-venereal
Non-venereal-OPHTHALMIA NEONATORUM
1% Silver nitrate-CREDE’S METHOD
1970-Global incidence-16million
Higher in B-blood group people.
Most common in the age group of years.
Risk factors include unprotected intercourse and multiple sexual partners.
Proctitis , throat infections , arthritis and disseminated infections can also be seen.

65. LAB DIAGNOSIS: SAMPLES TO BE COLLECTED:
MEN: Urethral discharge(acute cases), ‘morning drop’ of secretion in chronic cases. Exudate may be obtained after prostatic massage. Centrifuged deposits of urine may be used.
WOMEN: Urethral discharge, cervical swabs.Althogh repeated sampling of multiple sites is ideal, a single well taken endocervical swab will detect approximately 90% of gonococcal infections in women. High vaginal swabs not to be collected(1 in 3 infected women likely to be missed).

66. CONTD….
Other samples include rectal swabs, throat swabs, blood, swabs from skin lesions, pus aspirated from joints, conjuntival swab in neonatal ophthalmia.
Purulent material should be collected using dacron or rayon swabs because calcium alginate and cotton swabs may contain materials toxic for Gonococci.
Microscopy: Gram stained smear reveals Gram negative kidney shaped diplococci. If mostly intracellular then positive, if extracellular then equivocal and if not found then negative.

67. Contd….
CULTURE: Direct plating of the sample followed by immediate incubation at 36-37ºc in a moist atmosphere containing 5-10% CO2.
If direct plating and immediate incubation is impracticable several transport and culture systems are available.
Amies transport media and Stuart’s media.
Biochemical tests
Serology
Molecular methods

68. Treatment and prophylaxis:
Ceftriaxone+ doxycycline/tetracycline/azithromycin.
If penicillin sensitive ( NON PPNG) then amoxycillin+probenecid and doxycycline.
No effective vaccine.
Health education, contact tracing , monogamy and use of condoms are the only preventive measures.

69. Other neisseria species:
N.elongata ssp nitroreducens
N.weaveri ssp nov
N.gonorrhoeae ssp kochii
N. flavescens
N.mucosa

70. MORAXELLA:
Moraxella sps were formerly called Neisseria and later Branhamella sps.
Separated from Neisseriae based on DNA base composition, fatty acid composition and inability to produce acid from carbohydrates.
Moraxellas may be involved in opportunistic infections in compromised patients.
These occur as components of the normal flora of the upper respiratory tract, the conjuntiva, the skin and genital tract.

71. Moraxella catarrhalis:
Oval Gram negative cocci about 0.8µm in size.
May occur singly, but majority of these are diplococci with adjacent sides flattened.
Sometimes these may occur in groups of 4.
May be found within polymorphonuclear leucocytes.
Aerobes
Non sporing, non capsulate and non motile.
Optimum temperature is 36ºc but many strains may grow at 22οc .
CO2 is not an absolute requirement.
Most srains grow on Nutrient agar.

72. Contd….
Some strains grow on selective media for pathogenic Neisseriae.
Colonies on Blood agar or Chocolate agar are 1-2 mm in size, non haemolytic, friable, white or greyish, convex with an entire margin later becoming irregular. After 48 hours colonies are larger, more elevated with a raised opaque centre.
Oxidase positive
Catalase positive
Do not utilize carbohydrates.

73. Contd…. Reduce nitrates to nitrites. Produce DNase.
More resistant than meningococcus and gonococcus.
Cultures may remain viable for several months at 20^c if prevented from drying. May survive in sputum for 3-4 weeks.
Susceptible to a wide range of antibiotics but many strains produce β-lactamase and are resistant to penicillin and ampicillin.

74. CONTD….
It is found in the respiratory tracts of % of healthy young and middle-aged adults, 26.5% of healthy elderly people and 50.8% of healthy children.
It is therefore recognized as a significant potential pathogen of the respiratory tract, including the sinuses, bronchi and larynx.
20% of the cases of sinusitis are due to M.catarrhalis.
Chronic bronchitis among smokers is often caused by M.catarrhalis.
Although laryngitis is often caused by this organism, about 90% of cases are due to viruses.

75. Lab diagnosis:
(1)Specimens collected are sputum and transtracheal aspirates.
(2) Microscopy
(3) Culture(some strains on selective media used for pathogenic Neisseriae).
(4) Oxidase test, RCUT test, test for β-lactamase production.

76. Other moraxella species
Moraxella lacunata: Causes purulent conjuntivitis(angular blepharoconjuntivitis).
Moraxella lacunata and M. atlantae require serum for growth.
Loefflers medium supports the growth of M.lacunata and M.liquifaciens.
Moraxella lacunata produces gelatinase.

77. OLIGELLA:
Recently delineated from Moraxella on the basis of DNA:Rrna hybridization and serological tests.
O. urethralis is a rare cause of septic arthritis.
Can be misidentified as N.gonorrhoeae as it is non-motile,Gram negative diplococcus, both oxidase and catalase positivewhich will grow on Thayer-Martin medium. However, Oligella will grow on Mac Conkeys agar.

78. References: Saunder’s text and review series by: T.Stuart Walker.
Mackie & Mc Cartney Practical Medical Microbiology-14th edition
Jawetz Medical Microbiology-24th edition.
Bailey & Scott’s Diagnostic Microbiology-12th edition.
Textbook of Microbiology by Ananthanarayan and Paniker-8th edition.
Color atlas & textbook of diagnostic microbiology(Elmer.W.Koneman)-5th edition

79. contd….
Todar’s online textbook of bacteriology
Internet