Gram_Negative_Diplococci

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1. GRAM NEGATIVE DIPLOCOCCI

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

3. Neisseriae

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

5. Gram Negative diplococci Aerobes and facultative anaerobes. Non sporing Non motile Optimum temperature- 35-36ºc Optimum ph= 7.0-7.4 5-10% CO2 enhances growth. Fatidious in growth requirements. GENERAL FEATURES:

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

9. 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

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

11. -Gram Negative cocci -Oval/Spherical -Capsulated -0.6-0.8 µm in size. -Arranged in pairs(adjacent sides are flat). -Non-motile. -Generally intracellular/may be extracellular. NEISSERIA MENINGITIDIS MORPHOLOGY

12. Cultural characteristics: Exacting growth requirements. Blood,serum,ascitic fluid media. Strict aerobes. Opt. temperature 35°c-36°c(25-42^c). Opt. pH 7.0-7.4. Growth facilitated by 5%-10% CO2.

13. COLONIES -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

14. - Blood agar-weak haemolysis -Liquid media –Granular Turbidity

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

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

17. -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).

18. CLASSIFICATION - Based on capsular polysaccharide .

19. 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).

20. 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.

21. 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.

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

23. Nasopharyngeal acquisition & colonization transmission Pneumonia Bloodstream invasion Meningitis Meningitis+ Meningococcemia Meningococcemia

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

25. FEATURES Nausea Vomiting Headache Neck stiffness Lethargy Confusion MENINGITIS

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

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

28. Clinical features Chills Malaise Prostration Petechial rashes Metastatic involvement of joints,ears,eyes,lungs,adrenals. 10% pneumonia.

29. WATER HOUSE FRIDERICHSEN SYNDROME -Fulminant meningococcemia -Fatal condition -Shock -DIC -Multisystem failure -Deficiency of C 5 -C 9 -Pathogenic component-LPS(endotoxin) -Adrenal haemorrage and shock

30. CSF COLLECTION LAB DIAGNOSIS:

31. CSF examination: General appearance: Purulent Biochemical parameters: Glucose <35mg/100ml , proteins 80-500mg/100ml. Cytology: 400-20,000 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.

32. 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.

33. - 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.

34. 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.

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

36. NEISSERIA GONORRHOEAE - 1879-Albert.L.S.Neisser 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.

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

38. CULTURAL CHARACTERISTICS Aerobes/ facultative anaerobes pH -7.2-7.6 Temp. 35ºc-36ºc 5-10% CO 2 Chocolate agar,Mueller-hinton agar,Thayer martin media, Modified TM medium, Modified NYCM.

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

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

41. 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.

42. 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.

43. P1: Principal outer membrane protein. P2:OPA P3: Binds with P1 to form porin channels

44. 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.

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

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

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

48. 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)

49. 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

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

51. 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).

52. 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.

53. 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

54. 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.

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

56. 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.

57. 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 36 º c but many strains may grow at 22 ο c . CO2 is not an absolute requirement. Most srains grow on Nutrient agar.

58. 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.

59. 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.

60. CONTD…. It is found in the respiratory tracts of 1.5-5.4% 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.

61. 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.

62. 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.