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Neisseria Bacteria: Pathogenesis, Classification, and Clinical Relevance
- 1. NEISSERIA PRESENTER : MOHDSHAHZEB M.Sc. Medical Microbiology Sharda University, SMS&R
- 2. CLASSIFICATION Kingdom • Bacteria Phylum• Proteobacteriaceae Class • Betaproteobacteria Order • Neisseriales Family • Neisseriaceae Genus • Neisseria Species • meningitidis, gonorrhoeae, lactamica, mucosa, etc
- 3. HISTORY Discovered Neisseria meningitidis {1887} AntonWeichselbaum Isolated from spinal fluid of patient.
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- 7. COMMENSAL NEISSERIA SPECIES •Several species of Neisseria are harmless commensals (N. cinerea, N. lactamica, N. elongata, and N. mucosa) of human respiratory tract. Can be differentiated from pathogenic Neisseria by various ways, such as: o Can grow on basal media, such as nutrient agar o Can grow at 22°C Mostly, o Don’t grow on selective media (except N. lactamica) o Not capnophilic (CO2 is not required)
- 8. DIFFERENCE BETWEEN N. meningitidisand N. gonorrhoea N. meningitidis N. gonorrhoea Capsulated Non-capsulated Lens-shaped/ half moon-shaped (diplococci with adjacent slides flattened) Kidney shaped (diplococci with adjacent sides concave) Ferments glucose and maltose Ferments only glucose Rarely have plasmids Usually possesses plasmids, coding for drug- resistant genes Exist in both intra-and extracellular forms Predominantly exist in intracellular form Colony- circular Colony- varies in size with irregular margin Habitat- nasopharynx Habitat-genital tract( urethra, cervix), rarely pharynx Essentials of Medical Microbiology, Apurba S Sastry, 2nd edition
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- 10. Morphology Gram negative Oval/spherical diplococci 0.6-0.8 µm Beanshaped Encapsulated Arranged in pairs (adjacent sides flattened)
- 11. VIRULENCE FACTORS On thebasis of specificity of capsular polysaccharide antigens Meningococci divided into 13 serogroups: A, B, C, D, X, Y, Z, W-135, 29-E, H, I, K and L Serogroups A, B, C, X, Y, W-135 : most commonly associated with meningococcal disease Group A : Epidemics Group C : Localized outbreaks Group B : both epidemics & outbreaks
- 12. Based on theouter membrane protein serogroups further divided into serotypes: • PorA • PorB Both show antigenic variability and are responsible for sero- typing (PorB) and serosubtyping (PorA) of meningococci.
- 13. LPS AND ENDOTOXINS: Endothelialinjury is central to many clinical features of meningococcaemia, such as : Increased vascular permeability leading to loss of fluid and shock Intravascular thrombosis leading to disseminated intravascular coagulation(DIC). Myocardial dysfunction. IgA proteases : cleave mucosal IgA Transferrin binding proteins : help in uptake of iron from transferrin. Adhesins : include opacity proteins and pili..
- 14. EPIDEMIOLOGY Natural habitat &reservoir Human nasopharynx Nasopharyngeal Carriers 5-10% adults asymptomatic carriers Modes of infection Direct contact or respiratory droplets from the nose and throat of infected people Prevalence of meningitis is highest in sub-Saharan belt of Africa. Nearly 5 lakh cases of meningococcal disease occur each year, and 10% of those die.
- 15. PATHOGENESIS Incubation period: 3-4days They replicate and migrate to subepithelial spaces In meninges, organism are internalised into phagocytic cells Local invasion and spread from nasopharynx to meninges through blood stream (directly along perineural sheath of olfactory nerve, cribriform plate to subarachnoid space) Adherence of organism to nasopharyngeal mucosa Inhalation of contaminated droplets Steps
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- 18. Laboratory Diagnosis Specimen: CSF,blood, skin scrapping, nasopharyngeal swab CSF Sample First Portion Centrifuged Supernatant Capsular antigen detection by latex agglutination test Biochemistry Sediment Gram stain Second portion Culture on Blood agar, Chocolate agar Thayer martin agar Third Portion Inoculation in BHI Sub Cultured on Blood, Chocolate agar
- 19. Meningococci in CSFsmear
- 20. Normal CSF • Clear,colourless • 0-5 lymphocytes • Sterile • 15-45 mg/dL protein • 50-80 mg/dL glucose CSF in bacterial meningitis • Turbid • 100-10,000/mm3 • Bacteria in gram stain • >45 mg/dL protein • <40mg/dL glucose
- 21. BLOOD CULTURE :Blood should be immediately injected into blood culture bottles (brain-heart infusion broth, automated systems such as BacT/Alert) and incubated; subcultures are made onto blood agar plate and colonies grown are processed. Nasopharyngeal swabs, pus or scrapings from rashes should be carried in transport media (such as Stuart's medium) and inoculated onto selective media, such as Thayer martin medium or New York City medium.
- 22. Cultural characteristics • MediaUsed Selective Media : Modified Thayer-Martin agar, New York City medium Non selective Media : Sheep blood agar, Chocolate agar Colony characteristics Grey Round 1mm Smooth Convex Translucent Butyrous
- 23. Neisseria meningitidis growing onchocolate agar Neisseria meningitidis growing on Sheep blood agar
- 24. Biochemical tests Biochemicals Oxidase positive Catalasepositive Ferments glucose and maltose with acid production Doesn’t ferment lactose, sucrose and fructose Nitrate negative Colistin Resistant Gamma-glutamyl aminopeptidase positive Mackie & McCartney Practical Medical Microbiology, 14/e
- 25. SEROLOGY: Antibodies to capsularantigens can be detected by ELISA. This helps: • Retrospective diagnosis of disease • Know the response to vaccination • Diagnosis of chronic meningococcaemia MOLECULAR DIAGNOSIS : • PCR detects earlier than culture and also helps in serogroup identification • Real time PCR • Multiplex real-time PCR(e.g. BioFire FilmArray) can be used for simultaneous detection of common agents of pyogenic meningitis • Common genes targeted include: ctr A ( capsule transport gene) and sod C (Cu-Zn superoxide dismutase gene).
- 26. Prophylaxis • Chemoprophylaxis : Ceftriaxone Rifampicin Ciprofloxacin Vaccine Prophylaxis : • Bivalent vaccine containing capsular polysaccharides of serotypes A & C : for infants below 2 years • A quadrivalent vaccine constituted by polysaccharides of serotypes A, C, Y & W- 135 : for children & adults Conjugate Vaccine: • Addition of a protein carrier (adjuvant) increases the immunogenicity of the capsular vaccine.
- 27. Vaccine for groupB (Men B vaccine) Vaccine contains four recombinant proteins: • Adhesin A • Heparin binding antigen • Factor H binding protein • Outer membrane vesicles (OMV). Schedule: two doses given IM route 1 month apart Indication: 16-25 years
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- 29. Neisseria gonorrhoeae • Causessexually transmitted disease Gonorrhoea • First described by Neisser in 1879 in gonorrhoeal pus • Resembles meningococci very closely in many properties.
- 30. Morphology Gram negative Oval/spherical diplococci Found within thepolymorphs Kidney shaped Posses pili on their surface Arranged in pairs (adjacent sides concave)
- 31. VIRULENCE FACTORS 1. Pili 2.Lipo-oligosaccharide : Endotoxic 3. Outer membrane proteins : a. Protein I (Por) - A porin & helps in adherence. b. Protein II (Opa) – helps in adherence. c. Protein III (Rmp) – Associated with protein I. 4. IgA1 protease : Splits & inactivates IgA
- 32. EPIDEMIOLOGY Natural habitat &reservoir Urogenital tract Anal canal Source Adults asymptomatic female carriers & Patients Modes of infection • By sexual contact • From mother to baby during birth Gonorrhoea is an exclusively human disease, there are no animal reservoirs
- 33. PATHOGENESIS Incubation period: 2-8days Leads to clinical Manifestations Reach the sub-epithelial connective tissue & causes inflammation Penetrate through the intercellular space Gonococci adhere to epithelial cells of urethra or other mucosal surface through pili Mechanism
- 34. CLINICAL Manifestations The infectionmay spread to the periurethral tissues, causing abscesses & multiple discharging sinuses (water-can perineum) In some it may become chronic urethritis leading to stricture formation Extends to the prostrate, seminal vesical & epididymis The disease starts as an acute urethritis with mucopurulent discharge In Males
- 35. Rarely peritonitis maydevelop with perihepatic inflammation (Fitz- Hugh-Curtis syndrome) The infection may extend to Bartholin’s glands, endometrium & fallopian tubes causing Pelvic Inflammatory Disease (PID) The initial infection is urethritis & cervicitis but vaginitis doesn’t occur in adult females (vulvovaginitis can occur in prepubertal girls) In females
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- 37. In pregnant woman Prolonged ruptureof the membranes Premature deliveries Chorioamnioniti s Sepsis in infant
- 38. • In neonates: Characterized by purulent eye discharge, occurs within 2-5 days of birth. Transmission occurs during birth from colonized maternal genital flora. • Disseminated gonococcal infection(DGI): Occurs rarely following gonococcal bacteraemia. DGI is characterized by polyarthritis and rarely dermatitis and endocarditis. Most commonly associated with PorB. • In HIV-infected persons: Nonulcerative gonorrhoea enhances the transmission of HIV by 3-5folds, possibly because of increased viral shedding.
- 39. Culture characteristics • Fastidiousorganism don’t 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. • Essential to provide 5-10% CO2
- 40. Laboratory Diagnosis Sample Collection InMales Urethral Discharge (swab) Discharge after prostatic massage In Females Urethral discharge Cervical swab
- 41. Transport media : •Stuart’s media • Amies media
- 42. Methods of examination •Direct microscopy: Gram staining : Reveals gram-negative intracellular kidney-shaped diplococci. It is highly specific and sensitive in symptomatic men but not in women.
- 43. • CULTURE : •Media Used Selective Media : Thayer-Martin medium Non selective Media : Chocolate agar, Muller-Hinton agar, Modified New York City medium Colony characteristics Grey Irregular small Smooth Convex Translucent Finely granular surface
- 44. Neisseria gonorrhoea growing onThayer martin agar Neisseria gonorrhoea growing on new york city agar
- 45. Biochemical tests • Oxidasetest : Positive • Ferments only glucose but not maltose and sucrose • Tested by rapid carbohydrate utilization test (RCUT)
- 46. • MOLECULAR DIAGNOSIS: PCR assay is available for detection of N. gonorrhoeae from the clinical specimens targeting 16s or 23s rRNA gene.
- 47. treatment DRUG OF CHOICE: Third generation cephalosporins currently are the mainstay of therapy for uncomplicated gonococcal infection. Ceftriaxone (250 mg given IM, single dose) Cefixime (400 mg given orally, single dose) If coexisting chlamydial infection is present, then azithromycin or doxycycline can be added to the regimen.
- 48. DRUG RESISTANCE INNEISSERIA GONORRHOEAE • Gonococci were initially susceptible to most antibiotics, such as sulfonamides, penicillins, quinolones, but because of their continual usage, resistance has emerged over the time. • Various resistant strains have evolved in due course of time. The strains are often resistant to many drugs at a time. • Though third-generation cephalosporins are the drugs of choice for gonococcal infections at present, some strains show reduced susceptibility to ceftriaxone and cefixime (termed as cephalosporin intermediate/ resistant strains), which may be due to altered penicillin binding protein 2.
- 49. Antibiotic resistance inNeisseria gonorrhoeae: origin, evolution, and lessons learned for the future By Magnus Unemo1 and William M. Shafer2 et al; IN 2011
- 50. Prophylaxis • Early detectionof cases • Treatment of both partners • Tracing of contacts • Health education about safe sex practices, such as use condoms. • There is no vaccination available for gonococci.
- 51. references • Essentials ofMedical Microbiology, Apurba S Sastry, 2nd edition • Mackie & McCartney Practical Medical Microbiology, 14/e • Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future By Magnus Unemo1 and William M. Shafer2 et al; IN 2011
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