Biology 216 - Lect 15/16 Neisseria I. Neisseriaceae (family) Oxidase test: cytochrome oxidase or indophenol oxidase (di (tetra) methyl-p-phenylene diamine -> indophenol (reduced)) A. Acinetobacter - common in soil, sewage, common lab contaminant. Gm- diplobacilli - easily confused with Neisseria Oxidase-; growth on MacConkey. Disease - Rarely UTI, URT. B. Moraxella (used to be Branhamella) - normal mucosal flora - catarrhal (mucus) Gm- diplococci, Ox+, DNase+, non-fermentative Disease - pharyngitis, otitis media, conjunctivitis C. Neisseria - exclusively human mucosal organism Gm- diplococci, kidney bean, Ox+ Normal mucosal flora - moist medium colonies (grey to yellow) 2 pathogens we will discuss in detail. II. N. gonorrhoeae = gonococcus (GC) A. Bacteriology 1. Surface - pili (attachment to urogenital columnar epithelia) Can't bind squamous epithelia of vagina Multiple serotypes - antigenic shift by structural gene rearrangement (Cross over between cassettes of pilE(expressed) and pilS (silent) Constant region embedded in outer membrane (N-terminus). 2 stage attachment - pili, then Opa (PII) outer membrane (close binding) (Opa = opacity) 2. Products - Siderophores; IgAase (against IgA1); B-lactamase - first in 1976; from Haemophilus; plasmid; Far East;1 - 5 %. 3. Colony types 1 - 4 based on macroscopic colony morphology
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Types 1, 2 |
Types 3, 4, 5 |
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Pinpoint |
Large granular |
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Primary isolation |
Subculture |
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Virulent |
Avirulent |
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Pilus + |
Pilus -- |
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Siderophore + |
Siderophore -- |
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Resist Phagocytosis |
Less Resistant |
4. Disseminated gonococcal isolates (DGI) (1%) from sinovial, heart, meninges Resist serum cidal activity; Auxotypes (arg, uracil, hypoxanthine) Arthritis, petechia at joints. B. General Pathogenesis - pyogenic 1. Transmission - direct mucosa -> mucosa (venereal, oral, anal) Organisms do not survive long outside body (toilet seat exonerated) 2. Attachment - to columnar urogenital epithelia by pili Endocytosis - multiply in epithelial cells 3. Penetration - intracellular spaces to connective tissue (not active process) 4. Inflammation - LOS highly inflammatory, PMN, TNF, pus, occlusion, scarring. 5. Uncomplicated - local, self resolving 6. Animal model for study - subcutaneous chamber in mice (same for Staph TSS) C. Clinical - the clap (clapoir - brothel) Prostitution is not major factor - disease for everyone. 1. General - VD/STD #2 reportable bacterial disease in US (300,000/yr), second to Chlamydia Probably 3 million due to asymptomatic Chlamydia higher, now reportable to CDC (non-gonococcal urethritis) 1960 - 1980 - increase 150 to 500/100,000 (1990 dropped to 350, probably due to safe sex) 2. Male gonorrhea (uncomplicated) - anterior urethra 20 % chance of acquisition 2 - 8 day incubation - burning urination, yellow discharge w/ PMNs Usually local, self resolution 1 % complication - ascension -> urethral stricture; epididymitis, prostatitis. 10 % asymptomatic 3. Female gonorrhea (uncomplicated) - vaginal tract High chance of acquisition (near 100 %) Burning urination, discharge abdominal pain Up to 80 % asymptomatic (cervical) 10 % PID (pelvic inflammatory disease) - fever, abdominal pain. Ascension - fallopian tube (occlusion/anaerobes/ectopic pregnancy in the tube, rupture, shock), Scarring uterus -> infertility, peritonitis 4. Other sites - rectal, pharyngitis (like strept throat) 5. Gonococcal ophthalmia neonatorum Acquired at birth -> conjunctivitis -> blindness (used to be major cause) Prophylaxis - all states require erythromycin (used to be 1 % Ag NO3) (Silver not effective against Chlamydia which can cause same disease) D. Diagnosis (symptoms suggest GC or Chlamydia) 1. Direct exam of specimen (male with Gm- intracellular diplococci) Not as reliable in female due to saprophytic Neisseria. 2. Culture and ID Specimen - exudate, scrapping, swab, urine sediment a. Processed immediately - GC susceptible to drying, cold, FA on cotton swab (alginate or polyester swabs used) b. Culture - rich media, selective against normal flora, CO2 Chocolate (sterile site) Thayer Martin (VCN) - Vancomycin, Colistin, Nystatin Selective for pathogenic Neisseria. Other media similar (NY City medium) c. ID - Gm- diplo, Ox+ CHO fermentation - tubes, kits, Rapid ID (bugs + 20% sugar + indicator) (1 - 4 hours checks for preformed enzymes) Unique enzymes - artificial chromogenic substrate Latex agglutination tests 3. Serology - no tests available (local IgA immunity only) 4. DNA probe (to rRNA) - chemiluminescent probe ->photodetector Used to test urethral swab, cervical swab, colonies isolated on plates. E. Treatment 1. Penicillin + Probenecid (old method to prevent excretion of penicillin) Ceftriaxone (B-lactamase resistant cephalosporin + Doxycyline Works on PPNG (penicillinase producing N. gono) 2. Antibiotics different than used for Chlamydia F. Prevention and Control 1. Condoms and foams (lower incidence since AIDS). 2. Case contact tracing - expensive (used in AIDS since life-threatening) Skin testing (LH) tested feasibility with MIF assay. 3. Early detection and treatment to prevent spread. 4. Vaccines - none yet; local immunity; multiple serotypes; Exptl vaccines induce circulating cidal Ab - so what. Vaccine candidate - conserved PBP-3 (penicillin binding protein 3) 5. Major problems with control: - Contagious before symptoms appear. - Short term local immunity and multiple serotypes -> repeat infection. - Asymptomatic population. III. N. meningitidis - meningococcus (MC) A. Bacteriology - 4 majors you should know 1. Capsule - antiphagocytic 8 serogroups (A,B,C responsible for most; B responsible for 50 % of disease GpB capsule = E. coli K1 capsule 2. Pili - nasopharyngeal attachment 3. OMP - Ab is cidal to MC Possible GpB vaccine Ag since Gp CHO is not immunogenic 4. Endotoxin (LPS) - LPS blebs -> shock and most of symptoms B. Pathogenesis and Clinical - epidemic meningitis #1 major cause of meningitis in US ~4000/yr. Was #2 before 1995. Peak incidence 6 - 24 months and 20 yr old (military) 1. Transmission - Carriage 5 - 15 %; Crowding and stress -> 90 % carriage (nose) Transmitted by respiratory droplets. Endogenous (predisposition); Exogenous transmission. 2. Bacteremia - low immunity; deficiency in C3; new capsule exposure; spleen dysfunction; dry climate -> Blood -> meninges, joints, skin. 3. Major pathogenesis by LPS blebs- Petechia, fever, DIC, meningitis, shock. 4. Rapidly fatal (no other disease kills so quickly) 10 % fatal with treatment; 90 % fatal without treatment. C. Lab diagnosis - treatment before results from lab. 1. Isolation - specimen - petechia, CSF, joint, blood. Rich media (chocolate), CO2. Culture from nasopharynx is irrelevant. 2. ID - Biochemicals, Gm stain, oxidase, CIE, Fl-Ab, Latex agglutination. D. Treatment - antibiotics (large dose, immediately, iv or intrathecal) + SAIDs Symptoms - Cerebral edema (urea or mannitol) DIC - heparin, glucocorticosteroids. SAIDs - stabilize lysosome, decrease IL-1, PMN, capillary permeability. E. Prevention and Control 1. Carriers - eliminated from mucosa by rifamycin (used in outbreaks and household contacts). 2. Vaccine - Groups A,C,Y,W Capsular vaccine (50 ug); Single dose good for 3 years. Vaccine groups (not general public): Military during outbreaks. Travelers to endemic areas (arid and high carriage rates) Not immunogenic to most vulnerable group (6 - 24 month) Serogroup B vaccine is not immunogenic to any groups. Exptl OMP vaccine trials: Clinical trials show safe and immunogenic in adults. Cidal antibody when injected with alum into children. Inconclusive results in 2 year olds in Africa. Problem several serotypes of OMP.