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Gram-negative Cocci and Coccobacilli
Learning Objectives
For the following G-:
  • Neisseria meningitidis
  • Neisseria gonorrhoeae
  • Haemophilus influenzae
  • Bordetella pertussis
  • Pasteurella multocida
You should know:
  • general characteristics
  • major virulence factors
  • epidemiology
  • pathologenesis and disease
  • laboratory diagnisis
  • treatment and prevention
N. meningitidis vs N. gonorrhoeae
N. meningitidis


  • Glucose +

  • Maltose +

  • Capsule +

  • causes meningitis

  • coffee bean morphology


N. golorrhoeae


  • Glucose +

  • Maltose -

  • Capsule -

  • Causes gonorrhea

  • coffee bean morphology

N. gonorrhoeae
N. gonorrhoeae Major virulence factors
  • Pili: mediate attachment; undergo antigenic morphology (change morphology of pili so that the host has difficulty developing an immune response)
  • Por protein: prevention of phagolysosomal fusion
  • Lipo-oligosaccharide (LOS) = endotoxin (similar to LPS)
  • IgA protease: common in many bacteria; break up IgA which is present on mucous membranes
N. gonorrhoeae Epidemiology

  • Humans are the only host

  • Second most common STD

  • High prevelance in adolescents (62% of cases between ages 15-24)

  • Asymptomatic carriage: women > men

  • Disease: men > women

Pathogenesis and Clinical Manifestations of N. gonorrhoeae
  • primary site: Urethra, vagina
  • Men: Urethritis
  • Women: Inflammation of the endocervix, purulent vaginal discharge, irregular intermenstrual bleeding, infection of fallopian tubes>PIS>Scarring>INfertility
  • Babies: Ophthalmia neonatorum
Gonococcal Conjunctivitis (Ophthalmia neonatorum)
gonococcal urethritis
disseminated gonococcal infection
other sites of infection N. gonorrhoeae
  • anus/rectum: Protitis
  • Throat: Pharyngitis
  • Joints: Arthritis, especially in young, sexually active females (particularly in the knees and ankles)
Laboratory Diagnosis of N. gonorrhoeae


  • gram stain of the urethral/vaginal discharge

  • culture: Thayer-Martin agar (VPN)

  • Glu+, mal-, sucrose-

  • DNA probe

  • picture (a) chocolate medium overgrowth (grows all rectal bacteria)

  • picture (b) Thayer-martain Medium grows Neisseria only: chocolate agar + antibiotic that kill off normal rectal flora

N. gonorrhoeae Treatment and Prevention

  • Ceftriaxone--3rd generation cephalosporin

  • Mixed infection with Chlamydia trachomatis--add tetracycline

  • Prevention: nonspecific (e.g., condoms)

N. meningitidis: Major Virulence factors
  • Capsule = a polysaccaride that coats bacteria in order to evade immune response: antigen that defines 13 serogroups (epidemic serogroups A, B, C, W135, X and Y); antigen detected in CSF; antigen included in vaccine
  • Outer membrane protein (OMP)
  • Endotoxin (LOS--Lipooligosaccharide, similar to enteric LPS): major cause of infection
  • IgA protease--evasion of mucosal immunity
N. meningitidis Epidemiology
  • Asymptomatic carriage in the nasopharynx: ~5-10% (unclear as to why): lower in children; 25% in teenagers (particularly common on college campuses
  • children (6 mo-2yrs) are more susceptible
  • Serogroup B: the most prevalent in the U.S.
  • 2001: 1,094 cases
N. meningitidis Pathogenesis
  • Capsule and LOS
  • LOS-mediated damage to ciliated epithelial cells--generation of pro-inflammatory cytokines
  • invasion of submucosa--Type IV pilus (like a hook they can project and reel back in), N.m. adhesion A (NAD)
N. meningitis associated diseases

  • Meningitis, bacterium crosses the BBB: Fever, Headache, Stiff Neck, INcrease PMNs in the CSF (fatal in 20% of humans)

  • Meningococcemia: DIC (diffuse intravascular coagulopathy; as it travels through blood, LOS is relased), shock, waterhouse-friderichsen syndrome (very deadly, typically die within hours of symptoms appearing)

meningococcemia
meningococcemia
meningococcemia
Wterhouse-Friderichsen Syndrome (WHS): N. meningitidis evaded the adrenal glands; if a patient reaches this point of infection, he will almost certainly die because he can no longer secrete hormones
Laboratory Diagnisis of N. meningitidis:
  • Specimen: CFS (blood)
  • Direct exam: G-, kidney-shaped diplococci
  • Rapid detection: latex (bead) agglutination
  • Culture: BAP, CHOC
  • Ox+, Glu+, Mal+
Treatment and Prevention of N. meningitidis



  • Penecillin + 3rd generation cephalosporin

  • Quadrivalent Vaccine with group A, C, Y, and W135 polysaccharides (4/6 epidemic strains):

  • Meningococcal conjugate vaccine (MCV4)--Unvaccinated college freshman

  • Meningococcal polysaccharide vaccine (MPSV4)--2years of age and adolescents

  • group B is excluded due to low immunogenicity

Haemophilus influenzae general characteristics
  • gram-nevative
  • plomorphic
  • nonmotile cocobacillius
  • X (hemin) and V (NAD) factor requirement
  • two major categories:l encapsulated and unencapsulated (non-typable) strains
Haemophilus influenzae Gram-stain
Haemophilus influenzae Virulence factors

  • LPS: highly diverse glycoform structure--contributes to both commensal and pathogenic lifestyle

  • outer membrane proteins (OMPs)

  • adhesion proteins (allow it to stick to epithelial cells)

  • capsule: antigen that defines serotypes--6 major classes; antigen detected in CFS; antigen included in vaccine

  • IgA protease--probably does not play a major role in this particular case

Epidemology of Haemophilus influenzae
  • part of normal microbiota
  • incidence in the past: 8000 cases/year
  • incidence in 2008: 2,886 cases
  • Children < 5 y-old: 437 cases
  • decreased dramatically due to Hib vaccine
Pathogenesis and Disease of H. influenzae:
From nasopharynx H. flue spreads to:
  • middle ear - otitis media
  • sinus - sinusitis
  • bronchi - bronchitis
  • alveoli - pneumonia
  • epiglottis - epiglottitis
  • meninges - meningitis
Non-Respiratory Diseases (H. Influenzae)
  • cellulitis
  • arthritis
Laboratory Diagnosis of H. Influenzae
  • Culture: X and V factor requirement--H. influenzae is a difficult organisim to culture--reduced sensitivity of detection
  • Catalase+, Oxidase+
  • Antigen detaction--latex particle agglutination teat (LAT)
  • Polymerase chain reaction (PCR) (most sensitive)--used in clinics; most reliable
Treatmant and Prevention--H. Influenzae
  • Otitis media and sinusitis: Ampicilli, Augmentin, Trimethoprim-sulfamethoxazole
  • Meningitis: 3rd Generation Cephalosporin, i.e., ceftriaxone, cefotaxime
  • Hib Vaccine (very effective): Polysaccharide-protein conjugate vaccine; expensive, limited usage in the Developing World
Gram-negative Coccobacilli
  • Pasteurell multocida
  • Bordetella pertussis
Pasteurella multocida:
  • relatively uncommon
  • Short coccobacillus
  • encapsulated
  • grows well on BAP or CHOC at 37 degrees C
  • Gram negative, stains bipolarly
  • part of the normal flora in the mouths od domestic dogs and cats
  • transmission: through bites
Virulence Factors Pasteurella multocida
  • capsule
  • endotoxin
  • exotoxin (PMT): disrupts cellular metabolison in the skin, connective tissue, fat, bane (causes cells to become hyperactive vs. killing them)
Clinical Manifestations of Pasteuraella multocida
  • Cellulitis at the bite site--not to be confused with cat scratch disease (Nartonell app.)
  • Osteomyelitis--PMT causes bone degradation
Pasteurella multocida
  • bipolar staining
  • Indole+
  • "Sick" triple sugar iron (TSI) test
Pasteurella multocida
  • penicillin
  • fluoroquinolones
  • tetracyclines
Bordetella pertussis:
  • small coccobacillus
  • encapsulated
  • obligate human pathogen
  • Transmission: via aerosol
  • Highly contagious
  • worldwide distribution
  • occurs primarily in infants and young children (adults are carriers but are usually asymptomatic)
  • In the US, 2008: 13,278 cases
Virulence Factors: Bordella pertussis
  • Filamentous hemagglutination (FHA)
  • Pertacin
  • Pertussis toxin --> eukaryotic Adenylate cyclase increase --> cAMP increase
  • Adenylate cyclase toxin --> cAMP increase
  • Tracheal cytotoxin --> inhibits cilia --> promotes coughing fits --> expels bacteria
  • does not want to kill  us because they (bacteria) would die as well
Pathogenesis of Bordella pertussis
  • attachment to the cillia of teh epithelial cells in teh upper airways: with the help of FHA Pertactin
  • Tracheal cytotoxin
  • Non invasive
  • Pertussin toxin causes: mucuc secretion; lymphocytosis--increase in the number or proportion lymphocytes in the blood
  • Adenylate cyclase/Pertacin: Helps prevent invasion/uptake of teh bacterium
Bordella pertussis Clinical Manifestations
  • Tracheobronchitis
  • Severe proxysmal cough
  • Inspiratory "whoop" and vomiting after coughing--halmarks of pertussis infection
  • Duration: 1-4 weeks
  • Death ususally due to pneumonia, caused by secondary bacterial infection
Bordella pertussis Lab Diagnosis
  • Nasopharyngeal Swabs
  • Bordet-Gengou medium--selective for Bordetella spp., small pearl-like colonies
  • agglutination in specific antiserum
  • DFA--Firect Fluorescent antibody test (bacteria glow green if they are present)
Bordella pertussis treatment and Prevention
  • Erythromycin (sensitive to wide variety of patients but only antimicrobial that actually works in patients)
  • Amoxicillin, tetracycline, fluoroquinolones and cephalosporins--god in intro activities, not suitable for in vivo treatment
  • DaPT (Diptheria, acellular Pertussis and Tetanus) Vaccine: Acellular pertussis vaccine; mixture on 5 antigens (generally modified pertussis toxoid etc.)
Case Study 1: A 10-year old girl is brought in to Green Meadows Clinic because of redness, pain, and swelling of the left hand. According to her mother, the day before the onset of her symptoms, the child was accidentally bitten by her cat. Physical exam reveals temperature of 37.8 degrees C, enlarged axillary lymph nodes and erythematous and swollen left had that was hot to touch. The pain now has spread upward and involved the upper arm. An atending physician notics early sins of abcess formation and accumulation of pus at the bite site. A presumptive diagnosis is made and the purulent exudate sent for culture.
  • What is the most likely diagnosis?
  • What is the most likely cause?
  • Cat-bite cellulitis
  • Pasteurella multocida
Case 2: A 2-month-old male infant was brought to the peditrician because of coughing and chocking spells for two weeks. At the end on the coughing spell, teh baby often vomited and sometimes seemed to have difficulty catching his breath. The nurse in the clinic noted that the cough was paroxysmal. The mother related that she had had coughing spells followed by choking and near vomiting for three weeks. She had recieved a course of amoxicillin withour relief. ON evaluation, the baby's temperature was 37.7 degrees C (↑); pulse, 200/min (↑); respiration 72/min (↑); WBC, 140,000/mm3 (↑↑↑) of which 50% were lymphocytes (↑↑). Chest  x-ray revealed upper lobe infiltrates bilaterally and patchy infiltrates in teh middle lobes consistent with pneumonia. The infant was admitted to pediactric ICU where he was intubated and placed on a mechanical ventilator.
  • What is the most likely diagnosis?
  • What is the most likely cause?
  • What are virulence factors?
  • What is the treatmet choice?
  • How can teh disease be prevented?
  • Whooping cough (pertussis)
  • Bordetella pertussis
  • filamentous hemagglitinin, adenylate cyclase, pertussis toxin
  • Erythromycin
  • Immunization (DaPT)
Haemophilus influenzae:
  • small, grayosh, mucoid, dewdrop-like colonies
  • "Musty basement" odor
  • Pleomorphic--many forms
  • Fastisious--requires A (hemin) and V (NAD) factors, present in choclate agar
Identification of H. influenzae--XV test
Satellite test is poor mans version of the XV test
Bacillus anthracis general characteristics:
  • in culture: "bamboo-like rods" large gram+ rods (squared ends) with centrally, poorly stained oval endospores
  • in tissue: short chains, capsule, NO spore
  • colonies: rough, comma shaped projections from colony edge: "medusa head"
bacillus anthracis virulence factors
  • encoded on two plasmids (essential)
  • capsule (poly-D-glutamic acid, protein, NOT polysaccharide)
  • anathrax toxin (tripartite toxin): lethal factor (LF, protease); edema factor (EF, adenylate cyclase); protective antigen (PA)
  • PA binds and helps EF and LF enter the cell
  • PA + EF = edema toxin, causes edema
  • PA + LF = lethal toxin, (dominant virulence factor) causes cell death
Anthrax toxin:
  • EF + CaM + ATP --> ↑cAMP --> edema
  • LP --> ↓MAPKs --> necrosis hypoxia
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