medical microbio-MODULE 9. GRAM NEGATIVE COCCI (GNC)

Classify the genus Neisseria on the basis of:

a.     Morphology

b.     Staining reaction

c.     Cultural requirements: growth optimum temp, CO2 req, suseptible to changes in what

• enriched media used (2 antibiotics names and why needed)

a.     Morphology: Kidney bean shaped Gram-negative diplococci

b.     Staining reaction:  gram neg

c.     Cultural requirements: Fastidious bacteria but will grow if optimized for temperature at 37° C and if enhanced by 2 to 8% CO2 

•  pathogenic strains will not grow below 25° C or room temp

•  are quite sensitive to cold temperatures and will die for refrigerated or sample is plated onto media just removed from the refrigerator

• Incubator with CO2 filled as a chamber from external Source or candle Extinction jar is often used

•  highly susceptible to adverse conditions such as drying, chilling, sunlight, pH extremes

• Of utmost importance there must be minimal to no delay of culture of samples after collection 

• Enriched media is required: chocolate agar ( blood agar that has been heated to humalyze rbc, free hemoglobin contains Fe which require is required for growth) or chocolate agar-based media

  • commonly used media for isolates of pathogenic neisseria is Taylor Martin (TM) agar→ Chocolate-based agar with some antimicrobics added as in VCN agar which is another similar concept that only allows pathogenic bacteria to grow 

  • VCN agar→Chocolate agar modified by addition of antibiotics: Vancomycin to inhibit gram-positive bacteria, colistin to inhibit gram-negative bacteria rods, nystatin to inhibit yeast

Biochemical reactions: Critical criteria of neisseria

• media

• gram stain

• oxidase

• CTA

  • moraella catarrhalis difference from neisseria

• growth on media specific for pathogenic strains of neisseria such as ™/ thayer martin agar and VCN agar

• gram stain=kidney bean shaped Gram-negative diplococci

• Oxidase production= indicates presence of cytochrome oxidase which in the reduced state, the reagent is colorless/ oxidase negative;  in the oxidized state, the reagent is dark purple / oxidase positive therefore patient has neisseria 

• CTA (cystine tryptic agar) sugars to differentiate neisseria species

  • Yellow color due to acid production of metabolism os sugar = positive, red is negative

  • If only glucose pos→gonorrhoeae

  • If glucose and maltose pos→meningitis 

  • Moraella catarrhalis is all neg even though other biochem reactions are all same to neisseria

For laboratory diagnosis of pathogenic Neisseria, outline the three definitive criteria for diagnosis and the final biochemical tests done to complete the identification.

To find if pathogenic neisseria (genus): 1. Grow on VCN or TM agar, 2. Gram neg diplococci, 3. Oxidase positive 4–(species). CTA sugar test

N. meningitis and N. gonorrhoeae are genetically ____ but clinically manifest…

N. meningitis and N. gonorrhoeae are genetically similar but clinically manifest very diff

Gonorrhea is caustve agent of what

• most prevalent of the ____ diseases

• __% occur in aldolescnets

• ____to the CDC

Causative agent of gonorrhea,

most prevalent of the classical venereal diseases;

33% of all infections occur in adolescence,

continues to be one of the most commonly diagnosed infections that is reportable to the CDC

Meningitidis

• infection of (3) body parts and cause of what disease

  • effects of this worldwide

• beyond perinatal period, commonly transmitted how

• major causes of bac meingitis (2 important) and in kids (1 important)

  • what does this overall mean in lab/clinic

• is an infection of the membrane, the meninges, and CSF surrounding the brain and spinal cord;

• is a major cause of death and disability worldwide.

• Beyond the perinatal period, bacteria commonly transmitted from person to person through exchange of respiratory secretions are responsible for most cases of bacterial meningitis 

  • Causative agent of meningococcal meningitis, which has the potential to occur as epidemics 

  • Major causes of bacterial meningitis in adults are streptococcus pneumoniae which is a gram positive diplococcus, neisseria meningitis which is a gram-negative diplococcus, hemophilus influenza which is a small gram negative rod;

    • Major causes of bacterial meningitis in newborns and children are streptococcus agalactidae which is Group B strep that is a gram positive coccus, listeria monocytogenes which is a gram-positive rod=>All to say that gram stain results determine what a disease could be within minutes so that way the first antibiotic can be administered

• procedure if CSF is suspected of contam of meningitis

  • additional causes of meningitis

  • why important to know causative agent

• When CSF comes in and suspected of contamination of meningitis, made stat

• Spinal fluid tap collection to get CSF From between the subarachnoid space and Pia matter

• Additional causes of meningitis are viruses, fungi and yeasts, and parasites as well so pretty much any microbe

• knowing the cause is important because severity of disease can be different, and treatment of illness can be different as determined by the patient's age, patience immune status, bacterial route of transmission

other species of Neisseria relative pathogenicity and location (2)

There are other species of Neisseria  In humans, most are considered non-pathogenic and are part of the normal flora of the upper respiratory tract and other mucosal surfaces

Ex) path to ID N.gonorhoeae—4 steps

1. Gram neg diplococci on gram stain

2. Growth on media specific for neisseria (chocolate or TM/VCN media)

3. Oxidase positive (purple)

4. CTA sugars shows only glucose positive

only known natural reservoir of pathogenic neisseria is

humans

Clinical infection occurrence and transmission of N. meningitis

• Peak occurrence of disease in children

• only known reservoir—> and carrier

• spread how

• pop at risk

• Is worldwide and varies between sporadic cases and epidemic cases

•  Peak occurrence of disease in children from 6 to 24 months of age

•  human nasopharynx is the only known reservoir

  •  adult nasopharyngeal carrier

•   meningococci spread from person to person by Airborne droplets of infected nasopharyngeal secretions

•  others at risk are the military or those in boarding institutions / individuals living in close proximity to each other 

Clinical infection and occurrence of N. gonorrhoeae

• pathological determinant

• natural host

• most common disease of what and how many cases reported/ age group

• The presence of pili on virulent forms seems to be the only pathologic determinant

• Humans are the only natural host (as with whole genus)

• Is world wide and most common classic vernal disease with 1 mil cases reported annually

• Disease is most common in indiv 20-24 yr

Transmission N. gonorrhoeae: (adults and newborns)

• most common transmission is by sexual contact

• Newborn infants can become infected during passage through the birth canal

N. meningitis portal of entry

• where do infections usually stop —>what does this mean for patient

• what if infection proceeds (incubation time and mech of spread)—>symptoms

  • body locations (3)

• Invasion: Meningococci enter the body via the upper respiratory tract and establish themselves in the membrane

  •  most infection stop in the nasopharynx and the outcome of the host becomes an asymptomatic carrier

  •  if the infection precedes, and incubation period is a few days, mangococci are spread via the bloodstream / septicemia, which results in metastatic lesions in various areas like the skin, meninges, and eyes 

N. gonorrhoeae Invasion: primary infection begins where

• other possible portals (4)

• Primary infection usually begins at the epithelium of the urethra

  • Cervix, conjunctiva, rectal and pharyngeal mucosa may also serve as portals of entry

Morbidity and mortality N. meningitis: 

• death rate (developed and developing countries)

• % left with disability

  • 3 types of disabilities

• On an annual basis, 1 million cases of bacterial meningitis occurs globally and 200,000 of these die

•  case fatality rates vary with age of patient and time of illness and strain of bacterium causing infection

•  case fatality rates are 3 to 19% and developed countries and higher in developing countries with fatality rates of 37 to 60%

•  up to 54% of survivors are left with a disability due to The Infection Colon deafness, intellectual disabilities, neurological sequelae

Determinants of N. meningitidis pathogenicity: (2)

• Capsule: Contribute to the invasive properties of the meningococci, inhibits phagocytosis 

•  endotoxin from the gram-negative cell wall which has LPS

  • Is released when cell dies, especially Gram-negative

  •  is responsible for the extensive vascular damage that is a component of the disease process

  •  while many gram negative bacteria produce endotoxin, the levels produced by meningococcal bacteria are 100 to 1,000 times greater than normal

  •  endotoxin targets the heart, affecting its ability to pump; and no toxin also causes blood vessel leakage throughout the body, as every vessel starts to hemorrhage, major organs like the lungs kidneys and adrenals are damaged and eventually destroyed 

Symptoms of N. meningitis:

slight, moderate, severe (emboli meaning)

• 6 infections accompanying severe symps

  • waterhouse-friderichsen syndrome

• Vary from slight to moderate to severe

• slight = mild febrile disease that can be accompanied by pharyngitis

• Moderate = systemic disease characterized by fever, exhaustion 

• severe= Abrupt onset, headache, stiff neck, fever, petechial  eruptions or pinpoint hemorrhages developing into larger Hemorrhage areas causing emboli of mangococci

  •  emboli= a mass, such as a clump of bacteria or detached blood clot, foreign material like air bolus, that travels through the bloodstream and lodges and blood vessels, blocking it 

  • Severe may be accompanied by: 

1. Meningitis– inflammation of the meninges / membranes that envelop the brain and spinal cord 

2. Septecemia– bacterial infection of the blood

3. Arthritis– inflammation of the joints

4.  pericarditis– inflammation of membranes surrounding the heart 

5. Involvement of any organ system is possible

6.  may cause disseminated intravascular coagulation (DIC) followed by death as rapidly as one hour from onset of symptoms 

   1. Or waterhouse-friderichsen syndrome (adrenal gland involvement)

Complications N. meningitidis—mortality rate of treated vs untreated

after effects (5)

• Mortality is 85% if left untreated, 10% if treated early

  • Time is of the essence

• Some patients may survive meningococcal meningitis with no after affects

• Others may have lifelong pathologies such as deafness, CNS damage, necrosis of large areas of tissue requiring grafts, amputation, blindness

Pathogenesis of N. gonorrhoeae

• Invasion: primary infection portals of entery and —>human response (what becomes obstructed and why)

• Primary infection begins at portal of entry (epithelium of the urethra, cervix, conjunctiva, rectal and pharyngeal mucosa)

• Result of infection is inflammatory response with dense infiltration of neutrophils

  • In some individuals, ducts and glands become obstructed by the cellular exudate forming cysts or abscesses

N. gonorrhoeae effects on males (% aquire, incubation, % asymp, complications-4- and symp), females (% asymp, 4 symp, complication and 2 mech of this), children (3 infections)

• Males:

  • During unprotected intercourse–25% chance of acquiring gonorrhea from infected indiv (dec w/ condom use)

  • Incubates for 2-7 days

  • 10% remain asymptomatic, others present with burning on urination and yellow pus discharge

  • Complications include urethral stricture (narrowing of urethral tube), prostatitis, sterility, occlusion of vas deferens with scar tissue

• Females: 

  • 20-80% may be asymptomatic

  • Symptoms include burning or freq urination, vaginal discharge, fever, abdominal pain

  • Complications include PID (pelvic inflammatory disease) resulting from ascending infection w/ fallopian tubes and ovaries

    • This type of infection is a major cause of sterility by 2 mech:

1. Scar tissue causes partial obstruction of fallopian tubes such that fertilized ovum becomes trapped causing ectopic preg (doesn’t go to full term)

2. Scar tissue completely closes off lumen of fallopian tubes

• Children 3 infects: 

  • gonococcal ophthalmia neonatorum 

  • Neonatal gonococcal arthritis

  • Gonorrheal vulvovaginitis 

N. meningitis immunity

• antibodies aquired how and rel amt of defense of adults vs children (what age is most at risk)

• how do kids and adults aquire antibodies

• Antibodies present in the blood of very young infants is presumably acquired through the placenta

•  the lowest antibody titer present in infants is those between the age of 6 and 24 months due to loss of antibody provided passively by mother and inability / immature ability to make their own antibodies

• Adults have moderate immunity and antibody titers develop during the carrier state

treatment N. meningitidis and gonorroheae

N. meningitidis:

• For many years penicillin. Now dual therapy of azithromycin and ceftriaxone

N. gonorrhoeae 

• Usually penicillin, however new resistance emerging with beta-lactamase positive

prevention N. meningitidis: (2)

• what is used during epidemics to eradicate carrier state—why

• what is used for close contacts

• how are vaccines made (2) in relation to seriotypes

• what group meningococci are only modestly effective

• Antimicrobial prophylaxis 

  • Rifampin is used during epidemics to eradicate the carrier state (adult nasopharyngeal colonization)

  • Rifampin remains the drug of choice for prevention due to low cost, ease of admin, and well established record among infants/children

  • Rec is for chemoprophylaxis of household members and other close contacts of persons w/ invasive meningococcal disease

• Immunization

  • Immunity following meningococcal infection is serogroup specific

  • Internationally marketed meningococcal vaccines are based on combos of group specific capsular polysaccharides (A and C serotypes for eg) or conjugates between group C specific polysaccharide and a protein carrier

  • Polysaccharide vaccines are safe and highly immunogenic

  • Vaccines currently on market against group B meningococci have shown only modest efficacy in children and adults

• Commonly used media for isolates of pathogenic neisseria (2)

• what is a transport system specific for neisseria like in free clinics

• male vs female lab diag (where sample taken and outcome)

• Commonly used media for isolates of pathogenic neisseria is Taylor Martin (TM) agar→ Chocolate-based agar with some antimicrobics added as in VCN agar which is another similar concept that only allows pathogenic bacteria to grow 

• VCN agar→Chocolate agar modified by addition of antibiotics: Vancomycin to inhibit gram-positive bacteria, colistin to inhibit gram-negative bacteria rods, nystatin to inhibit yeast

• Transport system specific for a neisseria such as the jembec plate system complete with CO2 generating tablets and TM plates and bag for more affordable incubation

Lab diag in N. gonorrheae

• male=>urethral discharge can see gram neg diplococci and leukocytes

• female=> cervical smear with many epithelial cells, other bac similar to nisseria thus need more testing

Name the component of N. meningitidis responsible for the characteristic skin and tissue hemorrhaging in meningococcal meningitis. Discuss the importance of this virulence factor to the pathogenesis of Neisseria meningitidis.—>severe symptoms of n. meningitidis

Severe symptoms of N. meningitis= 

• petechial eruptions or pinpoint hemorrhages developing into larger Hemorrhage areas causing emboli of mangococci

  • emboli= a mass, such as a clump of bacteria or detached blood clot, foreign material like air bolus, that travels through the bloodstream and lodges and blood vessels, blocking it 

Severe may be accompanied by: 

1. Meningitis– inflammation of the meninges / membranes that envelop the brain and spinal cord 

2. Septecemia– bacterial infection of the blood

3. Arthritis– inflammation of the joints

4.  pericarditis– inflammation of membranes surrounding the heart 

5. Involvement of any organ system is possible

6.  may cause disseminated intravascular coagulation (DIC) followed by death as rapidly as one hour from onset of symptoms 

   1. Or waterhouse-friderichsen syndrome (adrenal gland involvement→turns red bc blood build up and no longer properly functional)

Name the most prevalent of the classical venereal diseases and identify what population has the highest incidence of infection.

Neisseria gonorrhoeae is causative agent of gonorrhea, most prevalent venereal disease; adolescents (⅓ of cases) occur in this group 

• Is world wide and most common classic vernal disease with 1 mil cases reported annually

• Disease is most common in indiv 20-24 yr 

Briefly explain the Waterhouse-Friderichsen Syndrome; include symptoms, cause, treatment, diagnosis.

• body part involved

• from what infection og

• leads to what organ failure

• how characterized

• what if not treated immediately

• progression of disease (~7)

• Adrenal gland involvement

•  severe meningococcal infection (N. meningitidis) 

• Fulminant disease state that occurs suddenly and of great intensity

•  adrenal gland failure due to bleeding  into adrenal gland

•  characterized by acute adrenal gland insufficiency, profound shock

•  fatal if not treated immediately

•  events of acute fatal meningococcal infection include: sudden onset with severe headache, rapid progression of disease, fever, coma, collapse, cyanosis or bluish pallor due to decreased oxygen intake, hemorrhaging of skin and mucous membranes 

“gonococcal ophthalmia neonatorum”

• due to what

• most common manifestation

• severe conjunvtivitis symp

• treatment (3 antibi and type of admin and percents of soln)

  • % of school of blind due to no treatment before

“Neonatal gonococcal arthritis”

• highly destructive form of what

• onset and acute or subacute

“gonorrheal vulvovaginitis”

• age group

• may cause what

• gonococcal ophthalmia neonatorum is infant gonorrhea and is due to infection acquired during passage through birth canal

  • Infection of the eye is most common manifestation (28% develops this if born to mom w/ infection)

  • Conjunctivitis is usually severe: N. gonorrhoeae can penetrate intact corneal epithelium and cause microbial keratitis, ulceration, perforation

  • Installation of 1% silver nitrate solution or antibiotic ointment (.5% erythromycin or 1% tetracycline) into conjunctival sac of newborn soon after birth is done to prevent blindness

    • Prior to routine use of Ag nitrate, 50% of children in schools for blind were bc of gonococcal ophthalmia neonatorum

• Neonatal gonococcal arthritis

  • Highly destructive form of arthritis

  • Acute w/ onset at 1-4 weeks of life (later mani)

• Gonorrheal vulvovaginitis 

  • Usually occurs in older children (girls 2-8 yrs) in cases suspected of sexual abuse

  • May cause sterility

Describe disseminated intravascular coagulation (DIC) in terms of its progression, organs involved and patient prognosis,

•  A severe symptom of meningitis

•  can result in death as rapidly as 1 hour from onset of symptoms

• Is always a secondary problem

•  is pathogenic activation of coagulation or blood clotting mechanisms

•  in other words, it is abnormal clotting (coagulopathy)  within blood vessels (intravascular)  that is widespread throughout the body (disseminated)

•  the causes are widespread formation of clots in the blood where abnormal bleeding occurs, clots disrupt blood flow within organs which leads to kidney failure, multiple organ failure, hypovolemia (not enough volume of blood to circulate), shock, and death

State a benefit the capsule provides to Neisseria meningitidis and to us as clinitians

• sero group that accounts for >90% mengio disease

• # of sero groups and on basis of what

 Serological groups→capsule used for ID: 

• 12 groups of N. meningitis have been ID on basis of immunological activity of capsular polysaccharides

• Serogroups A, B, C account for >90% of meningococcal disease 

Virulence factor: harder to phagocytize by neutrophils or macrophages

State the only known pathogenic determinant of Neisseria gonorrhoeae.

 The presence of pili on virulent forms seems to be the only pathologic determinant

Discuss the most significant infections caused by Moraxella catarrhalis and what population of people is most affected by this bacteria.

• cell morphology, gram stain, aerobic/anaerobic, oxidase

• 2 previous names

• pathogenic?

• most sig infections occur where and cause what 2 infections

• __most common cause of those 2 infections

• infections in what pop are more common

• Gram neg diplococcus w/ kidney bean appearance, aerobic, oxidase pos, (all just like genus neisseria)

  • Why previously known as neisseria catarrhalis and then changed to branhamella catarrhalis

• Previously considered to have no sig pathogenic conseq→recently associated w/ infection but not common

• Most sig infections are in upper respiratory tract infections, specifically:

  • Otitis media (ear) and sinusitis in children

  • Lower respiratory tract infections in adults 

• 3rd most common cause of otitis media and sinusitis in children following strep pneumoniae ad hemophilus influenzae 

• Infections in adults are more common if underlying conditions are present, esp in elderly 

why does neisseria need chocolate blood agar plates as opposed to BAP

need free Fe in hemoglobin to grow

what is responsible for the extensive vascular damage that is a component of N. meningitidis

endotoxin release 100-1000x greater than norm

most meningitis infections enter__ and stop—

but if proceeds incubates ands spreads via bloodstream where

the upper resp tract

stop in memb of the nasopharynx

skin, meninges, eyes