SEH Microbiology week 3

Name the five members of the Neisseriaceae family.

• Neisseria

• Kingella

• Eikenella

• SImonsiella

• Alysiella

What are the common characteristics of most Neisseria species

• Optimal growth in a humid atmosphere

• Gram negative diplococci, kidney bean shaped

• Cytochrome oxidase and catalase positive

List the two species of Neisseria that are considered the primary human pathogens.

• N. gonorrhea

• N. meningitidis

What are the common characteristics for N. gonorrhea and N. meningitidis

• Fastidious

• Require enriched media for optimal recovery

• Require iron for growth

  • They both compete with their human host by binding human transferrin to specific surface receptors

List the virulence factors and their function(s) for pathogenic Neisseria species

• Receptors for human transferrin—Allow it to compete for iron

• Capsule (N. meningitidis)—prevent phagocytosis

• Cell membrane proteins—Antigenic variation, causes production of useless antibodies, bind to pathogen and prevent binding of effective antibodies

• Major outer membrane porin proteins

  • Por A/B—Affective against inflammatoru response and complement killing

  • Protein II (Opa for opacity)—Facilitate adherence to phagocytic and epithelial cells

  • Protein III—Block host serum bactericidial action (IgG)

• Pili—Evade the immune systema nd aids in attachment, prevents phagocytosis

• Endotoxin

• IgA protease—Cleaves IgA on mucosal surfaces

What is gonorrhea and what parts of the body can be infected

• Meaning “flow of seed”, also called clap, reportable disease

• parts of body infected: genital tract, rectum, and throat, and it can also infect the eyes

What are the differences between men and women with gonorrhea infections

Men

• 90% of infected men show symptoms of acute infection

• Symptoms: Dysuria, urethral discharge

Women

• 50% are asymptomatic

• Symptoms include Lower abdominal pain, Cervical discharge, and Dysuria

How does gonorrhea infections affect extragenital infections, disease of the newborn, disseminated blood borne

Disseminated blood borne

• Fever, intermittent bacteremia, and rash on the extremities may be present

• Mostly from arginine, hypoxanthine, and uracil strains affecting women

• Gonococcal arthritis

Extragenital infections

• Oropharyngeal infections

  • Pharyngitis

Disease in newborns

• Ophthalmia neonaturum

• Infection is preventable via eye drops

What are appropriate specimens, swab(s), preferred transport media for gonorrhea testing

Specimens

• Genital sites (urethra in males, Endocervix in females)

  • Collect purulent discharge

• Rectal culture

  • Insert swab 4-5cm into anal canal

• Oral/pharyngeal

• Eye

• Blood/joint fluids

Swabs

• Dacron or rayon

• Calcium alginate and cotton swabs are inhibitory to N. gonorrhea

Transport media

• Gonococcal- selective media

• JEMBEC, Gono-Pak, Transgrow

• Plate within 6 hours if using Amies transport medium

Which N. gonorrhea specimen type can have a direct gram stain performed?

Urogenital specimen

What is the expected gram stain results from urethral discharge from a symptomatic male?

• Kidney bean-shaped gram negative intracellular diplococci, when present 89% likely to be gonococcal infection

• Avirulent form lacks pili and has extracellular gram negative diplococci

What would the gram stain result be to suggest nongonococcal urethritis

More than five PMNs per field with no bacteria

What is the preferred media for isolation of N. gonorrhoeae? Atmosphere?

• Thayer Martin, MTM, NYC, Martin-Lewis, GC-LECT

• Incubate inoculated culture media at 35°C in 3%-5% CO2

• CO2 incubator, generating pouch, or candle jar

List the colony characteristics for N. gonorrhoeae from MTM agar

• Small, gray to tan

• Translucent, raised

T1, T2 colony types

• Small and raised

• Appear bright in reflected light

T3-T5 colony types

• large and flat

What are the results for N. gonorrhea biochemical tests – oxidase, carbohydrate utilization

• Oxidase—purple/black color indicates positive

• Carbohydrate utilization—yellow color is positive result, (N. Gonorrhea is only positive for glucose)

List some advantages and disadvantages of nucleic acid assays for N. gonorrhoeae

Advantages

• Increased sensitivity and ability to test with noninvasive 1st morning urine specimens

Disadvantages

• Not approved for use as a test of cure

• Not approved for pharyngeal or rectal specimens

• Do not allow for recovery of an organism to be used for susceptibility testing

In fluorescent antibody testing, what do the fluorescent monoclonal antibodies attach to on the N. gonorrhoeae cell wall?

outer membrane proteins IA and IB (porins)

Describe the multi-test method.

• Conventional—chromogenic enzyme methods combine enzyme substrate tests with other biochemical tests

• Allows for identification of strains isolated on selective or nonselective media

• Can also identify other genera such as Haemophilus

Coagglutination immunologic assays will use dead S. aureus cells, how is the sensitivity and is there the possibility of false positives

• Generally high sensitivity

• False positive results may occur due to cross-reaction with other Neisseria spp.

What is the treatment of N. gonorrhea currently recommended from the CDC

• a single-dose intramuscular (IM) injection of ceftriaxone

• If chlamydial infection cannot be excluded, oral doxycycline should also be administered

What age population has the highest incidence of developing meningococcal disease

Infants and adolescents

What type of living conditions could contribute to the spread of meningococcus

• Crowded living conditions

  • Military recruits

  • College students

List the physical and clinical characteristics of meningitis

Physical characteristics

• Found only in humans

• May be commensal or an invasive pathogen

• Important etiologic agent of epidemic meningitis and meningococcemia

• Rarely associated with pneumonia, purulent arthritis, or endophthalmitis

• has been urogenital and rectal sites

Clinical characteristics

• Frontal headache, stiff neck

• Confusion, photophobia

• Nausea and vomiting

• Sometimes fever

• Purulent spinal fluid

Fulminant disease of meningococcus goes by what other name and what happens to the adrenal glands

• Waterhouse—Friderichsen syndrome

• Hemorrhage in the adrenal glands

What are the specimens of choice for meningococcus

• CSF

• Blood (non-SPS-containing blood collection system)

• Nasopharyngeal swabs or aspirates

• Joint fluids

• Less commonly sputum and urogenital sites

What anti-coagulant will inhibit N. meningitides

SPS

What would a direct gram stain from a CSF specimen of suspected meningitis

Presence of intracellular and extracellular gram-negative diplococci

Identification of meningitis is based on what three criteria

• Colony morphology, microscopic morphology, and the oxidase test

What are the colony characteristics found for meningitis?

• Colonies grow within 18-24 hours

• Medium-sized, gray, convex

• Encapsulated strains are mucoid

• Blood underneath colonies tend to have green tinge

Seletive agars

• Colorless to gray, convex, smooth

What are the biochemical results for meningitis: oxidase, carbohydrate utilization, DNase, and butyrate esterase

• Oxidase—positive

• Carbohydrate utilization—uses glucose and maltose (Mnemonic: MNM)

• DNase—Negative

• Butyrate esterase—Negative

What are the antibiotics used for treatment of meningtidis

• Ampicillin

• Penicillin G

• Chemoprophylaxis with ceftriaxone

Who should receive the vaccine for meningitis

• Individuals entering high school and college freshmen living in dorms

• Military recruits

• Asplenic patients older than 2 years of age

Moraxella catarrhalis can be found in two different clinical infections, what are they?

• Upper Respiratory Tract

• Lower Respiratory Tract

List the four predisposing factors for M. catarrhalis infection

• Advanced age

• Immunodeficiency

• Neutropenia

• Chronic debilitating diseases

What are other infections found with M. catarrhalis

• Acute otitis

• Sinusitis

• Endocarditis

• Meningitis

• Bacterial tracheitis

List the appropriate specimens for collection to isolate M. catarrhalis

• Middle ear effusion

• Nasopharynx

• Sinus aspirates, sputum, or bronchial aspirates

What are the colony characteristics for M. catarrhalis? What does the colony resemble

• Smooth, opaque, gray-to-white colonies

• Resembles hockey puck, older colonies resemble wagon wheel

Which antibiotics are resistant to M. catarrhalis

• Ampicillin

• Amoxicillin

Which antibiotics are used against M. catarrhalis

• Augmentin (Amoxicillin and Clavulanic acid)

• Extended-spectrum cephalosporins

• Azithromycin

• Quinolones

• Trimethoprim-sulfamethoxazole

Where is Neisseria lactamica found

Nasopharynx of infants and children

What other Neisseria species is N. lactamica colony characteristics similar to

N. meningitidis

Describe the gram stain appearance of Haemophilus

• Gram-negative pleomorphic coccobacilli or rods

• Coccobacilli in direct smears

• Rods with occasionally long filamentous rods from colony growth

What are the biochemical reactions of Haemophilus – Motility, oxidase, catalase, carbohydrate utilization?

• Nonmotile

• Oxidase positive

• Catalase positive

• Ferments carbohydrates

What are the three pathogenic species of Haemophilus

• H. influenza

• H. aegyptius

• H. ducreyi

What does the X and V factors stand for?

• X factor—Hemin, hematin (X for “unknown”)

• V (for Vitamin) factor is nicotinamide adenine dinucleotide (NAD)

What factors does H. influenzae need for growth?

Produces X factor, requires V factor

What factors does H. parainfluenza need for growth?

Require only V factor

Describe satellitism.

Growth of fastidious organisms around other bacteria that release the necessary growth factors or break down toxic products

List the virulence factors for H. influenzae.

• Capsule

• IgA proteases

• Adherence by fimbriae and other structures

• Outer membrane proteins and LPS

List the medical conditions caused by encapsulated and non-encapsulated strains of H. influenzae.

Encapsulated

• Septicemia, meningitis, arthritis, epiglottitis, tracheitis, pneumonia

Non-encapsulated

• Conjunctivitis, sinusitis, otitis media with effusion (middle ear infection)

What two medical conditions are caused by H. influenzae biogroup aegyptius

• Conjuctivitis

• Brazilian purpuric fever

What is H. ducreyi

a small, Gram-negative coccobacillus bacterium that primarily acts as an obligate human pathogen

What infections are associated with H. parainfluenza

• Otitis media

• Acute sinusitis

• Endocarditis

• Some cases of pharyngitis

Describe the media used and growth requirements for H. influenzae, H. aegyptius and H. ducreyi.

H. influenzae

• Chocolate agar with bacitracin (300mg/L)

H. aegyptius

• Chocolate agar supplemented with 1% IsoVitale X or Vitox

H. ducreyi

• GC agar, enriched chocolate, or Mueller Hinto agar with 5% chocolatized lysed horse blood

• Nairobi biplate

  • GC agar base+2% bovine hemoglobin+5% fetal calf serum on one half

  • Mueller Hinton agar with 5% chocolatized horse blood on other

  • Both sides contain 3mg/L vancomycin

What are the colony characteristics for H. influenzae

• Translucent, tannish, moist, smooth, convex

• Moist or bleach-like odor

• Encapsulated strains appear more mucoid

• No growth on MAC agar

• Beta-hemolytic on BAP

What do H. influenzae and H. ducreyi look like in a gram stain

H influenzae

• Small gram negative coccobacilli or small rods to long filaments; clear nonstaining areas (halos) may be seen

H. ducreyi

• Coccobacilli arranged in groups resembling “school of fish,” railroad tracks, or fingerprints

What guidelines are used for the identification of H. influenzae according to CLSI

• Gram negative bacilli or coccobacilli that exhibits colonies greater than 1mm on chocolate agar

• No growth or satellitism on SBA

• Negative porphyrin

What are the choice of antibiotics for H. influenzae and H. ducreyi

H. influenzae

• Cefotaxime or ceftriaxone

• Trimethoprim sulfamethoxazole, imipenem, ciprofloxacin, and chloramphenicol with ampicillin

H. ducreyi

• Azithromycin, ceftriaxone, ciprofloxacin, or erythromycin

Resistance for what antibiotic is confirmed by the beta lactamase test for Haemophilus?

Increased ampicillin resistance

List the organisms that make up the initials HACEK

H—haemophilus spp.

A—Aggregatibacter actinomycetemcomitans

C—Cardiobacterium hominis

E—Eikenella corrodens

K—Kingella spp.

What are the general characteristics for the HACEK group?

• gram negative bacilli

• Fastidious nutritional requirements

• Require an increased CO2 (5%-10%) environment

• Significant cause of endocarditis

• Can be normal biota of the oral cavity

• Opportunists and generally require a compromised host

Describe the colony characteristics for A. aphrophilus. Where is it commonly found

• Yellow, granular, convex colonies with opaque zone near centers on chocolate agar

• Key causative agent of endocarditis

Which organism is a major contributor to periodontitis

A. actinomycetemcomitans

What are the biochemical tests for A. actinomycetemcomitans – catalase, oxidase, X and V factors, urease, indole, esculin and citrate, growth on MacConky

• Catalase—positive

• Oxidase—negative/variable

• X & V factors—Negative

• Urease, indole, esculin, and Nitrate—Negative

• NO GROWTH ON MAC PLATES

What antibiotics are used for treatment of A. actinomycetemcomitans?

• Aminoglycosides, third generation cephalosporins, quinolones, chloramphenicol, tetracycline

What is the gram stain description of C. hominis

• Pleomorphic, nonmotile, fastidious, gram negative bacillus

• Sometimes appear false positive on Gram stained smears

• Show rosettes, swellings, or long filaments

What medical condition is C. hominis known for

Endocarditis

What are the biochemical test results for C. hominis – oxidase, catalase, indole, urease, nitrate, gelatin, esculin and carbohydrate utilization

• Oxidase—Positive

• Catalase—Negative

• Indole—Positive

• Urease, nitrate, gelatin, esculin—negative

• Carbohydrate utilization—fermenter, serum might be needed due to weak reaction

Which antibiotics are used for C. hominis

Penicillin and an aminoglycoside

Where is E. corrodens commonly found as normal flora

Oral and bowel cavities

• FIght and bite wound

• Opportunistic

What odor does E. corrodens have

Bleach-like odor

What kind of atmosphere condition and factor does E. corrodens require for growth

• Under increased CO2

• Factor X

What are the biochemical tests results for E. corrodens – motility, carbohydrate utilization, oxidase, catalase, lysine, ornithine, arginine

• Motility—nonmotile

• Carbohydrate utilization—Nonfermenter

• Oxidase—Positive

• Catalase—Negative

• Lysine, ornithine, arginine—Negative

List the general characteristics for Kingella species

• Important in pediatric population

• Causes osteoarthritis infection in children less than 4 years old

• Predilection for bones and joints

What criteria must be met before calling an organism Kingella kingae per CLSI?

• Gram-negative short coccoid bacilli forming large white to beige beta-hemolytic colonies on SBA

• No growth on MAC

• catalase positive

• Oxidase positive

Describe the colony characteristics for Kingella kingae.

• Spreading, corroding colony

• Smooth, convex, and beta-hemolytic colony

  • Hemolysis may appear beneath colony or in close proximity after 24 hours

  • Hemolysis more prevalent after 48 hours

What are the biochemical results for Kingella kingae – carbohydrate utilization

• Glucose and maltose—weak fermentation

• Sucrose—negative

Describe the two colony types for Kingella denitrificans

• Smooth, convex type

• Spreading corroding type

What organism may resemble Fusobacterium

Capnocytophaga

What are the biochemical reactions for Capnocytophaga – carbohydrate utilization, indole, bile esculin, TSI?

• Carbohydrate utilization—Ferments sucrose, glucose, maltose, and lactose

• Indole—Negative

• Bile esculin—Hydrolyzes

• TSI—May be negative without enrichment

What causes pasteurellosis?

Pasteurella

What are the most common clinical presentations for Pasteurella?

• ANIMAL BITES

• Systemic infections—septicemia, arthritis, endocarditis, osteomyelitis, meningtidis

• Pneumonic forms possible

• Soft tissue cutaneous infections

• Cutaneous infections

What are the biochemical reactions for Pasteurella – motility, catalase, oxidase, carbohydrate utilization?

• Motility—Nonmotile

• Catalase—positive

• Oxidase—positive

• Carbohydrate utilization—Ferment glucose with weak to moderate acid production without gas

How is Brucella spread/exposure?

• Aerosol, percutaneous, oral

• Sexual contact, breast-feeding—reported but rare

List the clinical stages for Brucella

• Acute—1-4 weeks after exposure

• Subchronic—within one year of exposure

• Chronic—Around 1 year after exposure

Which Brucella species are the most common cause of human brucellosis

Brucella melitensis

What are the colony characteristics of Brucella?

Under optimal conditions—smooth, raised, and translucent colonies

What are the biochemical test results for Brucella – oxidase, catalase, urease, hydrogen sulfide production, factors for growth?

• Oxidase— positive

• Catalase—positive

• Urease—positive within 2 hours

• Hydrogen sulfide production— Both positive and negative, depending on Brucella species

• Factors for growth— X & V factor testing maybe helpful in ID

What is the causative agent for tularemia?

F. tularensis

Who has an increased risk of having tularemia

individuals with specific occupations, recreational habits, or demographic profiles that bring them into contact with infected wildlife or ticks

What are the colony characteristics and gram stain for Francisella

Colony characteristics

• Strictly aerobic

• Slow grower

• Gray-white, raised smooth colonies

Gram stain

• Nonmotile, small gram-negative bacilli or coccoid bacteria

What tests are used to make presumptive ID for Francisella

DFA, immunohistologic staining with monoclonal antibody, PCR, slide agglutination, or single serology test

List the ways Legionella can be detected

• Isolation on special media

• MALDI-TOF mass spectometry

• Urine antigen detection

• DFA detection

• PCR multiplex

• Serology

What are the three febrile diseases caused by Legionella?

• Legionnaires’ disease

• Pontiac fever

• Asymptomatic infection

What kind of sources can have Legionella growing?

• Most aquatic sources—lakes, rivers, springs, man-made water treatment systems

• Specialized sources—fountains, heating and cooling towers of buildings

How is Legionnaires’ disease best diagnosed? Pontiac fever?

Legionnaires’ disease

• Using a combination of culture and urine antigen detection

Pontiac fever

• Lab findings, environmental studies, and epidemiology

Describe the gram stain morphology for Legionella

Pleomorphic, thin, weakly staining, gram negative bacilli, extend safranin stain to at least 10 minutes can enhance staining intensity of organisms

What are the colony characteristics for L. pneumophila

• Colonies appear grayish white or blue-green, convex and glistening on BYCE alpha agar

What antibiotic(s) are used for treating L. pneumophila

• Macrolide

• Ketolide

• Doxycycline

What are the general characteristics for Bordetella

• Small gram negative bacilli or coccobacilli

• Obligate aerobic bacteria that grow best at 35 to 37°C

• Do not ferment carbohydrates

• Oxidize amino acids

• Produce catalase

• Require special collection, transport systems, and media to grow