Home

Courses

Research

Biology Dept

CSU, Stanislaus

Letters of Recommendation Request Form

Diagnostic: Gram negative pathogens

Reference: http://education.med.nyu.edu/courses/old/microbiology/courseware/infect-disease/

Laboratory Diagnosis

p Biochemical tests and serological tests must be done in parallel

p Commercial kits commonly used

Gram Negative Aerobic Cocci

p Neisseria, Moraxella and Acinetobacter.

p two important human pathogens in the genus Neisseria

Neisseria gonorrhoeae

p fragile organism

n cannot survive in environment

n no animal reservoir

p Gram-negative diplococcus

n kidney bean shaped

n often intracellular in clinical samples

p Causes gonorrhea

p Other diseases:

n urethritis, cervicitis, salpingitis, proctitis, pharyngitis, conjuntivitis, pharyngitis, PID - pelvic inflammatory disease, arthritis, disseminated disease

Neisseria meningitidis

p Meningitis (epidemic CNS fever)

n septicemia, pneumonia, arthritis, urethritis

n acute inflammatory changes due to immune responses

n Septic manifestations include

p disseminated intravasuclar coagulation

p damage leads to shock.

n Laboratory Diagnosis:

p culture from blood, CSF, or transtracheal aspirates isolation.

p Previously considered harmless,

n Moraxella catarrhalis

p recently shown to cause human infections

p bronchitis and bronchopneumonia

p Most Neisseria, Moraxella and Acinetobacter

n can grow on simple media at varying temperatures.

p N. meningitidis and N. gonorrhoeae

n sensitive to fatty acids and trace metals in simple media

n requirements for growth

p enriched medium containing blood or blood products.

p carbon dioxide atmosphere

p incubation at 37 °C

Properties useful in distinguishing among the Gram negative cocci

p Oxidase test

n detects presence of cytochrome oxidase using a dye.

p dye is colorless in reduced state

p purple in the oxidized state.

p Neisseria and Moraxella

n Give positive oxidase test

n Commonly used for presumptive identification of species.

2. Ability to grow on NYC media –

-an enriched media containing antibiotics ( vancomycin, colistin, nystatin, and trimethoprim)

-permits growth of N. meningitidis and N. gonorrhoeae but not nonpathogenic Neisseria, Moraxella catarrhalis and other gram negative organisms

p Neisseria meningitidis on NYC agar

p Neisseria gonorrhoeae on NYC agar

p carbohydrate degradation –

p oxidative breakdown of sugars glucose, maltose and sucrose

p can help distinquish among N. meningitidis, N. gonorrhoeae, and M. catarrhalis.

Uninoculated sugar media

p Neisseria meningitidis sugar utilization

n + glucose, maltose

n - sucrose

p Neisseria gonorrhoeae sugar utilization

n + glucose

n - maltose, sucrose

p Moraxella catarrhalis sugar utilization

n - for all 3 sugars

Gram Negative Bacilli

p organisms in this group

n Enterobacteriaceae

p family containing many important pathogenic genera

n Vibrio, Campylobacter and Helicobacter

n Opportunistic organisms

n Pseudomonas and others

n Haemophilus and Bordetella

Examples of organisms and diseases they cause

p Escherichia coli

n diarrhea, urinary tract infection, meningitis in newborn, and range of nosocomial infections

p Shigella species

n invasive diarrheal disease - dysentery

p Salmonella typhi

n typhoid fever - enteric fever

p Yersinia pestis

n Plague

p Vibrio cholerae

n cholera

p Campylobacter jejuni

n enterocolitis

GROWTH on MacConkey agar

p Selects against some bacteria:

n Haemophilus, Bordetella, Campylobacter, and Helicobacter do not grow

n Enterobacteriaceae, Pseudomonas, and Vibrio grow readily

p differentiates among most Gram negative bacilli

n ability to utilize lactose

Enteric Gram-negative rods

p Lactose-fermenters

n Escherichia coli

n Klebsiella pneumoniae

p Non-lactose fermenters

n Salmonella enterica

n Shigella spp.

n Proteus mirabilis

n Pseudomonas aeruginosa

p Enterobacteriaceae (enterics)

n facultative Gram-negative rod-shaped bacteria

n inhabit intestinal tracts of humans and many animals.

p coliforms

§ ferment lactose (lactose positive)

§ nonnpathogenic in the intestinal tract

§ Escherichia coli, Enterobacter aerogenes.

§ Note: E. coli O157:H7 - implicated in causing bloody diarrhea.)

p enteric pathogens Salmonella and Shigella

§ unable to ferment lactose (lactose negative).

Organisms which DO NOT GROW on MacConkey agar

Fastidious Gram-negative rods

p Often pleomorphic coccobacilli

p Require enriched media (i.e. chocolate agar) for growth

p Do not grow on MacConkey agar

p Growth enhanced by incubation with carbon dioxide

p Examples:

n Haemophilus influenzae

n Bordetella pertussis

Haemophilus species

p Haemophilus influenza DISEASES

n Pneumonia, meningitis, epiglottitis, septic arthritis, cellulitis, otitis media, conjunctivitis, neonatal infection, and other clinical syndromes

n Source: Human respiratory and genital tract by direct contact or inhalation.

n Pathogenesis: Infection results in a primary bacteremia followed by acute pyogenic infection.

n Laboratory Diagnosis: cultures from blood, CSF, joint fluid, and biopsy material.

p Infections by other Haemophilus species

n Diseases: Conjunctivitis, genital ulcer, endocarditis, brain abscess, pneumonia, and other syndromes.

n Etiologic Agents: H. parainfluenza and other species.

n Source: Human respiratory and genital tract by direct contact or inhalation.

n Pathogenesis: Acute pyogenic infections

n Laboratory Diagnosis: Isolation of the organism from blood, sputum, tissue or mucosal swab.

Chocolate agar

p nutrient medium for culturing fastidious organisms

n Species of Haemophilus and Neisseria.

p comprised of sheep blood that provides

n factors X (hemin)

n V (nicotinamide adenine dinucleotide [NAD])

n special growth factor requirements of Haemophilus influenzae, Neisseria meningitidis and Neisseria gonorrhoeae.

p primary plating medium for any specimen which may contain fastidious organisms

n spinal fluids, eye cultures, gonococcal cultures.

p does not reveal hemolysis data

n differentiation among species of Haemophilus uses other tests.

Haemophilus influenzae

p growth on chocolate agar

p moist, smooth colonies.

Neisseria gonorrhoeae

p growth on chocolate agar

p waxy, tan colonies.

Properties Useful in Distinguishing Haemophilus Species

p growth factor requirements

n species differ in their requirement for X factor and/or V factor for growth

p H. influenza requires both X and V factors.

p Haemophilus parainfluenzae only requires X factor.

Bordetella pertussis

p encapsulated, strict aerobes.

p sensitive to environmental conditions.

p causes whooping cough

p diagnosis by nasal swab

p Growth is

n very slow (4 - 7 days)

n requires presence of a high concentration of blood (20 - 30%).

n colonies like mercury droplets on selective agar

p Special medium for isolation:

n Bordet-Gengou medium

p selective medium used for isolation and growth of Bordetella pertussis from clinical specimens

§ Diagnosis of whooping cough

p Contains

§ blood (provides nutrients)

§ starch (absorbs and neutralizes inhibitors of growth)

§ Penicillin to inhibit other respiratory flora

p Specific identification employs immunologic techniques

n agglutination or fluorescent antibody.

Campylobacter jejuni

p does not grow on MacConkey agar

n require special media and high carbon dioxide concentrations for growth

p acquire by eating undercooked chicken or drinking contaminated milk and water.

n Infection à gastrointestinitis (fever, cramps, and bloody diarrhea).

p curved rods with characteristic "sea gull" and wavy morphology

p Microaerophilic

n need high concentration CO2, low concentration O2

p Thermophilic

n optimal growth 42°C

p Fastidious

n blood or serum normally required

p needs antibiotic-containing selective medium

p grows in colonies resembling water droplets

p Isolated using Campy blood agar

Campy-BAP

p blood agar

p contains several antibiotics

n inhibit growth of Gram-positive and most Gram-negative bacteria

p Procedure:

n plate is inoculated

n placed in a microaerophilic environment

p sealed bag containing high carbon dioxide concentrations and low oxygen concentrations.

Organisms which GROW on MacConkey agar

Properties Useful in Distinguishing among the Enterobacteriaceae and the Enterobacteriaceae from Pseudomonas

p Properties observed on simple media:

n Enterobacteriaceae and Pseudomonas

p Both grow on simple media and on a selective/differential media

p MacConkey agar

§ differentiates among bacterial species based on their ability to utilize lactose

Other Properties

p PIGMENT PRODUCTION

n Some bacilli produce characteristic pigments which aid in their identification

p Pseudomonas aeruginosa

p Serratia marcescens

p ENCAPSULATION

p SWARMING

Pseudomonas aeruginosa

p Major cause of nosocomial infections.

n Targets immunocompromised individuals, burn victims, and individuals on respirators or with indwelling catheters.

p Colonize lungs of cystic fibrosis patients.

p Most frequently isolated non-fermenter

p Distinguish from other species of Pseudomonas:

n Can grow at 42 °C

n Produces a greenish pigment (pyocyanin) and a fluorescent pigment

n Has characteristic fruity odor

p Distinguish from Enterobacteriaceae

n None of the Enterobacteriaceae produces pigment at 37°C

p produces a green to blue, water-soluble pigment called pyocyanin.

p also produces a product called fluorescein

n fluoresce under short wavelength (254nm) ultraviolet light.

Serratia marcescens

p characteristic red colonies due to production of a water insoluble red pigment, prodigiosin

Encapsulation

p a number of enteric bacteria produce capsules

n resulting colonies may appear mucoid

Swarming

-many bacilli of the gastrointestinal tract are motile but the programmed wave-like movement of the bacteria is observed only with Proteus species

Properties observed on selective and differential agars

p MacConkey agar

n inhibitory for gram positive organisms

n contains lactose as a carbon source

p ability of various enteric bacteria to use lactose serves as a means of distinguishing among them.

n Lactose utilization

p detected by indicator dye

§ turns red below pH 7

§ colorless above pH 7)

Double Sugar Iron Agar Reactions

p Differential agar slants that contain

n more than one sugar (lactose and glucose in a 10:1 ratio)

n ferric salt

n Indicator

p phenol red ( yellow below pH7, red above).

p In these slants both respiration (aerobic) and fermentation (anerobic) growth can take place.

p very useful media for distinguishing among these organisms.

Double Sugar Iron Agar slant

p inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant