Unit 3 non framenters and API MICRO

What is the main carbohydrate source of carbon for fermentation and oxidation?

Glucose

What are the three biochemical pathways used to convert glucose to pyruvic acid?

EMP, ED, and HMP

What does EMP stand for?

Embden-Meyerhof-Parnas

Which biochemical pathway is anaerobic, used by facultative and obligate anaerobes, and produces strong acids?

EMP

In the EMP pathway, what serves as the final electron acceptor?

Organic compounds

Why is the EMP pathway easy for identification?

It produces strong acids

Which biochemical pathway is aerobic or oxidative, uses O2 as the final electron acceptor, and produces weak acids and water?

ED (Entner-Doudoroff)

What does ED stand for?

Entner-Doudoroff

What does HMP stand for?

Hexose Monophosphate Pathway (also Pentose Phosphate Pathway)

Which biochemical pathway is used by aerotolerant organisms that grow with or without oxygen, where growth aerobic doesn't mean oxygen is metabolized?

HMP

What are the four common clinical isolates of non-fermentative Gram-negative bacilli?

Pseudomonas aeruginosa, Acinetobacter spp., Burkholderia cepacia, Stenotrophomonas maltophilia

What is the habitat of non-fermentative Gram-negative bacilli?

Ubiquitous environmental organisms living in water and soil

Are non-fermentative Gram-negative bacilli ever normal flora?

Almost never

Where in hospital settings are non-fermentative Gram-negative bacilli isolated from?

Dialysate fluids, nebulizers, saline, catheters, and medical devices

What are the four risk factor categories for diseases caused by non-fermentative Gram-negative bacilli?

Immunosuppression, infused fluids, trauma, foreign body implantation

What are examples of immunosuppression risk factors for non-fermentative GNB infections?

Diabetes mellitus, cancer, steroids, transplantation

What are examples of infused fluids risk factors for non-fermentative GNB infections?

Dialysate, saline irrigations

What are examples of trauma risk factors for non-fermentative GNB infections?

Gunshot/knife/puncture wounds, surgery, burns

What are examples of foreign body implantation risk factors for non-fermentative GNB infections?

Catheters, prosthetic devices, corneal implants/contact lenses

What are the general characteristics of non-fermentative Gram-negative bacilli?

Thin GNR, non-spore forming, oxidase positive (some weak/variable), antimicrobial resistance to multiple classes, aerobic growth, never ferment CHO's, oxidative use of glucose

What is the OF reaction for non-fermentative GNB?

Reaction only in the open OF media tube

What is the TSI reaction for non-fermentative GNB?

K/K or K/nc

What are the clues that an isolate is metabolically non-fermentative?

Glucose non-fermenter on TSI, K/K or K/nc on API 20E, non-reactivity in 24 hours in commercial systems, fastidious organisms some NG on MAC, growth aerobic

What does the oxidase test determine?

The presence of the cytochrome oxidase system that oxidizes reduced cytochrome with oxygen

What is the reagent used in the oxidase test?

Tetramethylparaphenylene diamine dihydrochloride

How quickly must the oxidase test be read?

Within 10-30 seconds

What materials can cause a false positive oxidase test?

Stainless steel and nichrome

What should be used to pick colonies for the oxidase test?

Wooden applicator stick or platinum

Why does the oxidase test discolor media agar?

It kills the colony

What does the glucose utilization test determine?

If an organism uses carbohydrate oxidatively, fermentatively, or not at all

What is the name of the media used for glucose utilization testing?

Hugh and Liefson O/F media

What is the peptone concentration in O/F media?

0.2%

What is the carbohydrate concentration in O/F media?

1%

What is the agar concentration in O/F media?

0.3% (semisolid)

Why is the ratio of peptone to carbohydrate decreased in O/F media compared to TSI?

To detect small amounts of weak acids

Why is the peptone concentration lower in O/F media than in TSI?

Peptone utilization produces alkaline amines which can neutralize or mask weak acids

What is the indicator used in O/F media?

Bromthymol blue

What is the color of uninoculated O/F medium?

Green

What color is O/F medium in the presence of acid?

Yellow

What color is O/F medium in an alkaline environment?

Green to blue

How many tubes are inoculated for the O/F test?

2

How is the anaerobic tube prepared for the O/F test?

Overlaid with 1 cm of oil

At what temperature and time are O/F tubes incubated?

35°C for one to five days

What is the interpretation of O/F if both open and closed tubes are yellow?

Fermentative

What is the interpretation of O/F if the open tube is yellow and the closed tube is green?

Oxidative

What is the interpretation of O/F if both open and closed tubes are green?

Inert (asaccharolytic)

Where might aerobic organisms react in the O/F tube?

Near the top of the tube, not in the deep bottom

What sugars can be used in O/F medium?

Glucose (dextrose), lactose, maltose

What are the three methods for testing motility?

Stain (flagellar), tube media, wet mount/hanging drop

What are the four flagellar arrangements?

Monotrichous, lophotrichous, amphitrichous, peritrichous

Why is the top 4mm of motility tube media examined?

Because that is where motile organisms will spread

What is the age range of broth suspension used for a wet mount?

6-24 hours

What are the water-insoluble pigments produced by non-fermenters?

Carotenoids, violacein, phenazine

What color are carotenoids?

Yellow/orange

What color is violacein?

Violet/purple

What color is phenazine?

Red/maroon/yellow

What are the water-soluble pigments produced by non-fermenters?

Fluorescein (pyoverdin), pyocyanin, pyorubin, pyomelanin

What color is fluorescein (pyoverdin)?

Yellow/green or yellow/brown

What color is pyocyanin?

Blue

What color is pyorubin?

Red

What color is pyomelanin?

Brown/black

What is the characteristic green color of Pseudomonas aeruginosa due to?

Pyoverdin + Pyocyanin

What is the only non-fermentative GNB that produces pyocyanin?

Pseudomonas aeruginosa

What percentage of Pseudomonas aeruginosa do not produce pyocyanin?

4%

What must be present in media for pigment production to be demonstrated?

Divalent cations, Ca2+ and Mg2+

What media is used for pigment production?

Mueller Hinton agar and P agar

How is fluorescence visualized?

With UV/Woods lamp

Most Enterobacterales reduce nitrates to what?

Nitrites

Non-fermenters can take nitrate reduction a step further to produce what?

Nitrogen gas

What does a Durham tube collect in the nitrate reduction test?

Nitrogen gas

What are the two nitrate reagents?

Sulfanilic acid and (dimethyl) alpha naphthylamine

What does a red color after adding nitrate reagents indicate?

Nitrites produced due to nitrates being reduced

If there is no color after adding nitrate reagents, what are the two possibilities?

Nitrogen gas produced OR nitrates were not reduced at all

How is the nitrate reduction result clarified?

By adding zinc dust

What does zinc dust do in the nitrate reduction test?

Zinc ions reduce residual nitrates to nitrites producing a red color within 5-10 minutes

What is the interpretation if there is no color after reagents A and B, and no color after zinc dust?

Positive for nitrogen gas (N2)

What is the interpretation if there is no color after reagents A and B, but red color after zinc dust?

True negative (nitrates present still)

What is the interpretation if red color develops after reagents A and B?

Positive nitrite (NO2)

What does the urease test detect?

Enzyme that hydrolyzes urea to form alkaline byproducts (ammonia, H2O, CO2)

What is the indicator in the urea test?

Phenolphthalein or phenol red

What color is a positive urea test?

Pink/magenta

What does the indole test detect?

Degradation of tryptophane into indole, pyruvic acid, and ammonia

What is the reagent for the indole test?

p-dimethylaminobenzaldehyde (Kovacs or Ehrlich's reagent)

What color is a positive indole test?

Red

What amino acids are tested for amino acid decarboxylases?

Lysine, ornithine, and arginine

What is in the control broth for the amino acid decarboxylase test?

Glucose, no amino acid

What color is the control broth for a valid amino acid decarboxylase test?

Yellow

What is a positive result for amino acid decarboxylase test?

Test is purple, control is yellow

What is the indicator in the amino acid decarboxylase test?

Bromcresol purple

What is the TSI reaction for a non-fermenter?

K/K or K/NC

Why does a non-fermenter produce K/K or K/NC on TSI?

It cannot ferment lactose or glucose, and degrades peptones producing alkaline byproducts

What are the four categories of commercial identification systems for non-fermenters?

Commercial kits, traditional tube biochemicals, automated systems, nucleic acid sequencing or mass spec

What is MALDI-TOF?

Analysis of peptide spectra when separated in mass spectrophotometer

What are the factors contributing to difficulties in identification of non-fermenters?

Infrequently encountered, not familiar with non-fermenters, conventional media not suitable, grow slowly/weak reactions, experience required interpreting, specialized media outdates, low accuracy of packaged ID systems

When is a non-fermenter sent to a reference laboratory?

When it is "non-fermenter, not P. aeruginosa, Acinetobacter spp., Burkholderia spp., or S. maltophilia"

What are the categories of medically important non-fermenters based on motility and saccharolytic ability?

Motile strongly saccharolytic, motile weak or nonsaccharolytic, non-motile pigmented, non-motile oxidase negative, non-motile oxidase positive

What is the therapy for severe non-fermenter infections?

Combination therapy

What anti-pseudomonal penicillins are used for non-fermenter infections?

Carbenicillin, ticarcillin, piperacillin

What aminoglycosides are used for non-fermenter infections?

Amikacin, tobramycin, gentamycin

What are non-fermenters innately resistant to?

First and second generation pens and cephems, trimethoprim, and chloramphenicol