(F) Lesson 13: Non-fermenters and Miscellaneous Gram-Negative Bacilli (Part 1) Flashcards
The heart and soul of microbiology
Identification
If a patient is infected with an organism, one thing we can do is perform?
Culture and Sensitivity
- It is not a routine procedure inside the clinical premises.
- It is rarely done.
- It is only done when the disease is deadly or the infection is already severe.
- There is something wrong with the clinical intervention.
Culture and Sensitivity
In the Philippines instead of culture and sensitivity, we do what?
Empiric Therapy
- We look at the clinical presentation, not on the causative agent of the infection.
- A broad-spectrum antibiotic is prescribed
- We do trial-and-error
Empiric Therapy
Determines the best antibiotic that can be prescribed to the patient
AST
What do we do when we receive unknown samples?
- Gram-staining
- Culture with BAP, CAP, and MAC
- Biochemical Testing
- Comes 2nd in a clinical setting, but essential to do
- Do not report normal flora
Gram Stain
- The special media will be used for follow-up
- In these plates, we will observe the hemolytic pattern.
Culture using BAP, MAC, and CAP
Medium
- Will separate G(-) from G(+)
- Selective for G(-) due to an inhibitor
- Determines whether an organism is a lactose fermenter or not
- Non-fermenters have high diagnostic significance
MAC
Medium
- For fastidious organisms and gonococcal species
CAP
- After G/S or culture, subject it to ____ depending on the growth.
Biochemical testing
- Through ____, you can narrow down the possible causative agents of the infection.
Biochemical test results
- One of the biggest points of differentiation in G(-) organisms
TSI or carbohydrate fermentation test
- Causes difficulty in ID
- We use the wrong manner of testing
Non-reactive organisms or biochemically inert organism
They do not ferment any carbohydrates, rather they oxidize carbohydrates.
Non-fermenters
General Characteristics
- Grow much better in an ____ environment
Aerobic
General Characteristics
T or F: Non-fastidious organisms grow on regular media, even w/o supplementation.
T
General Characteristics
- Most non-fermenters are oxidase (positive/negative).
Positive
General Characteristics
This differentiates Enterobacteriaceae from non-fermenters since most Enterobacteriaceae are (-) oxidase.
Oxidase Test
The only (+) oxidase Enterobacteriaceae
Plesiomonas spp.
T or F: Non-fermenters are non-ubiquitous and are rarely found in most environments.
F (ubiquitous and found in most environments)
General Characteristics
Can withstand treatment with ____ and ____
Chlorhexidine (antiseptic solution) and quaternary ammonium compounds (QACs)
Risk Factors for Clinical Infections
- Diabetes mellitus
- Cancer
- Steroids
- Transplantation
Immunosuppression/Immunocompromisation
Risk Factors for Clinical Infections
- Gunshots, knife wounds, punctures
- Surgery
- Burns
Trauma
Risk Factors for Clinical Infections
- Catheters. urinary or bloodstream
- Prosthetic devices—joints, valves
- Corneal implants or contact lenses
Foreign Body Implantation
Risk Factors for Clinical Infections
- Dialysate
- Saline irrigations
Infused Fluids
Biochemical Characteristics and Identification
- Gram stain of non-fermenters?
Thin, gram-negative pleomorphic
Biochemical Characteristics and Identification
- Non-fermenters are usually oxidase (positive/negative).
Paulit-ulit tayo ha.
Positive
Biochemical Characteristics and Identification
- No acid production in the slant or butt of ____ or ____
TSIA or KIA
Biochemical Characteristic and Identification
- (Nonreactivity/Reactivity) in 24 hours in commercial multi-test kit systems used primarily for the identification of Enterobacteriaceae
Nonreactivity
Biochemical Characteristics and Identification
- (Resistance/Susceptibility) to a variety of classes of antimicrobial agents, such as aminoglycosides, third-generation cephalosporins, penicillins, and fluoroquinolone
Resistance
Identify the pigment produced by these organisms.
- Chryseobacterium and Elizabethkingia spp. (weak fermenters)
- Sphingomonas paucimobilis
- Pseudomonas (Chryseomonas) luteola
- Pseudomonas oryzihabitans
- Sphingobacterium spp.
- Pseudomonas stutzeri
Yellow
Identify the pigment produced by these organisms.
- Methylobacterium spp.
- Roseomonas spp.
Pink
Identify the pigment produced by these organisms.
- Acinetobacter spp.
Purple
Identify the pigment produced by these organisms.
- Pseudomonas aeruginosa
Blue-Green
Identify the pigment produced by these organisms.
- Chromobacterium violaceum
Violet
Identify the pigment produced by these organisms.
- Stenotrophomonas maltophilia
Lavender to lavender-green (blood agar)
Identify the pigment produced by these organisms.
- Pseudomonas stutzeri
- Shewanella putrefaciens
Tan
These species produce wrinkled colonies?
- Pseudomonas stutzeri
- Pseudomonas oryzihabitans
- Burkholderia pseudomallei
Identify the odor of the following organisms.
- Alcaligenes faecalis
- Myroides odoratus
- Pseudomonas aeruginosa (grapes)
Sweet
Identify the odor of the following organisms.
- E0-4
- Neisseria zoodegmatis
Popcorn
- Most nonfermenters are (motile/non-motile) even on plated media
Motile
- Serrated or spreading colonies can be suspected as?
Pseudomonas aeruginosa
- ____ colonies indicate that the organism is motile
Spreading
These species are non-motile.
- Acinetobacter spp.
- Moraxella spp.
- Chryseobacterium spp. and Elizabethkingia spp.
- Sphingobacterium spp.
- Oligella spp.
Most non-fermenters are oxidase-positive except?
- Acinetobacter spp.
- Stenotrophomonas maltophilia
- Pseudomonas luteola and P. oryzihabitans
- Burkholderia cepacia
This species produces blackening in the media when cultured.
Shewanella putrefaciens
- Tests whether organisms are glucose oxidizers or fermenters
- Medium: Hugh & Leifson
- Uses two tubes, 1 is topped with mineral oil to mimic an anaerobic environment since it tests the oxidation and fermentation capability of an organism when it comes to utilizing carbohydrates
- (+) yellow = acid
- (-) green = no acid
Oxidase and Fermentation Test (OF Test)
- High carbohydrate, low peptone
- Contents: 1% glucose, 1% agar, peptone
Hugh and Leifson
Hugh and Leifson Medium
Solidifies the medium, differentiating semi-solid from broth media
Agar
Hugh and Leifson Medium
Buffer for enteric bacteria, which can directly kill them
Peptone
- Indicator of Hugh and Leifson
- The original color of the medium is green since it is metachromatic
- It detects changes in pH
Bromothymol blue
OF Test
Fermentation requires what?
Mineral oil
OF Test
Changes in (anaerobic/aerobic) tube = (+) oxidation
Aerobic
OF Test
Changes in anaerobic tube = (+) (fermentation/oxidation)
Fermentation
Match the results with the organism.
- Escherichia coli
- Pseudomonas
- Alacaligenes
A. +/-
B. -/-
C. +/+
- C
- A
- B
Organisms that neither oxidize nor ferment.
Asaccharolytic organisms
- Uses two tubes subjected to different temperatures
- Temperature: 35°C and 42°C
Growth at 42°C
Identify the organism.
- Both tubes contain growth (at 35°C and 42°C).
Pseudomonas aeruginosa
Identify the organism.
- There is growth only in the 35°C tube.
Pseudomonas fluorescens
- ID of Pseudomonas from Enterobacteriaceae
- Not necessary since you can perform oxidase test
- Uses the Cetrimide agar which is a selective agar
Cetrimide Test
Cetrimide Test
If cetrimide media is unavailable, perform ____ as an alternative
Oxidase test
Cetrimide Test
Positive with growth?
Pseudomonas aeruginosa
Cetrimide Test
Negative with no growth?
Escherichia coli
Familiarize yourself with the prelimenaries of Non-fermenters.
- Gram Staining: Gram-negative coccobacilli
- TSI: K/K
- MAC: Positive/Growth on MAC
- Oxidase Test: Most are oxidase-positive
- O/F Test: Non-oxidizer can either mean the organism is a fermenter or asaccharolytic
- Genus: Pseudomonas
- Accounts for the largest percentage of all nonfermenters isolated from clinical specimens
Pseudomonas (Pseudomonads)
Fluorescent or non-fluorescent?
- P. aeruginosa
- P. fluorescens
- P. putida
- P. veronii
- P. mosselii
- P. monteilii
Fluorescent
Fluorescent or non-fluorescent?
- P. stutzeri
- P. mendocina
- P. alcaligenes
- P. pseudoalcaligenes
- P. luteola
- P. oryzihabitans
Non-fluorescent
- G(-) bacillus or coccobacillus
- Strictly aerobic metabolism
- Motile usually with polar or polar tufts of flagella
- (+) Oxidase (P. luteola, P. oryzihabitans)
- (+) Catalase
- (+) growth on MAC
- Usually an oxidizer of carbohydrates, but some species are asaccharolytic
Pseudomonads
Characteristics common to Pseudomonads
- Has a main character syndrome
- Most commonly isolated species (moist environments, pools, hot tubs, catheters, humidifiers)
- The leading cause of nosocomial (hospital-acquired) respiratory tract infections
- When isolated from a sterile body site, it constitutes a true infection
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Since many antibiotics are found in hospitals, their continuous exposure to those results in the development of ____ among organisms.
Resistance
What are the sterile parts of the body?
- Blood
- Pleural fluid
- Joint fluids or tissues
- Cerebrospinal fluid (CSF)
Clinical Infections of Pseudomonas aeruginosa
- Presented with ecthyma gangrenosum of skin
- Different from sepsis (sepsis when it multiplies)
- (+) bacteria in the blood
Bacteremia
Clinical Infections of Pseudomonas aeruginosa
What are the top 3 bacteremia-causing bacteria?
- Klebsiella pneumoniae
- Escherichia coli
- Pseudomonas aeruginosa
Clinical Infections of Pseudomonas aeruginosa
- Either you die of infection or airway obstruction
Pulmonary Disease
Especially among individuals with CF
Clinical Infections of Pseudomonas aeruginosa
- An inherited disorder, defect in mucus-producing gene resulting in very thick production of mucus that may attract organisms.
Cystic fibrosis
Clinical Infections of Pseudomonas aeruginosa
- Ear infection among swimmers or divers
Otitis externa
Clinical Infections of Pseudomonas aeruginosa
- Also called Jacuzzi or hot tub syndrome ( they thrive well even in higher temperatures (42 deg C))
Necrotizing Skin Rash
Clinical Manifestations of Pseudomonas aeruginosa
Familiarize yourself with the rest of the clinical manifestations of Pseudomonas aeruginosa.
- Wound infections
- Nosocomial urinary tract infections (UTIs)
- Endocarditis
- Infections following burns or trauma
- Meningitis
- Infections of the nail beds in people with artificial nails
- Keratitis
- Endophthalmitis
Clinical Manifestations of Pseudomonas aeruginosa
- One of the risk factors for infection
Immunocompromisation
Clinical Manifestations of Pseudomonas aeruginosa
T or F: The majority of nonfermenters produce pulmonary infections.
T
Virulence Factors of Pseudomonas aeruginosa
- A substance by P. aeruginosa to produce biofilm as armor
- Causes mucoid colonies in CF
Alginate
Virulence Factors of Pseudomonas aeruginosa
- Can be made from other substances aside from alginate
- This makes organisms resistant to antibiotics, even WBCs since they can’t be identified
- Destruction of this can expose the organism for it to be more susceptible to phagocytosis
Biofilm
Virulence Factors of Pseudomonas aeruginosa
- Like the Corynebacterium, it can also produce exo- and endotoxins
- Most important exotoxin
- Blocks protein synthesis
Exotoxin A
Pigments of Pseudomonas fluorescent group
- Yellow-green or yellow-brown pigment
- Water-soluble and fluoresces under short-wavelength UV light
Pyoverdin
Pigments of Pseudomonas fluorescent group
Blue
Pyocyanin
Pigments of Pseudomonas fluorescent group
Red
Pyorubin
Pigments of Pseudomonas fluorescent group
Brown
Pyomelanin
Pseudomonas aeruginosa
Growth on Sheep Blood Agar (SBA)
Beta-hemolytic, flat spreading colonies with a metallic sheen
Pseudomonas aeruginosa
The fruity, grape-like ordor is due to?
2-aminoceptophenone
Pseudomonas aeruginosa
It is (positive/negative) for:
- Denitrification of nitrate and nitrite
- Arginine dihydrolase (ADH)
- Growth at 42° C
- Citrate
- Acetamide utilization
Positive
Pseudomonas aeruginosa
Growth on ____ which is selective and differential for P. aeruginosa ID
Cetrimide agar
Pseudomonas aeruginosa
Detergent that inhibits bacteria
Cetrimide
Pseudomonas aeruginosa
- Medium enhances pigments produced by P. aeruginosa
Cetrimide agar
Pseuodomonas aeruginosa
Resistant or Susceptible?
- Penicillin
- Ampicillin
- First- and second-generation cephalosporins
- Trimethoprim-sulfamethoxazole
- Chloramphenicol
- Tetracycline
Resistant
Pseudomonas aeruginosa
- Aminoglycosides
- Semisynthetic penicillins (piperacillin and ticarcillin)
- Third- and fourth-generation cephalosporins (ceftazidime, cefepime)
- Carbapenem (except ertapenem)
- Fluoroquinolones
Susceptible
- Can develop resistance while undergoing treatment
- It would require ____
AST
Pseudomonas aeruginosa
____ for severe infections:
- Ceftazidime/cefepime/piperacillin/carbapenem (imipenem or meropenem) + Aminoglycoside (tobramycin or amikacin)
Combination therapy
Pseudomonas aeruginosa
- Can be fatal in critically ill patients
Multidrug resistant P. aeruginosa
- Very low virulence, rarely causing clinical disease (has very low infectivity rate)
- From respiratory specimens, contaminated blood products, urine, cosmetics, hospital equipment, and fluids
- Both are associated with transfusion-associated septicemia
- To differentiate against P. aeruginosa: (+) pyocyanin and growth at 42 deg C
- (+) Pyoverdin, xylose
- (-) Pyocyanin, no growth at 42°C, nitrate to nitrite
Pseudomonas fluorescens and Pseudomonas putida
Used to differentiate Pseudomonas fluorescens and Pseudomonas putida
Gelatin hydrolysis
Positive for gelatin hydrolysis?
Pseudomonas fluorescens
Negative for gelatin hydrolysis?
Pseudomonas putida
Pseudomonas fluorescens and Pseudomonas putida
Resistant or Susceptible?
- Carbenicillin
- SXT
Resistant
Pseudomonas fluorescens and Pseudomonas putida
Resistant or Susceptible?
- Aminoglycosides
- Polymyxin
- Piperacillin
Susceptible
- Associated with catheter-related sepsis in patients with cancer
Pseudomonas putida
- Pseudobacteremia
- Mistakenly identified as P. fluorescens: P. mosselii (2002), P. veronii, and P. montelilii
Pseudomonas fluorescens
Used for identification for Pseudomonas fluorescens?
MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight)
- Non-pigmented, non-hemolytic
- (+) oxidase, catalase, ADH, optimal growth at 30 deg C
Pseudomonas mosselii
- Rare isolate and even rarer pathogen in the clinical laboratory
- To differentiate against P. aeruginosa
- (+) starch hydrolysis, nitrate to nitrite (soil denitrifier), anaerobe
- (-) arginine dihydrolase (ADH)
- Colonies: wrinkled, leathery, adherent colonies that may produce a light-yellow or brown pigment
- Causes septicemia, meningitis in the HIV-infected patient, pneumonia (especially in CF and other immunocompromised patients), endocarditis, postsurgical wound infections, septic arthritis, conjunctivitis, and UTIs in an immunocompromised host
Pseudomonas stutzeri
Pseudomonas stutzeri
Resistant/Susceptible?
- Chloramphenicol
- First and second-generation cephalosporins
Resistant
Pseudomonas stutzeri
Resistant/Susceptible?
- Aminoglycosides
- SXT
- Ampicillin
- Polymyxin
- Tetracyclines
- Fluoroquinolones
- Third-generation cephalosporins
Susceptible
- In soil and water, rarely from human specimens
- Colonies: non wrinkled, flat colonies that may appear with a yellowish-brown pigment smooth buttery appearance
- (-) pyoverdin, acetamide, proteolysis, starch hydrolysis
(+) oxidase, arginine dihydrolase (ADH), motile (single polar flagella), glucose, xylose
Pseudomonas mendocina
Pseudomonas mendocina
Used as the differentiation for Pseudomonas aeruginosa
Pigment
T or F: Pseudomonas mendocina is a non-producer of pyoverdin.
T
- Contaminants when isolated from clinical specimens
- (+) oxidase, MAC, nitrate to nitrite, motile (polar flagella)
- (-) biochemical test
Pseudomonas alcaligenes and pseudomonas pseudoalcaligenes
T or F: Pseudomonas alcaligenes/pseudoalcaligenes are known to be really biochemically inert: comes out negative for the majority of the biochemical testing panel.
T
Identify the organism.
Positive for arginine dihydrolase and is a weak fructose fermenter.
Pseudomonas pseudoalcaligenes
- Only member of the Pseudomonas family that are (-) oxidase
- Both from soil and water, wounds, abscesses, blood cultures, peritoneal and chronic ambulatory peritoneal dialysis (CAPD) fluids
- Higher risk for infection by these organisms in the presence of foreign materials (e.g., catheters), corticosteroid use, and immunocompromised states
- Both are G(-), non-fermenter
- (-) oxidase
- (+) catalase, motile, glucose oxidizer, MAC growth
- Colonies: wrinkled or rough colonies that produce an intracellular non-diffusible yellow pigment
Pseudomonas luteola and Pseudomonas oryzihabitans
Used to differentiate Pseudomonas oryzihabitans and Pseumonas luteola
- Ortho-nitrophenyl-galactopyranoside or O’Nitrophenyl-β-D-galactopyranoside (ONPG) test
- Esculin hydrolysis
Positive for ONPG, Esculin hydrolysis or Ortho-nitrophenyl-galactopyranoside?
Pseudomonas oryzihabitans
Negative for ONPG, Esculin hydrolysis or Ortho-nitrophenyl-galactopyranoside?
Pseudomonas luteola
- Japanese rice paddies, hospital drains, and respiratory therapy equipment
- Possible from an octopus bite while swimming in the ocean
- From the eye of one patient with postoperative endophthalmitis
- Resistant: first- and second-generation cephalosporins
- Usually susceptible to penicillin
- (+) ONPG, esculin hydrolysis
Pseudomonas oryzihabitans
- Sole isolate from a case of prosthetic valve endocarditis and subdiaphragmatic abscess from multiple brain abscesses in a child
- Sensitive to all β-lactams.
- (-) ONPG, esculin hydrolysis
Pseudomonas luteola