(F) Lesson 13: Non-fermenters and Miscellaneous Gram-Negative Bacilli (Part 1) Flashcards by Kchianna Lauren Coronel

The heart and soul of microbiology

Identification

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If a patient is infected with an organism, one thing we can do is perform?

Culture and Sensitivity

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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

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In the Philippines instead of culture and sensitivity, we do what?

Empiric Therapy

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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

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Determines the best antibiotic that can be prescribed to the patient

AST

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What do we do when we receive unknown samples?

Gram-staining
Culture with BAP, CAP, and MAC
Biochemical Testing

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Comes 2nd in a clinical setting, but essential to do
Do not report normal flora

Gram Stain

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The special media will be used for follow-up
In these plates, we will observe the hemolytic pattern.

Culture using BAP, MAC, and CAP

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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

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Medium

For fastidious organisms and gonococcal species

CAP

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After G/S or culture, subject it to ____ depending on the growth.

Biochemical testing

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Through ____, you can narrow down the possible causative agents of the infection.

Biochemical test results

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One of the biggest points of differentiation in G(-) organisms

TSI or carbohydrate fermentation test

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Causes difficulty in ID
We use the wrong manner of testing

Non-reactive organisms or biochemically inert organism

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They do not ferment any carbohydrates, rather they oxidize carbohydrates.

Non-fermenters

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General Characteristics

Grow much better in an ____ environment

Aerobic

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General Characteristics

T or F: Non-fastidious organisms grow on regular media, even w/o supplementation.

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General Characteristics

Most non-fermenters are oxidase (positive/negative).

Positive

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General Characteristics

This differentiates Enterobacteriaceae from non-fermenters since most Enterobacteriaceae are (-) oxidase.

Oxidase Test

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The only (+) oxidase Enterobacteriaceae

Plesiomonas spp.

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T or F: Non-fermenters are non-ubiquitous and are rarely found in most environments.

F (ubiquitous and found in most environments)

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General Characteristics

Can withstand treatment with ____ and ____

Chlorhexidine (antiseptic solution) and quaternary ammonium compounds (QACs)

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Risk Factors for Clinical Infections

Diabetes mellitus
Cancer
Steroids
Transplantation

Immunosuppression/Immunocompromisation

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# 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**?

1. Pseudomonas stutzeri 2. Pseudomonas oryzihabitans 3. 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. 1. Escherichia coli 2. Pseudomonas 3. Alacaligenes A. +/- B. -/- C. +/+

1. C 2. A 3. 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.

1. **Gram Staining:** Gram-negative coccobacilli 2. **TSI:** K/K 3. **MAC:** Positive/Growth on MAC 4. **Oxidase Test:** Most are oxidase-positive 5. **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?

1. Klebsiella pneumoniae 2. Escherichia coli 3. **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.

# 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.

* 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.

# 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

(F) Lesson 13: Non-fermenters and Miscellaneous Gram-Negative Bacilli (Part 1) Flashcards

(125 cards)