Modified Gram Stain & OF Tests

Modified Gram Stain (Burke Modification)

• Utilizes methyl violet with NaHCO3, iodine, acetone, and safranin (2 min).

Glucose Non-Fermenter TSI Reaction

• Yields a K/NC (alkaline/no change) reaction with growth on Triple Sugar Iron (TSI) agar.

Oxidative-Fermentation (OF) Medium

• Contains 1% carbohydrates and peptones.
• Features a lower peptone concentration to prevent alkaline reactions from masking acid production.
• Has a reduced agar concentration for improved visualization.

OF Test: Acid Production in Both Tubes

• Indicates the organism is a fermenter.

OF Test: Acid Production Only in Open Tube

• Classifies the organism as a glucose oxidizer (non-fermenter).

OF Test: No Acid Production in Either Tube

• Indicates the organism is assachrolytic (unable to break down sugars).

Hugh-Leifson OF Indicator

• Uses bromthymol blue as an indicator.

King's OF Indicator

• Employs phenol red, which is less toxic.

OF Tube Color with Acid Production

• Turns yellow.

Embden-Meyerhoff Pathway

• Converts glucose to pyruvic acid, then to mixed and lactic acids (strong acids).

Entner-Duoderoff Pathway

• Converts glucose to pyruvic acid, then to the Krebs cycle (weak acid production).

Porphyrin Test

• Determines the X factor requirement in Haemophilus species.

Porphyrin Test and X-Independent Strains

• X-independent strains secrete enzymes in heme biosynthesis.

Porphyrin Test Interpretation

• Red color after adding Kovac's reagent indicates porphyrin presence; heme biosynthesis occurs, so the organism does not need X factor.

Leucine Aminopeptidase (LAP) Test

• A disk test, similar to PYR.
• Hydrolysis of substrate releases B-napthylamide.
• Combines with cinnamaldehyde reagents to form a pink color.

LAP Test Results

• Enterococcus and some other streptococci are LAP positive.
• Aerococcus is LAP negative.

Pseudomonas aeruginosa Positives (OMEGA U)

• Oxidase, motile, elastase, gelatinase, arginine dihydrolase, and urease positive.

Pseudomonas aeruginosa Fermentation Type

• Non-fermenter, utilizes glucose oxidation.

Pseudomonas aeruginosa Gram Stain Morphology

• Gram-negative, long, thin rods.

Pseudomonas aeruginosa Hemolysis and Odor

• Exhibits beta-hemolysis and produces a grape-like or tortilla-like odor.

Pyocyanin Characteristics

• Blue pigment, non-fluorescent, chloroform soluble, and water-soluble.

Pyoverdine Characteristics

• Yellow pigment, fluorescent, chloroform-insoluble, and water-soluble.

Pseudomonas aeruginosa Environmental Preference

• Thrives in moist environments such as sinks, catheters, sponges, and hot tubs.

Pseudomonas aeruginosa Virulence Factor

• Exotoxin A.

Pseudomonas aeruginosa Exotoxin A Similarity

• Similar to diphtheria toxin.

Pseudomonas aeruginosa Exotoxin A Mechanism

• Inhibits protein synthesis by ADP ribosylation.

Pseudomonas aeruginosa Exotoxin A Systemic Effects

• Can be expressed systemically but does not always cause systemic effects.

Pseudomonas aeruginosa Infections

• Affects the immunocompromised.
• Causes UTIs, septicemia, respiratory disease (CF), wound/sepsis, and osteomyelitis.

Pseudomonas aeruginosa Sepsis Manifestation

• Often presents with ecthyma gangrenosum.

Pseudomonas aeruginosa Folliculitis

• Associated with hot tubs and whirlpools.

Pseudomonas aeruginosa and Cystic Fibrosis (CF)

• Clogs the alveoli in CF patients.

Pseudomonas aeruginosa Antibiotic Sensitivity

• Highly resistant to antibiotics but sensitive to colistin/polymixin B.

Pseudomonas aeruginosa CF Isolates

• Mucoid, shorter rods.
• More sensitive to antibiograms.

Other Fluorescent Pseudomonas Species

• Pseudomonas fluorescens and Pseudomonas putida.

Differentiation of Pseudomonas aeruginosa

• P. aeruginosa grows at 42°C and reduces nitrate.

Pseudomonas stutzeri Characteristics

• Non-fluorescent, wrinkled, light-brown colonies.

Pseudomonas luteola & Pseudomonas oryzihabitan Characteristics

• Oxidase negative and produce an orange or yellow pigment.

Pseudomonas aeruginosa Typing

• Utilizes molecular fingerprinting for typing.

Important Pathogenic Burkholderia Species

• cepacia complex (sp cepacia), mallei, and pseudomallei.

Burkholderia Characteristics

• Oxidase variable, catalase positive, and motile.

Burkholderia Gram Stain Morphology

• Gram-negative straight or curved rods.

Burkholderia Growth

• Can grow on Blood Agar Plate (BAP) and MacConkey agar (MAC).

Burkholderia cepacia Pigment

• Yellow pigment.

Burkholderia cepacia Complex Genomovars

• Consists of 9 different genomovars.

Burkholderia Infections

• Causes respiratory disease (contributes to CF), septicemia, soft tissue infections, and ocular infections.

Burkholderia cepacia Complex in CF Patients

• Infections often occur after Pseudomonas infections.

Selective Agar for Burkholderia in CF Patients

• Burkholderia cepacia selective agar (BCSA).

Burkholderia cepacia Complex Septicemia

• More common in patients with granulomatous disease.

Burkholderia cepacia vs. Burkholderia gladioli

• B. cepacia: oxidase positive, sucrose positive.
• B. gladioli: oxidase negative, sucrose negative.

Burkholderia Antibiotic Resistance

• Is often resistant to antibiotics and forms biofilms, similar to Pseudomonas.

Burkholderia Antibiotic Susceptibility

• Is very important in non-CF patients.

Burkholderia Species as Bioterrorism (BT) Agents

• B. mallei and B. pseudomallei.

Burkholderia mallei & Burkholderia pseudomallei Infections

• Cause ulcers and respiratory infections, spread via inhalation, and various other routes.

Burkholderia mallei Characteristics

• No growth at 42°C, non-motile, oxidase variable, and nitrate negative.

Burkholderia pseudomallei Characteristics

• Grows at 42°C, motile, oxidase positive, and nitrate positive.

Burkholderia mallei Morphology

• Gram-negative coccobacilli, forming grey colonies.

Burkholderia pseudomallei Morphology

• Gram-negative bipolar bacilli, forming white colonies.

Burkholderia mallei & Burkholderia pseudomallei

• Are both negative for indole production.

Burkholderia Colistin/Polymixin B Resistance

• Burkholderia is resistant.

Stenotrophomonas maltophilia

• Was previously named Pseudomonas maltophilia.

Stenotrophomonas maltophilia Characteristics

• Oxidase negative, glucose variable.

Stenotrophomonas maltophilia Growth

• Grows on BAP and MAC.

Stenotrophomonas maltophilia Prevalence

• The third most common non-fermenter in clinical labs.

Stenotrophomonas maltophilia Infections

• Causes septicemia, respiratory tract infections, UTIs, skin and soft tissue infections, endocarditis, meningitis, and ocular infections.

Stenotrophomonas maltophilia Patients Affected

• Primarily affects immunocompromised and hospitalized patients.

Stenotrophomonas maltophilia Septicemia

• Associated with catheter use.

Stenotrophomonas maltophilia Infections Mortality Rate

• Infections are rare, but have a high mortality rate when they occur.

Stenotrophomonas maltophilia Color and Odor

• Lavender-green color on BAP and produces an ammonia odor.

Selective Media for Stenotrophomonas maltophilia

• Utilizes VIA, SM2i, and GNSA.

Stenotrophomonas maltophilia Antibiotic Resistance

• Extremely resistant to antimicrobials, more so than Pseudomonas.

Stenotrophomonas maltophilia Unusual Characteristics

• Oxidase negative, maltose positive, lysine positive, DNase positive, gelatinase positive, and ONPG positive.

Acinetobacter General Characteristics

• Non-motile, oxidase negative, catalase positive, and difficult to decolorize during Gram staining.

Acinetobacter Growth and Pigment

• Grows on BAP and MAC, and produces no pigment.

Acinetobacter Biochemical Activity

• Relatively biochemically inert among non-fermenters.

Acinetobacter Major Pathogen

• Acinetobacter baumannii.

Acinetobacter baumannii Complex

• Part of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

Acinetobacter Infections

• Causes pneumonia, septicemia, wounds, UTIs, and meningitis.

Acinetobacter Infection Risk Factors

• Surgery, hospitalization, and medical implantation devices.

Acinetobacter Pneumonia Risk Factors

• Alcohol abuse, smoking, COPD, and diabetes.

Acinetobacter Septicemia Risk Factors

• Include a high APACHE (acute physiology & chronic health evaluation) score.

Acinetobacter Wound Infections

• Seen in military and burn patients.

Acinetobacter Growth and Odor

• Grows well on BAP and MAC, producing a fishy odor.

Acinetobacter Negative Reactions

• Negative for all decarboxylases, nitrate reduction, motility, and oxidase production.

Acinetobacter Glucose Use

• Glucose variable.

Acinetobacter baumannii Differentiation

• Can be differentiated by growth at 41°C and glucose oxidation.

Acinetobacter Memory Device

• Acineto = inert: No pigment, decarboxylases, motility, nitrate, or oxidase.

General Non-Fermenter Characteristics

• Catalase positive and Gram-negative.

General Non-Fermenter Habitat

• Widespread in the environment and found in many hospital sources.

General Non-Fermenter Environmental Preference

• Tend to like moist environments forming biofilms.

General Non-Fermenter Infections

• Tend to include respiratory infections, septicemia, wound infections, and UTIs; Acinetobacter and Stenotrophomonas can cause meningitis.

Most Common Gram-Negative Non-Fermenters

• Pseudomonas > Acinetobacter > Stenotrophomonas.

Oxidase and Motility of Each Gram-Negative Non-Fermenter

• Pseudomonas: Oxidase positive, motile.
• Burkholderia: Oxidase variable, motile.
• Stenotrophomonas: Oxidase negative, motile.
• Acinetobacter: Oxidase negative, non-motile.

Most Resistant Gram-Negative Non-Fermenter

• Stenotrophomonas shows multi-drug resistance.

Colistin/Polymixin B Sensitivity

• Pseudomonas is sensitive.
• Burkholderia is resistant.

Haemophilus General Characteristics

• Gram-negative rods (possibly filamentous), oxidase positive, non-motile, facultative anaerobe, fastidious.
• Requires X and/or V factors from blood.

Haemophilus vs. Enterobacteriaceae

• Haemophilus is oxidase positive.

Haemophilus Normal Flora

• Part of the normal flora in the nasopharynx.

Haemophilus influenzae Infections

• Meningitis, pneumonia, sinusitis, otitis media, and cellulitis.

Meningitis Reporting

• Meningitis is a reportable disease.

Haemophilus influenzae Transmission

• Via droplet infection.