Non-Fermentative Gram-Negative Bacilli - PART 1

NON-FERMENTATIVE GRAM-NEGATIVE BACILLI

• Found in most environments typically in soil and water, plants and decayed vegetation, and many food stock. Prefer moist environments in the hospital.

• These can be isolated from nebulizers, dialysis fluids, saline, catheters, and other devices

• They can stand treatment with chlorhexidine and quaternary ammonium compounds.

• Do not ferment carbohydrates

• Fail to acidify an oxidative fermentative (OF) medium.

• Prefer to grow in aerobic environment

Oxidase (+)

This is a feature that differentiates Non-fermentative bacteria from Enterobacteriaceae

Fail to ferment carbohydrates

no change in the butt and slant in Triple Sugar Iron Agar (TSIA) or Kligler Iron Agar (KIA)

• Growth on MacConkey Agar

• Oxidase Reaction

• Glucose Oxidase Fermentative Test

CLASSIFICATION SYSTEM DEVISED FOR GROUPING NON-FERMENTERS

• Pseudomonas aeruginosa

• Acinetonacter spp

• Burkholderia spp

• Stenotrophonas maltophilia

Non-fermenters

PSEUDOMONAS

Accounts for the largest percentage of all non-fermenters isolated from clinical specimens

PSEUDOMONAS

Strictly aerobic
Catalase (+)
Oxidase (+)
Grows on MacConkey Agar

Discoloration

Pseudomonas on MacConkey Agar has a characteristic _?

PSEUDOMONAS AERUGINOSA

• Most commonly isolated species from clinical specimens.

• Found in moist environments, pools, hot tubs, catheters, and humidifiers in hospitals.

• In hospital environments, it will dwell in the sink, shower, and respiratory equipment.

• Causes nosocomial infection/hospital-acquired infection

• Opportunistic pathogen

• HABITAT/RESERVOIR: soil, water, and plants.

PSEUDOMONAS AERUGINOSA

• Survives well in domestic environments such as hot tubs, whirlpools, contact lens solutions.

• Rarely part of the normal flora of healthy humans.

Opportunistic pathogen

It will cause a disease or sickness to your patient, especially if the host’s immune system is down.

PSEUDOMONAS AERUGINOSA

causes folliculitis, otitis externa, osteomyelitis, endocarditis, respiratory infection, bacteremia, UTI

Folliculitis

Infection on the skin.

Endocarditis

common among IV drug users

PSEUDOMONAS AERUGINOSA

Virulence factors includes production of slime polysaccharide, endotoxin, proteases and inactivates components of complement and has growth at 42 ℃

Endotoxin

lipopolysaccharide

Exotoxin

● Proteases ● Hemolysins ● Lecithinase ● Elastase ● Deoxyribonuclease

Exotoxin A

Most important exotoxin. Functions similarly as diphtheria toxin by:
● Blocking protein synthesis, which kills the host cells.
● Promotes cellular damage and tissue invasion
● Toxic for macrophages

Proteolytic enzymes and hemolysins

destroys cells and tissues

Alginate

• A polysaccharide polymer that inhibits phagocytosis and contributes to the infection potential in patients with cystic fibrosis.

• Causes mucoid colonies and production of biofilm

Pili

on bacterial surface that mediates attachment

Pyoverdine pigment

Yellow green, water soluble and fluoresces under short-wavelength UV light.

Pseudomonas aeruginosa.

Producing pigments is one of the identifying characteristics of

Pyocyanin

water soluble, bright bluish phenazine pigment and damages cells by producing REACTIVE OXYGEN SPECIES

Pseudomonas aeurginosa

the only non-fermenter that produces pyocyanin

Catalase

The reactive oxygen species are also bactericidal to the organism. In order to protect itself from destruction, the organism produces the enzyme _

Pyorubin

red; water soluble

Pyomelanin

brown/dark; water-soluble

Ecthyma gangrenosum

The Pseudomonas aeruginosa may invade and destroy cell walls of the subcutaneous blood vessels, resulting in the formation of cutaneous papules that become black and necrotic

Jacuzzi or Hot Tub Syndrome

In swimmers and divers, a necrotizing rash develops and users in recreational facilities are infected with Pseudomonas aeruginosa.

Nail Infection

In artificial nails, the nail beds become infected with Pseudomonas aeruginosa. It can cause greenish discoloration.

Pseudomonas aeruginosa

• Beta-hemolytic (flat spreading colonies) on BA and colorless on MacConkey

• Produces fruity grape-like odor caused by the presence of 2-aminoacetophenonol.

• Alkaline slant over alkaline butt, no gas, no 𝐻2S

Cetrimide agar

selective and differential medium for the identification of P. aeruginosa

Cetrimide

acts as a detergent and help inhibit bacteria, as well as, enhance the production of pigments

Pseudomonas putida

• has been associated with catheter-related sepsis in patients with cancer.

• Low virulence, rarely causes clinical disease

• Gelatin hydrolysis (-)

• Produce acid from xylose

PSEUDOMONAS FLUORESCENS

• Pseudobacteremia related to contaminated catheters and catheter-related devices

• Gelatin hydrolysis (+)

• Produce acid from xylose

PSEUDOMONAS FLUORESCENT GROUP

P. putida and P. fluorescens

Pseudomonas stutzeri

• (+) can grow in anaerobic environment in nitrate-containing media, which then differentiates it from other pseudomonas.

• Rarely isolated, rarely causes infection

Pseudomonas stutzeri

• Wrinkled, leathery, adherent colonies that may produce a light-yellow or brown pigment

• ADH (-)

• Starch hydrolysis (+)

PSEUDOMONAS MENDOCINA

• Produces non wrinkled, flat colonies, may appear with a yellowish-brown pigment

• Oxidase (+)

• ADH (+)

• Acetamide (-)

PSEUDOMONAS ORYZIHABITANS

• Found in Japanese rice paddies.

• Has isolated from hospital drains and respiratory therapy equipment.

• Has been isolated from the eye of one patient with post-operative endopthalmitis.

PSEUDOMONAS LUTEOLA

Has been recovered as the only isolate from a case of: ● Prosthetic valve endocarditis ● Subdiaphragmatic abscess ● Multiple brain abscess in a child

● O-nitrophenyl-beta-D-galactopyranoside (ONPG) test AND Esculin Hydrolysis

Pseudomonas luteola can be differentiated from Pseudomonas oryzihabitans by the:

P. aeruginosa

Only _ grows at 42 ℃. (+)

oxidize xylose

Pseudomonas monteilli can be distinguished from Pseudomonas putida by its inability to

Pseudomonas fluorescens

Only can liquefy gelatin (+).

arginine dihydrolase

Pseudomonas stutzeri can be differentiated from Pseudomonas mendocina by the

ACINETOBACTER SPP

• Short, rod shaped to spherical Gram-negative bacilli

• Inhabit the environment.

• Can be found in fomites, animal food products.

• These organisms are capable of surviving in inanimate objects for extended periods.

• Opportunistic pathogen

• Strictly aerobic

ACINETOBACTER SPP

Can resist decolorization and retain the crystal violet stain → leading to misidentification ● making it Gram-positive cocci in smears made from blood culture bottles

ACINETOBACTER SPP

This organism accounts for 1-3% of all nosocomial infections, second only to Pseudomonas aeruginosa.

ACINETOBACTER SPP

• Can grow on MacConkey Agar

• Catalase (+)

• Oxidase (-)

• Non-motile

ACINETOBACTER SPP

gives a purplish hue on MacConkey

Carbapenem-Resistant-Acinetobacter Baumanii

Only susceptible to colistin and tigacycline.

STENOTROPHOMONAS MALTOPHILIA

Risk factors for colonization or infection: neutropenia

STENOTROPHOMONAS MALTOPHILIA

• 3rd most common non-fermentative, gram-negative bacilli isolated in the clinical laboratory Nosocomial pathogen

• Found in the environment

• Not part in the normal flora in humans, but it can quickly colonize the respiratory tract of hospitalized patients.

• Non-motile

STENOTROPHOMONAS MALTOPHILIA

MacConkey Agar: colonies may appear bluish.
Oxidase (-)
DNAse (+)
Catalase (+)
Esculin and Gelatin Hydrolysis (+)
Lysine decarboxylase (+)

STENOTROPHOMONAS MALTOPHILIA

Lavender-green to light purple pigment on BA
Oxidizes maltose
Brown pigment on HIA with tyrosine
Blue on MAC

Trimethoprim-Sulfamethoxazole (SXT)

DOC for STENOTROPHOMONAS MALTOPHILIA

BURKHOLDERIA SPP.

Non-pathogenic
Plant, soil, water-reservoir
All are motile except Burkholderia mallei (polar flagella)
Nosocomial infection Aerobic
Non-spore forming

BURKHOLDERIA CEPACIA COMPLEX

Grows well on most laboratory media, but may lose viability on Sheep Blood Agar in 3 to 4 days without transfers.

BURKHOLDERIA CEPACIA COMPLEX

Dark pink to red on MAC
Non-wrinkled
Non fluorescing yellow or green pigment
Almost all oxidize glucose
Dirt-like odor on BA

BURKHOLDERIA CEPACIA COMPLEX

Lysine Decarboxylase (+)
ONPG (+)
Ornithine decarboxylase (-)
Reduction of nitrate to nitrite (-)

BCSA

Most effective in reducing overgrowth while maintaining good recovery of Burholderia cepacia.

OFPBL: Oxidative-Fermentative Base-Polymyxin B-Bacitractin-Lactose

It can isolate Burkholderia cepacia from respiratory secretions of patients with cystic fibrosis.

Burkholderia cepacia

ferments lactose and appears yellow, while non-fermenters appear green.

PC agar

Contains: ■ Crystal violet ■ Bile salts ■ Polymyxin B ■ Ticarcelin ○ These inhibit gram-positive and rapid-growing gram-negative organisms. ○ Pyruvate and Phenol red are also added.

Burkholderia cepacia

breaks down the pyruvate, creating an alkaline pH and resulting in a color change of the pH indicator from yellow to pink.

BURKHOLDERIA MALLEI

• Causes Glanders

• respiratory tract zoonosis

• Primarily affecting livestock such as horses, mules, and donkeys

• Rare infection in humans

• May cause severe local suppurative or acute pulmonary infections

• Considered by government agencies to be a potential bioterrorism agent

BURKHOLDERIA MALLEI

Nonmotile
Growth on MAC
Variable glucose oxidation
ADH (+)
Reduce nitrates to nitrites (+)

BURKHOLDERIA PSEUDOMALLEI

Causes Melioidosis - “Whitmore’s Disease”

BURKHOLDERIA PSEUDOMALLEI

• Acquired via inhalation or contact through cut or abraded skin

• An aggressive, granulomatous, pulmonary disease caused by

• ingestion, inhalation, or inoculation of the organisms through direct contact with contaminated soil and surface water, with metastatic abscess formation in lungs and other viscera

• Southeast Asian Countries : Vietnam, Thailand, Northern Australia and Mexico

BURKHOLDERIA PSEUDOMALLEI

Overwhelming septicemia can occur.
Local infections: orbital, cellulitis, dacryocystitis.
Most common: Pneumonia

BURKHOLDERIA PSEUDOMALLEI

Nonfermentative wrinkled colony
Bipolar staining on Gram stain
Ashdown medium: selective medium, supplemented with colistin

BURKHOLDERIA PSEUDOMALLEI

Deep pink colonies because of the absorption of neutral red in the medium
Colonies exhibit an earthy odor

BURKHOLDERIA GLADIOLI

• Resembles Burkholderia cepacia complex

• Found in patients with cystic fibrosis and chronic granulomatous disease

• Recovered from the blood and tissue of immunocompromised patients (CF px) who had lung transplant

BURKHOLDERIA GLADIOLI

Yellow pigment may be produced after 48-72 hrs of incubation
One or two polar flagella
Catalase (+)
Urease (+)
Oxidase (-)
Grows on MAC
Oxidizes glucose
Mannitol (+)
Decarboxylase (-)

AEROMONAS

• Facultative anaerobes

• Widely distributed in freshwater, estuarine, and marine environments

• Frequently isolated from retail produce sources and animal meat

  products

• Fish, reptiles, amphibians, mammals, and humans
  Enteric pathogens

AEROMONAS

Motile
Reduce nitrates to nitrite (+)
Oxidase (+)
Glucose-fermenting
Typically grows from 10-42℃

AEROMONAS

has to groups (Mesophilic and Psychrophilic)

Mesophilic Group

Optimal growth @ 37˚C
All are motile by means of a single polar flagellum..
A. Hydrophilia complex
A. veronii complex
A. caviae complex

Psychrophilic group

Non-motile
Optimal growth @ 22˚C
A. salmonicida - Fish pathogen
are not considered human pathogens.

AEROMONAS

Intestinal Infections:
Recognized as enteric pathogens.
5 diarrheal presentations:
acute, secretory diarrhea
Acute, dysenteric form of diarhea
Chronic, diarrhea
Cholera-like diseases
Nebulous syndrome

Nebulous Syndrome

Commonly referred to as traveler’s diarrhea (similar to Enterotoxigenic E. coli). Most cases are self-limiting, but in the pediatric, geriatric, and immunocompromised population, supportive therapy and antimicrobials.

AEROMONAS

are recognized as enteric pathogens, but not in the same manner as the more common enteric pathogens: Salmonella, Shigella, and Vibrio cholerae.

AEROMONAS

The pattern of the virulence is more like the multifactorial patterns of the various E. coli subgroups associated with enteric disease: ● Enterotoxigenic E. coli ● Enterohemorrhagic E. coli.

AEROMONAS

Severe watery diarrhea is associated with heat labile and heat stable enterotoxin.

Aeromonas caviae

Most frequently associated with gastrointestinal infections especially in neonate and pediatric populations.

Aeromonas hydrophila and Aeromonas veronii

Other species associated with diarrhea. There are more serious complications from infections of these organisms, like it cause Hemolytic Uremic Syndrome or Kidney Disease.

Aeromonas veronii biovar sobria

Has also been linked to cholera-like disease characterized by abdominal pain, fever and nausea.

Septicemia and Wound Infections

Most common for aeromonas. Involves a recent traumatic aquatic exposure, such as boating or fishing accident, and generally occurs on the extremities. Most common presentation: cellulitis, more cases or few cases of myonecrosis and necrotizing fasciitis, and a rare case of eczema gangrenosum or eczema gangrenosum associated with sepsis

Aeromonad sepsis

• Most invasive type of aeromonas infection.

• The species associated are: Aeromonas veronii biovar sobria. Aeromonas jandaei. Aeromonas hydrophila

• Such patients are most likely to be immunocompromised and have a history of:
  liver disease or dysfunction
  hematologic malignancies
  hepatobiliary disorders or traumatic injuries
  

AEROMONAS

uses combined use of ampicillin SBA and a modified cefsulodin-Irgasin-novobiocin (CIN) II plate ○ With 4 μg of cefsulodin highest recovery of aeromonads. ○ pink-centered colonies from the fermentation of mannitol.

Aeromonas Agar

Uses D-xylose ○ It is a relatively new alternative medium by using D-xylose as a differential characteristic.

Oxidase test (+)

Distinguishes Aeromonas from Enterobacteriaceae except for Plesiomonas shigelloides.

Spot Indole test (+)

Most clinically relevant Aeromonas are indole positive.

String test

: can be used to differentiate Vibrio species from Aeromonas species since in this test, organisms are emulsified in 0.5% sodium deoxycholate, which lyses Vibrio cells but not those of Aeromonas species. Cell lyses releases DNA which can be pulled up into a string with an inoculating loop.

API 2OE strip

DEFINITIVE IDENTIFICATION OF AEROMONAS: