Non-Fermentative Gram-Negative Bacilli

So lonngggggggg

soil, water, plants, decaying vegetation, and many foodstuffs

Where are non-fermentative gram-negative bacilli commonly found in the environment?

Moist environment

What kind of environments do non-fermentative gram-negative bacilli prefer?

Nebulizers, dialysate fluids, saline, catheters, and other medical devices

Where can non-fermentative gram-negative bacilli be found in the hospital settings?

Chlorhexidine and quarternary ammonium compounds (QACs)

What disinfectant can non-fermentative gram-negative bacilli withstand?

No, they do not ferment carbohydrates

Do non-fermentative gram-negative bacilli ferment carbohydrates?

Aerobic environment

What type of environment do non-fermentative gran-negative bacilli prefer for growth?

Oxidizers and non-oxidizers (asaccharolytic)

What are the two metabolic classifications of non-fermentative gram-negative bacilli?

They oxidize carbohydrates to derive energy for metabolism

How do oxidizers derive energy?

They do not break down carbohydrates at all

How do non-oxidizers (asaccharolytic) obtain energy?

Oxidase positive

What is the typical oxidase reaction of non-fermentative gram-negative bacilli?

They are oxidase positive and do not ferment carbohydrates

How can non-fermentative gram-negative bacilli be differentiated from enterobacteriaceae?

No change in the butt and slant (no acid production)

Result of a Triple Sugar Ion (TSU) or Kligler iron sugar?

Growth on McConkey Agar, Oxidase reaction, Glucose oxidase-fermantative test

Three main criteria for classifying non-fermentative gram-negative bacilli

Aminoglycosides, third-generation cephalosporins, penicillins, Fluoroquinolones

What classes of antimicrobial agents are non-fermentative gran-negative bacilli resistant to ?

Pseudomonas aerugunosa,

Acinetobacter species,

Burkholderia species,

Stenotrophomonas maltophilia

These group of Non-fermenters, make up most isolates routinely seen in clinical laboratories.

Pseudomonas

Gram-negative bacilli or coccobacilli, strictly aerobic and motile by polar flagella, oxidase positive except for P. luteolus and P. oryzihabitans, catalase positive , Grows on MacConkey Agar, Oxidizers of carbohydrates

soil, water, and plants

Where is Pseudomonas aeruginosa commonly found in nature?

Hot tubs, whirlpools, and contact lens solutions

Domestic setting where Pseudomonas aeruginosa survives

Sinks, showers, and respiratory equipment

In hospital environments, where can Pseudomonas aeruginosa be found?

No, it is rarely part of the normal flora of healthy humans.

Is Pseudomonas aeruginosa nornally part of the human flora ?

It primarily causes disease in immunocompromised individuals

Why is Pseudomonas aeruginosa considered an opportunistic pathogen?

Nosocomial infections, including bacteremia and urinary tract infections (UTIs)

What type of infections can Pseudomonas aeruginosa cause in hospitals?

Folliculitis , otitis externa, respiratory infections

What type of infections can Pseudomonas aeruginosa cause in the community?

Folliculitis

What skin infection is associated with P. aeruginosa?

Otitis externa

An infection of the external ear canal and can be caused by P. aeruginosa

Immunocompromised state, trauma, IV drug use, and cystic fibrosis

Which conditions make individuals more susceptible to P. aeruginosa infections?

Osteomyelitis

Which bone infection can be caused by P. aeruginosa following trauma?

Endocarditis

Heart condition caused by P. aeruginosa in IV drug users

Respiratory infections in cystic fibrosis patients

Respiratory condition commonly affected by P. aeruginosa

• Exotoxin A

• Endotoxin (lipopolysaccharides)

• Proteolytic enzymes

• Alginate

• Pili

• Motility (flagella)

Virulence Factors

Exotoxin A

Most important exotoxin, Functions similarly to diptheria toxin by blocking protein synthesis, which kills the host cells

Proteolytic Enzymes and Hemolysins

Capable of destroying cells and tissues. On the bacterial surface, pili mediate the attachment of the organism to host cells.

Alginate

A polysaccharide polymer. When there is overproduction, it will cause mucoid colonies and production of biofilm, therefore contributing to the infection potential of patients with cystic fibrosis. Will also allow the organism to inhibit phagocytosis.

Pseudomonas aeruginosa

Bacterial specie that produce pyoverdin

Pseudomonas fluorescent group

To which bacterial group does P. aeruginosa belong based on pigment production?

Pigment

an important identifying characteristics of P. aeruginosa

Yellow-green

What color is Pyoverdin ?

Water-soluble

Pyoverdin water solubility?

Short-wavelength ultraviolet (UV) light

Under what type of light does Pyoverdin fluoresce?

Acts as siderophore, helping the bacteria acquire iron from the environment

Function of Pyoverdin in P. aeruginosa

Pyocyanin

A blue phenazine pigment, Water - soluble, combining with pyoverdin

• Its presence used specifically to identify P. aeruginosa

Reactive oxygen species

What harmful substances does Pyocyanin produce that can damage cells?

They cause cellular damage

What effect do reactive oxygen species have on cells?

By producing the enzyme catalase to neutralize Reactive oxygen species

How does Pseudomonas aeruginosa protect itself from the harmful effects of its own Pyocyanin production?

Pyorubin

A Red pigment

Pyomelanin

A brown or black pigment

Ecthyma Gangrenosum

A condition caused by P. aeruginosa wherein there is black discoloration on the skin surrounding the wound / formation of cutaneous papules that become black and necrotic.

Jacuzzi or Hot tub syndrome

In swimmers or divers, a necrotizing rash develops in users in recreational facilities that are infected with P. aeruginosa

Nail infection

If artificial nails are present, the nail bed can become infected with the organism, causing greenish discoloration.

Bloodd Agar and MacConkey Agar

On which routine laboratory media does P. aeruginosa grow well?

Beta-hemolysis

What type of hemolysis does P. aeruginosa exhibit on sheep blood agar?

Flat, spreading colonies with pigment discoloration

Appearance of P. aeruginosa colonies on BA

A fruity, graoe-like odor due to 2-aminoacetophenone

What kind of odor does P. aeruginosa produce, and what causes it?

Pseudomonas aeruginosa

Identify the causative organism:

• Gram staining: Gram-negative coccobacilli or bacilli

• Blood agar: Beta-hemolytic, flat, spreading colonies with a bluish-green discoloration

• MacConkey agar: Colorless colonies (non-lactose fermenter)

• Odor: Fruity, grape-like smell (due to 2-aminoacetophenone)

• Pigments: Pyocyanin (blue-green) and Pyoverdin (yellow-green fluorescent)

• Biochemical tests:

  • Oxidase: Positive

  • Citrate: Positive

  • Arginine dihydrolase: Positive

  • Growth at 42°C

  • Acetamide utilization: Positive

• Penicillin

• Ampicillin

• first and second-generation cephalosporins

• Trimethoprim sulfamethoxazole (SXT)

• Chloramphenicol, and Tetracyclines

P. aeruginosa is resistant to Antimicrobials?

• Aminoglycosides

• Piperacillin and Ticarcillin

• Third and fourth-generation cephalosporins (Ceftazidime and Cefepime)

• Carbapenems (except ertapenem)

• fluoroquinolones

P. aeruginosa is susceptible to Antimicrobials?

• Pseudomonas aeruginosa

• Pseudomonas fluorescens

• Pseudomonas putida

• Pseudomonas veronii

• Pseudomonas monteilii

• Pseudomonas mosselii

Pseudomonas Fluorescent Group

• Pseudomonas stutzeri

• Pseudomonas mendocina

• Pseudomonas pseudoalcaligenes

• Pseudomonas alcaligenes

• Pseudomonas luteola

• Pseudomonas oryzihabitans

Pseudomonas Non-Fluorescent Group

Pseudomonas aeruginosa

This is the only Fluorescent Pseudomonas group that grows at 42 degrees celsuis.

P. aeruginosa, P. stutzeri, P. mendocina

Nitrate reduction positive

P. fluorescens

Gelatin Liquified positive

Arginine Dihydrolase

positive for all fluorescent group

negative for P. stutzeri and positive for P. mendocina

Lysine decarboxylase

negative for all Pseudomonas

Urea Hydrolysis

Viable result (for all) Pseudomonas

Oxidizes Glucose

positive result for all Pseudomonas

P. fluorescens and putida are viable,

the rest are negative

Oxidizes Lactose

P. stutzeri

Oxidizes Mannitol

All Pseudomonas except for P. monteilii

Oxidizes Xylose

Pseudomonas stutzeri

Can grow in an anaerobic environment in nitrate-containing media, which differentiates it from other Pseudomonas species.

P. putida and P. fluorescens

rarely cause clinical due to their low virulence

P. putida

has been associated with catheter-related sepsis in cancer patients

P. oryzihabitans

It has been found in Japanese rice paddies and has been isolated from hospital drains and respiratory therapy equipment, This organism has been isolated from the eye of one patient with post-operative endophthalmitis.

P. luteola

It has been recovered as the only isolate from a case of prosthetic valve endocarditis, and subdiaphragmatic abscess and multiple brain abscess in a child.

Can be differentiated from oryzihabitans by the ONPG test and esculin hydrolysis.

Acinetobacter spp.

member of Moraxellaceae, only has 11 species officially named and approximately 21 species or stains have been identified.

Acinetobacter baumannii and Acinetobacter lwoffii

Most commonly seen Acinetobacter species in clinical specimens

Acinetobacter spp.

Gram-negative coccobacilli,

Inhabit the environment,

Can be found in formites, animal food product, Opportunistic pathogen, strictly aerobic, can resist decolorization and retain crystal violet stain.

Oxidase (-)

Catalase (+)

Nonmotile

Can grow on MacConkey Agar

Acinetobacter baumannii

Saccharolytic (Glucose oxidizing)

Acinetobacter lwoffii

Asaccharolytic (Non-glucose oxidizing spp.)

• Penicillin

• First and second-generation cephalosporins

• Fluoroquinolones

Acinetobacter spp. are often resistant to What antimicrobials?

• Aminoglycosides

• B-lactam plus B-Lactamase Inhibitor combinations (e.g., ampicillin-sulbactam, piperacillin-tazobactam)

• Carbapenems

Acinetobacter spp. are has a Variable susceptibility to What antimicrobials?

Crabapenem-resistant Acinetobacter baumannii

What is CRAB ?

Colistin and Tigecycline

antibiotics for carbapenem-resistant Acinetobacter baumanni (CRAB)

Stenotrophomonas maltophillia

3rd most common non-fermentative gram-negative bacillus isolated in the lab.

Stenotrophomonas maltophillia

Non-lactose fermenters but colonies appear bluish in MacConkey Agar

Blood Agar : lavander-green to light purple pigment

Oxidase (-)

Catalase (+)

DNAse (+)

Esculin and Gelatin Hydrolysis (+)

Lysine decarboxylase (+)

Oxidizes maltose faster than Glucose,

Produces brown pigment in Heart Infusion Agar that contains Tyrosine

Trimethoprim sulfamethoxazole

Drug of choice for Stenotrophomonas maltophillia

• Ticarcillin-clavulanate

• Levofloxacin

• Tigecycline

Stenotrophomonas maltophillia is susceptible to ?

• Burkholderia cepacia complex

• Burkholderia pseudomallei

• Burkholderia mallei

• Burkholderia gladioli

Burkholderia spp.

Burkholderia spp.

non-pathogenic, found in plants, soil, and water, are all motile except for B. mallei, cause nosocomial infection, Aerobic, non-spore-forming gram- negative bacilli.

3-4 days without transfer

Burkholderia cepacia complex may lose viability on Sheep blood agar within how many days?

Burkholderia cepacia complex

Blood Agar : smooth, slightly raised, and have a dirt-like odor colonies

MacConkey Agar : dark pink to red colonies (non-lactose fermenter)

Lysine decarboxylase (+)

ONPG (+)

Ornithinedecarboxylase (-)

Fail to reduce nitrate to nitrite

• Burkholderia cepacia-selective agar (BCSA)

• Oxidative-fermentative base-polymyxin B-bacitracin-lactose agar (OFPBL)

• Pseudomonas cepacia agar (PC)

Some culture media that are specifically used or can optimally isolate and grow Burkholderia cepacia

• Burkholderia cepacia-selective agar (BCSA)

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

Oxidative-fermentative base-polymyxin B-bacitracin-lactose agar

It can isolate Burkholderia cepacia from respiratory secretions of patients with cyctic fibrosis. It incorporates bacitracin as an added fermentation to differentiate isolates. B cepacia ferments lactose and appears yellow, while non-lactose appear green.

Pseudomonas cepacia Agar

Contains crystal violet, bile salts, polymyxin B, and ticarcillin to inhibit Gram-negative organisms. Pyruvate and phenol red are also added. Burkholderia cepacia breaks down pyruvate, creating an alkaline pH, which results in a color change of the pH indicator from yellow to pink.

Burkholderia mallei

Causes Glanders (affects horse, donkeys), considered by government agencies to be a potential bioterrorism agent, nonmotile, Grown on MAC.

Oxidase production: varibale

Oxidizes glucose

Reduces nitrates to nitrites

ADH positive

Melioidosis / Whitemore’s disease

A Burkholderia pseudomallei disease that is 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 the lungs and other viscera.

Burkholderia pseudomallei

Overwhelming septicemia can occur, cause local infections, including orbital cellulitis, the crycystitis, most common presentation is Pneumonia, Grows non-fermentative wrinkled colonies.

Bipolar staining on gram-stained smears.

Ashdown Medium : earthy-odor

Ashdown Medium : supplemented with colistin

Colonies in this agar are deep pink because of the absorption of neutral red in the medium.

Contains crystal violet and gentamicin as selective agents to suppress the growth of contaminating organisms.

Burkholderia gladioli

Plant pathogen, Produce yellow pigment after 48-72 hours incubation, motile by means of one or two flagella, Grows on MAC oxidizes glucose.

Catalase (+)

Urease (+)

Mannitol (+)

Oxidase and Decarboxylase (-)

Aeromonas

widely distributed in freshwater, estuarine, and marine environments.

Isolated from retail produce sources and animal meat products.

Fish, reptiles, amphibians, mammals and humans

Grows from 10-42 °C.

Oxidase +

Glucose-fermenting