Nonfermentative Gram-negative Bacilli

• Nonfermentative Gram-negative Bacilli


  Group of Gram negative bacilli that do not ferment glucose and other sugars
• Nonfermenters


  • Mostly environmental bacteria - they can be found in water, soil, vegetables, plants and also in hospital surfaces
  • Not part of the normal flora of the human body
  • Most of these organisms are oxidase positive except for Acinetobacter spp.
  • Frequently resistant to many of the antibiotics that are effective against the members of the Enterobacteriaceae
• Pseudomonas


  • Strictly aerobic
  • Catalase-positive
  • Oxidase-positive (except P. luteolus and P. oryzihabitans)
  • Motile - some have polar flagella
• Pseudomonas aeruginosa


  Most commonly isolated species from clinical specimens
• Pseudomonas aeruginosa


  • Found in moist environments, pools, hot tubs, catheters, and humidifiers in hospitals
  • Ubiquitous - these are found everywhere, especially in the hospital environments
  • Causes nosocomial infection
  • Opportunistic pathogen
  • Can be found on medical equipment, disinfectant solutions and soap
  • Not part of the normal flora of healthy people/ human normal flora
• Nosocomial infections


  Infections that can be acquired through hospitalization

  
• Immunosuppressed


  Most patients in the hospitals
• Pathogenesis of Pseudomonas aeruginosa

  1. Produces slime polysaccharide, endotoxin, and proteases inactivate components of complement

  2. Exotoxin A - promotes cellular damage and tissue invasion and is toxic for macrophages and blocks protein synthesis

  3. Pili - found on bacterial surface and mediate attachment to host cells

  4. Alginate - a polysaccharide polymer that inhibits phagocytosis and contributes to the infection potential in patients with cystic fibrosis
• Cystic fibrosis


  • Genetic (inherited) disease that causes thick, sticky mucous to build up in organs including the lungs and the pancreas
  • Thick mucous will clog the airways and will make it difficult to breath
  • Cystic fibrosis patients = immunosuppressed since there is production of thick mucous which could be a breeding ground for these organisms = more prone to get opportunistic pathogens
• Pigments produced by Pseudomonas aeruginosa

  • Pyocyanin: water-soluble pigment, bright bluish phenazine pigment that damages cells by producing reactive oxygen species
  • Pyoverdin: green, water-soluble and fluoresces under short-wavelength ultraviolet light
  • Pyorubin: red pigment (rubin - red)
  • Pyomelanin: brown pigment (melanin - brown)
• Ecthyma gangrenosum


  Caused by Pseudomonas aeruginosa
• Jacuzzi or hot tub syndrome


  Necrotizing rash in divers and swimmers caused by Pseudomonas aeruginosa
• Gram stain of Pseudomonas aeruginosa

  • Gram-negative bacilli (rods)
• Pseudomonas aeruginosa on Blood Agar

  • Produces beta-hemolytic, rough or ground glass appearance
• Pseudomonas aeruginosa on MacConkey Agar

  • There is a slight discoloration on the MacConkey agar due to the pigment
• MacConkey Agar


  • Distinguishes lactose fermenters from non-lactose fermenters
  • Lactose fermenters: produces pink colonies
  • Non-lactose fermenters: will have colorless colonies
• Cetrimide Agar


  • Selective media for isolation of Pseudomonas aeruginosa
  • Cetrimide will act as a detergent and will inhibit most bacteria as well as enhance the production of pigments
• Fluorescent pseudomonads

  • P. aeruginosa
  • P. fluorescens
  • P. putida
  • P. veronii
  • P. monteilii
  • P. mosselii
• Nonfluorescent pseudomonads

  • P. stutzeri
  • P. mendocino
  • P. pseudoalcaligenes
  • P. alcaligenes
  • P. luteola
  • P. oryzihabitans
• Acinetobacter spp.


  • Short, rod shaped to spherical Gram negative bacilli
  • Non motile
  • Strictly aerobic - needs air in order for it to grow (needs oxygen)
  • Difficult to decolorize in Gram stain - if it is difficult to decolorize, it can retain the Gram positive result = purple color

  
• Acinetobacter baumannii


  Will have purplish colonies on MacConkey agar
• Some species under Acinetobacter

  • Acinetobacter calcoaceticus
  • Acinetobacter baumannii complex
  • Acinetobacter baumannii: Saccharolytic - glucose oxidizing and non-hemolytic
  • Acinetobacter lwoffii: Non-glucose oxidizer = non-saccharolytic
  • Acinetobacter haemolyticus: Non-saccharolytic and produces hemolysis (is hemolytic)
• Antimicrobial susceptibility of Acinetobacter baumannii complex

  Susceptible to doxycycline, SXT (Trimethoprim Sulfamethoxazole), quinolones, ureidopenicillins, imipenem, ampicillin, sulbactam, ceftazidime as well as carbapenems (except ertapenem)
• Antimicrobial susceptibility of Acinetobacter baumannii

  • Resistant to beta lactam and aminoglycoside antibiotics and susceptible to carbapenems
  • The resistance to aminoglycosides is caused by plasmid-mediated acetyl adenylyl and phosphotransferases
  • Carbapenems are higher grade types of antibiotics
  • There are also strains of Acinetobacter baumannii that will develop resistance = Carbapenem-resistant Acinetobacter baumannii (CRAB): These strains are resistant to all classes of antibiotics except colistin and tigecycline
• Stenotrophomonas maltophilia


  • Third most common nonfermentative, Gram negative bacilli isolated in the clinical laboratory
  • Nonmotile
  • Nosocomial pathogen - hospital acquired infection
• Risk factors for colonization or infection with Stenotrophomonas maltophilia

  • Mechanical ventilation
  • Use of broad-spectrum antibiotics
  • Catheterization
  • Neutropenia - having a low WBC count
• Laboratory Diagnosis of Stenotrophomonas maltophilia

  • MacConkey Agar - colonies may appear bluish but in the picture it appears colorless since it is non-fermentative it will not ferment carbohydrates
  • Blood agar plate - Lavender green colonies (ideal growth) but not all the time can be isolated in a clinical setting
  • Oxidase +, DNAse +, Catalase +, Esculin and gelatin hydrolysis +, Lysine decarboxylase +

  
• Antimicrobial Susceptibility of Stenotrophomonas maltophilia

  • Susceptible: levofloxacin, trimethoprim sulfamethoxazole, ticarcillin-clavulanate and minocycline
  • Trimethoprim-sulfamethoxazole is the antibiotic of choice
  • Resistant: cephalosporins, penicillins, carbapenems, and aminoglycosides - Intrinsically resistant
• Burkholderia

  • Aerobic, non-spore-forming
  • All are motile (polar flagella) except Burkholderia mallei
  • Nosocomial infection - Most nonfermentative gram-negative bacilli are opportunistic pathogens especially to those immunosuppressed patients or with weak immune systems
• Burkholderia pseudomallei


  • Causes Melioidosis - an aggressive, granulomatous, pulmonary disease caused by ingestion, inhalation, or inoculation of the organisms, with metastatic abscess formation in lungs and other viscera
  • Melioidosis is most prevalent in Southeast Asia and Australia

  
• Burkholderia cepacia complex


  • Complex comprises at least nine species (B. cenocepacia, B. multivorans, B. vietnamienesis, B. cepacia, and B. doloda)
  • Nosocomial pathogen associated with contaminated equipment, medications, disinfectants
  • Can cause bacteremia, UTI, septic arthritis, respiratory tract infection
  • Important pathogens in patients with cystic fibrosis and Chronic granulomatous disease

  
• Chronic granulomatous disease


  Genetic disorder in which WBCs are unable to kill certain types of bacteria and fungi. A malfunction in the immune system.
• Burkholderia pseudomallei under the microscope

  • Nonfermentative wrinkled colony
  • Bipolar staining on Gram stain
  • Ashdown medium: selective medium, supplemented with colistin (Colistin allows the inhibition of contaminants and other organisms)
  • Deep pink colonies because of the absorption of neutral red in the medium
  • Colonies exhibit an earthy odor
• Burkholderia cepacia

  • Grows well on most lab media
  • Burkholderia cepacia-selective agar (BCSA): selective media
• Burkholderia


  Nosocomial pathogen associated with contaminated equipment, medications, disinfectants
• Burkholderia


  • Can cause bacteremia, UTI, septic arthritis, respiratory tract infection
  • Important pathogens in patients with cystic fibrosis and Chronic granulomatous disease

  
• Burkholderia pseudomallei


  • Nonfermentative wrinkled colony
  • Bipolar staining on Gram stain
  • Grows on Ashdown medium: selective medium, supplemented with colistin
  • Deep pink colonies because of the absorption of neutral red in the medium
  • Colonies exhibit an earthy odor
• Burkholderia cepacia


  • Grows well on most lab media
  • Burkholderia cepacia-selective agar (BCSA)- MOST effective in reducing overgrowth while maintaining good recovery of B. cepacia
  • Pseudomonas cepacia selective agar (PC)
  • Oxidation-fermentative base-polymyxin B, bacitracin lactose agar (OFBL)
  • MacConkey agar
  • Sheep blood agar
• Burkholderia cepacia biochemical reaction

  • Oxidase positive
  • Lysine decarboxylase positive
  • ONPG positive
  • Ornithine decarboxylase negative
  • Oxidizes glucose, maltose, lactose, and mannitol
  • Fail to reduce nitrate to nitrite
• 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
  • 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