Nonfermenting Gram Negative Bacilli Notes

Nonfermenting Gram Negative Bacilli

  • MacConkey Positive, Nonfermenters (Oxidative)
    • Oxidase Negative: Acinetobacter and Stenotrophomonas
    • Oxidase Positive: Pseudomonas

General Characteristics

  • Growth in MacConkey as colorless colonies (lactose negative)
  • Fail to acidify O-F Media, overlaid with mineral oil
    • Hugh-Leifson O/F Basal Medium
      • Fermentative: Change in color in both tubes. Enterobacteriaceae, Vibrio, Aeromonas, Plesiomonas and Chromobacterium
      • Oxidative: Change in color in tubes w/out mineral oil (Open tube). Acinetobacter, Stenotrophomonas, Pseudomonas, Burkholderia
      • Nonsaccharolytic : No change in color in both tubes. Alcaligenes, Oligella, Moraxella
  • Fail to acidify TSI Agar
  • Most isolates are Oxidase Positive (except Acinetobacter and Stenotrophomonas)
  • Opportunistic pathogens, not usually found as indigenous microflora of the human body
  • Obligate aerobe organisms

Clinically Significant Species

  • Pseudomonas aeruginosa
  • Acinetobacter spp.
  • Stenotrophomonas maltophilia
  • Burkholderia spp.

Pseudomonas aeruginosa

  • Clinical significance
    • Most commonly isolated species of the genus in clinical specimens.
    • Infections:
      • Bacteremia, often presenting with ecthyma gangrenosum
      • Wound infections
      • Pulmonary disease: VAP, especially among individuals with CF
      • Healthcare-associated urinary tract infections (UTIs)
      • Endocarditis
      • Bone infections
      • Eye infections, including keratitis, ulcers, and endophthalmitis
      • Infections following burns or trauma
      • Rare cases, central nervous system infections, including meningitis
      • Otitis externa, particularly in swimmers or divers
      • Necrotizing rash, referred to as Jacuzzi or hot tub syndrome
    • Biofilm production related to the overproduction of alginate and the mucoid phenotype isolated from CF patients is associated with serious infections.
    • Forms microcolonies in tissue that are associated with quorum-sensing, biofilm-producing strains, which indicates that quorum sensing is also linked to the formation of microcolonies below the surface in severe wounds.
    • Can survive harsh environmental conditions and displays intrinsic resistance to a wide variety of antimicrobial agents
  • Virulence Factors
    • Exotoxins
      • Protease, hemolysins, lecithinase, elastase and Dnase
      • Exotoxin A - most important exotoxin; blocks protein synthesis
    • Endotoxin (LPS), motility, pili and capsule
    • Alginate
      • Polysaccharide polymer in mucoid strains
    • Inherently resistance to a number of antimicrobial agents.
  • Identifying Characteristics
    • Strict aerobic
    • Pigmented
    • Fruity, Grapelike or “corn tortilla-like” Odor (2-aminoacetophenone)
    • Growth at 42°C
    • Grows in Cetrimide Agar
    • Acetamide Positive
  • Pigments
    • Fluorescein (Pyoverdin) – yellow-green / yellow-brown- P.fluorescens/P. putida
    • Pyocyanin – blue
    • Pyorubin – red
    • Pyomelanin – brown/black (P. stutzeri)
  • Treatment
    • P. aeruginosa is usually susceptible to the aminoglycosides, semi-synthetic penicillins such as piperacillin and ticarcillin, third- and fourth-generation cephalosporins (ceftazidime and cefepime, respectively), carbapenems (except ertapenem), and the fluoroquinolones.
    • The incidence of resistance is much higher in healthcare-associated strains of P. aeruginosa.
    • Treatment of severe P. aeruginosa infections usually requires combination therapy, often with ceftazidime or cefepime, piperacillin, or a carbapenem (imipenem or meropenem) with an aminoglycoside (tobramycin or amikacin).

Pseudomonas fluorescens and Pseudomonas putida

  • are of very low virulence, rarely causing clinical disease
  • P. putida has been associated with catheter-related sepsis in patients with cancer; isolation of P. fluorescens from blood culture bottles in asymptomatic patients has been responsible for clusters of pseudobacteremia, probably related to contaminated catheters and catheter-related devices.
  • Both species can grow at 4° C and have been linked to transfusion-associated septicemia.
  • Produce pyoverdin, but neither produces pyocyanin or grows at 42° C
  • cannot reduce nitrate to nitrogen gas, but they can produce acid from xylose
  • Gelatin hydrolysis can be used to differentiate the two species from each other; P. putida is negative and P. fluorescens is positive.
  • They are usually susceptible to the aminoglycosides, polymyxin, and piperacillin, but are resistant to carbenicillin and SXT

Pseudomonas stutzeri

  • a rare isolate and even rarer pathogen in the clinical laboratory, is usually easily recognizable because of its characteristic macroscopic appearance of wrinkled, leathery, adherent colonies that may produce a light-yellow or brown pigment
  • Isolates are ADH negative and starch hydrolysis positive
  • has been reported to be responsible for diseases that include septicemia, meningitis in people with human immunodeficiency virus infection, pneumonia (especially in patients with CF and those who are immunocompromised), endocarditis, postsurgical wound infections, septic arthritis, conjunctivitis, and UTIs.
  • Isolates in vitro are usually susceptible to the aminoglycosides, SXT, ampicillin polymyxin, tetracyclines, fluoroquinolones, and third-generation cephalosporins (e.g., ceftazidime) but resistant to chloramphenicol and the first- and second- generation cephalosporins.

Pseudomonas mendocina

  • found in soil and water but is rarely isolated from human specimens; when it is, it is often considered a contaminant.
  • produces nonwrinkled, flat colonies that may appear with a yellowish-brown pigment, smooth buttery appearance.
  • It is oxidase and ADH positive

Pseudomonas pseudoalcaligenes and Pseudomonas alcaligenes

  • often considered contaminants when isolated from clinical specimens.
  • They are oxidase positive and biochemically negative in many tests for which other Pseudomonas spp. test positive.
  • They grow on MAC agar and are variable in the reduction of nitrates to nitrites or nitrogen gas.

Pseudomonas luteola and Pseudomonas oryzihabitans

  • These two pseudomonads are rarely isolated from humans but have been isolated from wounds, abscesses, blood cultures, peritoneal and chronic ambulatory peritoneal dialysis (CAPD) fluids, and other sources.
  • They have also been implicated in cases of peritonitis and possibly meningitis, although often in association with each other or with other bacteria.
  • Both of these pseudomonads are gram-negative, nonfermentative, oxidase-negative bacilli. They are catalase positive and motile, oxidize glucose, grow on MAC agar, and often produce an intracellular nondiffusible yellow pigment. Both species typically produce wrinkled or rough colonies at 48 hours
  • P. luteola can be differentiated from P. oryzihabitans by positive o- nitrophenyl-β d-galactopyranoside (ONPG) and esculin hydrolysis tests.

Acinetobacter spp.

  • Clinical significance
    • Associated with ventilators, humidifiers, catheters infection
    • Opportunistic (1-3% of all nosocomial infection, 2nd most commonly isolated non-fermenter); burns, trauma
    • About 25% of adults present in skin; 7% in pharynx
    • Ubiquitous; found in soil, water, foodstuffs
    • Related infections: UTI, Pneumonia, endocarditis, meningitis and cellulitis
  • Identifying Characteristics
    • A. baumannii
      • Saccharolytic (glucose oxidizing), nonhemolytic strains.
    • A. lwoffii
      • Asaccharolytic, nonhemolytic strains.
    • A. haemolyticus
      • Asaccharolytic , β-hemolytic strains.
    • Acinetobacter spp. are plump coccobacilli that tend to resist alcohol decolorization, thus sometimes appearing gram positive.
    • A. baumannii may exhibit purplish hue (due to lactose oxidation)
    • A. baumannii colony appears with a blue-grey (cornflower blue) center in EMB.
    • Catalase + and Oxidase -
  • Approach to Identification
    • commercial identification systems including MicroScan and Vitek 2. These automated identification systems can readily identify A. baumannii and S. maltophilia.
    • MALDI-TOF mass spectrometry has been shown to reliably and accurately identify Acinetobacter species where other conventional (i.e., biochemical) methods are unable to do so.
    • Nucleic acid–based methods, including amplification of the 16S ribosomal RNA (rRNA) gene, are routinely used to identify Acinetobacter spp.

Stenotrophomonas maltophilia

  • Clinical significance
    • When S. maltophilia is isolated from clinical specimens, it is initially regarded as a saprophyte or colonizer.
    • can quickly colonize the respiratory tract of hospitalized patients
    • endocarditis, especially in a setting of prior intravenous drug abuse or heart surgery; wound infections, including cellulitis and ecthyma gangrenosum; bacteremia; and, rarely, meningitis and UTIs
    • S. maltophilia is rarely associated with lower respiratory tract infections, although it has been isolated from 6.4% to 10.2% of patients with CF.
    • The most important risk factors in affected individuals were immunosuppression, hospitalization, especially in an ICU, and the presence of a central venous catheter.
  • Identifying characteristics
    • Oxidase negative
    • Catalase, DNase positive
    • Esculin and Gelatin hydrolysis (+)
    • Lysine decarboxylase positive
    • lavender green to light purple pigmentation in BAP and Ammonia-like smell
    • MAC – Colonies exhibit blue color
    • Oxidizes glucose: W(+/-)
    • Oxidizes maltose: S(+)
    • susceptible to SXT, so this is the drug of choice for most infections

Antimicrobial Resistance

  • Acinetobacter spp. and S. maltophilia, are a significant health care concern because of their intrinsic mechanisms of antimicrobial resistance, which impart broad-spectrum antibiotic resistance
  • The World Health Organization has designated carbapenem-resistant A. baumannii (CRAB) as one of the critical priority pathogens for new antimicrobial drug development.
  • Acinetobacter spp. are classified as MDR or extremely drug resistant (XDR) if they have chromosomally encoded AmpC beta- lactamases, mutations in porin channels, and overexpression of bacterial efflux pumps.
  • S. maltophilia exhibits resistance to a wide range of antibiotics, including beta- lactams, cephalosporins, aminoglycosides, tetracyclines, and polymyxins, through reduced membrane permeability, various enzymes, or efflux pumps.

Burkholderia

  • B. cepacia
  • B. pseudomallei
  • B. gladioli
  • B. mallei

Burkholderia cepacia

  • Clinical significance
    • Pneumonia in patients with cystic fibrosis or chronic granulomatous disease
    • Isolated from irrigation fluids, anesthetics, nebulizers, detergents and disinfectants.
    • Onion bulb rot in plants and foot rot in humans
    • Intrinsic resistance to antibiotics
  • Identifying characteristics
    • Smooth, non-wrinkled and slightly raised; dirtlike odor in BAP; Produces a non-fluorescing yellow/green pigment
    • NLF; become dark pink to red after 4-7 days in MacConkey Agar
    • Most strains are ONPG positive
    • Specific selective media, such as Burkholderia cepacia selective agar (BCSA), Pseudomonas cepacia (PC) agar, or oxidative– fermentative base–polymyxin B–bacitracin– lactose (OFPBL) agar, may be used to isolate B. cepacia complex and Ralstonia spp. from the respiratory secretions of CF patients

Burkholderia pseudomallei

  • Clinical significance
    • “Melioidosis” an aggressive, granulomatous, pulmonary disease caused by ingestion, inhalation, or inoculation of the organisms, with metastatic abscess formation in lungs and other viscera
  • Identifying characteristics
    • Bipolar staining (safety pin) in Gram Stain
    • Smooth to wrinkled colonies in BAP
    • Wrinkled and deep pink in Ashdown media
    • “Earthy odor”
    • “Sniffing” of plate is discouraged (work on BSC)
    • Although isolates may be susceptible in vitro to many antimicrobial agents, including SXT, chloramphenicol, tetracycline, semi-synthetic penicillins, and ceftazidime, the clinical response to therapy is usually slow, and relapses are common.

Burkholderia mallei

  • Glanders- zoonosis affecting horses,mules,donkeys. Formation of nodular lesions in lungs. Coughing, fever and release of infectious nasal discharge.
  • Potential bioterrorism agent
  • Severe local suppurative or acute pulmonary infections
  • Non-motile; non-pigmented colonies; no distinct odor
  • The drugs to consider testing for both B. mallei and B. pseudomallei should be ceftazidime, imipenem, doxycycline, and tetracycline.

Other Nonfermenters

Alcaligenes faecalis

  • moist hospital environment, a-hemolytic with fruity odor similar to apples and strawberries on BAP (feather-edged, non pigmented)
  • Grows well on MAC and motile by peritrichous flagella
  • Grows in 6.5% NaCl broth; oxidase and catalase positive
  • Becoming nosocomial alert due to isolation of antibiotic resistant strains

Oligella

  • Colonize distal urethra and may cause serious and active infection
  • Cannot grow on MacConkey Agar
  • urethralis and ureolytica – common species; the latter is motile and urease positive; both are oxidase positive

Moraxella lacunata

  • Angular conjunctivitis (catarrhal conjunctivitis)
  • BAP – small colonies and pit the agar; no growth on MAC
  • Catalase, oxidase and nitrate reduction positive

Shewanella

  • Shewanella algae and S. putrefaciens
  • have been recovered from various human specimens, including specimens from abscesses and traumatic ulcers, otitis media, ocular infections, osteomyelitis, peritonitis, and septicemia, but are usually present in mixed culture.
  • Environmental sources, such as stagnant water, natural gas (petroleum), brine, and spoiled dairy products, meat, and fish may contain S. putrefaciens. S. algae is more frequently isolated from clinical specimens than S. putrefaciens, whereas S. putrefaciens is more frequently isolated from environmental sources.
  • Colonies of Shewanella spp. are often mucoid and can produce a tan to brown pigment causing greenish discoloration of SBA.
  • Both species are motile, ornithine decarboxylase and nitrate reductase positive, and produce profuse H2S in TSIA, resembling H2S producers of the order Enterobacterales.
  • S. algae requires NaCl (halophilic) and is asaccharolytic, whereas S. putrefaciens is nonhalophilic and saccharolytic.

Sphingomonas

  • Sphingomonas paucimobilis can be isolated from many water sources, including swimming pools, as well as from hospital equipment and laboratory supplies.
  • S. paucimobilis and S. parapaucimobilis.
  • septicemia, meningitis, leg ulcers, empyema, and splenic and brain abscesses
  • The yellow-pigmented S. paucimobilis does not grow on MAC agar and requires more than 48 hours for culture on SBA
  • Isolates are weakly oxidase positive (some strains may be negative), motile at 18° to 22° C but not at 37° C, indole negative, and oxidizers. S. parapaucimobilis resembles S. paucimobilis, except that isolates of S. parapaucimobilis are H_2S positive by the lead acetate method, Simmon citrate positive, and DNase negative.

Identifcation Methods

  • Acetamide utilization
    • Determines the ability of an organism to use acetamide as the sole source of carbon Indicator: Bromthymol blue (acetamide → ammonia)
    • Positive: Deamination of acetamide resulting in a blue color Pseudomonas aeroginusa
    • Negative: No color change Stenotrophomonas maltophia, Pseudomonas fluorescens, Pseudomonas putida
  • Growth at 42°C
    • Tests the ability of an organism to grow at 42°C
    • Positive: Good growth at both 35°C and 42°C Pseudomonas aeroginusa, Stenotrophomonas maltophilia
    • Negative: No growth 42°C but good growth at 35°C Pseudomonas fluorescens, Pseudomonas putida