03-Non-Fermentative Gram-Negative Bact Eria v1- 3 2

Non-Fermentative Gram-Negative Bacteria

Dr. John R. WarrenDepartment of PathologyNorthwestern University

Feinberg School of MedicineJune 2007

Non-Fermentative Gram-Negative Bacteria

• Nonfermentative for glucose (TSI = alkaline over no reaction)

• Oxidative for glucose (Hugh-Leifson O-F glucose positive)

• Asaccharolytic for glucose (Hugh-Leifson O-F glucose negative)

• Cytochrome oxidase positive or negative

Hugh-Leifson OF versus TSI Medium

• TSI AGAR SLANT

Total protein = 2.6 g%

Total carbohydrate = 2.1 g%

Protein to carbohydrate (w/w) = 1.2• OF BROTH MEDIUM

Total protein = 0.2 g%

Total carbohydrate = 1.0 g%

Protein to carbohydrate (w/w) = 0.2

Growth of Gram-Negative Non-Fermenters on TSI Agar Slants

• Non-fermentative gram-negative bacteria grow abundantly within 16-18 hours of inoculation on the surface of TSI agar slants.

• Non-fermentative gram-negative bacteria neither grow in nor acidify the deep of TSI slants.

Growth of Oxidative Non-Fermenters in

Hugh-Leifson OF Broth

• Growth with acidification of broth in Hugh-Leifson tube not sealed by mineral oil (oxidative tube)

• No growth in Hugh-Leifson tube sealed by a layer of mineral oil (fermentative tube)

• Substrates utilized: glucose, lactose, maltose, xylose, mannitol, sucrose, adonitil, dulcitol

Growth of Asaccharolytic Non-Fermenters in Hugh-Leifson OF Broth

• Growth without acidification but with alkalinization of broth in Hugh-Leifson tube not sealed by mineral oil (oxidative tube)

• No growth in Hugh-Leifson tube sealed by a layer of mineral oil (fermentative tube)

Most Common Non-Fermentative Gram-Negative

Bacteria

• Pseudomonas aeruginosa (most common)

• Acinetobacter species (second most common)

• Stenotrophomonas maltophilia (third most common)

Other Clinically Prevalent Gram-Negative Fermenters

• Pseudomonas stutzeri

• Burkholderia cepacia

• Burkholderia pseudomallei

• Moraxella

• Achromobacter xylosoxidans

Classification of Clinically Prevalent Gram-Negative Non-Fermenters

PseudomonadsPseudomonas aeruginosaPseudomonas stutzeriBurkholderia pseudomalleiBurkholderia cepaciaStenotrophomonas maltophilia

Non-PseudomonadsAcinetobacter baumannii MoraxellaAchromobacter xylosoxidans

Classification of Pseudomonads1

Pseudomonas aeruginosa (family Pseudomonadaceae)rRNA Group ICytochrome oxidase +, OF glu +, motile, produces fluorescent water-soluble pigment (pyoverdin), polymyxin B susceptible

Pseudomonas stutzerirRNA Group I (family Pseudomonadaceae)Cytochrome oxidase +, OF glu +, motile, does not produce pyoverdin, polymyxin B susceptible

1Pseudomonads are separated into five taxonomically distinct ribosomal RNA homology groups.

Classification of Pseudomonads1

Burkholderia cepacia (family Burkholderiaceae)rRNA Group IICytochrome oxidase +, OF glu +, motile, polymyxin B resistant

Burkholderia pseudomallei (family Burkholderiaceae)rRNA Group IICytochrome oxidase +, OF glu +, motile, polymyxin B resistant

Stenotrophomas maltophilia (family Xanthomonadaceae)rRNA Group VCytochrome oxidase –, OF glu +, OF maltose ++, motile, polymyxin B susceptible

1Pseudomonads are separated into five taxonomically distinct ribosomal RNA homology groups.

Classification of Non-Pseudomonads

Acinetobacter baumanii (family Moraxellaceae)Cytochrome oxidase –, OF glu +, OF lactose ++, non-motile

Moraxella (family Moraxellaceae)Cytochrome oxidase +, OF glu – (asaccharolytic), non-motile

Achromobacter xylosoxidans (family Alcaligenaceae)Cytochrome oxidase +, OF glu +, OF xylose +, motile

Pseudomonas aeruginosa: Natural Habitats

• Aqueous environments: disinfectants, soaps, eye drops, irrigation fluids, dialysis fluids, hydrotherapy baths, and showerheads (nosocomial sources)

• Surface of raw fruits and vegetables (GI colonization of profoundly immunosuppressed individuals with bacteremia)

Pseudomonas aeruginosa: Natural Habitats

• Swimming pools, hot tubs, contact lens solution, cosmetics, illicit injectable drugs (community sources)

• Infrequently as microbial flora of human skin and mucosal surfaces in healthy individuals

Pseudomonas aeruginosa: Modes of infection

• Exposure to contaminated solutions and medical devices

• Introduction by penetrating wounds

• Person-to-person transmission (presumed)

• Ingestion of contaminated raw fruits or vegetables

Pseudomonas aeruginosa: Types of infectious disease

• Nosocomial infections--pneumonia (leading cause), wound infection (especially burn wounds with high rate of bacteremia and mortality), urinary tract infection, peritonitis (chronic ambulatory peritoneal dialysis), bacteremia

• Malignant otitis externa (diabetics and elderly with destruction of underlying soft tissue and bone)

Pseudomonas aeruginosa: Types of Infectious Disease

• Corneal ulcers due to contaminated contact lens solution

• Osteomyelitis of foot heel in children secondary to a puncture wound

• Endocarditis in intravenous drug users

• Mucoid (alginate-producing) variants in chronic airway infection of cystic fibrosis patients

Pseudomonas fluorescens and P. putida: Types of Infectious Disease• Fluorescent group of Pseudomonas = P.

aeruginosa, P. fluorescens, P. putida• P. fluorescens and P. putida generally not

significant clinically• P. fluorescens and P. putida associated with

transfusion acquired bacteremia (source=skin of blood donor)

• P. fluorescens associated with pseudobacteremia due to infusion of contaminated solutions

Pseudomonas aeruginosa: Microbiological Properties

• Straight or slightly curved, slender gram-negative rods

• Oxidize rather than ferment D-glucose (OF glucose +)

• Grow anaerobically using nitrate or arginine as terminal electron acceptors (occasional strains)

Pseudomonas aeruginosa: Microbiological Properties

• Growth on 5% sheep blood or chocolate agar in carbon dioxide or ambient air

• Colonies spreading and flat with a pearl-like sheen (pearlescent) and grape-like odor

• Positive for cytochrome oxidase and catalase

Pseudomonas aeruginosa: Microbiological Properties

• Yellow-green or yellow-brown water-soluble pigment pyoverdin

• Blue water-soluble pigment pyocyanin (P. aeruginosa only fluorescent species that produces pyocyanin)

• Pyoverdin and pyocyanin combined give a distinctive bright green color

• Growth at 42oC (P. aeruginosa only fluorescent species that grows at 42oC)

Pseudomonas fluorescens and P. putida:Microbiological

Properties

• No distinctive colony morphology on sheep blood agar

• No pyocyanin production

• No growth at 42oC

• P. fluorescens liquefies gelatin, P. putida does not

Acinetobacter Species: Natural Habitats

• Widely distributed including the hospital environment

• Able to survive on moist and dry surfaces including human skin

• More frequently colonizing than infecting

Acinetobacter Species: Modes of Infection

• Colonization of hospital patients by environmental sources

• Introduction of organisms into normally sterile sites by medical instrumentation (intravenous or urinary catheters, endotracheal tubes or tracheostomies, respiratory care equipment) in debilitated hospital patients (antibiotic treatment, surgery, intensive care units, surgery)

Acinetobacter Species: Types of Infectious Disease

• Nosocomial infections of the respiratory tract, urinary tract, and wounds (including catheter wounds) often with progression to bacteremia

• Sporadic cases of ambulatory peritoneal-dialysis related peritonitis, endocarditis, meningitis, arthritis, and osteomyelitis

Acinetobacter Species: Microbiological Properties

• Gram-negative coccobacillary rods occurring singly and in Neisseria-like pairs

• Oxidize rather than ferment D-glucose(OF glucose +)

• Neither oxidize nor ferment D-glucose(OF glucose –)

• A. baumannii complex/OF glu + OF lac +, non-hemolytic

• A. lwoffii/OF glu – OF lac –, non-hemolytic• A. haemolyticus/OF glu – OF lac –, β-hemolysis on

sheep blood agar

Acinetobacter: Genomospecies

• Twenty-one different genomospecies based on DNA-DNA hybridization

• Genomospecies 1, 2, 3, and 13: A. calcoaceiticus-baumanii complex (A. baumanii1)

• Genomospecies 8/9: A. lwoffi2

• Genomospecies 4: A. haemolyticus3

1Saccharolytic, non-hemolytic2Non-Saccharolytic, non-hemolytic3Non-Saccharolytic, β-hemolytic

Acinetobacter Species: Microbiological Properties

• Non-lactose fermenter but colonies on MacConkey show distinctive pinkish hue

• Growth on 5% sheep blood or chocolate agar in carbon dioxide or ambient air (MacConkey incubated in ambient air)

• Smooth, opaque, gray-white colonies, slightly smaller than Enterobacteriaceae

• Cytochrome oxidase negative

• Non-motile

Stenotrophomonas maltophilia: Natural Habitats

• Widely distributed including moist hospital environments (respiratory therapy equipment)

• Colonizer of human respiratory tract in a hospital setting

Stenotrophomonas maltophilia: Modes of Infection

• Colonization of hospital patients by environmental sources

• Introduction of organisms into normally sterile sites by medical instrumentation (similar to Acinetobacter)

Stenotrophomonas maltophilia: Types of Infectious Disease

Nosocomial including bacteremia,

meningitis, urinary tract infection,

endocarditis, ambulatory peritoneal-

dialysis related peritonitis, and soft tissue

infections

Stenotrophomonas maltophilia: Microbiological Properties

• Short to medium-size, straight gram-negative rods

• Glucose oxidizer (OF glu +) with occasional negative strains (~15%)

• Strong maltose oxidizer (OF mal +) (100%) (more intense than OF glu + reaction)

• Colonies on sheep blood agar rough and lavender-green with ammonia odor

Stenotrophomonas maltophilia: Microbiological Properties

• Cytochrome oxidase negative

• Positive for DNase (unlike most other glucose-oxidizing gram-negative bacilli)

• Positive for lysine decarboxylase (unlike most other glucose-oxidizing gram-negative bacilli)

• Resistant to most antibiotics except trimethoprim-sulfamethoxazole

Other Clinically Important Gram-Negative Fermenters

• Pseudomonas stutzeri

• Burkholderia cepacia

• Burkholderia pseudomallei

• Moraxella

• Achromobacter xylosoxidans

Pseudomonas stutzeri: Natural Habitats and Clinical Infections

• Environmental sources (especially aqueous) as with Pseudomonas aeruginosa

• Bacteremia and meningitis reported in immunosuppressed individuals

• Pneumonia in alcoholics• Endophthalmitis following cataract surgery

and bacteremia due to contaminated hemodialysis fluid (iatrogenic infections)

Pseudomonas stutzeri: Microbiology

• Cytochrome-oxidase positive gram-negative rods forming distinctive dry, wrinkled colonies (1-6 mm) on blood agar

• Key reactions: OF glucose + and OF lactose –, arginine dihydrolase –, ability to grow in 6.5% NaCl broth, gas from nitrate, and no growth with cetrimide (growth of P. aeruginosa cetrimide-resistant)

Burkholderia cepacia: Natural Habitats and Clinical Infections

• Soil and environmental water• Unpasteurized dairy products• Contaminated respiratory therapy equipment,

disinfectants, medications, and mouthwash• Nosocomial pathogen causing bacteremia (most

often associated with indwelling vascular catheters and polymicrobial), respiratory infections (ventilator-associated pneumonia), septic arthritis, urinary tract infections

Burkholderia cepacia: Clinical Infections

• Second leading cause of bacteremia and third most common cause of pneumonia in chronic granulomatous disease of childhood

• Chronic pneumonia in cystic fibrosis (3-7%) with rapid decline in lung function, transmissibility of infection via close personal contact (nosocomial spread), and poor outcome with lung transplantation

• “Cepacia Syndrome” Rapid and fatal clinical deterioration with necrotizing granulomatous pneumonia

Burkholderia cepacia: Clinical Infections

• Skin and soft tissue infections with burn or surgical wounds, in soldiers with prolonged foot immersion in water

• Isolation from blood cultures of multiple patients over short period of time should be investigated for “pseudobacteremia” (contaminated infusion or disinfectant fluid)

Burkholderia cepacia: Microbiology

• Burkholderia cepacia complex (BCC): nine genomic species (genomovars) including B. cepacia (genomovar I)

• DNA PCR and microarray technology under development for laboratory identification

• Cytochrome-oxidase positive gram-negative rods forming smooth, round, opaque, and yellow colonies (genomovar I) on blood agar

• Wet, runny, and mucoid colonies when recovered for cystic fibrosis sputum (requires at least 3 days for appearance)

Burkholderia cepacia: Microbiology

• Bright pink colonies on MacConkey agar after 4 days of incubation due to lactose oxidation

• Positive for lysine decarboxylation (genomovar I) (DNase negative, vs. Stenotrophomonas maltophilia that is DNase positive)1

• Saccharolytic with OF glu + and OF xyl + (100%), OF lac + and OF suc + (91%) (acidify slant and deep of TSI slant after 4-7 days be oxidation of glucose, lactose, and sucrose)

• ONPG +1Among non-fermentative gram-negative bacteria, only B.

cepacia and S. maltophilia lysine positive

Burkholderia cepacia: Use of Selective Agar

• Pseudomonas cepacia agar (PCA): selective containing crystal violet, polymyxin B, and bacitracin; differential with B. cepacia forming a pink-red color due to pyruvate metabolism.

• Utilized to recover B. cepacia from cystic fibrosis sputum

• Isolation from PCA by single colony pick and ID by Vitek-2 but ~15% misidentification

• Confirmation of Vitek-2 ID by manual identification (? Role of PCR for ID of genomovariants)

Burkholderia pseudomallei: Natural Habitats and Clinical

Infections• Primarily in tropical and subtropical areas (Southeast

Asia, tropical Australia, Indian subcontinent, China)• Prevalent in rice-growing regions of Thailand and the

Top End of the Northern Territory of Australia• Human infection and disease (melioidosis) is by

inhalation of the organism or through contact with broken skin (cut or abrasion)

• Pneumonia most common type of infection with progressive pulmonary consolidation in severe cases

• Mortality increases with bacteremia• Pneumonia without septic shock has a reported 4%

mortality, pneumonia with septic shock 65% mortality

Burkholderia pseudomallei: Clinical Infections

• Disseminated abscess formation with skin pustules, septic arthritis, osteomyelitis, spleen, liver, kidneys, and prostate

• Silent asymptomatic infection for years and then reactivation (like TB) to active disease documented for as long as 29 years

• “Vietnamese time bomb” in returned US soldiers

• Travelers to and emigrants from endemic areas

Burkholderia pseudomallei: Microbiology

• Cytochrome oxidase-positive gram-negative rod forming dry wrinkled colonies similar to Pseudomonas stutzeri after a few days on blood agar

• Saccharolytic (OF glu +) including OF lac + (P. stutzeri OF lac –), arginine dihydrolase + (P. stutzeri –), + gas from nitrate (P. stutzeri –), and lysine decarboxylase – (Burkholderia cepacia +)

Moraxella: Natural Habitats and Clinical Infections1

• Saprophytic on human skin and mucous membranes

• Most freqently isolated species by culture M. nonliquefaciens is a component of normal respiratory flora

• Ocular pathogens (conjunctivitis, keratitis) and unusual causes of invasvie infection (meningitis, bacteremia, endocarditis, and arthritis)

1Excludes Moraxella catarrhalis (identified in the laboratory using Neisseria protocols)

Moraxella: Microbiology

• Cytochrome-oxidase positive gram-negative or gram-variable Neisseria-like diplococci, forming small (0.5-1mm) colonies on blood agar (24-48 hr), smooth, translucent to semiopaque in appearance, occasional strains show pitting of agar

• Non-motile, indole negative, and asaccharolytic• Species identification generally not performed

because given the similarity of pathogenic signficance of all species

Psychrobacter (Moraxella) phenylpyruvicus

• Exceedingly rare cause of infection BUT commercial identification systems can misidentify Brucella as Psychrobacter (Moraxella) phenylpyruvicus

• Brucella an obligate pathogen (facultative intracellular parasite) and requires accurate identification

Psychrobacter phenylpyruvicus versus Brucella Brucella P. phenylpyruvicus

Oxidase – – Motility – –Urease + +

Gram stain Tiny coccobacilli Broad diplococci OF Xylose +1 –1

PDA – +1Brucella also OF glu +, P. phenylpyruvicus is

uniformly asaccharolytic

Achromobacter xylosoxidans: Natural Habitats and Clinical

Infections• Environmental organisms (soil and water) and

component of endogenous flora of GI tract• Most commonly reported infection:

bacteremia, often associated with intravascular catheters

• Also causes biliary tract sepsis, meningitis, pneumonia, peritonitis, osteomyelitis, prosthetic knee infection, and prosthetic valve endocarditis

• Nosocomial outbreaks due to contaminated solutions (IV, irrigation, hemodialysis)

Achromobacter xylosoxidans: Microbiology

• Cytochrome oxidase positive, indole negative, motile, saccharolytic non-fermenter

• OF glu + and OF xylose + (OF negative for mannitol, lactose, sucrose, and maltose)

• Nitrate reduction + (60% gas producing), citrate +, urease –

Recommended Reading

Winn, W., Jr., Allen, S., Janda, W., Koneman,

E., Procop, G., Schreckenberger, P., Woods,

G.

Koneman’s Color Atlas and Textbook of

Diagnostic Microbiology, Sixth Edition,

Lippincott Williams & Wilkins, 2006:• Chapter 7. The Nonfermentative Gram-Negative

Bacteria

Recommended Reading

Murray, P., Baron, E., Jorgensen, J., Landry, M. Pfaller, M.Manual of Clinical Microbiology, 9th Edition, ASM Press, 2007:• Blondel-Hill, E., Henry, D.A., and Speert, D.P. Chapter 48.

Pseudomonas• LiPuma, J.J., Currie, B.J., Lum, G.D., and Vandamme, P.A. Chapter

49. Burkholderia, Stenotrophomonas, Ralstonia, Cupriavidus, Pandoraea, Brevundimonas, Comamonas, Delftia, and Acidovorax.

• Schreckenberger, P.C., Daneshvar, M.I., and Hollis, D.G. Chapter 50. Acinetobacter, Achromobacter, Chryseobacterium, Moraxella, and Other Nonfermentative Gram-Negative Rods