NonFermentative GramNegative Bacteria

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Non-Fermentative Gram-Negative Bacteria

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

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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

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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

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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.

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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

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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)

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Most Common Non-Fermentative Gram-Negative
Bacteria

• Pseudomonas aeruginosa (most common)
• Acinetobacter species (second most common)
• Stenotrophomonas maltophilia (third most common)

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Other Clinically Prevalent Gram-Negative
Fermenters

• Pseudomonas stutzeri
• Burkholderia cepacia
• Burkholderia pseudomallei
• Moraxella
• Achromobacter xylosoxidans

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Classification of Clinically Prevalent
Gram-Negative Non-Fermenters

• Pseudomonads
• Pseudomonas aeruginosa
• Pseudomonas stutzeri
• Burkholderia pseudomallei
• Burkholderia cepacia
• Stenotrophomonas maltophilia
• Non-Pseudomonads
• Acinetobacter baumannii
• Moraxella
• Achromobacter xylosoxidans

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Classification of Pseudomonads1

• Pseudomonas aeruginosa (family Pseudomonadaceae)
• rRNA Group I
• Cytochrome oxidase , OF glu , motile, produces
  fluorescent water-soluble pigment (pyoverdin),
  polymyxin B susceptible
• Pseudomonas stutzeri
• rRNA 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.

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Classification of Pseudomonads1

• Burkholderia cepacia (family Burkholderiaceae)
• rRNA Group II
• Cytochrome oxidase , OF glu , motile,
  polymyxin B resistant
• Burkholderia pseudomallei (family
  Burkholderiaceae)
• rRNA Group II
• Cytochrome oxidase , OF glu , motile,
  polymyxin B resistant
• Stenotrophomas maltophilia (family
  Xanthomonadaceae)
• rRNA Group V
• Cytochrome oxidase , OF glu , OF maltose ,
  motile, polymyxin B susceptible
• 1Pseudomonads are separated into five
  taxonomically distinct ribosomal RNA homology
  groups.

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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

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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)

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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

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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

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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)

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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

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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 (sourceskin of
  blood donor)
• P. fluorescens associated with pseudobacteremia
  due to infusion of contaminated solutions

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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)

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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

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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)

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Pseudomonas fluorescens and P. putidaMicrobiologi
cal Properties

• No distinctive colony morphology on sheep blood
  agar
• No pyocyanin production
• No growth at 42oC
• P. fluorescens liquefies gelatin, P. putida does
  not

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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

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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)

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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

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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 , ß-hemolysis on
  sheep blood agar

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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-hemolytic
• 2Non-Saccharolytic, non-hemolytic
• 3Non-Saccharolytic, ß-hemolytic

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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

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Stenotrophomonas maltophilia Natural Habitats

• Widely distributed including moist hospital
  environments (respiratory therapy equipment)
• Colonizer of human respiratory tract in a
  hospital setting

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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)

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Stenotrophomonas maltophilia Types of Infectious
Disease

• Nosocomial including bacteremia,
• meningitis, urinary tract infection,
• endocarditis, ambulatory peritoneal-
• dialysis related peritonitis, and soft tissue
• infections

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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

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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

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Other Clinically Important Gram-Negative
Fermenters

• Pseudomonas stutzeri
• Burkholderia cepacia
• Burkholderia pseudomallei
• Moraxella
• Achromobacter xylosoxidans

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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)

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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)

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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

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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

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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)

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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)

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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

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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)

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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

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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

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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
  )

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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)

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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

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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

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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

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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)

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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

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Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W., Koneman,
• E., Procop, G., Schreckenberger, P., Woods,
• G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 7. The Nonfermentative Gram-Negative
  Bacteria

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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