Nonfermenters

NON FERMENTERS

MAJ INAM DANISH KHAN

PROLOGUEBacterial metabolismIntroduction to nonfermentersClassificationIdentification

PseudomonasAcinetobacterStenotrophomonasBurkholderiaMoraxellaSphingomonasOther nonfermenters

NonfermentersEpidemiologyTransmissionClinical FeaturesDiagnosisTreatmentResistance

BACTERIAL METABOLISM• Aerobic respiration• Oxygen as exogenous electron acceptor• High energy yield (Electron transport)• Glycolysis, Krebs Cycle, Electron transport chain

• Anaerobic respiration• Other molecules as electron acceptors • Limited energy yield (Electron transport)

• Fermentation (Mostly anaerobic)• Endogenous electron acceptor• Limited energy made available• Enterobacteriaceae, Aeromonas, Vibrio

Growth atmosphere Property ExampleObligate aerobe Requires atmospheric O2 Pseudomonas aeruginosa

Mycobacterium tuberculosisMicrococcus

Obligate anaerobe Will not tolerate O2 Bacteroides fragilisClostridium haemolyticumClostridium novyi type B

Facultative anaerobe Grows aerobically but can grow anaerobically

Staphylococcus aureusE. coli

Aerotolerant anaerobe Anaerobic but tolerates O2 Clostridium perfringensClostridium carnisClostridium histolyticum

Microaerophilic Prefers reduced O2 levels Campylobacter jejuniHelicobacter pyloriStreptococcus intermediusBorrelia burgdorferiLactobacillus

Capnophilic Prefers increased CO2 Neisseria

BACTERIAL METABOLISM

AEROBIC METABOLISM• Embden Meyerhoff Parnas pathway• Entner Duodoroff pathway• Pseudomonas• Agrobacterium• Rhizobium

• Pentose phosphate pathway• E coli• Bacillus subtilis• Enterococcus faecalis• Leuconostoc mesenteroides

CARBOHYDRATE CATABOLISM

NONFERMENTERS• Heterogenous - Aerobic non sporing GNB• 15 families - Evolving taxonomy

• Ubiquitous in environment• Human body – Dry surfaces (Skin), oropharynx• Environment - Sink, showerheads, floor mop, swimming

pools, hot tubs, flowers, raw fruits, vegetables• Hospital – Moist surfaces (soap, disinfectant, respiratory

equipment, eye drops, contact lens solution, irrigation fluids, hydrotherapy baths)

NONFERMENTERS• Predominantly opportunistic/nosocomial• Septicemia, meningitis, osteomyelitis, wound infections• Immunosuppression, foreign body implantation, trauma,

prolonged antimicrobial therapy, underlying disease

• Numerous virulence factors• Resistant to antimicrobials/disinfectants• Beta lactams, aminoglycosides, carbapenems• No CLSI criteria, E test/MIC values given• Facultative anaerobes & Microaerophiles 70-80%• Aerobic nonfermenters 10-15%• Hemophilus & related organisms 10-15%• Unusual bacilli <1%

PseudomonasAcinetobacter

StenotrophomonasBurkholderia

Moraxella

SphingomonasBrevundimonas

ComamonasFlavimonas

Roseomonas

AlcaligenesAcidovoraxBalneatrix

ChryseobacteriumFlavobacterium

MyroidesAgrobacterium

OchrobacteriumMethylobacteriumSphingobacterium

WeeksellaShewanella

OligellaDelftia

Pandoraea

AchromobacterPsychrobacter

Micrococcus luteus

CLASSIFICATION• Oxidative NF

• Growth + acid in OX tube• Oxidize glucose, lactose, maltose,

xylose, mannitol , sucrose, adonitol, dulcitol

• Asaccharolytic NF• Growth + alkaline in OX tube

• Pseudomonads• rRNA gp I

• Pseudomonas• rRNA gp II

• Burkholderia• Ralstonia

• rRNA gp III• Comamonas• Acidovorax• Delftia

• rRNA gp IV• Brevundimonas

• rRNA gp V• Stenotrophomonas

CLASSIFICATION• Oxidase negative group

• Acinetobacter• Bordetella• Massilia

• Oxidase +, Indole -, asaccharolytic, coccoid NF• Moraxella• Psychrobacter• Oligella

• Oxidase +, Indole -, asaccharolytic, rod shaped NF• Alcaligenes• Achromobacter• Myroides• Neisseria• Ralstonia

• Oxidase +, Indole -, motile, saccharolytic, rod shaped NF• Achromobacter xylosoxidans• Ochrobactrum• Rhizobium• Agrobacterium• Sphingomonas• Herbaspirillum

CLASSIFICATION• Oxidase +, Indole -, nonmotile,

saccharolytic, coccoid/ rod shaped NF• Sphingobacterium• Pedobacter• Psychrobacter immobilis

• Halophilic NF• Shewanella• Alishewanella• Halomonas

• Pink pigmented NF• Methylobacterium• Roseomonas

• Oxidase +, Indole +, motile/nm, yellow pigmented NF• Flavobacterium• Chryseobacterium• Myroides• Empedobacter• Bergeyella• Weeksella• Balneatrix

IDENTIFICATION• Collection of samples• Human• Autopsy• Environmental

• Microscopy• Morphology• Motility

• Culture• Agar cultures

• Differential media• Selective media• Colony characteristics• Pigment

• Blood cultures• Broth cultures

• Biochemical tests• Oxidase• Carbohydrate utilisation• TSI• Indole• Citrate • Urease• OF tests• Amino acid decarboxylation• Esculin hydrolysis• Gelatin hydrolysis• Phenylalanine deaminase

IDENTIFICATION• Antimicrobial sensitivity• Disk diffusion• Molecular probes• Automated systems

• Immunological tests• Card agglutination• Immunochromatography• EIA• Immunofluorescence assay

• Molecular microbiology• Amplification methods

• PCR• qPCR• Nested PCR• TMA• bDNA• NASBA

• Non amplification methods• Nucleic acid hybridization

• Automated DNA sequencing

• Automated systems• Vitek• Vitek 2• Microscan• Phoenix• Vitek MS (MALDI-TOF)• Remel• Crystal• API

• Detection of resistance• Phenotypic• Genotypic

• Animal inoculation

• Typing• Phenotypic• Serotyping• Biotyping• Antibiograms• Bacteriocin typing• Protein typing• Phage typing• MALDI TOF mass spectrometry• FAME gas chromatography

• Genotypic• PFGE• Ribotyping• Multilocus sequence typing• RAPD analysis• Repetitive sequence based PCR• RFLP• Chromosomal sequence FP

IDENTIFICATION

IDENTIFICATION: CDC• McConkey• Oxidase• Hugh Leifson OF test• Motility• Nitrate reduction/denitrif• Urease• Esculin hydrolysis• Indole• Decarboxylation reactions• Pigment production• Phenylalanine deaminase• Growth in cetrimide• Growth at 420 C

• Poor growth, lactose -• +/- (Plesiomonas)• (Stenotrophomonas -/-)• +/-, polar flagella• +/- (Pseudomonas)• +/-• +/-• +/-, Ehrlichs more sensitive• Lysine, arginine, ornithine• +/-• +/-• +/-• +/- (Pseudomonas)

E. coli, Yersinia, Aeromonas, Vibrio  A/A Salmonella, Shigella, Edwardsiella K/A Nonfermenters such as Pseudomonas and others  K/K Erroneous inoculation of a Gram + or some other organism A/K

IDENTIFICATION• Pigment production

• Pseudomonas aeruginosa• Pseudomonas fluorescens• Pseudomonas putida• Stenotrophomonas (yellow, green)• Moraxella (yellow)• Sphingomonas (yellow)• Agrobacterium (yellow colony)• Chryseobacterium (yellow colony)• Delftia

• Weakly motile• Sphingomonas• Moraxella• Ralstonia• Pandoraea

• Nonmotile• Acinetobacter

• Oxidase negative• Pseudomonas luteola• Pseudomonas oryzihabitans• Burkholderia cepacia• Burkholderia gladioli• Stenotrophomonas• Acinetobacter baumanii• Acinetobacter lwoffi• Sphingomonas

• ONPG positive• Pseudomonas• Burkholderia• Rhizobium

IDENTIFICATION• Urease positive

• Pseudomonas stutzeri• Psychrobacter• Rhizobium• Ochrobacter• Achromobacter• Bergeyella (Rapid)• Myroides (Rapid)• Oligella (Rapid)

• Esculin hydrolysis• Pseudomonas luteola• Ochrobactrum anthropi• Achromobacter• Rhizobium radiobacter• Brevundimonas vesicularis• Alishewanella

• Gelatin hydrolysis• Weeksella• Myroides• Oligella

• Nitrate to nitrite• Pseudomonas• Achromobacter• Comamonas• Sphingobacterium• Psychrobacter

• Nitrate to gas• Pseudomonas stutzeri• Ochrobactrum anthropi• Achromobacter xylosoxidans

IDENTIFICATION• Asaccharolytic

• Moraxella• Psychrobacter• Oligella• Alcaligenes• Achromobacter• Myroides• Neisseria• Ralstonia

• Lysine decarboxylase • Stenotrophomonas• Burkholderia

• Arginine decarboxylase• Burkholderia pseudomallei• Ochrobactrum anthropi• Achromobacter

• Polymyxin B resistance• Burkholderia• Ralstonia• Pandoraea

PSEUDOMONAS• Nosocomial UTI• Nosocomial RTI• Peritonitis (CAPD)

• Bacteremia• Endocarditis• Osteomyelitis• Arthritis• Meningitis• Brain abscess• Otitis externa• Ocular infections

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• Wound infections• Ecthyma gangrenosum• Burn wounds

• Neutropenia• Cystic fibrosis• Chronic lung disease • Acute leukaemia• Diabetes mellitus• Transplant recipients• iv drug users

PSEUDOMONAS• Strict aerobe (can grow anaerobically – nitrate/arginine)• Beta hemolytic colony with metallic sheen• Usually grows on McConkey• Pyocyanin, pyoverdin, pyomelanin, pyorubin• Fruity odour (Also Alcaligenes, Myroides), rarely foul odour• Motile with polar flagella (single/tufts)• Oxidase +, catalase +• OF glucose +• Mucoid – poor biochemical reactions• Aeromonas – similar colonies, no odour, indole +

PSEUDOMONAS• Fluorescent group

• Pseudomonas aeruginosa• Pseudomonas fluorescens• Pseudomonas putida • Pseudomonas veronii• Pseudomonas monteilli

• Nonfluorescent group• Pseudomonas stutzeri• Pseudomonas mendocina• Pseudomonas alcaligenes • Pseudomonas pseudoalcaligenes• Pseudomonas luteola• Pseudomonas oryzihabitans

OTHER PSEUDOMONADS• P. putida – Transfusion acquired bacteremia (donor skin)• P. fluorescens – Infusion associated pseudobacteremia,

liquefies gelatin• P. mendocina - similar to nonpigmented P. aeruginosa• P. luteola – Ox-• P. stutzeri – Environmental sources, wrinkled colony•Otitis media, pneumonia, endocarditis, meningitis,

conjunctivitis, osteomyelitis• P. oryzihabitans – Ox-, wrinkled colony, esculin – • Septicemia, prosthetic valve endocarditis

ACINETOBACTER• Family Moraxellaceae• 25 genomospecies (DNA-DNA hybridization) – 10 named• Ubiquitous including hospital environment• Normal flora of skin, faeces, pharynx (25%)• Easy colonization in hospitalized patients• Nosocomial UTI, RTI, bacteremia, enodocarditis,

meningitis, septicemia, osteomyelitis, arthritis, peritonitis, wound, burn, eye, ENT infections

• Resistant to penicillins, chloramphenicol• Aminoglycosides, doripenem, tigecycline, colistin

ACINETOBACTER• Grow on McConkey, weakly acid/purple colony• Nonhemolytic on blood agar• Ox –• Nonmotile• OF glu +, OF lactose ++, • Rapid glucose utilization, xylose• A. baumanii – OF glu +, OF lactose +, nonhemolytic• A. lwoffi – OF glu -, Of lac -, nonhemolytic, less virulent• A. hemolyticus – OF glu -, OF lac -, beta hemolytic• Insignificant - urine, feces, respiratory, vaginal fluids

STENOTROPHOMONAS• Family Xanthomonadaceae• Ubiquitous including hospital environment• Not recognized as normal flora• Easy colonization in hospitalized patients• Nosocomial UTI, RTI, bacteremia, endocarditis, meningitis,

wound infections, cellulitis, ecthyma gangrenosum• Cystic fibrosis, cardiac surgery, iv drug abuse• Causes outbreaks

STENOTROPHOMONAS• Sheep BA – Rough, lavender green, ammonia odour• Trypticase soy agar - Yellow tan pigment• Lysine +, Ox –, catalase +, • Grows at 420C• OF glu +, OF maltose ++• ONPG +, DNAse +, nitrate reduction –• Esculin hydrolysis +, gelatin hydrolysis +• Natural resistance to aminoglycosides, beta lactams• SXT, tetracycline, chloramphenicol, ceftazidime, ticarcillin-

clav, aztreonam• Regarded as saprophyte or colonizer when isolated• Colonizes blood collection equipment• Pseudoinfections through contaminated collection tubes

BURKHOLDERIA• Family Burkholderiaceae, 9 genomovars• Low grade nosocomial pathogen• Contaminated water, irrigation fluids, anesthetics,

nebulizers, detergents, disinfectants, dairy products• Nosocomial UTI, RTI, bacteremia, endocarditis,

osteomyelitis, wound infections, CGD, cystic fibrosis• Resistant to disinfectants• Ceftazidime, meropenem, minocycline, SXT (Resistance ↑)• Bioterror agent• Grows on all media, BCSA (crys v, polymyxin B, bacitracin)• Lactose +, oxidase + (weak), motile by polar flagella• OF glu +, OF xyl +, OF suc +• Lysine +, ONPG +, nitrate +

BURKHOLDERIA• B. cepacia• Glanders (RTI, septicemia)• Pneumonia, cystic fibrosis, septic arthritis, peritonitis

• Cepacia syndrome• Fatal necrotizing granulomatous pneumonia

• Skin, soft tissue infections, surgical/burn wound infections• Pseudobacteremia – infusion/disinfectant fluid• Nat resis - Penicillins, cephalosporins, aminoglycosides, colistin

BURKHOLDERIA• B. mallei• Glanders in equines, occasionally in humans

• B. pseudomallei (Whitmore’s bacillus)• Melioidosis – Glanders like disease• Pneumonia, honeycomb abscess, bacteremia, septicemia

• Acute septicemia – Typhoid like disease• Transmitted via respiratory route, rat fleas• Vietnamese time bomb – silent asymptomatic infection for years• Rough convoluted colonies in 3 days, lactose +

• B. gladioli

A. baumanii S. maltophilia B. cepacia

MORAXELLA• Weakly motile• Ox +• OF glu – (asaccharolytic)• Bright yellow pigment• Isolated from body sites, equipment, hospital environment• Bronchitis, bronchopneumonia in COPD in elderly• Sinusitis, otitis in healthy people• Conjunctivitis, keratitis in malnourished alcoholics,

meningitis, bacteremia, endocarditis, arthritis• M. lacunata - Bacteremia, peritonitis• M. nonliquefaciens – normal respiratory flora• Tetracycline, chloramphenicol, aminoglycoside, SXT

SPHINGOMONAS• Weakly motile• Bright yellow pigment• Isolated from body sites, equipment, hospital environment• Bacteremia, peritonitis• Tetracycline, chloramphenicol, aminoglycoside, SXT

OTHERS• Brevundimonas – Requires vitamins for growth, bacteremia in

hemodialysis, sickle cell, immunocompromised patients

• Achromobacter – Ox +, OF glu +, OF xyl +, motile, nitrate +, citrate +, • Nosocomial outbreaks• Bacteremia, RTI, meningitis, peritonitis, osteomyelitis, prosthetic

valve endocarditis, prosthetic knee infections

• Chryseobacterium – Ox+, Nosocomial meningitis, bacteremia• Comamonas – Ox+, cat+, orange indole – anthanilic acid,

uncertain pathogen

OTHERS• Psychrobacter – Ox -, OF – (asaccharolytic), nonmot, urease +, • Alcaligenes - Common in water, no fermentation/oxidation • Ralstonia – Pinpoint colonies, weakly motile, rare • Pandoraea – Weakly motile, fatty acid profiling• Delftia – Ox+, grows on McConkey, pigment• Rhizobium – Ox+, oxidise lactose• Acidovorax – Curved rod, uncertain pathogen• Myroides – Bacteremia• Methylobacterium & Roseomonas – Grow better on SDA

REFERENCES• Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger

P, Woods G. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 6th Ed, Lippincott Williams & Wilkins 2006: Chapter 7. The Nonfermentative Gram-Negative Bacteria

• Ananthnarayan R, Paniker CKJ.Text book of Microbiology, 7th Ed 2005

• Mackey & McCartney – Practical medical microbiology• Murray-text book of microbiology

Thank You

Pseudomonas coloniesOF testKonemanPhenylalanine deaminaseONPG

Flavobacterium