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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
a
• 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