non-fermenting and miscellaneous gram negative bacilli
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NON-FERMENTING AND MISCELLANEOUS GRAM NEGATIVE BACILLI
GROUP 6, 3GMTPeralta, Ana Eryka Elaine
Pilares, Felicia AnnRafols, Kate Syvel
Sales, Reneepearl KimSato, Mikie
Sison, Jacqueline Anne
ClassificationPseudomonads
Pseudomonas aeruginosa (most common)Pseudomonas stutzeriBurkholderia pseudomalleiBurkholderia cepaciaStenotrophomonas maltophilia (also common)
Non-PseudomonadsAcinetobacter baumannii MoraxellaAchromobacter xylosoxidans
General Characteristics• Obligate aerobes• All are motile (except Burkholderia mallei)• Non-fermentative for glucose• TSI reaction: K/K
• Oxidative for glucose (except Moraxella – asaccharolytic)• Hugh-Leifson O-F glucose positive
• Cytochrome oxidase positive (except Acinetobacter and
S. maltophilia)
General Characteristics
Cytochrome oxidase
OF glucose Motility Pyoverdin* production
Susceptibility to Polymixin B
P. aeruginosa**
+ + + +(also P.
fluorescens and P. putida)
+
P. stutzeri + + + - +
Burkholderia cepacia
+ + + -
Burkholderia pseudomallei
+ + + -
Stenotrophomas maltophilia
- +(also OF
maltose ++)
+ +
Pseudomonads
*Pyoverdin is a fluorescent, yellow-green or yellow-brown water-soluble pigment**Of the Fluorescent group, P. aeruginosa solely produces Pyocyanin, a blue water soluble pigment;
P. fluorescens and P. putida cannot
General CharacteristicsNon-Pseudomonads
Cytochrome oxidase
OF glucose Motility
Acinetobacter baumanii
- +(also OF
lactose ++)
-
Moraxella + -(asaccharolytic*)
+/-
Achromobacter xylosoxidans
+ +(also OFXylose +)
+
*Assacharolytic means that it alkalinizes (rather than acidifies) the broth in Hugh-Leifson tube unsealed with mineral oil
Colony Characteristics
Colony Characteristics
Pseudomonas aeruginosa
Spreading and flat with a pearl-like sheen (pearlescent) & grape-like odorGrow at 42˚C
Pseudomonas stutzeri
Produce distinctive dry, wrinkled colonies (1-6mm) on BAP
Burkholderia cepacia
Smooth, round, opaque, & yellow colonies on BAPBright pink colonies on MAC (due to lactose oxidation)
Stenotrophomonas maltophilia
Rough, lavender-green colonies with ammonia odor on Sheep Blood Agar
Pseudomonads
Colony Characteristics
Colony Characteristics
Acinetobacter spp.
Smooth, opaque, gray-white colonies (slightly smaller than Enterobacteriaceae)
A. baumanii – non-hemolyticA. iwoffii – non-hemolyticA. haemolyticus – beta hemolytic on Sheep Blood Agar
Moraxella Small (0.5-1mm), smooth, translucent to semi-opaque colonies on BAP
Non-Pseudomonads
Microscopic Appearance
Microscopic Appearance
Pseudomonas aeruginosa
Straight or slightly curved, slender, Gram (-) rods
Burkholderia cepacia Gram (-) rods
Stenotrophomonas maltophilia
Short to medium sized, straight, Gram (-) rods
Acinetobacter spp. Gram (-) coccobacillary rods occuring singly and in Neisseria-like pairs
Moraxella Gram (-) or Gram-variable Neisseria-like diplococci
Virulence factors: Fluorescent: Pseudomonas aeruginosa
• Endotoxin from cell wall (lipopolysaccharide)• Motility (Flagella)• Pilli(Type 4)• Neuraminidase- facilitate binding pilli• Capsule• Exotoxins: protease (LasBelastase, LasAelastase, and alkaline protease),
hemolysins (Phospholipase C and Rhamnolipid), lecithinase, elastase, DNAse, exotoxin A, exoenzyme S
• Alginate- polysaccharide polymer, produces mucoid colonies• Resistant to aminoglycosides, ъ-lactam antibiotics, fluoroquinolones• Pyocyanin• Pyoverdin
Fluorescent: Pseudomonas fluorescens and Pseudomonas putida
• Very low virulence• Resistant to cabernicillin
Nonfluorescent group:• P. stutzeri• Resistant to Chloramphenicol and Cephalosporin
• P. mendocina• Flagellum, oxidise glucose and xylose, positive for oxidase and
ADH, nonproteolytic• P. pseudoalcaligenes, P. alcaligenes• Oxidase positive
Stenotrophomonas maltophilia
• Resistant to Cephalosporins, Penicillins, Carbapenems, Aminoglycoside• Positive for:• DNase• Esculin• Gelatin Hydrolysis• Lysine decarboxylase
Acinetobacter (A. baumanii& A. lwoffii)• S-form LPS- major stimulator of immune
response; resistance to the natural bactericidal action of human serum• Adhesins- formation of biofilm• Lipase(s)- damage tissue lipids• Elaboration of siderophores- to
scavenge iron and promote survival in vivo
Burkholderia
• Burkholderiacepacia:• Flagella• Lysine decarboxylase positive• o-nitrophenyl-β-d-galactopyranoside (ONPG)
positive• Burkholderia gladioli• one or two polar flagella• Catalase and urease positive• Resistant to polymyxin B
Burkholderia
• Burkholderiamallei• Burkholderiapseudomallei:• Secretion (type III protein secretion system)• Lipopolysaccharide• Capsule• Exoproteins (Phospholipase C, putative
collogenase, MucD Ser protease homologs)• Adhesins
Flavobacterium and Chryseobacterium
• Flavobacterium• Chryseobacterium• DNase• Fibrinolysin• Gelatinase• Lecithinase• Lipase• Proteinase
CultivationP. AREUGINOSA
• Carbon Source- ACETATE• Nitrogen Source – AMMONIUM SULFATE• 37 degrees, able to grow at temperatures as high as 42 degrees• resistant to high concentrations of salts and dyes, weak antiseptics,
and common antibiotics• 1st - large, smooth, with flat edges, elevated• 2nd - a mucoid appearance, attributed to the production of alginate
slime
P. STUTZERI• Freshly isolated colonies - adherent, wrinkled appearance,
reddish brown• Shapes of colonies are neither uniform nor necessarily
constant: they change appearance with time• After repeated transfers: may become smooth, butyraceous,
pale in color (colonial dissociation)• 30°C and stored at 4°C for 24 hours
B. PSEUDOMALLEI• Ashdown’s medium• Suppressors: gentamicin, crystal violet• 96 hours, flat wrinkled, purple colonies• Enriched with 4% glycerol• The cost of each plate is only US$0.04 = Php. 1.75
STENOTROPHOMONAS MALTOPHILIA• smooth, glistening, with entire margins and are white to pale
yellow• on blood agar there may be a greenish discoloration of the
medium around confluent growth• on clear media, some strains may exhibit a brownish
discoloration• optimal at 35°C• methionine or cystine is required for growth by most but not
all strains
ACOLIGENES XYLOSOXIDANS• flat, spreading and rough colonies• blood agar and MAC agar
Biochemical Identification of Species
• All the nonfermenters will not yield acidic reactions in the anaerobic portion of media such as TSI (Triple Sugar Iron) or KIA (Kligler’s Iron Agar)
• Thin, gram-negative bacilli or coccobacilli on gram stain may indicate the presence of nonfermenter.
• For clinically significant isolates of P.aeruginosa, S. malthophilia, Acinetobacter spp., and Burkholderia cepacia, the CLSI recommends broth dilution or Kirby Bauer disk diffusion assay to be performed.
Pseudomonas aeruginosa; Pseudomonas stutzeri;
Burkholderia cepacia; Burkholderia pseudomallei
•TSI (K/K) •Positive in MacConkey agar•Positive in Oxidase test
P.Aeruginosa• Unipolar motility
Moraxella atlantae; Moraxella osloensis
•TSI (K/K)•Positive in MacConkey agar.•Positive in Oxidase test
Acinetobacter baumanii
•TSI (K/K) •Positive in MacConkey agar•Negative in Oxidase test•Usually nitrate negative•Non motile
Stenotrophomonas maltophilia
•TSI (K/K)•Positive in MacConkey•Negative in Oxidase test
Clinical Infections and DiseasesNon fermenters account for about 15% of all gram (-) bacilli
isolated clinical specimens
Non Fermenters can be responsible for infection: o Septicemia o Meningitidiso Osteomyelitis o Wound infections (develops after trauma or surgery)
Risk Factors for Diseases cause by Nonfermentative Gram- Negative Bacilli
ImmunosuppressionTraumaForeign body implatationInfused fluids
Diseases Produced
(Jawetz, Melnick & Adelberg,2007)Mahon, Lehman & Manyselis,2010)
(Jawetz, Melnick & Adelberg,2007)Mahon, Lehman & Manyselis,2010)
(Jawetz, Melnick & Adelberg,2007)Mahon, Lehman & Manyselis,2010)