NON-FERMENTING AND MISCELLANEOUS GRAM NEGATIVE

GROUP 6, 3GMT Peralta, Ana Eryka Elaine Pilares, Felicia Ann Rafols, Kate Syvel Sales, Reneepearl Kim Sato, Mikie Sison, Jacqueline Anne

Classification
Pseudomonads Pseudomonas aeruginosa (most common) Pseudomonas stutzeri Burkholderia pseudomallei Burkholderia cepacia Stenotrophomonas maltophilia (also common) Non-Pseudomonads Acinetobacter baumannii Moraxella Achromobacter 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
Pseudomonads
Cytochrome OF glucose oxidase Motility Pyoverdin* production Susceptibility to Polymixin B

P. aerugino sa** P. stutzeri
Burkholderi a cepacia

+

+

+

+
(also P. fluorescens and P. putida)

+

+ +

+ +

+ +

-

+ -

Burkholderi + + + a pseudomall overdin is a fluorescent, yellow-green or yellow-brown water-soluble pigment ei Of the Fluorescent group, P. aeruginosa solely produces Pyocyanin, a blue water soluble pigment;
P. fluorescens and P. putida cannot Stenotrophoma s maltophilia

-

+

+

+

General Characteristics
Non-Pseudomonads

Cytochrom OF glucose e oxidase
Acinetobacter baumanii Moraxella Achromobacte r xylosoxidans

Motility

+ +

+
(also OF lactose ++)

+/+
acidifies) the broth in

(asaccharolytic*)

+
(also OF Xylosethan (rather +)

*Assacharolytic means that it alkalinizes Hugh-Leifson tube unsealed with mineral oil

Colony Characteristics
Pseudomonads

Colony Characteristics Pseudomona Spreading and flat with a pearls aeruginosa like sheen (pearlescent) & grape-like odor Grow at 42˚C Pseudomona Produce distinctive dry, wrinkled s stutzeri colonies (1-6mm) on BAP
Burkholderia cepacia

Smooth, round, opaque, & yellow colonies on BAP Bright pink colonies on MAC (due

Colony Characteristics
Non-Pseudomonads

Colony Characteristics Acinetobact Smooth, opaque, gray-white er spp. colonies (slightly smaller than Enterobacteriaceae) A. baumanii – non-hemolytic A. iwoffii – non-hemolytic A. haemolyticus – beta hemolytic on Sheep Blood Agar Small (0.5-1mm), smooth, translucent to semi-opaque colonies on BAP

Moraxella

Microscopic Appearance
Microscopic Appearance Pseudomonas Straight or slightly curved, aeruginosa slender, Gram (-) rods Burkholderia Gram (-) rods cepacia Stenotrophom Short to medium sized, onas straight, Gram (-) rods maltophilia Acinetobacter Gram (-) coccobacillary rods spp. 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

Cultivation P. 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
blood agar and MAC agar

flat, spreading and rough colonies

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)

Biochemical Identification of Species

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 Diseases
q q

Non fermenters account for about 15% of all gram (-) bacilli isolated clinical specimens Non Fermenters can be responsible for infection: Septicemia Meningitidis Osteomyelitis Wound infections (develops after trauma or surgery)

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o

q q q q

Risk Factors for Diseases cause by Nonfermentative GramImmunosuppression Negative Bacilli
Trauma Foreign body implatation Infused fluids

Diseases Produced

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level

hon, Lehman & Manyselis,2010) wetz, Melnick & Adelberg,2007)

hon, Lehman & Manyselis,2010) wetz, Melnick & Adelberg,2007)

Click to edit Master text styles Second level ● Third level ● Fourth level ● Fifth level

hon, Lehman & Manyselis,2010) wetz, Melnick & Adelberg,2007)

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