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)