Non-fermenting and Miscellaneous Gram-Negative Rods

Review well < 3

• Fail to acidify an oxidative-fermentative (OF) medium when it is – (with mineral oil)

• Fail to acidify triple sugar iron agar (TSIA) butts.

• Grows in aerobic environment

• Some members oxidize carbohydrates – oxidizers; others are non-oxidizers or asaccharolytic

• Oxidase positive

• All species except B. mallei (Burkholderia mallei) are motile, having one or several polar flagella

• For non-fermenters, optimum temperature range: 30 to 37 degrees Celsius (mesophilic)

• Acidovorax facilis is the only MacConkey negative

• Pseudomonas spp. are catalase positive

• Fail to acidify an oxidative-fermentative (OF) medium when it is – (with mineral oil)

• Fail to acidify triple sugar iron agar (TSIA) butts.

• Grows in aerobic environment

• Some members oxidize carbohydrates – oxidizers; others are non-oxidizers or asaccharolytic

• Oxidase positive

• All species except B. mallei (Burkholderia mallei) are motile, having one or several polar flagella

• For non-fermenters, optimum temperature range: 30 to 37 degrees Celsius (mesophilic)

• Acidovorax facilis is the only MacConkey negative

• Pseudomonas spp. are catalase positive

General Characteristics of Non-fermenters

Biochemical Characteristics and Identification

• Oxidase ****__ reaction, although reaction can be weak and variable

• Nonreactivity in 24 hours in commercial multi-test kit systems used primarily for the identification of Enterobacteriaceae

• No acid production in the slant or butt of TSIA or KIA

• Resistance to a variety of classes of antimicrobial agents, such as aminoglycosides, third-generation cephalosporins, penicillin, and fluoroquinolones

positive

Clinically Significant Non-fermentative, Gram-negative Bacilli

• Gram-negative bacillus or coccobacillus

• Strictly aerobic metabolism

• Motile usually with polar flagellum or polar tuft of flagella

• Oxidase positive (except P. luteolus and P. oryzihabitans)

• Catalase positive

• Usually grows on MAC agar

• Usually an oxidizer of carbohydrates, but some species are asaccharolytic

• Produce water-soluble pigments, yellow green pigment (pyoverdine), blue pigment (pyocyanin)

• when the pyocyanin has been combined with pyoverdine it will result in a bright green color

A. Pseudomonas

Clinically Significant Non-fermentative, Gram-negative Bacilli

What group is this?

• Pseudomonas aeruginosa

• Pseudomonas fluorescens

• Pseudomonas putida

• Pseudomonas monteiliii

• Pseudomonas veronii

• Pseudomonas mosseliii

pseudomonas are classified as a fluorescent group and non fluorescent group

able to produce a yellow green or yellow brown pigment and that is what we called “Pyoverdine”. Pyoverdine is a water-soluble pigment

aside from pyoverdine, again, pseudomonas aeruginosa also produce pyocyanin

Pseudomonas Fluorescent Group

Clinically Significant Non-fermentative, Gram-negative Bacilli

What group is this?

• Pseudomonas stutzeri

• Pseudomonas mendocina

• Pseudomonas alcaligenes

• Pseudomonas pseudoalcaligenes

• Pseudomonas luteola

• Pseudomonas oryzihabitans

Pseudomonas non-fluorescent group

Pseudomonas Fluorescent Group

• Motile and rod shaped

• Obligate aerobe

• Producing a sweet or grape- like or corn taco-like odor (due to the presence of 2- aminoacetophenone)

• Produces the fluorescent bluish pigment, pyocyanin

• Produces the fluorescent pigment pyoverdine

• May also produce a dark red pigment (pyorubin) or a brownblack pigment (pyomelanin)

Pseudomonas aeruginosa

What agar is this?

Culture media: ______________-selective and differential

• this medium also enhances the production of 2 pigments which is pyoverdine and pyocyanin.

Cetrimide Agar

• acts as a detergent

• their growth at 42 degree C can help to differentiate it from other pseudomonas spp or other fluorescent group

• for the identification of P.A usually we base on colonial morphology with the presence of pigment, with the oxidase positivity, and their growth at 42 C

Cetrimide

• Can grow at 4° C

• Produce pyoverdine

• Cannot reduce nitrate to nitrogen gas

• Can produce acid from xylose

• Gelatin hydrolysis:

  P. putida: negative

  P. fluorescens: positive

• these organisms can be isolated from respiratory specimens, contaminated blood products, urine, and other hospital equipment

Pseudomonas fluorescens and Pseudomonas putida

Pseudomonas Nonfluorescent Group

• Wrinkled, leathery, adherent colonies

• Light-yellow or brown pigment

• ADH negative

• Starch hydrolysis positive

• Can grow in an anaerobic environment in nitrate-containing media --- nitrogen gas

• Septicemia, meningitis in the human immunodeficiency virus-infected patient, pneumonia (especially in CF and immunocompromised patients), endocarditis, postsurgical wound infections, septic arthritis, conjunctivitis, and UTIs

• usually the isolates are ADH negative and starch hydrolysis positive can be used to distinguish the Pseudomonas stutzeri from most other Pseudomonas species; this is one of their characteristics

• they are also able to produce nitrogen gas (can also be used to distinguish Pseudomonas stutzeri from most other pseudomonas species

Pseudomonas stutzeri

Pseudomonas Nonfluorescent Group

• contaminant

• produces nonwrinkled, flat colonies that may appear with a yellowish-brown pigment

• pigmentation can be a variable trait but many exhibit a smooth buttery appearance.

• many exhibit a smooth buttery appearance.

• oxidase and ADH positive

• does not produce pyoverdin – that is why they classified it as nonflourescent group.

• acetamide negative.

• Motile with the means of single polar flagellum

• oxidizes glucose and xylose

• nonproteolytic and does not hydrolyze starch

Pseudomonas mendocina

Pseudomonas Fluorescent Group

• nonpigmented and nonhemolytic

• positive for oxidase, catalase, and ADH

• optimal growth occurs at 30 C

Pseudomonas mosselii

Pseudomonas Nonfluorescent Group

• both are oxidase positive

• biochemically negative in many tests

• they are often considered as contaminants, if they have been isolated from clinical specimens

• Grow on MAC agar

• motile by means of polar flagellum

Pseudomonas pseudoalcaligenes and Pseudomonas alcaligenes

Pseudomonas Nonfluorescent Group

• ADH positive and will weakly ferment fructose

P. pseudoalcaligenes

Pseudomonas Nonfluorescent Group

• gram-negative, nonfermentative, oxidase- negative bacilli.

• catalase positive and motile, oxidize glucose, grow on MAC agar

• produce an intracellular nondiffusible yellow pigment.

• Produce wrinkled or rough colonies at 48 hours

• ONPG test, we used it to detect the enzyme beta galactosidase, this beta galactosidase is present in late lactose fermenters

Pseudomonas luteola and Pseudomonas oryzihabitans

• aerobic, catalase-positive, oxidase negative, Gram- negative bacteria

• may also appear as diplococci on smears and then resemble Neisseria

• grow best at 35°C to 37°C

Acinetobacter

• most commonly isolated in clinical laboratories

• glucose-oxidizing, nonhemolytic strain

Acinetobacter baumannii

• described to colonize human skin and cause occasionally infections in immunocompromised patients.

Acinetobacter radioresistens

• Glucose negative nonhemolytic strain.

Acinetobacter Iwoffii

Acinetobacter

Clinical Infections

• primarily affect patients with weakened immune systems and coexisting diseases

• usually, when we talk about Acinetobacter, they are able to affect the immunocompromised patients.

• UTIS; pneumonia, tracheobronchitis, or both; endocarditis

• septicemia; meningitis

• Cellulitis – from the contamination from catitters or from introduction of foreign body.

• ______________________- eye infections: such as endophthalmitis, conjunctivitis, and corneal ulcerations

Acinetobacter baumannii

Acinetobacter

Identifying Characteristics

• can resist decolorization and retain the crystal violet

• development of purplish hue produced by some species on MacConkey agar

• ___________ is saccharolytic____, and ___________is asaccharolytic

• CRAB (carbapenem resistant A. baumannii)- only susceptible to colistin and tigecycline

• Acinetobacter baumannii on MacConkey a organisms saccharolyn, which may cause to re lactose fermenting organism

A. baumannii, A. Iwoffii

• third most common non-fermentative, gram- negative bacillus in the clinical laboratory

• Colonies may appear bluish on MAC agar positive for catalase, DNase, esculin and gelatin hydrolysis, and lysine decarboxylase

• this has been known because of the characteristic bluish on MAC agar.

Stenotrophomonas maltophilia

Burkholderia

• usually, they contain plant pathogens, but they can also affect humans. They can be classified as opportunistic organism.

• weak, slow, positive oxidase reaction.

• oxidize glucose, and many will oxidize maltose, lactose, and mannitol.

• lysine decarboxylase and ONPG positive

• ornithine decarboxylase negative and fail to reduce nitrate to nitrite

• motile by means of polar tufts of flagella

• Causes onion bulb rot in plants and foot rot in humans

Burkholderia cepacia Complex

Burkholderia

• old name is Pseudomonas mallei

• Smooth and cream to white colonies in BAP and weakly oxidase (+) .

• The only non-motile among all pseudomonads

• CAN'T GROW in 42 C

• Causes GLANDER'S DISEASE - infectious disease of horses, goats, sheep, & donkey. Rare cause of human infection acquired by direct contact, trauma, or inhalation.

• this is respiratory tract zoonosis.

• FARCY- disseminated form of glander's disease

• considered as a potential bioterrorism agent- meaning those are pathogenic organism that are usually used in order to produce death____.

Burkholderia mallei

Burkholderia

• Cream to tan wrinkled colonies in BAP

• Motile via polar tuft flagella

• ADH (+)

• Highly oxidative- glucose, maltose lactose, mannitol

• causes MELIOIDOSIS- This is an pulmonary disease; transmitted via ingestion, inhalation or in inoculation in the organism. Glanderslike disease which has a long latent period.

• Manifestation of symptoms takes years hence the (it has been called) synonym Vietnam Time Bomb

Burkholderia pseudomallei

Burkholderia

• produces yellow pigment after 48-72 hours of incubation

• motile by means of one or two polar flagella

• catalase and urease positive

• Grows on surface of MAC agar

• Oxidizes glucose

• mannitol positive and decarboxylase negative, and is negative for oxidase, although some strains are weakly positive.

Burkholderia gladioli

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• both of these isolates can be found on water, like swimming pool, tap water and dialysis fluid, they are usually resistant to disinfectants.

• obligately aerobic gram-negative bacilli

• both posses peritrichous flagella

• may produce a fruity odor

• on the surface of Sheep blood Agar agar plate, they can exhibit green discoloration.

• Oxidation-Fermentation media: nonoxodative, produce a deep blue color at the top, except for Achromobacter xylosoxidans- old name “Achromobacter xylosoxidans variety denitrificans”

Alcaligenes and Achoromobacter

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Brevundimonas

• Motile and possess a single polar flagellum

• Oxidize glucose

• Oxidase positive

• Most strains grow on MAC agar

B. diminuta

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Brevundimonas

• Slender rod, with polar flagella

• Only about 25% grows on MAC agar

• Most strains produce an orange intracellular pigment

• Oxidase positive and oxidizes glucose and maltose

• Esculin Hydrolysis: to differentiate B. diminuta (rarely positive, contaminant) and B. vesicularis (positive)

B. vesicularis

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Oxidase positive

• Nonmotile, saccharolytic coccobacilli that grow weakly, if at all, on MAC agar

• All oxidize glucose and xylose

• EO-3 and many EO-4 isolates have a yellow non- diffusible pigment

• EO refers to Eugonic oxidizer

• P. yeei (EO-2): coccoid or O-shaped cells on gram stain

CDC Groups EO-3, EO-4 and Paracoccus

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Chromobacterium

• Motile, facultative anaerobe, oxidase positive

• Rare cause of human infection

• Found in soil and water

• Unique because of the violet pigment that it produces – VIOLACEIN

Chromobacterium violaceum

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Straight to slightly curved rods

• Produce alkalinity in OF media

• Catalase and oxidase positive

• Motile by multitrichous polar flagella

• Reduce nitrate to nitrite

Comamonas and Delftia

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Have been reported to cause nosocomial bacteremia

Comamonas testosteroni and Comamonas terrigena

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Associated with keratitis in soft contact lens wearers and nosocomial infections including bacteremia and endocarditis

Delftia acidovorans (Comomonas acidovorans)

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Associated with catheter-related bacteremia

Delftia tsuruhatensis

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Balaneatrix, Bergeyella, Chryseobacterium, Elizabethkingia, Empedobacter, Myroides, Weeksella, Wautersiella, Sphingobacterium spp.

• Non-motile except Balaneatrix alpaca

• Lavender green discoloration of the agar on the media with blood

• Fruity odor

Flavobacteriaceae

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• Meningitis or septicemia in a newborn

• produce acid from glucose, maltose, and mannitol

• hydrolyze esculin, indole positive

• growth on MacConkey is variable

Elizabethkingia meningoseptica (Chryseobacterium meningosepticum)

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• First isolated in 1987 during an outbreak of pneumonia and meningitis linked to individuals attending hot springs spa

• Curved to straight rod

• The only Motile by polar flagella

• Pale yellow, becoming Brown with age

• P__ositive for oxidase and indole__

• Saccharolyticus, oxidizing glucose, mannose, fructose, and other sugars

Balaneatrix alpaca

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• linked to nosocomial infections - bacteremia

Chryseobacterium indologenes

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• Endophthalmitis following cataract surgery

Empedobacter brevis

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• environmental, low-virulence, nonfermenting, gram negative bacillus

• UTIS, endocarditis, ventriculitis, and cutaneous infections in immunocompromised patients

Myroides odoratimimus

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• asaccharolytic, indole and oxidase positive, and fail to grow on MAC agar

Weeksella

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• found in genitourinary specimens

• grows on modified Thayer-Martin medium or other media selective for Neisseria gonorrhoeae

Weeksella virosa

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Flavobacteriaceae

• cellulitis, tenosynovitis, septicemia, pneumonia, and meningitis, in particular in association with dog and cat bite wounds

Bergeyella (Weeksella) zoohelicum

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Pink pigmented colonies

• Nonvacuolated, coccoid bacteria, forming pairs and short chains

Methylobacterium and Roseomonas

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Ralstonia and Cupriavidus

• Slow growers

• Oxidase and catalase positive

• Grows on MAC agar, reduces nitrate, oxidizes glucose and xylose, and is motile by means of a single polar flagellum

Ralstonia pickettii

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Ralstonia and Cupriavidus

• Motile (peretrichous flagella), oxidase positive, catalase positive, asaccharolyticus, gram negative bacillus

• Most strains grow on MAC agar

Cupriavidus pauculus

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

• Mucoid

• Produce a Tan to Brown pigment causing greenish discolorization of SBA

• Motile, ornithine decarboxylase and nitrite reductase positive, and produce profuse H2S I TSI agar

• S. algae – requires NaCl (halophilic) and is asaccharolyticus

• S. putrifaciens – non-halophilic and saccharolyticus

Shewanella

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Sphingomonas

• Yellow pigmented

• does not grow on MAC agar

• requires more than 48 hours for culture on SBA

• weakly oxidase positive

• motile at 18° to 22°C but not at 37°C, indole negative, and oxidizers.

S. paucimobilis

Less Commonly Encountered Nonfermentative, Gram- Negative Bacilli

Sphingomonas

• H2S positive by the lead acetate method____, Simmon citrate positive, and DNase negative

S. parapaucimobilis