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**__

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***

\
* Y**ellow 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 a**__**cetate method**____,__ **Simmon citrate positive**, and **DNase negative**

*S. parapaucimobilis*