Gram-negative Bacilli

Non-fermentative

Leanne Jessa G. Gumahad, RMT

• Pseudomonas
• Acinetobacter
• Stenotrophomonas
• Burkholderia
 General characteristics
• Does not ferment any carbohydrate
• Fail to acidify OF media when it is laid w/ mineral oil
• Fail to acidify triple sugar of TSI butts
• Some oxidize carbs to derive energy for their metabolism (oxidizers)
• Others do not break down carbs & are inert and biochemically
inactive (non-oxidizers/ asaccharolytic)
• Oxidase (+) though reaction may be weak & variable
• Found in most environments & in hospitals, they can be isolated from
nebulizers, dialysate fluids, saline, catheters, & other devices
• Will not yield acidic reaction in the anaerobic portion of media
(TSI/KIA)
• Thin, gram-negative bacilli or coccobacilli on gram stain
• Can withstand treatment with chlorhexidine & quaternary
ammonium compounds
• Rarely a normal flora but easily colonize hospitalizes patients
• May be resistant to multiple classes of antibiotics
• Common disease: septicemia, meningitis, osteomyelitis, & wound
infections following surgery/ trauma
Oxidative –Fermentative
(OF) Test
• Differentiates Enterobacteriaceae from
Pseudomonas species
• Determines ability of organism to utilize a
substrate, which produces acid byproducts
in the presence/ absence of oxygen
• Semi-solid medium: OF medium/ Hugh and
Leifson medium
• Oxygen barrier: Mineral oil/ melted paraffin
• pH indicators: Andrade’s acid fuchsin, https://microbiologyinfo.com/wp-content/uploads/2019/04/of-test-graph.jpg

bromcresol purple, bromthymol blue, phenol

red
Pseudomonas species
• Gram-negative bacillus or coccobacillus which are motile usually
with polar tufts or flagella
• Strict aerobes, non-sporeforming
• Oxidase (+) except P. luteolus & P. oryzihabitants
• Catalase (+)
• MAC- round, flat, colorless
• Oxidizer of carbs (but some species are asaccharolytic)
• May be found in cosmetics, swimming pools, hot tubs, & inner
part of shoes
Pseudomonas species
• Fluorescent group- P. aeruginosa, P.
fluorescens, P. putida, P. veronii, P. mosselii,
P. monteilii
• Produce pyoverdin- yellow-green or
yellow-brown pigment
• Fluoresces under short-wavelength UV
light
• P. aeruginosa also produces pyocyanin (blue-
water soluble pigment)
• Pyoverdin and pyocyanin will result to the
green color characteristics of P. aeruginosa
colonies
https://www.shutterstock.com/shutterstock/photos/524153776/display_1500/stock-photo-pseudomonas-aeruginosa-colonies-as-test-on-macconkey-agar-plate-contains-small-light-
grains-focus-524153776.jpg
Pseudomonas species

• Culture media: MAC, BAP, CAP,

Sellers medium (promotes pigment
production), Cetrimide agar, Irgasan,
C390
• Cetrimide agar- differential &
selective medium for P. aeruginosa
Pseudomonas
aeruginosa

• BAP: beta hemolytic, flat spreading colonies

with metallic sheen
• Odor: Fruity like/ grape-like/ corn tortilla
• Caused by the presence of 2-
aminoacetophenone
• (+) growth @ 42 dec C
• (+) acetamide & citrate utilization (blue color)
• (+) gluconate production
• (+) arginine dihydrolase https://www.shutterstock.com/shutterstock/photos/1910251387/display_1500/stock-photo-pseudomonas-aeruginosa-colonies-with-beta-hemolysis-on-blood-agar-plate-
1910251387.jpg

• VF: Endotoxin, pili, alginate, exotoxins

(lecithinase, elastase, protease)
Pseudomonas aeruginosa
• Agent of blue pus
• Accounts for 15% of nosocomial infections (pneumonia &
bacteremia)
• Leading cause of nosocomial respiratory tract infections
• 3rd most common cause of gram-negative bacillary
bacteremia after E. coli & K. pneumoniae
• Can colonize mucosal surface such as oropharynx
• Common cause of ventilator-associated pneumonia
• Causes otitis externa in swimmers/divers, Jacuzzi or hot tub
syndrome (necrotizing skin rash) in recreational facilities, &
infections of the nail beds in people with artificial nails,
keratitis, & endophthalmitis
https://scontent-mnl1-1.xx.fbcdn.net/v/t1.18169-9/10314520_876515679025177_4833553569801887579_n.jpg?_nc_cat=105&ccb=1-
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VcngC&_nc_ohc=GidXXrAIJ_wAb5dgHgV&_nc_ht=scontent-mnl1-1.xx&oh=00_AfCgrfNsWtmx6fek9iYqGW9SYyTR8JSW-
cVZUg_pHYLosw&oe=664C08E4
Pseudomonas fluorescens
Pseudomonas putida
• Isolated from contaminated blood products, respiratory specimens,
urine, cosmetics, hospital equipment, & fluids
• P. putida- catheter related sepsis in patients with cancer
• Isolation of P. fluorescens blood culture bottles in asymptomatic
patients has been responsible for clusters of pseudo bacteremia
• Both can grow in 4 deg C & linked to transfusion-associated septicemia
• Both produces pyoverdin but not pyocyanin
• Cannot reduce nitrate to nitrogen gas
• Can produce acid from xylose
• Gelatin hydrolysis
• Pseudomonas fluorescens (+)
• Pseudomonas putida (-)
Acinetobacter species
• Family Moraxellaceae
• Strictly aerobic, gram-negative coccobacilli or even gram-negative
cocci on gram stain
• Can appear as gram-positive cocci in smears made from culture
bottles
• Non-motile, oxidase (-), catalase (+)
• MAC- purple colonies
• BAP: Gummy colonies
• A. baumannii- glucose-oxidizing, non-hemolytic strain
• A. lwofii- glucose-oxidizing, non-hemolytic strain
• A. haemolyticus- non-glucose-oxidizing, hemolytic strain
• Infections: UTI, pneumonia, endocarditis, meningitis, cellulitis
Stenotrophomonas maltophilia
• Short to medium, gram-negative rods
• 3rd most common isolate of non-fermentative gram-
negative bacilli
• Strictly aerobic, motile, & can grow at 42 deg C
• Oxidase (-)
• Catalase, DNase, esculin & gelatin hydrolysis, & lysine
decarboxylase (+)
• MAC- blue colonies
• BAP- lavender-green to light purple pigmentation & odor
like ammonia/ ammonia-cyanide smell
• Infections: Endocarditis, Bacteremia, & wound infections
https://microbe-canvas.com/uploads/image/bacterien/stenotrophomonas-maltophilia/stenotrophomonas-maltophilia_nf16_bk_ba_24-
1_f-350x220.jpg
 Burkholderia species
• Obligate aerobe & motile by polar flagella except B. mallei
• Non-pathogenic
• Acquired through contact with heavily contaminated medical devices
• Medium-sized, gram-negative rods
• Oxidase & catalase (+)
 Burkholderia cepacia
• Opportunistic pathogens usually associated with pneumonia in patients
with cystic fibrosis or chronic granulomatous disease
• Other disease: endocarditis (IV drug-abusers), pneumonitis, UIT,
osteomyelitis, dermatitis & wound infections from contaminated water
• Isolated from irrigation fluids, anesthetics, nebulizers, detergents, &
disinfectants
• Grows well on BAP/MAC but losses viability of not sub-cultured in 3-4
days
• BAP- non-wrinkled yellow or yellow-green
• LDC & ONPG (+)
• Weak positive oxidase reaction
 Burkholderia mallei
• Non-motile, gram-negative coccobacilli that produces non-pigmented
colonies in BAP 2 days
• Causes glanders (AKA: Farcy disease) which primarily affects horses,
mules, & donkeys
• Rare in humans
• Variable growth in MAC
• Oxidase production is variable
Burkholderia pseudomallei
• Causes melioidosis (AKA: Vietnamese Time Bomb)- aggressive,
granulomatous, pulmonary disease caused by ingestion, inhalation,
or inoculation of organisms, w/ metastatic abscess formation in
lungs & other viscera (overwhelming septicemia)
• BAP/CAP: Smooth, creamy at 24h; may display metallic sheen on
BAP, CAP at 48 h; some become either mucoid or dry and wrinkled
after 48-72h
• Pink on MAC/EMB agar at 24-48h; will become dry and wrinkled
after 48-72h
• Presence of bipolar bodies
• Can grow at 42 deg C at 48 hours; earthy odor
• Ashdown medium with colistin: dry, wrinkled, & deep pink colored
colonies

https://www.researchgate.net/publication/268036180/figure/fig4/AS:392084822478870@1470491714540/Burkholderia-pseudomallei-
colonies-grown-on-Ashdowns-agar-after-2-days-incubation.png
Other Gram-negative Bacteria

• Alcaligenes faecalis
• Obligate aerobic, gram-negative bacillus; motile by peritrichous flagella
• Asaccharolytic, producing alkaline reactions in carbohydrate media
• Grow well on MAC
• BAP: feather-edges, non-pigmented, alpha hemolytic, & have fruity odor
of apples or strawberry
• (+) Oxidase; grows well on 6.5% NaCl
• Chromobacterium violaceum
• Only species in genus
Chromobacterium; motile
• Opportunistic pathogen that
causes neutrophil deficiency to
immunocompromised patients
• Grows on MAC at 42 deg C
• Exhibits violet pigmentation
(violacein pigment)
• Gram-negative curved bacilli
https://www.researchgate.net/profile/Alexey-
Shatalov/publication/331954463/figure/fig1/AS:739232431763459@1553258154866/Growth-of-
Chromobacterium-violaceum-on-MacConkey-left-and-5-Sheep-blood-agar-right.png
• Shewanella putrefaciens
• Isolated from water, dairy products, petroleum gas, & other
environmental sources
• Motile, strong H2S producer, saccharolytic
• Isolated from humans with ocular infections, otitis media, & septecimia
• BAP- mucoid & greenish
• Oxidase & ornithine decarboxylase (+)
References
• Aryal, S. (2022e, August 10). OF (Oxidation-Fermentation) Test – Principle, Procedure, Uses and
interpretation. Microbiology Info.com. https://microbiologyinfo.com/of-test/
• Ciulla, A. P., & Lehman, D. C. (2009). Success! in Clinical Laboratory Science: A Complete Review. Prentice
Hall.
• Graeter, L., Hertenstein, E., Accurso, C., & Labiner, G. (2014). Elsevier’s Medical Laboratory Science
Examination Review - E-Book. Elsevier Health Sciences.
• Mahon, C., Lehman, D., & Manuselis, G. (2015). Textbook of Diagnostic Microbiology, 5th edition (5th ed.).
Saunders Elsevier.
• Rodriguez, M. T. (2016). Review Handbook in Diagnostic Bacteriology. In Review Handbook in Diagnostic
Bacteriology. C & E Publishing.
• Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology Fourteenth Edition.
http://125.212.201.8:6008/handle/DHKTYTHD_123/3190