NON FERMENTATIVE GRAM (-) BACILLI OF (Oxidation-Fermentation) Medium

DISEASE MANIFESTATIONS: Hugh and Leifson Formulation

1. Septicemia  Useful in determining an organism’s

2. Meningitis type of CHO utilization whether
3. Osteomyelitis oxidative, fermentative or inert
4. Wound infections (usually after surgery
 Very important in the identification of
or trauma)
nonfermenting gram(-)rods

 Contains the same amount of CHO(1%)

RISK FACTORS FOR DISEASES CAUSED
found in TSI and KIA, but with lower
BY
amount of peptone (0.2%)
NONFERMENTATIVE GRAM (-) BACILLI
 In TSI and KIA, which contain large
1. Immunosuppression: Diabetes Mellitus, amount of peptone(2%), whatever acid
Cancer, steroids, transplantation produced are neutralized or masked by
2. Trauma: Gun shot, knife wounds, the alkaline reaction from peptone
punctures surgery, burns utilization
3. Foreign body implantation: Catheters
(urinary or bloodstream), prosthetic  Used to detect small amounts of acid
devices (joints,valves), corneal implants produced, whether oxidatively or
fermentatively
or contact lenses
4. Infused fluids: Dialysate, saline
 The lower concentration of agar makes
irrigations it semisolid which permits acid to be
formed on the surface to diffuse
INITIAL CLUES FOR NONFERMENTERS throughout the medium

1. Gram (-) bacilli or coccobacilli  pH indicator for acid detection:

2. Oxidase (+) Bromthymol blue
3. TSI nonreactive (K/K or K/NC)
4. Resistance to antibiotics: Oxidative-fermentative test with glucose:
Aminoglycosides, Cephalosporins, oxidative result of Pseudomonas aeruginosa
Imipenem, Penicillins after a 24-hour incubation at 37°C.
Note no growth or color change in the oil-
CATEGORIZED according to its:
covered (anaerobic) tube and acid production
1. Oxidizers/ Saccharolytic and growth in the top portion of the open
a. Breaks down CHO oxidatively
2. Nonoxidizers/Asaccharolytic/ (aerobic) tube.
Nonsaccharolytic/Biochemically Inert
a. Not able to break down CHO
either fermentatively or
oxidatively
 I. GENUS PSEUDOMONAS/BURKHOLDERIA LABORATORY DIAGNOSIS
 BAP: β-hemolytic, green sheen due to
 Motile with polar or polar tufts of flagella the pigment pyocyanin
(except Pseudomonas mallei)  MCA: blue-green colonies
 Oxidase (+)  Many strains produce fruity, grape-like
 Catalase (+) odor (overripe grapes or corn tortillas)
 Usually grows on MCA due to aminoacetophenone
 Usually oxidizes CHO  Produces pyocyanin and pyoverdin
 Grows at 42°C
A. PSUEDOMONAS AERUGINOSA  Cetrimide (+)
 Citrate (+)
 Single most common and reported  Alcohol Dehydrogenase (+) (ADH)
isolate of the genus  Acetamide Utilization (+)
 Uncommon part of the normal flora  The mucoid (alginate-producing strains
(only 4 –12% of humans carry it as part isolated primarily from patients with
of the normal fecal flora) cystic fibrosis may be blue green or pale
 Accounts for 5 –10% of all nosocomial yellow green, they are extremely
infections mucoid
 Most frequently isolated o May slide across the surface of
the plate when plate is tipped

CLINICAL INFECTIONS PYOCYANIN

 A turquoise blue or bright bluish or blue
a) Bacteremia with erythema
water-soluble and chloroform
gangrenosum of the skin as hallmark
extractable pigment
b) Wound infections
 No other bacteria produces pyocyanin
c) Pneumonia especially among those with
o Therefore its detection is
cystic fibrosis
d) Nosocomial UTI sufficient for identification that
e) Rarely, CNS disease including meningitis the isolate is P. aeruginosa
f) Endocarditis  Easy to detect on most media, but can
g) Otitis externa in swimmers or divers be specifically determined in Seller’s
h) Infections after burns Media and Fluorescent-Nitrate (FN)
i) Jacuzzi or hot tub syndrome, a Media
necrotizing skin rash  About 4% of clinical strains of P.
aeruginosa are apyocyanogenic
VIRULENCE FACTORS
1. Endotoxin PYOVERDIN
2. Slime  A fluorescent green pigment
3. Coagulase  Can be detected on Seller’s Media or FN
4. Proteases Media
5. Lecithinase  Produced by P. aeruginosa and other
6. Dnase species like P. fluorescens and P. putida
7. Hemolysin
8. Elastase OTHER PIGMENTS PRODUCED BY SOME
9. Exotoxin STRAINS OF PSEUDOMONAS AERUGINOSA

1.PYORUBIN
 Red pigment
 2.PYOMELANIN  Ofloxacin
 Brown pigment  Norfloxacin
PSEUDOCEL / CETRIMIDE AGAR B. BURKHOLDERIA CEPACIA
 Used to select for P. aeruginosa in  Formerly Pseudomonas cepacia,
specimens with mixed flora Pseudomonas multivorans,
 Inhibits other Pseudomonas species Pseudomonas kingii
(except P. fluorescens) and closely  A plant pathogen
related organisms  Low grade nosocomial pathogen
 Contains Cetrimide (cetyl-trimethyl  Pneumonia in cystic fibrosis patients
ammonium bromide or hexadecyl-  Endocarditis
trimethyl ammonium bromide) which is  UTI
highly inhibitory and has been used as  Has been isolated from irrigation fluids,
an antiseptic anesthetics, nebulizers, detergents,
 If organism can tolerate Cetrimide, it disinfectants
will grow on the medium  Preferred temperature is 30°C
 Magnesium Chloride and Potassium  Produces a non -fluorescent yellow or
Sulfate stimulate the production of green pigment
pyocyanin  Colonies are non-wrinkled (to
differentiate it from P. stutzeri which
also produces yellow pigment)
ANTIMICROBIAL SUSCEPTIBILITY TESTING
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Group A GROUP A
 Ceftazidime  Trimethoprim-sulfamethoxazole
 Gentamicin
 Tobramycin GROUP B
 Piperacillin  Ceftazidime
 Meropenem
Group B  Minocycline
 Amikacin  *Chloramphenicol
 Aztreonam  *Levofloxacin
 Cefepime  *Ticarcillin-Clavulanate
 Ciprofloxacin * MIC testing only
 Levofloxacin C. PSEDOMONAS STUTZERI
 Imipenem  Wrinkled, leathery, adherent colony that
 Meropenem may produce a light yellow or brown
 Piperacillin-tazobactam pigment (initial clue to identification)
 Ticarcillin  A saprophyte
 Produces diseases in immunosuppressed
Group O patients and surgical patients
 Ticarcillin-clavulanate
 Colistin D. SPHINGOMONAS PAUCIMOBILIS
 Polymyxin B  Formerly Pseudomonas paucimobilis
 Netilmicin  Yellow-pigmented pseudomonad
 Gatifloxacin  Does not grow on MCA
Group U
 Lomefloxacin
 Colonizers or contaminants in swimming  CDC considers this organism too
pools, hospital equipments and laboratory dangerous for routine laboratory study
supplies
 Can cause peritonitis, septicemia
E. BURKHOLDERIA PSEDOMALLEI
 Formerly Pseudomonas pseudomallei
 Causes melioidosis or Vietnamese Time G. FLOURESCENT PSEUDOMONADS
Bomb  Pseudomonas fluorescens
 Found in water and muddy soil in  Pseudomonas putida
Southeast Asia (including Vietnam and  Pseudomonas veronii
Thailand), Northern Australia and Mexico  Pseudomonas monteilii
 Produces wrinkled colonies
o B. pseudomallei oxidizes lactose
H. OTHER PSEUDOMONADS
o P. stutzeri is lactose (-)
 Demonstrates bipolar staining in gram-
 Brevundimonas diminuta
stained smear
o formerly Pseudomonas diminuta
MELIOIDOSIS
 Pseudomonas gladioli
 An aggressive granulomatous pulmonary  Pseudomonas mendocina
disease following ingestion, inhalation or
 Pseudomonas alcaligenes
inoculation of the organisms with further
metastatic abscess formation in lungs and  Brevundimonas vesicularis
other viscera
o formerly Pseudomonas vesicularis
 A pneumonia-like illness
 Maybe systemic  Shewanella putrefaciens
 Febrile illness or an acute or chronic
suppurative infection o formerly Pseudomonas putrefaciens
 Was found primarily among returning  Comamonas species
Vietnam veterans and Southeast Asian
immigrants o formerly Pseudomonas acidovorans or
F. BURKHOLDERIA MALLEI Pseudomonas testosteroni
 Formerly Pseudomonas mallei  Methylobacterium extorguens
 Causative agent of Glanders Disease o formerly Pseudomonas mesophilica
 Disease of horses (occasionally other
 Acidovorax delafieldii
animals such as goats, sheep, donkeys,
dogs) o formerly Pseudomonas dealfieldii
 Transmitted to humans by contact with
infected animals
 Acidovorax facilis
 My present as a systemic septicemic  Acidovorax temperans
infection
 Ralstonia mannitolilytica
 A local suppurative infection with
lymphadenopathy, acute pneumonia or o formerly Pseudomonas thomasii
chronic disease
 Non-motile  Ralstonia insidiosa
 Does not grow at 42°C  Ralstonia pickettii
o formerly Pseudomonas pickettii,
Burkholderia pickettii
 II. GENUS ACINETOBACTER
LABORATORY DIAGNOSIS
 Second only to Pseudomonas aeruginosa in  Gram (-) coccobacilli
frequency of isolation in the clinical  Oxidase (-)
laboratory
 Motility (-)
 Consists of 12 DNA hybridization groups  Catalase (+)
(genospecies)  Grows on MCA: purplish hue
 Acinetobacter baumannii
TWO (2) MOST COMMONLY ISOLATED SPECIES o saccharolytic-produces acid in open
1. Acinetobacter baumannii tube
 Formerly known as Acinetobacter
calcoaceticus var anitratus;  Acinetobacter lwoffii:
o Herellea vaginicola o asaccharolytic
2. Acinetobacter lwoffii o the purplish hue may cause the
 Formerly known as Acinetobacter species to resemble a lactose
calcoaceticus var lwoffii; fermenting organism in differential
o Mimapolymorpha media for gram (-) bacilli

 Ubiquitous in the environment: soil, water, ANTIMICROBIAL SUSCEPTIBILITY TESTING

milk, frozen soups GROUP A
 In the hospital: ventilators, humidifiers,  Ampicillin-sulbactam
catheters  Ceftazidime
 About 25% of adults have skin colonization  Ciprofloxacin
with an Acinetobacter species and 7% carry  Levofloxacin
the organism in the pharynx  Imipenem
 Resistant to many antimicrobials including  Meropenem
penicillins and first and second generation  Gentamicin
cephalosporins  Tobramycin
 Most of the diseases produced by
Acinetobacter are caused by A. baumannii GROUP B
 Acinetobacter lwoffii is less virulent  Amikacin
 Piperacillin-tazobactam
CLINICAL INFECTIONS  Ticarcillin-clavulanate
 Accounts for 1-3% of all nosocomial  Cefotoximeor Ceftriaxone
infections  Cefepime
 UTI  Tetracycline
 Doxycyline
 Septicemia
 Minocycline
 Pneumonia  Piperacillin
 Meningitis  Trimethoprim-sulfamethoxazole
 Tracheobronchitis
 Cellulitis GROUP O
 Ticarcillin
 Endocarditis
 Netilmicin
 Eye Infections  Colistin
 Polymyxin B
 Gatifloxacin
 III. GENUS STENOTROPHOMAS MISCELLANEOUS GRAM (-) RODS
Non-Fermenters with Peritrichous Flagella
STENOTROPHOMONAS MALTOPHILIA 1.Alcaligenes species
 Common in the hospital environment as a. Alcaligenes faecalis
contaminant in blood-drawing equipment, b. Alcaligenes odorans
disinfectants, transducers and similar c. Alcaligenes xylosoxidans
equipments 2.Agrobacterium species
 Not a part of the normal human flora 3.Oligella ureolytica
 Resistant to 4.Ochrobactrum anthropi
 cephalosporins
 penicillins Non-Motile
 aminoglycosides 1. Flavobacterium species
 imipenem a. Flavobacterium odoratum
 Nosocomial Infections: b. Flavobacterium meningosepticum
o Pneumonia c. Flavobacterium indologenes
o Endocarditis 2. Sphingobacterium species
o Wound Infections 3.Chryseomonas species
o Bacteremia 4.Flavimonas species
 Oxidase (-) 5.Moraxella species
 Esculin (+) a. Moraxella nonliquefaciens
 Catalase (+) b. Moraxella lacunata
 Lysine Decarboxylase (+) c. Moraxella phenylpyruvica
 DNAse (+) d. Moraxella atlantae
 Gelatin Hydrolysis (+)

ANTIMICROBIAL SUSCEPTIBILITY TESTING

GROUP A
 Trimethoprim-sulfamethoxazole

GROUP B
 Levofloxacin
 Minocycline
 *Ceftazidime
 *Chloramphenicol
 *Ticarcillin-Clavulanate
* MIC only