Leeds ACINETOBACTER MEDIum

Cat. no. G261 Leeds Acinetobacter Medium, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Leeds Acinetobacter Medium is recommended for the selective and differential isolation of Acinetobacter spp.

SUMMARY

Acinetobacter spp. are ubiquitous bacteria that have been isolated from patients, the environment, soil, and water. They are frequently responsible for episodes of nosocomial infection and have been isolated from nebulizers, gloves and dust. Skin colonization with A. baumannii in healthy individuals is common and may serve as a source of infection. Acinetobacter has been reported as responsible for 2-10% of all gram-negative bacterial infections in intensive care units in Europe and the United States.(1) Acinetobacter baumannii is often resistant to commonly used antibiotics including beta-lactams and aminoglycosides. Because of this, management of A. baumannii infection has become a public health concern in many countries. Some strains have become resistant to almost all currently available antibacterial agents, including carbapenems, which were once considered the drug of choice for treatment of A. baumannii infections.(1-6)

Outbreaks are often related to the virulence of the strain and most outbreaks occur in critical care settings involving chronically ill patients. Risk factors include those accompanying other hospital-acquired MDR infections and ventilator-associated pneumonia. Acinetobacter can be found in almost every type of environment and unknown mechanisms appear to impede dessication of the organism. Resistance of the organism to multiple classes of antimicrobial agents can also produce a reservoir of antibiotic resistance genes in the hospital environment.(2)

Several selective and differential media are available for the isolation of Acinetobacter spp. Mandel et al. developed a medium containing agar, soy peptone, pancreatic digest of casein, sodium chloride, lactose, maltose, bile salts, and bromocresol purple. This medium was modified to Herellea agar by substituting sucrose for maltose. Mandel's medium was further modified by Holton by substituting desiccated ox bile for the bile salts and ampicillin, cefsulodin and vancomycin were added. Holton also substituted fructose, mannitol, phenylalanine, and phenol red for lactose and bromocresol purple. Leeds Acinetobacter Medium (LAM) was developed at the University of Leeds by Jawad et al. In this formulation, ampicillin was removed and the concentrations of the other antibiotics were adjusted to obtain optimum performance.(1)

Hardy Diagnostics Leeds Acinetobacter Medium contains peptones to support cell growth. Fructose, sucrose, and mannitol are fermentable carbohydrates and phenylalanine is added to further differentiate those organisms that produce phenylpyruvic acid. Phenol red is the pH indicator that will turn yellow in the presence of acid and pink to mauve when high levels of alkalinity are produced in the medium by the growth of organisms. Selective agents are included to inhibit unwanted flora.

FORMULA

Ingredients per liter of deionized water:*

Casein Acid Hydrolysate 15.0gm
Soy Peptone 5.0gm
Sodium Chloride 5.0gm
Fructose 5.0gm
Sucrose 5.0gm
Mannitol 5.0gm
Phelylalanine 1.0gm
Ferric Ammonium Citrate 0.4gm
Phenol Red 0.02gm
Selective Agents** 0.035gm
Agar 12.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, specimens should be inoculated into the appropriate transport media and refrigerated until inoculation in a sterile container, or other appropriate means of transport. Consult appropriate references for specimen collection and transport.(1)

Method of Use: Prior to inoculation, the medium should be brought to room temperature. Inoculate the media with the specimen and streak for isolation. Incubate aerobically at 35ºC. for 24-48 hours.

INTERPRETATION OF RESULTS

Acinetobacter spp. will produce light pink mucoid colonies with a pink to mauve color diffused into the medium. The colonies are circular, convex, smooth, and opaque with entire margins of 1 to 2mm in diameter after 24 hours at 35 degrees C.

Stenotrophomonas maltophilia will produce light pink colonies with a pink to mauve diffused into the medium. The colonies are opaque and flat with rugose surfaces and crenated margins of 1 to 2mm in diameter after 24 hours at 35 degrees C.

Burkholderia cepacia will produce light pink colonies with a pink to mauve color diffused into the medium. 90% of B. cepacia strains are oxidase positive.

Citrobacter spp. will produce yellow colonies with a yellow color diffused into the medium. The organism is inhibited on MDR Acinetobacter Medium.

Providencia alcalifaciens will produce brown colonies with a brown-black color diffused into the medium.

Serratia marcescens will produce pink colonies with yellow margins.

LIMITATIONS

While the medium is differential and selective for Acinetobacter spp., other antimicrobial susceptibility and biochemical tests must be performed for complete identification.

Burkholderia cepacia and/or Stenotrophomonas maltophilia may grow on this medium producing pink colonies. Differentiation can by made by colonial morphology and/or an oxidase test.

White colonies present at 24 hours may turn pink after 48 hours of incubation. Further testing is needed to determine if these isolates are Acinetobacter spp.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Acinetobacter baumannii
ATCC® BAA-747**
A 24hr 35°C Aerobic Growth; pink mucoid colonies with pink to mauve color diffused into the medium
Citrobacter freundii
CV5
B 24hr 35°C Aerobic Partial inhibition; small yellow colonies with yellow color diffused into the medium
Burkholderia cepacia
ATCC® 25416
B 24hr 35°C Aerobic Partial inhibition; small pink colonies with pink to mauve color diffused into the medium
Escherichia coli
ATCC® 25922**
B 24hr 35°C Aerobic Inhibited
Enterococcus faecalis
ATCC® 29212
B 24hr 35°C Aerobic Inhibited
Candida albicans
ATCC® 10231
B 24hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

Physical Appearance

Leeds Acinetobacter Medium should appear clear to slightly opalescent, and light peach in color.

Acinetobacter baumanni growing on MDR Acinetobacter

Acinetobacter baumannii (ATCC® BAA-7477) colonies growing on Leeds Acinetobacter Medium (Cat. no. G261). Incubated aerobically for 24 hours at 35ºC.

Burkholderia cepacia growing on MDR Acinetobacter Medium

Burkholderia cepacia (ATCC® 25416) colonies growing on Leeds Acinetobacter Medium (Cat. no. G261). Incubated aerobically for 24 hours at 35ºC.



MDR Acinetobacter Medium

Uninoculated plate of Leeds Acinetobacter Medium (Cat. no. G261).

REFERENCES

1. Valentine, S.C., et al. 2008. Phenotypic and Molecular Characterization of Acinetobacter baumannii Clinical Isolates from Nosocomial Outbreaks in Los Angeles County, California. J. Clin. Microbiol.; 46:2499-2507.

2. Montefour, K., et al. 2008. Acinetobacter baumannii: An Emerging Multidrug-Resistant Pathogen in Critical Care. Critical Care Nurse; 28:15-25.

3. Richet, H. and E. Fournier. 2006. Nosocomial Infections Caused by Acinetobacter baumannii : A Major Threat Worldwide. Infect. Control Hosp. Epidemiol.; 27:645-646.

4. Bou, G., et al. 2000. Characterization of Nosocomial Outbreak Caused by a Multiresistant Acinetobacter baumannii Strain with a Carbapenem-Hydrolyzing Enzyme: High-Level Carbapenem Resistance in Acinetobacter baumannii Is Not Due Solely to the Presence of Beta-Lactamases. J. Clin. Microbiol.; 38:3299-3305.

5. Peleg, A.Y., Franklin, C., Bell, J.M., Spelman, D.W. 2006. Emergence of Carbapenem Resistance in Acinetobacter baumannii Recovered From Blood Cultures in Australia. Infect. Control Hosp. Epidemiol.; 27:759-761.

6. Park, Y.K., Peck, K.R., Cheong, D., Song, J., Ko, K.S. Emerging Infectious Diseases; 15(8): 1325-1326. (Letter)

7. Jawad, A., Hawkey, P.M., Heritage, J. and A.M. Snelling. 1994. Description of Leeds Acinetobacter Medium, a New Selective and Differential Medium for Isolation of Clinically Important Acinetobacter spp., and Comparison with Herellea Agar and Holton's Agar. J. Clin. Microbiol.; 32:2353-2358.

8. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

9. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

10. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

11. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

12. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

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