HIGH-LEVEL AMINOGLYCOSIDE RESISTANCE (HLAR) DIFFERENTIATION DISKS

Gentamicin, 120mcg 50 disks/cartridge
Streptomycin, 300mcg 50 disks/cartridge

INTENDED USE

Gentamicin and Streptomycin High-Level Aminoglycoside Resistance (HLAR) Differentiation Disks, are used to detect high-level aminoglycoside resistance in Enterococcus faecalis and E. faecium. These disks are designated for use for HLAR testing, in accordance with the NCCLS performance standards for susceptibility testing.(8,9)

SUMMARY

Enterococci are characteristically resistant to a wide variety of antimicrobial agents making single-drug therapy often ineffective. Systemic enterococcal infections, such as endocarditis, are usually treated with a combination of two antimicrobial agents: one specific action against the cell wall, such as a beta-lactam or a glycopeptide (i.e., penicillin, ampicillin or vancomycin) and an aminoglycoside, which inhibits bacterial protein synthesis (i.e., gentamicin or streptomycin). These agents act synergistically to enhance killing of the bacteria, since the aminoglycoside has increased uptake into the cell, after cell wall damage by the beta-lactam agent.(2,3,5)

All enterococci naturally have low-level resistance to aminoglycosides, which invalidates use of the disk test with usual concentrations of antimicrobial agents. HLAR is only meaningful for a testing method. When an enterococcal strain has high-level resistance to the aminoglycoside, there is no synergism and combination therapy with a beta-lactam drug will not have the desired bactericidal effect. Therefore, it is important to detect the presence of high-level resistance in order to predict aminoglycoside synergy.(2)

Strains that show HLAR to gentamicin, the most commonly used and best aminoglycosides against enterococci, possess one or more aminoglycoside-modifying enzymes. These enzymes may make them resistant to one or more of a variety of other aminoglycosides, including tobramycin, netilmicin, and amikacin, but not streptomycin.(5) Other HLAR enzymes are active against streptomycin, but not gentamicin. Thus, testing gentamicin and streptomycin is preferred to provide information on the two most active of the aminoglycosides, that do not show cross-resistance to each other. If a strain has high-level resistance to both gentamicin and streptomycin, tobramycin will not be effective. There are some strains that have emerged that will still be amikacin or netilmicin susceptible, however, and testing for high-level resistance in these agents may be indicated.(12)

Many isolates of E. faecalis and E. faecium have acquired high-level resistance to one or more of the aminoglycosides, while other enterococci and viridans streptococci have not yet acquired the genes for resistance. Performing in vitro susceptibility testing of clinical isolates of E. faecalis and E. faeciumfrom systemic infections is critical for determining which combination of agents may be effective therapy.(3)

The three most commonly used methods for HLAR detection are; agar dilution, broth microdilution, and disk diffusion (using high-concentration disks).(11) When performing the disk diffusion test to determine high-level resistance to aminoglycosides, it is important to remember that standard gentamicin and streptomycin disks (10mcg each) that are used for routine disk diffusion testing cannot be used. Only high-concentration disks can be used to determine aminoglycoside resistance.(5)

FORMULA

Each HLAR Differentiation Disks contains the following specified concentrations of the appropriate antibiotics on high quality 6mm diameter filter paper disks:

Gentamicin 120mcg
Streptomycin 300mcg

STORAGE AND SHELF LIFE

Storage: Upon receipt store at -20ºC. away from direct light. A small supply of disks for use within one week can be stored at 4ºC. The disks should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect from light, excessive heat, and moisture.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for the primary isolation of patient specimens. It should be used only with cultures of isolated organism. These are high-concentration disks; do not use standard susceptibility disks for HLAR testing.

1. Allow disks to equilibrate to room temperature.

2. Using a pure 18-24 hour culture, prepare a suspension (equivalent to a McFarland 0.5 opacity standard) of the organism to be tested.

3. Dip a sterile non-toxic cotton swab (Cat. no. 258061WC) into the organism suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. Evenly inoculate the dried surface of a Mueller Hinton Agar plate (Cat. no. H11 or G45) by streaking the swab over the entire surface of the plate in three directions, as for a routine disk diffusion test. (9)

4. Aseptically place one Gentamicin Disk (120mcg) and one Streptomycin Disk (300mcg) on the media surface, far enough away from each other to leave room for zones of inhibition (greater than 30mm apart). With sterile forceps, gently tap each disk to the media surface to ensure uniform diffusion of the antibiotic into the medium.

5. Invert plates, and incubate aerobically at 35ºC. for 18-24 hours.

6. Examine plates for confluent or almost confluent growth, and measure zones of inhibition for each disk. (If growth is unacceptable, the test cannot be interpreted). Reincubation of the plate for an additional 24 hours may be done to verify susceptibility of the strain to streptomycin.

INTERPRETATION OF RESULTS

Sensitive: Zone size is greater than or equal to 10mm. The organism is not an HLAR Enterococcus and synergy with the cell wall-active agent is likely.

Resistant: No zone of inhibition (6mm). The organism is an HLAR Enterococcus and synergy with the cell wall-active agent is not likely.

Intermediate: Zone sizes of 7 to 9mm are considered inconclusive, and should be retested by an alternative method (i.e., standard agar screen or broth microdilution methods).(2,4,5)

LIMITATIONS

Zone sizes of 7 to 9mm should be tested by an alternative method, as problems exist with detection of this resistance by commercial susceptibility systems.(6)

Standard gentamicin (10mcg) and streptomycin (10mcg) disks that are used for routine disk diffusion testing are not intended to be used for determining high-level aminoglycoside resistance (HLAR).(5)

Some isolates with HLAR to streptomycin may not demonstrate resistance until after 48 hours of incubation.(5)

All Enterococcus faecium strains produce a chromosomally encoded aminoglycoside acetyltransferase, which eliminates synergism between cell wall-active antimicrobials and the aminoglycosides tobramycin, kanamycin, netilmicin, and sisomicin. Performing further testing for resistance to these agents is not meaningful.(12)

Susceptibility to either gentamicin or streptomycin does not predict susceptibility to other aminoglycosides; erroneous therapeutic decisions can result if cross-susceptibility is assumed.

Strains that are high-level resistant to gentamicin are also resistant to tobramycin and kanamycin, but may still be susceptible to amikacin (or netilmicin, for E. faecalis). If the strain is resistant to both gentamicin and streptomycin, further testing for high-level resistance to amikacin and netilmicin in a reference laboratory may be indicated.(12)

Resistance to streptomycin does not predict resistance to any other aminoglycoside.

Mueller Hinton Agar supplemented with sheep blood is not appropriate for determining HLAR, since larger zones of inhibition were observed than those observed on unsupplemented agar, possibly causing false susceptibility, especially with streptomycin.(11)

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Zone Size
Time Temperature Atmosphere Gentamicin Streptomycin
Enterococcus faecalis
ATCC ® 51299
F 18-24hr 35°C Aerobic 6mm 6mm
Enterococcus faecalis
ATCC ® 29212
F 18-24hr 35°C Aerobic 16-23mm 14-20mm

User Quality Control

PHYSICAL APPEARANCE

HLAR Differentiation Disks are 6mm (in diameter) filter paper disks, and should appear white in color.

REFERENCES

1. 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.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

4. Isenberg, H.D. 1998. Chap. 5 - Antimicrobial Susceptibility Testing, p. 227-229. Essential Procedures for Clinical Microbiology. American Society for Microbiology, Washington, D.C.

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

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

7. Bantar, C.E., M. Micucci, L. Fernandez Canigia, J. Smayevsky, and H.M. Bianchini. 1993. Synergy Characterization for Enterococcus faecalis Strains Displaying Moderately High-Level Gentamicin and Streptomycin Resistance. J. Clin. Microbiol.; 31:1921-1923.

8. NCCLS, January 2003. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; approved standard 6th ed. M7-A6.

9. NCCLS, January 2003. Performance Standards for Antimicrobial Disk Susceptibility Tests; approved standard 8th ed. M2-A8.

10. Sahm, D.F., and C. Torres. 1988. High-Content Aminoglycoside Disks for Determining Aminoglycoside-Penicillin Synergy against Enterococcus faecalis. J. Clin. Microbiol.; 26:257-260.

11. Swenson, J.M., M.J. Ferraro, D.F. Sahm, N.C. Clark, D.H. Culver, F.C. Tenover, and The National Committee for Clinical Laboratory Standards study group on Enterococci. 1995. Multilaboratory Evaluation of Screening Methods for Detection of High-Level Aminoglycoside Resistance in Enterococci.J. Clin. Microbiol. ; 33:3008-3018.

12. Chow, J.W. 2000. Aminoglycoside Resistance in Enterococci. Clin. Infect. Dis. 31:586-589.


080516gr