Metallo-Beta-Lactamase (MBL) Neo-Sensitabs™

Cat. no. Z155 Metallo-Beta-Lactamase (MBL) Neo-Sensitabs™:
Imipenem (IMI10) and Imipenem + EDTA (IM10E)
2 cartridges x 50 tabs each

INTENDED USE

Hardy Diagnostics Metallo-Beta-Lactamase (MBL) Neo-Sensitabs™ are recommended for use in the imipenem-EDTA double-disk synergy test (DDST) for differentiating metallo-beta-lactamase (MBL)-producing Enterobacteriaceae, including New Delhi metallo-beta-lactamase (NDM-1)-producing strains. The product is intended for research use only.

SUMMARY

The following information was obtained from the manufacturer's product insert:

The worldwide spread of acquired metallo-beta-lactamases (MBLs) in gram-negative aerobes is of great concern. MBL production in clinical isolates of key gram-negatives; Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae should be carefully monitored.(2)

MBLs are classified into five major types: IMP, VIM, SPM, GIM, and SIM-type enzymes. In Enterobacteriaceae, only IMP and VIM enzymes have been found as of yet. MBLs hydrolyze most beta-lactams (carbapenems and large spectrum cephalosporins), except aztreonam. This phenotype of multiple beta-lactam resistance and aztreonam susceptibility may be helpful for identification of these strains in the laboratory. If the strain is resistant to aztreonam, it may be due to additional resistance mechanisms (efflux, other beta-lactams, ESBLs, etc.). Their expression is not inducible.

The MBL enzymes are resistant to beta-lactamase inhibitors and susceptible to chelating agents like EDTA (2-MPA) and diplicolinic acid (DPA).

Early detection of MBL-producing microorganisms is essential to prevent dissemination of these organisms.(21,23,24) Current research on MBLs, including strains of Enterobacteriaceae and non-fermenters producing MBLs, reveals that MBL-producers (particularly in Enterobacteriaceae) may show low MIC values against carbapenems, making it difficult for the laboratory to detect MBL-positive isolates, particularly with the use of automated systems.(12,18)

Suspicious isolates (resistant to ceftazidime showing no synergy between clavulanate and third generation cephalosporins and possibly showing reduced susceptibility to carbapenems) should be tested for carbapenemase activity using double-disk synergy test (DDST) methods.

The first metallo-beta-lactamase producing strain of Escherichia coli was detected in Barcelona, Spain using imipenem + EDTA Neo-Sensitabs™ and Etest®.(8,20) The first metallo-beta-lactamase producing strain of Klebsiella pneumoniae was found in France.(9) MBL-producing gram-negatives have now emerged in Australia, as well as other countries such as the United States.(3,14) The resistance gene blaIMP-4 appears highly mobile in the Australia strains, and this outbreak involved five different gram-negative genera. Consequently, diagnostic laboratories worldwide should be on high alert, because early detection may limit wide dispersal of MBL genes.

FORMULA

The imipenem (IMI10) Neo-Sensitab™ measures 9mm in diameter and contains 10ug of imipenem per tab. The imipenem + EDTA (IM10E) Neo-Sensitab™ measures 9mm in diameter and contains 10ug of imipenem and 750ug of EDTA per tab. Neo-Sensitabs™ are printed on both sides with a unique five character code for easy identification.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 15-30ºC. away from direct light. The tabs 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

Reliable detection of carbapenem resistance mechanisms may be determined by antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method and the modified Hodge test.(3,4)

Direct specimen testing is not recommended. It is recommended that isolated organisms, established isolation techniques, and tests for purity be performed before inoculating media for testing. Direct inoculation of clinical specimens will produce erroneous results.

For detection of carbapenemase-producing Pseudomonas spp. and Acinetobacter spp. isolates, consult listed references.(4,5,7,10,11,17-19,22)

The following information was obtained from the manufacturer's package insert:

Some resistance profiles may suggest MBL production. For example:

a) Pseudomonas aeruginosa, Pseudomonas spp. and Acinetobacter spp.
All isolates non-susceptible to carbapenems and resistant to either ticarcillin, ticarcillin+clavulanate or ceftazidime should be tested for MBL production.

b) Enterobacteriaceae
For Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella spp., and Shigella spp.: all carbapenem S-I-R isolates that are resistant to cefoxitin and amoxicillin+clavulanate, and are non-susceptible to ceftazidime (inhibition zone < 18mm) should be tested for MBL production. In all other cases, all isolates are non-susceptible to carbapenems.(5)

Preparation of inoculum with test and control cultures:(4)

1. Perform a Gram stain using a pure culture.

2. Select three to five similar, well isolated colonies and transfer with inoculation needle or loop into 4-5ml of a suitable broth such as Tryptic Soy Broth (Cat. no. K89).

3. Incubate the broth culture at 35ºC. for two to six hours to develop a turbidity that exceeds or is equivalent to a 0.5 McFarland Standard (Cat. no. ML05). Alternatively, make a direct broth or saline suspension of colonies selected from an overnight culture (a non-selective medium such as Blood Agar should be used).

4. Dilute to obtain turbidity equivalent to a 0.5 McFarland Standard (Cat. no. ML05). For diluent, use sterile broth or saline. The suspension should contain approximately 1 to 2x108 cfu/ml for E. coli, ATCC® 25922. Alternatively, standardize the inoculum photometrically to facilitate adjustment of rapidly growing microorganisms.
Note: Overnight broth cultures should not be used as inoculum.

Inoculation:(4)

1. Within 15 minutes, dip a sterile swab into the adjusted inoculum, rotate it several times and press firmly against the upper inside wall of the tube to express excess fluid.

2. Streak the entire surface of the Mueller Hinton (MH) Agar (Cat. no. G45 or H11) plate three times, turning the plate 60º between streaks to obtain an even inoculation.

3. The lid may be left ajar for three to five minutes, but no more than 15 minutes, to allow for any surface moisture to be absorbed before applying the drug-impregnated tabs.

4. Apply one imipenem + EDTA (IM10E) and one imipenem (IMI10) Neo-Sensitab™ directly to the surface of an inoculated Mueller Hinton plate using aseptic precautions. Deposit tabs so that the centers are at least 24mm apart.

Note: It is important to pay attention to how close the Neo-Sensitabs™ are to the edge of the plate, regardless of the number of Neo-Sensitabs™ used. If tabs are placed too close to the edge of the plate, zones may not form fully round diameters. Because some of the drug diffuses almost instantaneously, tabs should not be relocated once they have come into contact with the agar surface. Instead, place a new tab in another location on the agar. Press tabs down firmly with a sterile needle or forceps to ensure complete contact with the agar surface.

5. Within 15 minutes, place plates agar side up in a 35 +/- 2ºC. incubator.

6. Examine plates after 16 to 18 hours of incubation. Measure across the diameter of the zones using gross visual inspection and record the diameters of zones to the nearest millimeter. For further details in measuring zones of inhibition, consult the listed reference.(4) If only isolated colonies grow, the inoculum is too light and the test should be repeated.

INTERPRETATION OF RESULTS

Enterobacteriaceae - isolates displaying reduced susceptibility to imipenem, 10ug (producing a zone < 23mm or MIC 1ug/ml breakpoint) on Mueller Hinton Agar using a 0.5 McFarland standard inoculum should be suspected of possessing carbapenemases.

Pseudomonas aeruginosa - isolates displaying susceptibility to imipenem 10ug (producing a zone < 22mm) on Mueller Hinton Agar using a 0.5 McFarland standard inoculum should be suspected of possessing a carbapenemase. Most isolates possessing KPC or GES enzymes are highly resistant to ceftazidime. Confirmation with modified Hodge test is strongly suggested.

After overnight incubation, an imipenem + EDTA (10ug+750ug) zone 7mm or larger than the imipenem (10ug) zone indicates the presence of a metallo-beta-lactamase (MBL) and should be interpreted as EDTA-synergy positive.(10,11,15)

LIMITATIONS

In vitro susceptibility does not necessarily imply in vivo effectiveness.

Metallo-beta-lactamases are zinc-dependent. Most Mueller Hinton agar formulations contain physiological levels of zinc ions and should be used for carbapenem testing. Formulations containing low levels of zinc ions may yield false susceptibility results for carbapenems in the presence of MBLs.

Some strains of Acinetobacter baumannii producing certain oxacillinases may yield false-positive metallo-beta-lactamase results.

Use of the modified Hodge test in conjunction with double-disk synergy tests may improve carbapenemase detection.(11)

Some MBL-inhibiting agents, such as EDTA, may possess their own bactericidal activity, resulting in expanded zones of inhibition not associated with true MBL production. Zone sizes produced by beta-lactam/inhibitory metallo-beta-lactamase agent combinations may vary according to the way the inhibitory metallo-beta-lactamase agent is incorporated into the beta-lactam disk.(15)

Screening-positive, MBL-producing isolates by polymerase chain reaction (PCR) may be helpful in determining the genetic resistance mechanism. However, PCR results are solely dependent upon previously characterized genetic-resistance mechanisms.

Total or partial loss of MBL-producing cells may occur during room temperature storage of isolates. Loss of resistance mechanisms may be avoided by testing for imipenem susceptibility and MBL production concurrently.(11,19)

Neo-Sensitab™ performance and results depend on tab potency, proper use of inoculum and control cultures, functional plated media, proper storage conditions, and other factors.

The test applies primarily to rapidly growing aerobic pathogens.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, Tryptic Soy Broth (TSB, Cat. no. K89), Mueller Hinton Agar (Cat. no. G45 or H11), 0.5 McFarland Standard (Cat. no. ML05), incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Known positive Stenotrophomonas maltophilia ATCC ® 13636 and negative Pseudomonas aeruginosa ATCC ® 27853 controls should be used to monitor the accuracy of the tablets and inoculum.

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Stenotrophomonas maltophilia
ATCC ® 13636**
F 24hr 35 o C Aerobic Synergy positive; > 7mm increase in zone of inhibition using IM10E tablet
Pseudomonas aeruginosa
ATCC ® 27853**
F 24hr 35 o C Aerobic Synergy negative; zones of inhibition within acceptable range (4)

** Recommended QC strains for User Quality Control.

USER QUALITY CONTROL

Physical Appearance

The Metallo-Beta-Lactamase (MBL) Neo-Sensitabs™ package contains two cartridges of 9mm (in diameter) antimicrobial-containing tablets that should appear yellow in color. The Imipenem Neo-Sensitabs™ are printed with the letters IMI10 on both sides, whereas the Imipenem + EDTA Neo-Sensitabs™ are printed with the letters IM10E on both sides.

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. Castanheira, M, M.A. Toleman, R.N. Jones, F.J. Schmidt, and T.R. Walsh. Molecular characterization of a beta-lactamase gene blaGIM-1, encoding a new subclass of metallo-beta-lactamase. Antimicr. Ag. Chemother.; 48(12):4654-4661.

3. Centers for Disease Control and Prevention. 2010. Detection of Enterobacteriaceae isolates carrying metallo-beta-lactamase--United States. MMWR; 59(24):750 [cited 2011 February 15]. Available from: www.cdc.gov/mmwr/preview/mmwrhtml/mm5924a5.htm

4. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). Performance Standards for Antimicrobial Susceptibility Testing, Informational Supplement. M100. CLSI, Wayne, PA.

5. Cornaglia, G. et al. 2007. Metallo-beta-lactamases as emerging resistance determinants in gram-negative pathogens: open issues. Antimicr. Ag. Chemother.; 29:380-388.

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

7. Jesudason, M.V., A.J. Kandathil, and V. Balaji. 2005. Comparison of two methods to detect carbapenemase and metallo-beta-lactamase production in clinical isolates. Indian J. Med. Res.; 121:780-783.

8. Larrosa, M.N. et al. 2004. E. coli multi-resitente productora de una metalo-beta-lactamasa (Spanish). SEIMC Congress, Bilbao. May 16-19th.

9. Lartigue, M.F., L. Poirel, and P. Nordmann. First detection of a carbapenem-hydrolyzing metalloenzyme in an Enterobacteriaceae isolate in France. Antimicr. Ag. Chemother.; 48(12):4929-4930.

10. Lee, K., Y. Chong, H.B. Shin, Y.A. Kim, D. Yong, and J.H. Yum. 2001. Modified Hodge test and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing Pseudomonas and Acinetobacter species. Clin. Microbiol. Infect.; 7:88-91.

11. Lee, K., Y.S. Lim, D. Yong, J.H. Yum, and Y. Chong. 2003. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol.; 41(10):4623-4629.

12. Miriagou, V., C.C. Papagiannitsis, E. Tzelepi, J.B. Casals, N.J. Legakis, and L. S. Tzouvelekis. 2010. Detecting VIM-1 production in Proteus mirabilis by an imipenem-dipicolinic acid double disk synergy test. J. Clin. Microbiol.; 48:667-668.

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

14. Peleg, A.Y., C. Franklin, J.M. Bell, and D.W. Spelman. 2005. Dissemination of the metallo-beta-lactamase gene blaIMP-4 among gram-negative pathogens in a clinical setting in Australia. Clin. Infect. Dis.; 41:1549-1556.

15. Picao, R.C., S.S. Andrade, A. G. Nicoletti, E.H. Campana, G.C. Moraes, R.E. Mendes, and A.C. Gales. 2008. Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolated. J. Clin. Microbiol.; 46(6):2028-2037.

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

17. Renu, G., T. Rajeev, S. Smita. 2010. The existence of metallo-beta-lactamases in carbapenem susceptible gram-negative bacilli: a cause for concern. J. Clin and Diag. Res.; 4:2679-2684.

18. Shin, K.S, B.R. Son, S.B. Hong, and J. Kim. 2008. Dipicolinic acid-based disk methods for detection of metallo-beta-lactamase-producing Pseudomonas spp. and Acinetobacter spp. Diagn. Microbiol. Infect. Dis.; 62(1):102-105.

19. Takahashi, A., S. Yomoda, I. Kobayashi, T. Okubo, M. Tsunoda, and S. Iyobe. 2000. Detection of carbapenemase-producing Acinetobacter baumannii in a hospital. J. Clin. Microbiol.; 38:526-529.

20. Tortola, M.T., S. Lavilla, E. Miro, J.J. Gonzalez, N. Larrosa, M. Sabate, F. Navarro, and G. Prats. 2005. First detection of a carbapenem-hydrolyzing metalloenzyme in two Enterobacteriaceae isolates in Spain. Antimicr. Ag. Chemother.; 49(8):3492-3494.

21. U.K. Health Protection Agency. 2009. Multi-resistant hospital bacteria linked to India and Pakistan. Health Protection Report; 3(26).

22. Yong, D., K. Lee, J.H. Yum, H.B. Shin, G.M. Rossolini, and Y. Chong. 2002. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol.; 40(10):3798-3801.

23. Yong, D., M.A. Toleman, C.G. Giske, H.S. Cho, K. Sundman, K. Lee, and T.R. Walsh. 2009. Characterization of a new metallo-beta-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob. Agents and Chemo.; 53(12):5046-5054.

24. Zarfel, G., M. Hoenigl, E. Leitner, H.J.F. Salzer, G. Feierl, L. Masoud, T. Valentin, R. Krause, and A.J. Grisold. 2011. Emergence of New Delhi metallo-beta-lactamase, Austria. Emerg. Infect. Dis.; 17(1):129-130.


ATCC is a registered trademark of the American Type Culture Collection.
Etest is a registered trademark of AB BIODISK.
Neo-Sensitab is a trademark of Rosco Diagnostica.

This document is provided for general product information only. It does not replace the manufacturer's product insert. Always refer to the actual product insert for procedural use and for the most recent information.

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