Characterization of HMRZ-86: a novel chromogenic cephalosporin for the detection of extended-spectrum ß-lactamases

Hideaki Hanaki1, Ryoichi Kubo2,*, Tomota Nakano3, Makoto Kurihara3 and Keisuke Sunagawa4

1 Kitasato Institute, Infection Science Control Research Center, Tokyo; 2 Kanto Chemical Co., Inc., Life Science Department, Tokyo; 3 Kanto Chemical Co., Inc., Isehara Laboratory, Kanagawa; 4 Kitasato University, Department of Infectious Disease, Kanagawa, Japan

Keywords: ß-lactamase differentiation, clinical laboratory, disc tests

Sir,

HMRZ-86 is a new chromogenic cephalosporin. A carboxypropyloxyimino group bonded to the side chain at position 7 in the compound (Figure 1) protects the ß-lactam ring from a range of ß-lactamases, but not from extended-spectrum ß-lactamases (ESBLs) or metallo-ß-lactamases.1 Hydrolysis of the ß-lactam ring by these enzymes changes the wavelength absorbed by the conjugated double bond located at position 3, shifting the colour of the compound from yellow to red, in much the same way as occurs with nitrocefin.2 In this study, we determined the usefulness of the compound for identifying and differentiating a range of ß-lactamases.



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Figure 1. Structural formula of HMRZ-86; (7R)-7-[2-(aminothiazol-4-yl)-(z)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid trifluoroacetate, E-isomer.

 
Test strains and enzyme types are outlined in Table 1. ESBL-producing strains were cultivated overnight at 37°C on brain heart infusion agar (Oxoid Ltd) containing ceftazidime 2 mg/L.


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Table 1. Test result of ß-lactamase-producing microorganisms on each disc type
 
Discs (8 mm) were prepared as follows: nitrocefin (5 µg); nitrocefin/aztreonam (5 µg/5 µg); nitrocefin/aztreonam/clavulanic acid (5 µg/5 µg/20 µg); HMRZ-86 (5 µg); HMRZ-86/sodium mercaptoacetic acid (SMA) (5 µg/0.1 M SMA). Discs were stored under refrigeration and used within a few days of preparation. Discs are generally stable for several weeks if refrigerated, but change colour within days at room temperature.

Immediately before use, discs were moistened with phosphate buffer (20 µL 0.1 M, pH 7.0). Isolated colonies were applied to discs with a platinum loop; discs were left to stand at room temperature for 15–30 min and the colour of the disc was observed with the naked eye. A change in colour from yellow to red was taken as a positive result.

Results are shown in Table 1. All strains with Class A penicillinases gave a positive reaction with nitrocefin discs, and a negative reaction with HMRZ-86 and nitrocefin/clavulanic acid discs (clavulanic acid inhibits Class A-type activity). All strains with Class A ESBLs gave a positive reaction with nitrocefin, nitrocefin/aztreonam and HMRZ-86 discs: SMA did not affect ESBL activity. Strains with Class B metallo-ß-lactamases gave positive reactions with all discs except those containing HMRZ-86 plus SMA. However, we found that if SMA is allowed to react with HMRZ-86 for an extended period of time, a false-positive result may be obtained and therefore colour changes should be read within 30 min. All strains with Class C cephalosporinases gave positive reactions with nitrocefin discs, but negative reactions with the other four discs. All strains with Class D non-ESBLs gave positive reactions on nitrocefin, nitrocefin/aztreonam and nitrocefin/clavulanic acid/aztreonam discs, but negative reactions with HMRZ-86 and HMRZ-86/SMA discs. Strains with Class D ESBLs gave positive reactions with all discs; the activity of the enzymes was not inhibited by clavulanic acid and this distinguished them from the Class A-type ESBLs.3

Metallo-ß-lactamases4 also react with HMRZ-86, but are inhibited by certain chelating agents such as EDTA and SMA. Chelating agents such as these react with HMRZ-86 and may produce false-positive results after extended periods; such agents should be mixed with HMRZ-86 just before use. Class C enzymes do not react with HMRZ-86 and are inhibited by aztreonam.5 However, several pathogens from clinical specimens produce large amounts of cephalosporinase (AmpC), and other pathogens produce enzymes that are inhibitor resistant. It is difficult to differentiate these organisms from organisms producing Class D enzymes, which may or may not be ESBLs.6 Non-ESBL types react with nitrocefin, nitrocefin/aztreonam and nitrocefin/clavulanic acid/aztreonam, but not with HMRZ-86. On the other hand, ESBL types react with the combined compounds as well as HMRZ-86.

Traditional detection methods are labour intensive and time-consuming, making them difficult to implement as part of the routine of a small hospital. Our study confirmed that HMRZ-86 reacts with ESBLs and metallo-ß-lactamases, but not with traditional penicillinases and cephalosporinases. The reaction occurs within 30 min, and the result is indicated by a visible change in colour. Although the vividness of the colour depends on the strain, even weak reactions are readily visible within 30 min. We believe that HMRZ-86 is useful for differentiating ESBLs from traditional ß-lactamases. For this method to be effective, we found it necessary to culture strains on media containing a third-generation cephalosporin (ceftazidime or cefotaxime). Before testing, the cells of certain microorganisms that store ß-lactamase in their periplasmic space must be crushed to extract the enzyme, and we rubbed the colony against the paper disc for this purpose. The value of HMRZ-86 in detecting ESBLs requires further validation with clinical specimens. Nevertheless, we believe the compound will provide an effective screening test for ESBLs in the clinical laboratory.

Acknowledgements

We gratefully acknowledge the assistance of Takayoshi Kobayashi (Kanto Chemical Co., Inc.) and Tsuyoshi Koiso (Showa Yakuhin Kako Co. Ltd) in preparing the materials used in our study.

Footnotes

* Corresponding author. Tel: +81-3-3667-8061; Fax: +81-3-3667-6891; E-mail: kubor{at}gms.kanto.co.jp Back

References

1 . Kawashima, S., Yamazaki, H., Harada, H. et al. (2002). Cephem compounds and ESBL-detecting reagents containing the same. International Patent Information Number WO02/24707 A1.

2 . O’Callaghan, C. H., Morris, A., Kirby, S. M. et al. (1972). Novel method for detection of ß-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 283–8.[ISI][Medline]

3 . Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211–33.[Free Full Text]

4 . Payne, D. J., Bateson, D. A., Gasson, B. C. et al. (1997). Inhibition of metallo-ß-lactamases by a series of mercaptoacetic acid thiol ester derivatives. Antimicrobial Agents and Chemotherapy 41, 135–40.[Abstract]

5 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

6 . Jacoby, G. A. (1998). Epidemiology of extended-spectrum ß-lactamases. Clinical Infectious Diseases 27, 81–3.[ISI][Medline]





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