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Journal of Clinical Microbiology, March 2009, p. 827-829, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.02464-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

High Concentrations of Manganese in Mueller-Hinton Agar Increase MICs of Tigecycline Determined by Etest

Carlos Fernández-Mazarrasa,¹ Olav Mazarrasa,² Jorge Calvo,¹ Asunción del Arco,¹ and Luis Martínez-Martínez^1,3*

Service of Microbiology, University Hospital Marqués de Valdecilla,¹ Department of Molecular Biology, School of Medicine,³ University of Cantabria, and Occupational Safety and Health Center, Santander, Spain²

Received 22 December 2008/ Accepted 2 January 2009

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MICs of tigecycline determined by Etest were 4 to 12 times (three ATCC strains) and 2 to 8 times (50 clinical isolates) higher in Mueller-Hinton agar from Merck than in Mueller-Hinton agar from either Oxoid or Difco. This was related to a much higher concentration of manganese in the medium from Merck.

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Tigecycline is the first glycylcycline approved for clinical use (16). It can be oxidized when added to a broth medium that has been oxygenated during storage (13) or when tigecycline-containing broth is stored before inoculation. This can be solved using freshly autoclaved broth or recently prepared solid media (2) and is expected to be avoided in the disk diffusion or gradient diffusion (Etest) assays.

During a nation-wide study in Spain on the activity of tigecycline against clinical isolates using Etest strips (AB Biodisk, Solna, Sweden), the results in our center were up to 16 to 20 times higher than those obtained in other centers (A. Pérez, Wyeth Farma S.A., Spain, personal communication). After excluding the possibility that some technical error occurred in our laboratory, we hypothesized that these results could be related to differences in the composition of the Mueller-Hinton agar used in different laboratories, as the commercial source for this medium in our center (Merck) was different from that in many other laboratories in Spain.

In this study, the activity of tigecycline determined with Etest strips on two different lots of commercially available Mueller-Hinton agar from Oxoid (Basingstoke, Hampshire, England; lots 418599 and 456733), Difco (Difco Mueller-Hinton agar [BD, Sparks, MD]; lots 6093187 and 6177851), and Merck (Darmstadt, Germany; lots VL379337507 and VL546637607) was evaluated. We also determined the effect that differences in the concentrations of ions of the evaluated Mueller-Hinton agars may have on the in vitro activity of tigecycline.

In a preliminary study, five clinical isolates (selected among those we already tested in the multicenter study in Spain) and four reference strains, namely, Escherichia coli ATTC 25922, Pseudomonas aeruginosa ATCC 27953, Staphylococcus aureus 29213, and Enterococcus faecalis 29212, were evaluated. MICs of tigecycline for these two groups of organisms were consistently 4 to 12 times higher in the medium from Merck than in the medium from Difco or Oxoid, except for the P. aeruginosa ATCC strain (MICs of 64 to 128 µg/ml in the three media) (data not shown).

In a second phase, MICs of tigecycline for 50 clinical isolates (one per patient; clonally unrelated as assessed by repetitive extragenic palindromic PCR and/or pulsed-field gel electrophoresis) were determined in duplicate experiments on different days. Extended-spectrum β-lactamase (ESBL) production was confirmed by disk diffusion (4). Resistance to methicillin in S. aureus and to vancomycin in Enterococcus faecium was confirmed by PCR detection of the mecA (9) and vanA (12) genes, respectively. MICs of tigecycline were also determined by microdilution, according to CLSI guidelines (5) using fresh Mueller-Hinton broth (less than 12 h old) from Difco. MIC₅₀s and MIC₉₀s for bacteria grown in the medium from Difco or Oxoid were consistently lower than those found when the bacteria were grown in medium from Merck (Table 1). Considering the conservative breakpoints for resistance defined by the European Committee on Antimicrobial Susceptibility Testing (>2 µg/ml for enterobacteria; >0.5 µg/ml for Staphylococcus and Enterococcus) (8) and assuming resistance to Acinetobacter baumannii when the MIC of tigecycline was >2 µg/ml, the numbers of strains resistant to tigecycline changed dramatically, depending on the medium used. None of the ESBL-producing E. coli or Klebsiella pneumoniae, methicillin-resistant S. aureus, or E. faecium were resistant when tested in medium from Difco or Oxoid, while 10 to 30% of these isolates would be defined as resistant when tested in the medium from Merck. These differences were even higher for A. baumannii: 10%, 10%, and 70% of isolates were resistant in the media from Oxoid, Difco, and Merck, respectively.

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TABLE 1. MIC50s and MIC90s of tigecycline by Etest against five species (10 clonally unrelated isolates per species) in Mueller-Hinton agar from three manufacturersa

The ion concentrations in Mueller-Hinton agar from the three manufacturers were determined by atomic absorption spectroscopy (Perkin Elmer 1100) using nitrous oxide-acetylene (for calcium and magnesium) or air-acetylene (other elements) (10, 11). The concentration of manganese in the medium from Merck was 272 times higher than the concentration in the medium from Difco or Oxoid. Minor variations in the concentrations of magnesium and zinc were also variably observed for the three media (Table 2). When the activity of tigecycline was determined in agar medium from Difco supplemented with either manganese chloride (Sigma, Madrid, Spain) or sulfate chloride (Sigma) to concentrations of 630 µg/ml of free Mn²⁺ (similar to those observed in the medium from Merck), MICs increased against all tested organisms except P. aeruginosa ATCC 27853 (Table 3).

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TABLE 2. Ion content in Mueller-Hinton agar from three manufacturers

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TABLE 3. MICs (µg/ml) of tigecycline determined by Etest against 12 clinical isolates and 4 reference strains in four types of Mueller-Hinton agar

It has been reported that calcium and magnesium interfere with the activity of tetracycline, aminoglycosides, and colistin against P. aeruginosa and other gram-negative bacteria (7, 18). Magnesium also decreases the in vitro activity of fluoroquinolones by chelating these agents (1), and zinc interferes with the action of carbapenems against P. aeruginosa by decreasing the expression of the OprD porin (6). The results of this study show that high concentrations of manganese increase the MICs of tigecycline determined by Etest against microorganisms of clinical relevance. Similarly, it has been reported recently (17) that the inhibition zone diameters obtained on Mueller-Hinton agar from two different manufacturers (Oxoid and BD in Thailand) differed by a mean difference of 3.5 mm and a range from 1 to 6 mm, with the smaller zones obtained on the Oxoid medium, in which the manganese content was three times higher than in the BD medium. Another group in Spain has also observed that the MICs of tigecycline against A. baumannii determined by Etest using medium from bioMérieux were 4 to 16 µg/ml in comparison with values of 0.25 to 3 µg/ml obtained in medium from BBL or Biomedics (3). Our data suggest that differences in manganese may (at least in part) explain their results. We consider that MICs of tigecycline in media with low manganese content are more clinically relevant, as the concentration of manganese in human serum (0.8 to 1.2 µg/liter) (14, 15) is far from the high manganese concentrations found in Mueller-Hinton agar from Merck.

The mechanism by which manganese interferes with in vitro activity of tigecycline is presently unknown. It could be interesting to evaluate whether this is related to the formation of complexes between tigecycline and manganese and/or to the induction by manganese of RND (resistance-nodulation-cell division) efflux pumps involved in tigecycline resistance in clinical isolates. It would also be relevant to study the effect of different concentrations of manganese and other cations (Zn, Ca, Mg, Co, . . .) on the in vitro activity of tigecycline compared to tetracyclines. These studies may contribute to defining conditions more adequate for in vitro testing of tigecycline against clinical isolates.

 
This study was supported in part by a grant from Wyeth. Research in our laboratory is supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008).

 
* Corresponding author. Mailing address: Service of Microbiology, Hospital Universitario Marqués de Valdecilla, Avda. de Valdecilla s/n, Santander 39008, Spain. Phone: 34 942 202580. Fax: 34 942 203462. E-mail: lmartinez{at}humv.es

Published ahead of print on 14 January 2009.

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Journal of Clinical Microbiology, March 2009, p. 827-829, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.02464-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fernández-Mazarrasa, C. Articles by Martínez-Martínez, L. Search for Related Content PubMed PubMed Citation Articles by Fernández-Mazarrasa, C. Articles by Martínez-Martínez, L.