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Journal of Clinical Microbiology, June 2007, p. 2005-2008, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00578-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation of Moxalactam with the BD Phoenix System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci{triangledown}

H. Pupin,1 H. Renaudin,1 O. Join-Lambert,2 C. Bébéar,1 F. Mégraud,1 and P. Lehours1*

CHU de Bordeaux, Hôpital Pellegrin, Laboratoire de Bactériologie, Place Amélie Raba Léon, 33076 Bordeaux, France,1 Hôpital Necker Enfants Malades, Service de Microbiologie, 149 rue de Sèvres, 75015 Paris, France2

Received 15 March 2007/ Returned for modification 22 March 2007/ Accepted 3 April 2007


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ABSTRACT
 
The performance of moxalactam with the BD Phoenix system for the detection of methicillin resistance in coagulase-negative staphylococci was evaluated by use of a collection of 186 strains. Moxalactam was a better drug as an indicator of methicillin resistance for mecA-positive strains than oxacillin and cefoxitin were. For strains other than Staphylococcus saprophyticus, a moxalactam MIC >16 µg/ml was indicative of methicillin resistance.


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TEXT
 
Coagulase-negative staphylococci (CoNS) represent an important etiology of nosocomial infections and especially bloodstream infections. In the United States, data compiled by SCOPE (Monitoring and Control of Pathogens of Epidemiologic Importance) indicate that between March 1995 and September 2002, 60% of cases of hospital bacteremia were due to gram-positive bacteria. All of the antibiotic resistance mechanisms described for Staphylococcus aureus have also been found in CoNS, in particular, the production of penicillinase and also the production of an additional penicillin binding protein (PBP), named PBP 2a, encoded by the mecA gene. The inducible character of methicillin resistance of CoNS (8) and its heterogeneous expression hinder its detection in the laboratory. The French Committee for Antibiotic Susceptibility Testing of the French Society of Microbiology (CA-SFM) recommends the use of three ß-lactams, i.e., oxacillin, cefoxitin, and moxalactam, to determine the methicillin resistance of Staphylococcus species. However, although the interpretation of oxacillin MICs has been well defined by both the CA-SFM (susceptible, ≤0.25 µg/ml; resistant, >2 µg/ml) and the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) (susceptible, ≤0.25 µg/ml; resistant, ≥0.5 µg/ml), no breakpoints for cefoxitin and moxalactam MICs were proposed. The use of cefoxitin was introduced in the 2004 guidelines of the CA-SFM (10). Felten et al. described a breakpoint that can be used to detect S. aureus mecA-positive strains, that is, a breakpoint for resistance of >4 µg/ml (2). The use of a moxalactam disk was subsequently introduced in the CA-SFM recommendations in 2005 (11), but to date, no MIC breakpoint has been published. The relevance of the moxalactam disk is slightly better for mecA-positive strains, for which Felten et al. obtained a better sensitivity compared to that achieved with cefoxitin (97% and 94%, respectively) (3). Moreover, Join-Lambert et al. demonstrated that the moxalactam disk better differentiated heteroresistant CoNS isolates from mecA-negative isolates (7).

Our study concerns the evaluation of moxalactam with the BD Phoenix system for the detection of methicillin resistance in CoNS. Indeed, the new "combo" panel, ID/AST Staphylococcus (PMIC/ID52), which combines assays for Staphylococcus species identification and antibiotic susceptibility testing, has been available in Europe since 2005. The concentration ranges of the three ß-lactams included in this panel are 0.25 to 4 µg/ml for oxacillin, 2 to 8 µg/ml for cefoxitin, and 2 to 16 µg/ml for moxalactam.

One hundred eighty-six CoNS strains (95 mecA-negative strains and 91 mecA-positive strains) were investigated. Determination of the presence or the absence of the mecA gene was performed for all strains by real-time PCR (TaqMan; Roche Diagnostics, Meylan, France), as previously described by Fang and Hedin (1). The strains were divided into two sets, as described below.

We proceeded first by determining an MIC breakpoint for moxalactam which would distinguish between mecA-positive and mecA-negative CoNS strains, using a set of 51 strains (set A): 20 mecA-positive strains (18 Staphylococcus epidermidis strains, 1 Staphylococcus warneri strain, 1 Staphylococcus hominis strain) and 31 mecA-negative strains (6 S. epidermidis strains, 7 Staphylococcus lugdunensis strains, 12 Staphylococcus saprophyticus strains, 3 S. warneri strains, 1 Staphylococcus capitis strain, 1 S. hominis strain, 1 Staphylococcus gallinarum strain) selected on the basis of the heterogeneity of methicillin susceptibility. All of the mecA-positive strains had moxalactam MICs >16 µg/ml. Twenty-five of the 31 mecA-negative strains had moxalactam MICs <16 µg/ml. There was thus an overlap between the mecA-positive and the mecA-negative populations constituted by 6 of the 11 Staphylococcus saprophyticus mecA-negative strains. Given that S. saprophyticus is known to be less susceptible to oxacillin than other CoNS, we decided to use a breakpoint of >16 µg/ml for moxalactam. By applying this breakpoint, the sensitivity (the percentage of correctly characterized methicillin-resistant CoNS strains) and the specificity (the percentage of correctly characterized methicillin-susceptible CoNS strains) were 100% and 80.6%, respectively (Table 1). When the six S. saprophyticus strains were excluded, the specificity and the sensitivity reached 100% each. Finally, oxacillin showed the lowest performance (85% sensitivity and 45% specificity) (Table 1). This lack of specificity was expected due to the presence of 17 mecA-negative CoNS strains with MICs between 0.5 and 2 µg/ml (11 S. saprophyticus strains, 5 Staphylococcus lugdunensis strains, and 1 Staphylococcus gallinarum strain). This phenomenon has been well described in the literature, mainly for S. saprophyticus strains (4, 6, 9, 12). When the breakpoint defined by the CLSI was applied, the sensitivity and the specificity were 100% and 45%, respectively; three mecA-positive CoNS strains with MICs between 0.5 and 2 µg/ml were correctly categorized. No mecA-positive strain had an oxacillin MIC ≤0.25 µg/ml, and no mecA-negative strain had an oxacillin MIC >2 µg/ml. In conclusion, the CLSI breakpoints were better adapted to mecA-positive strains but overestimated resistance, mainly for mecA-negative S. saprophyticus strains. Finally, when the cefoxitin breakpoint for resistance of >4 µg/ml defined for S. aureus by Felten et al. (2) was considered, the sensitivity and the specificity for cefoxitin were 75% and 100%, respectively (Table 1).


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  		TABLE 1. Comparison of sensitivity and specificity of oxacillin, cefoxitin, and moxalactam for CoNS (set A and set B) according to susceptibility testing method used

In the second part of our study, the moxalactam breakpoint was evaluated with a larger number of CoNS strains (set B). One hundred thirty-five nonduplicate CoNS strains (71 mecA-positive strains and 64 mecA-negative strains) were investigated: 82 Staphylococcus epidermidis strains, 24 Staphylococcus hominis strains, 7 Staphylococcus haemolyticus strains, 12 Staphylococcus capitis strains, 4 Staphylococcus warneri strains, 2 S. lugdunensis strains, 1 Staphylococcus schleiferi strain, 1 Staphylococcus caprae strains, 1 Staphylococcus simulans strain, and 1 unidentified species. These strains were not selected on the basis of their heterogeneous resistance to oxacillin but, rather, on the basis of the fact that they represent a global picture of the CoNS strains isolated in our laboratory over a 1-year period. The distribution of the oxacillin MICs obtained by the BD Phoenix system according to mecA status is shown in Fig. 1A: 65 mecA-positive strains had oxacillin MICs >2 µg/ml, and 6 mecA-positive strains had MICs ranging from 0.5 to 2 µg/ml (which is the intermediate zone, according to the CA-SFM recommendations). Sixty-two mecA-negative strains had MICs ≤0.25 µg/ml, and two strains had an MIC of 0.5 µg/ml. The sensitivity and the specificity were therefore 91.5% and 96.8%, respectively. When the breakpoint for oxacillin defined by the CLSI was applied, the sensitivity and the specificity were 100% and 96.9%, respectively.


Figure 1
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  		FIG. 1. Oxacillin, cefoxitin, and moxalactam MICs for 135 CoNS strains in set B (71 mecA-positive and 64 mecA-negative) obtained with the BD Phoenix system. Black columns, mecA-positive strains; white columns, mecA-negative strains; broken lines, (i) the current breakpoint for CoNS strains for oxacillin defined by the CA-SFM recommendations (susceptible, ≤0.25 µg/ml; resistant, >2 µg/ml) or for cefoxitin defined by Felten et al. (2) (resistance, >4 µg/ml) and (ii) the current breakpoint for CoNS strains for moxalactam defined in this study (resistance, > 16 µg/ml); the number of strains categorized as methicillin susceptible (S) or resistant (R) according to these breakpoints is indicated at the top of each panel on each side of the broken lines.

Considering cefoxitin, the majority of mecA-positive strains (66/71) had an MICs >4 µg/ml, but 5 strains had MICs ≤4 µg/ml. All of the mecA-negative strains had MICs ≤4 µg/ml (Fig. 1B). The sensitivity with cefoxitin was 93%, indicating that 7% of the mecA-positive strains in this set could not be detected if the cefoxitin MICs were considered alone, which could be a critical factor for patient management. However, the specificity was excellent (100%), indicating that all of the mecA-negative strains were properly categorized.

Concerning the moxalactam MICs obtained with the BD Phoenix system, when the breakpoint defined with set A (resistance breakpoint, >16 µg/ml) was applied, all mecA-positive strains had MICs >16 µg/ml. The 64 mecA-negative strains had MICs ≤16 µg/ml (Fig. 1C). The sensitivity and the specificity were 100% each; however, no S. saprophyticus strain was present in this set of strains.

Finally, considering the data obtained with set A and set B, we conclude that the methicillin susceptibility of CoNS cannot be ideally determined by using a single molecule. Therefore, we focused on the latter part of our study, to integrate the ß-lactam MICs obtained with the BD Phoenix system as validation rules (Fig. 2). First, the strains were categorized based on oxacillin MIC breakpoints of >2 µg/ml for resistance and ≤0.25 µg/ml for susceptible, as defined by the CA-SFM. If the MICs were in the intermediate range for oxacillin (>0.25 to ≤2 µg/ml), the moxalactam MICs were considered by applying a resistance breakpoint of >16 µg/ml. Then, the results for the strains in sets A and B were a sensitivity and a specificity of 100% and 93.7%, respectively. All mecA-positive strains were well categorized, but six mecA-negative S. saprophyticus strains in set A (moxalactam MICs, >16 µg/ml) were incorrectly categorized. By applying a decisional tree that integrated cefoxitin (resistance, >4 µg/ml) instead of moxalactam, the sensitivity and the specificity would have been 92.3% and 100%, respectively; all mecA-negative strains but not all mecA-positive strains (7.3%) were well categorized. We believe that an oxacillin-moxalactam association is better than an oxacillin-cefoxitin association, because all of the mecA-positive strains could be detected. Another option would be to consider the association of cefoxitin-moxalactam. However, oxacillin rather than cefoxitin was preferred as the starting point of our decisional tree, because it is the only ß-lactam for which the CA-SFM and the CLSI have proposed specific breakpoints for CoNS. Our decisional tree is valid for every CoNS species except mecA-negative S. saprophyticus, which could be misclassified. The main interest in the use of the CA-SFM recommendations compared to those of the CLSI is to define an intermediate zone for oxacillin which could be considered a security measure for CoNS strains with heterogeneous methicillin susceptibilities or naturally less susceptible species, such as S. saprophyticus. mecA-positive S. saprophyticus strains are rare, and moreover, in our study, all mecA-negative S. saprophyticus isolates had cefoxitin MICs ≤4 µg/ml. This result is in line with the conclusion of the study by Higashide et al., who suggested that the cefoxitin disk diffusion method could be more suitable than the oxacillin disk diffusion method for the detection of mecA-mediated resistance in S. saprohyticus (5). Therefore, we assumed that the ß-lactam MICs of mecA-positive S. saprophyticus strains would show a marked increase. In order to check this, eight mecA-positive S. saprophyticus strains provided by M. Higashide (University of Tsukuba, Tsukuba, Japan) (5) were tested at the end of the study. Indeed, the corresponding MICs obtained with the BD Phoenix system were >2 µg/ml for oxacillin, >4 µg/ml for cefoxitin, and >16 µg/ml for moxalactam. In conclusion, this species could be categorized by using the same criteria adopted by Felten et al. for S. aureus, i.e., the oxacillin and cefoxitin MICs.


Figure 2
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  		FIG. 2. Decisional tree for determination of methicillin susceptibilities of CoNS (except for Staphylococcus saprophyticus) on the basis of the oxacillin and moxalactam MICs obtained with the BD Phoenix system. methi-S, methicillin susceptible; methi-R, methicillin resistant.

In this study the BD Phoenix system showed good reliability. The use of three ß-lactams (oxacillin, cefoxitin, and moxalactam) in the same antibiotic susceptibility testing panel offers a great advantage to microbiologists because susceptibility to each antibiotic can be analyzed at the same time without having to perform different techniques. The decisional tree that we propose can be integrated into the validation software of the BD Phoenix system (BDXpert) and, in parallel, into expert software. Our laboratory currently uses this decisional tree routinely.


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ACKNOWLEDGMENTS
 
This study was supported with a grant from BD Diagnostics.

We thank the Bordeaux Hospital Microbiology Laboratory staff, especially Josiane Brunet, Marie-Jo Bonici, and Stéphane Beteille, for their collaboration in this study. We thank M. Higashide for providing mecA-positive S. saprophyticus strains and Gioia Babini for fruitful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Bactériologie, CHU Pellegrin, Place Amélie Raba Léon, 33076 Bordeaux cedex, France. Phone: 33 5 57 57 12 86. Fax: 33 5 56 51 41 82. E-mail: philippe.lehours{at}chu-bordeaux.fr Back

{triangledown} Published ahead of print on 11 April 2007. Back


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Journal of Clinical Microbiology, June 2007, p. 2005-2008, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00578-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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