Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus. sp.: Evaluation of the Broth Microdilution and Etest Methods

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1 JCM Accepts, published online ahead of print on 22 September 2010 J. Clin. Microbiol. doi: /jcm Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 2 Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus sp.: Evaluation of the Broth Microdilution and Etest Methods Rodrigo M. Paiva 1*, Alice B. Mombach Pinheiro Machado 1, Alexandre P. Zavascki 2, +, Afonso L. Barth 1,3, +. + Both Senior authors, who have contributed equally. Running title: Vancomycin MIC for MRCoNS. 1 Unidade de Microbiologia e Biologia Molecular, Serviço de Patologia Clínica, Hospital de Clínicas de Porto Alegre; 2 Serviço de Medicina Interna, Hospital de Clínicas de Porto Alegre; 3 Departamento de Análises, Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul *Corresponding author: Rodrigo M. Paiva, Unidade de Microbiologia e Biologia Molecular, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, 2350 Ramiro Barcelos Street, Porto Alegre, RS, Brazil, , Phone: / Fax: , rpaiva@hcpa.ufrgs.br 1

2 ABSTRACT Vancomycin MIC results were determined by broth microdilution method (BMD) and by Etest on 130 methicillin-resistant coagulase-negative staphylococci bloodstream isolates from a tertiary hospital. The majority (98.5%) of MIC results by BMD were 1 µg/ml in contrast to MIC by Etest (72.3% was 1.5 µg/ml). The MICs obtained by the Etest were, in general, one to twofold higher than MICs by BMD

3 Coagulase-negative staphylococci (CoNS) have emerged as important nosocomial pathogens during the last decade, particularly in nosocomial bloodstream infections (4). Resistance to methicillin in CoNS is very common among isolates recovered from hospitalized individuals (4, 19). For this reason, vancomycin is usually the drug of choice for treatment of infections by methicillin-resistant CoNS (MRCoNS) (17). A reduction in the efficacy of vancomycin has been described in studies with methicillin-resistant Staphylococcus aureus (MRSA) infections treated with this antibiotic, and it has been suggested that slight increases in vancomycin MICs between 1 and 2 µg/ml, which are within the susceptible range, may be related to suboptimal clinical outcomes (12, 16). Therefore, the determination of MIC of vancomycin has been recommended for these pathogens (3). Some of these studies have used the broth microdilution method (BMD) for determining vancomycin MICs, while others have used the commercial Etest technique (AB Biodisk, Solna, Sweden). It has been reported that Etest provides higher MICs than those obtained with BMD in S. aureus, mainly MRSA (14, 15). Nevertheless, there is no study comparing both methodologies for determination of MIC of vancomycin in CoNS. Considering the increasing incidence of MRCoNS, the need for MIC determination for vancomycin and the absence of studies assessing the performance of Etest in these organisms, we aimed to compare the Etest and BMD for determination of the MIC of vancomycin in MRCoNS isolates. 130 clinical isolates of CoNS recovered from blood of patients hospitalized from May/2004 to August/2005 at Hospital de Clínicas de Porto Alegre were analyzed. Only one isolate per patient were included. Blood cultures were performed using BacT/Alert (biomérieux, Marcy L Etoile, France). The colony morphology, Gram stain reaction, catalase test and absence of the coagulase enzyme were used to identify CoNS. Isolates 3

4 were identified as Staphylococcus epidermidis by PCR, with primers for tuf (10). The isolates that were not identified as S. epidermidis by PCR were identified using the API ID 32 STAPH (biomérieux, Marcy L Etoile, France) semi-automated system, according to the instructions of the manufacturer. Results yielding a quality of identification of 85% or higher were accepted. The presence of meca gene was assessed by PCR using specific primers (13). MICs of vancomycin were determined in duplicate by the reference BMD as recommended by CLSI, using in-house-prepared panels. The following dilutions of vancomycin: 16, 8, 4, 2, 1, 0.5, 0.25 and µg/ml were tested. The standard Etest procedure was performed using Mueller-Hinton agar (Becton Dickinson, Sparks, Md.) with an inoculum density equivalent to 0.5 McFarland. Vancomycin Etest strips were placed onto the agar with sterile forceps. The cultures were incubated for 24h at 35ºC. S. aureus ATCC was used for quality control (3). The Wilcoxon test was used to compare the MICs obtained by Etest and BMD. Two sets of comparisons were done: one with the exact MIC value by Etest and the other rounding the MIC determined by Etest up to the next dilution. All 130 isolates of CoNS proved to be meca positive and were identified as follows: 87 (66.9%) S. epidermidis, 13 (10.0%) S. haemolyticus, 12 (9.2%) S. hominis and 11 (8.5%) S. capitis. Seven isolates (5.4%) were not identified to the species level. The MICs of vancomycin ranged from 0.25 to 2 µg/ml by BMD and from 0.38 to 3 µg/ml by Etest. No discrepancies were observed in duplicates performed by BMD. The MIC 50 and MIC 90 for vancomycin were both 1 µg/ml by BMD and 1.5 and 2 µg/ml, respectively, by Etest (P < for both comparisons). Most MICs determined by Etest were 1.5 µg/ml (94 isolates, 72.3%) while most MICs were 1 µg/ml (128 isolates, 98.5%) by BMD. One hundred thirteen (86.9%) and five (3.8%) isolates presented a MIC by Etest one and twofold dilutions higher than by BMD, respectively. 4

5 Only 10 isolates (7.7%) presented the same MIC using the two methods (Fig. 1). When the Etest MICs 0.38, 0.75, 1.5, and 3 µg/ml were converted to 0.5, 1, 2, and 4 µg/ml, respectively, there was an even higher discrepancy between the methods. In this case, almost all MRCoNS had MICs 1 µg/ml (124 isolates, 95.4%) (Fig. 2). MICs determined by Etest were higher than those determined by BMD for all MRCoNS species, when analyzed separately (Table 1). No isolate had MIC higher than 4 µg/ml regardless the method used. In the last two decades, a worldwide increase in the number of CoNS with decreased susceptibility to glycopeptides (mainly vancomycin) has been described (6, 7). The emergence of decreased vancomycin susceptibility among staphylococci has led to evaluations of susceptibility tests performed by clinical laboratories to avoid imprecise vancomycin MIC determinations. Although the BMD is the reference method for MIC determination, the Etest is an attractive option as it is easy to perform. However, the results of the present study demonstrate that Etest provides MICs of vancomycin higher than MICs determined by CLSI reference method (BMD). We found that the MICs by Etest were one to twofold dilutions higher than by BMD. These findings have been reported for S. aureus, indicating discrepancies among different methods for MIC determinations (8, 9, 11, 14, 15, 18). However, studies comparing MIC methods for CoNS were lacking. It is of note that the same discrepancies between Etest and BMD methods described for S. aureus were also observed in this study for CoNS. Moreover, the differences between the MIC methods were confirmed independently of the CoNS species identified. A limitation of this study is that MICs of vancomycin for MRCoNS from our report were lower than MICs described in other studies (2, 5), so we could not precisely evaluate the performance of Etest in organisms with higher MICs. Actually, most 5

6 (86.2%) S. epidermidis presented MICs by BMD = 1 µg/ml and only 1.1% MIC = 2 µg/ml. Among S. haemolyticus, most MICs by Etest were around 1 to 2 µg/ml, despite the fact that members of this species are usually more resistant to glycopeptides (1). In conclusion, the commercial Etest method overestimates MIC values when compared to BMD, as described for S. aureus. Although relatively infrequent, MRCoNS with reduced susceptibility to vancomycin are a potential concern if they present the same impact in clinical outcomes, as for S. aureus. ACKNOWLEDGEMENTS This study was supported by a Grant from Fundo de Incentivo à Pesquisa e Eventos (FIPE) Hospital de Clínicas de Porto Alegre. Vancomycin Etest strips were kindly provided by Pfizer. A.L.B. (309410/2007-0) and A.P.Z. (301829/2008-0) receive research fellow from CNPq. 6

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