Postepy Hig Med Dosw. (online), 2013; 67: 921-925
Original Article
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The need to assay the real MIC when makingthe decision to eradicate Staphylococcus aureus with vancomycin
Konieczność oceny MIC rzeczywistego przy podjęciu decyzji o eradykacji Staphylococcus aureus wankomycyną
Hanna Tomczak1  ABDEF, Edyta Szałek2  DEF, Wioleta Błażejewska1  BC, Katarzyna Myczko1  BF, Anna Horla1  BF, Edmund Grześkowiak2  G
1Central Laboratory of Microbiology, University Hospital, Poznań, Poland
2Department of Clinical Pharmacy and Biopharmacy, Karol Marcinkowski University of Medical Sciences, Poznań, Poland
Corresponding author
Hanna Tomczak Ph.D. Central Laboratory of Microbiology, University Hospital, ul. Przybyszewskiego 49, 60-355 Poznań, Poland; e-mail: hannatomczak@interia.pl

Authors' Contribution:
A - Study Design, B - Data Collection, C - Statistical Analysis, D - Data Interpretation, E - Manuscript Preparation, F - Literature Search, G - Funds Collection

Received:  2012.11.12
Accepted:  2013.06.26
Published:  2013.09.04

Summary
Purpose: The aim of the study was a comparison of the MIC (minimal inhibitory concentration) evalu­ated in the automatic system Vitek 2 and the real MIC of vancomycin by the Etest method for S. aureus strains isolated from clinical materials.
Material and Methods: Over a twelve-month study period we compared the results obtained with two commercial methods - the automatic system VITEK 2 and the real MIC by Etest - for 359 strains of S. aureus isolated from clinical materials.
Results: Most of the strains of S. aureus were cultured from wounds (84), the ear (60) and nose (42). MSSA (methicillin-sensitive Staphylococcus aureus) was isolated in 342 cases and MRSA (methicillin­-resistant Staphylococcus aureus) in 17 cases. The test with the Vitek automatic method showed that vancomycin had MIC values of =< 1.0 µg/ml in more than 96% and 2.0 µg/ml in over 3% of cases. Using the Etest technique MIC =< 1.0 µg/ml was obtained in only 16.4% of cases and values of >1.0 µg/ml in 83.6% of cases.
Discussion: In view of such big differences between the MIC values obtained with the two methods the authors suggest that the Etest method of assaying the real MIC is more useful than the auto­matic method.
Key words: MIC, vancomycin, S. aureus




Introduction
Vancomycin is a glycopeptide antibiotic with the molecular mass of about 1500 Daltons. Due to poor drug absorption from the alimentary tract it is administered intravenously [25]. Vancomycin is a nephrotoxic antibiotic. Therefore, its concentrations in the serum need to be monitored, per­mitting one to modify the dose in order to obtain desirable concentrations [5,8,10,25]. The increase of S. aureus strains with reduced susceptibility to vancomycin forces doctors to administer high doses in order to obtain trough levels of 15 to 20 mg/L [9]. In severe sepsis it is necessary to increase the dose of vancomycin in order to achieve the steady-state concentration of Ctrough 15-20 mg/l [3]. The generally accept­ed regimen comprises the loading dose of 15 mg/kg with subsequent continuous infusion of 15-40 mg/kg in order to obtain higher concentrations. Continuous infusions do not increase toxicity, cause smaller fluctuations of concentra­tions in the blood and reduce costs [1,4,12].
Among most clinical strains of Gram-positive microbes, re­sistance to vancomycin is very rare [25]. However, recently the phenomenon of gradual increase in the value of mini­mal inhibitory concentration (MIC) of glycopeptides for S. aureus has been observed, which is defined in the literature as MIC creep [21].
The study is a comparison of the MIC assayed with the auto­matic system Vitek 2 from bioMerieux company and the real MIC of vancomycin by the Etest method for S. aureus strains isolated from clinical materials in the Central Microbiologi­cal Laboratory, H. Święcicki Clinical Hospital, Poznań Uni­versity of Medical Sciences. Both of the procedures applied in the studies are consistent with the recommendations re­lated to test choice in estimation of bacterial sensitivity to antibiotics and chemotherapeutics of agents edited by the National Reference Centre for Antimicrobial Susceptibility, where the Etest is an obligatory technique when S. aureus is isolated from the clinical material [14].
Material and methods
The research material was collected from November 2010 to December 2011. S. aureus was cultured 359 times during the period. The pathogen under analysis was mostly cultured from wounds (84), from the ear (60), nose (carried in the na­sal atrium) (42), blood (35), pus (29), BAL (bronchoalveolar lavage) (25), throat (23), skin lesions (16) and fistulas (9). The other materials were individual cases.
The strains of S. aureus under analysis generally came from patients diagnosed in laryngology and dermatology clinics (88) and from hospital departments: laryngology (74), der­matology (44), nephrology (40), ICU (intensive care units) (37), surgery (27), gastroenterology (13) and maxillo-facial surgery (12).
MSSA (methicillin-sensitive Staphylococcus aureus) was iso­lated in 342 cases and MRSA (methicillin-resistant Staphy­lococcus aureus) in 17 cases.
The microbiological investigations of the materials under analysis were carried out according to the current procedu­res of clinical microbiology, recommended by the National Reference Centre for Antimicrobial Susceptibility. Each ma­terial was cultured and routinely assayed both for bacteria and fungi. The microorganisms were identified by means of the systems Vitek (bioMerieux) and ATB (bioMerieux). If staphylococci were obtained from the material under inve­stigation, Slidex Staph Plus (quick agglutination test) (bio­Merieux) was used to determine if they were coagulase po­sitive (S. aureus) or coagulase negative. In each case the MIC of vancomycin for S. aureus was assayed, both for MSSA and MRSA, by means of two methods - with the automatic sys­tem Vitek 2 (bioMerieux) and the real MIC of vancomycin by Etest technique (bioMerieux). The Etest method is ba­sed on the quantitative gradient of concentration, used for estimation of the real MIC value of a studied drug against the tested microbes, in µg/ml. The range of 15 consecutive double dilutions gives a possibility to label the MIC betwe­en the conventional dilutions very precisely. The gradient of the drug on plastic strips remains stable for 18-24 hours, which covers the critical times of microbial growth. In the automatic system Vitek it is possible to obtain the result as early as within 6-8 hours, whereas it takes about 24 hours in the Etest (only after that time can mechanisms of resi­stance be revealed).
Results
S. aureus was cultured in 359 materials. When the automatic method Vitek was applied, in 256 cases (71.3%) the MIC of vancomycin for S. aureus was =< 0.5 µg/ml, in 91 cases it was 1.0 µg/ml (25.3%) and in 12 cases it was 2 µg/ml (3.3%) (Ta­ble 1). The real MIC assayed with the Etest was 0.5 µg/ml in 1 case, in 9 cases it was 0.75 µg/ml, in 49 cases 1.0 µg/ml, in 140 cases 1.5 µg/ml, in 159 cases 2.0 µg/ml, and in 1 case MIC was 4.0 µg/ml (Table 2). Out of 256 S. aureus strains, where the MIC of vancomycin assayed with the automatic method Vitek was =<0.5 µg/ml, the same result, 0.5 µg/ml, was not obtained in any case when the Etest was applied. In 7 cases the value was 0.75 µg/ml, in 38 cases it was 1.0 µg/ml, in 96 cases 1.5 µg/ml, and in 115 cases it reached as much as 2.0 µg/ml. On the other hand, in the group of 91 strains whe­re the MIC of vancomycin was assayed as 1 µg/ml with the Vitek automatic method, the Etest resulted in the following values: in 1 case the MIC was 0.5 µg/ml, in 2 cases it was 0.75 µg/ml, in 11 cases 1.0 µg/ml, in 40 cases 1.5 µg/ml, and in 38 cases 2.0 µg/ml. In the group of 12 cases where the Vitek system revealed the MIC value of 2.0 µg/ml, the Etest sho­wed the MIC of 1.5 µg/ml in 4 cases, in 7 cases it was 2 µg/ml, and in 1 case 4.0 µg/ml (Table 3). In the group of MRSA strains the MIC of vancomycin assayed with the automatic method was =<0.5 µg/ml in 9 cases, in 6 cases it was 1.0 µg/ml, and in 2 cases it was 2.0 µg/ml. When the Etest was applied, the following results were obtained: in 2 cases the MIC was 1.0 µg/ml, in 2 cases it was 1.5 µg/ml, and in 5 cases it was 2.0 µg/ml (Tables 1 and 2).
Table 1. The MIC of vancomycin for S. aureus assayed with the automatic method Vitek

Table 2. The real MIC of vancomycin for S. aureus assayed with the Etest method

Table 3. The MIC of vancomycin for S. aureus - the two methods compared

The assay with the Vitek automatic method showed MIC va­lues of vancomycin amounting to =<1.0 µg/ml in more than 96% of cases and 2.0 µg/ml in over 3% of cases. Using the Etest technique MIC values =<1.0 µg/ml were obtained in only 16.4% of cases and values >1.0 µg/ml in 83.6% of cases. Thus, assuming that a given strain will respond to vancomy­cin treatment at MIC =<1.0 µg/ml, it should be expected that vancomycin can be administered in almost (Fig. 1).
Figure 1. MIC of vancomycin for S. aureus evaluated by automatic Vitek technique and real MIC estimated using Etest technique

Discussion
In recent years the proportion of S. aureus strains with de­creased sensitivity to vancomycin has been increasing [9]. Due to the decrease in clinical effectiveness of vancomycin towards strains of S. aureus, for MIC >4.0 µg/ml, in 2006 the CLSI (the Clinical and Laboratory Standards Institute, USA) decreased the breakpoint of bacteria susceptibility from 4 to 2.0 µg/ml and the breakpoint of resistance from 32.0 µg/ ml to 16.0 µg/ml. The growing tendency of the percenta­ge of strains with reduced sensitivity to glycopeptides can still be observed, which gives rise to a discussion on further reduction of the breakpoints which have been designated so far [9]. Wang et al. observed a significant increase in the percentage of S. aureus strains with the vancomycin MIC of 1 µg/ml from 19.9% to 70.4% between the years 2000 and 2004. The number of strains with the MIC >=2.0 µg/ml also incre­ased [23]. The main cause of the phenomenon is probably the exposure of bacteria to doses of antibiotics in concen­trations defined as sub-MIC levels [15]. It has been proved that even a slight increase in the MIC below the sensitivity breakpoint may affect the clinical efficacy of glycopeptides [15]. At present there are suggestions that S. aureus strains with the vancomycin MIC of >1.0 µg/ml exhibit poor reac­tion to treatment with this antibiotic [7,8,15,16,20,24]. In such cases in order to avoid therapeutic failure it is neces­sary to consider inclusion of an alternative but usually more expensive antibiotic [7,8,15,16,20,24].
Before implementation of treatment with vancomycin in each case the real MIC of this antibiotic should be establi­shed (e.g. with the Etest method) for the isolated strain. Ac­cording to the guidelines of the National Reference Centre for Antimicrobial Susceptibility, in each case it is necessary to label the real MIC (which can be done e.g. with the Etest method) for S. aureus [14]. The reliability of automatic me­thods applied to measure the MIC of glycopeptides for S. aureus must be treated with due caution. On the basis of our results we observed considerable differences between the MIC of vancomycin for S. aureus assayed with the automatic method and the real MIC. The investigation with the Vitek automatic method showed MIC values of vancomycin =<0.5 µg/ml in 71%, 1 µg/ml in 25%, and 2.0 µg/ml in about 3% of cases. On the other hand, when the Etest was applied, the MIC of 0.5 µg/ml was obtained only in 0.3% of cases, 0.75 µg/ml in 2.5% of them, and 1.0 µg/ml in 13.6%. However, in as many as 39% of cases the real MIC of 1.5 µg/ml was obtained, in 44.3% it was 2.0 µg/ml, and the MIC of 4.0 µg/ ml was obtained in 0.3% of cases. Furthermore, for the strains where the MIC values of =<0.5 µg/ml were labelled with the Vitek automatic method in as many as 256 cases the real MICs were higher (i.e. 100% of the values). Thus, if we assume that a particular strain will respond to tre­atment with vancomycin, where the MIC is =<1.0 µg/ml, it would be necessary to assume that the antibiotic could be implemented in nearly 96% of all cases under investigation on the basis of the results in the Vitek system. However, it could be implemented only in 16% of cases if we assume the real MIC values obtained from the Etest method. Due to such big differences in the results obtained by means of both methods and according to reports in the literatu­re, which show that there is a higher chance for clinical success when the MIC of vancomycin does not exceed the value of 1.0 µg/ml [7,8,15,16,20], the authors underline the fact that the real MIC assayed by means of the Etest is more useful than the Vitek automatic method. It is worth noting that only the Etest enables detection of S. aureus heteroresistance to vancomycin (the drug gradient on the strip remains stable for 18-24 hours, which covers the mi­crobial growth during the investigation) [25]. Furthermo­re, the results we present, where for more than 84% of S. aureus strains investigated with the Etest the MIC exceeded 1 µg/ml, may prove the global tendency of the bacteria to exhibit growing resistance to glycopeptides. However, it is impossible to draw a conclusion about the presence or absence of differences in the MIC for MSSA and MRSA due to the small number of MRSA isolates.
The application of vancomycin for eradication of MSSA is not recommended. Doing so may contribute to selection of strains with reduced sensitivity [7,8]. Clindamycin plays an important role in treating S. aureus infections. As a protein synthesis inhibitor it inhibits toxin production [6].
In each case when vancomycin is applied to eradicate infec­tions with S. aureus aetiology, special care is recommended, especially in view of the results of sensitivity to vancomycin labelled by means of an automatic system.
Selection of the method of pathogen sensitivity labelling may significantly influence the final result [2,11,13,17,18,19,22] and thus therapeutic decisions. The antibiogram based on the real MIC assay should be an essential element when vancomycin therapy is included.
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The authors have no potential conflicts of interest to declare.