Combined effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms | Enfermedades Infecciosas y Microbiología Clínica

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José" "apellidos" => "Medina-Pascual" ] 4 => array:2 [ "nombre" => "Claudia" "apellidos" => "Valenzuela" ] 5 => array:2 [ "nombre" => "Manuel" "apellidos" => "López-Brea" ] 6 => array:2 [ "nombre" => "Juan Antonio" "apellidos" => "Sáez-Nieto" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0213005X12004387?idApp=UINPBA00004N" "url" => "/0213005X/0000003100000010/v1_201312040018/S0213005X12004387/v1_201312040018/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Combined effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "655" "paginaFinal" => "659" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Bruna Leite, Fernanda Gomes, Pilar Teixeira, Clovis Souza, Elisabeth Pizzolitto, Rosário Oliveira" "autores" => array:6 [ 0 => array:3 [ "nombre" => "Bruna" "apellidos" => "Leite" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 1 => array:3 [ "nombre" => "Fernanda" "apellidos" => "Gomes" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Pilar" "apellidos" => "Teixeira" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "Clovis" "apellidos" => "Souza" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "Elisabeth" "apellidos" => "Pizzolitto" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] 1 => array:2 [ "etiqueta" => "c" "identificador" => "aff0015" ] ] ] 5 => array:4 [ "nombre" => "Rosário" "apellidos" => "Oliveira" "email" => array:1 [ 0 => "roliveira@deb.uminho.pt" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "¿" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Department of Biotechnology, Federal University of São Carlos, São Carlos, São Paulo, Brazil" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Faculty of Pharmaceutical Sciences, São Paulo State University, Araraquara, Brazil" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Efecto combinado de linezolid y N-acetilcisteína contra Staphylococcus epidermidis biofilms" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 799 "Ancho" => 3079 "Tamanyo" => 132388 ] ] "descripcion" => array:1 [ "en" => "Effect of linezolid (LIN) and/or NAC on the metabolic activity of biofilm cells of S. epidermidis 9142 and 1457, after 24h of contact with linezolid (MIC and PS) and NAC (4mgml−1 and 40mgml−1), alone (A) and in combination (B), after 24h of treatment expressed as XTT absorbance (cellular activity). Error bars represent standard deviation." ] ] ] "textoCompleto" => "IntroductionStaphylococcus epidermidis is a commensal inhabitant of the healthy human skin and mucosa. However, this gram-positive bacterium has developed interesting strategies to change into a notorious pathogen,<a class="elsevierStyleCrossRef" href="#bib0005">1</a> being now one of the leading causes of prosthetic device infections. This capacity appears to be due to its ability to colonize both the host's skin as part of the commensal flora and prosthetic materials via its ability to adhere to the surface of indwelling medical devices and to form biofilms.<a class="elsevierStyleCrossRef" href="#bib0010">2</a> This feature is the main virulence factor associated with S. epidermidis infections and is a major clinical problem, mainly due to the high level of biofilm tolerance/resistance to antibiotics.<a class="elsevierStyleCrossRef" href="#bib0015">3</a> In fact, resistance to antibiotics by pathogenic S. epidermidis isolates is an increasing trait.<a class="elsevierStyleCrossRefs" href="#bib0020">4,5</a> When in biofilms, cells can be up to 1000 times more tolerant to antibiotics than their planktonic counterparts.<a class="elsevierStyleCrossRef" href="#bib0030">6</a> This coagulase negative staphylococci is the cause of a great number of infections,<a class="elsevierStyleCrossRefs" href="#bib0035">7,8</a> namely endocarditis and infections of any type of indwelling medical devices, such as peripheral or central intravenous catheters (CVCs).<a class="elsevierStyleCrossRef" href="#bib0040">8</a>In the beginning of this decade, linezolid (an oxazolidinone) was approved as a new antibiotic, offering a new option for the treatment of, for example, complicated skin and skin structure infections caused by resistant gram-positive pathogens<a class="elsevierStyleCrossRef" href="#bib0045">9</a> and has been claimed to be 100% efficient.<a class="elsevierStyleCrossRef" href="#bib0050">10</a> Linezolid is a protein synthesis inhibitor and is active against gram-positive microorganisms, including enterococci, streptococci, and Staphylococcus aureus.<a class="elsevierStyleCrossRefs" href="#bib0055">11,12</a> Nowadays, this synthetic antibiotic plays a very important role against infections caused by multiresistant gram-positive pathogens<a class="elsevierStyleCrossRefs" href="#bib0065">13,14</a> and has excellent activity against most staphylococci, although resistance has begun to emerge.<a class="elsevierStyleCrossRefs" href="#bib0075">15,16</a> Nevertheless, when tested against S. aureus and S. epidermidis biofilms it was not as efficacious as expected.<a class="elsevierStyleCrossRef" href="#bib0085">17</a>On the other hand, N-acetylcysteine (NAC) is a non-antibiotic drug that decreases/prevents biofilm formation ability and adherence to biomaterials devices of a variety of bacteria<a class="elsevierStyleCrossRefs" href="#bib0090">18–20</a> and reduces the production of the extracellular polysaccharide matrix,<a class="elsevierStyleCrossRef" href="#bib0105">21</a> while promoting the disruption of mature biofilm.<a class="elsevierStyleCrossRefs" href="#bib0080">16,18,20</a> NAC is widely used in medical practice.<a class="elsevierStyleCrossRefs" href="#bib0115">23,24</a>The main goal of this study was to determine the susceptibility of S. epidermidis biofilm cells to a new antibiotic (linezolid) in combination with a non-antibiotic drug (NAC), both with different modes of action, searching for possible synergistic interactions. For that, we assessed the inhibitory effect of these antimicrobial agents through the enumeration of biofilm viable cells, metabolic activity and total biofilm biomass.MethodsBacterial strains and growth conditionsTwo clinical isolate strains of S. epidermidis, 9142 and 1457 were used in this study because they produce very thick biofilms with an abundant amount of extracellular matrix.<a class="elsevierStyleCrossRefs" href="#bib0125">25,26</a> Both strains were provided by Dr. G. B. Pier, Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.The culture media used, tryptic soy broth (TSB) and tryptic soy agar (TSA), were prepared according to the manufacturer's instructions. All strains were inoculated into 15ml of TSB from TSA plates not older than 2 days and grown for 18 (±2) h at 37°C in an orbital shaker at 130rpm. Cells were harvested by centrifugation (for 5min at 9500×g and 4°C) and resuspended in TSB with the final suspension adjusted to an optical density (640nm) equivalent to 1×109CFUml−1, to be used in the subsequent assays. The experiments were performed with S. epidermidis cultures in the log-phase of growth.The stock solution of linezolid was prepared in Milli-Q water and diluted in TSB in order to be used as working solution (at MIC and peak serum concentrations). The working solutions of NAC (MIC and 10×MIC) were prepared in TSB. Both antimicrobial agents tested were pure substances (Sigma–Aldrich, St. Louis, MO).Minimum inhibitory concentration (MIC)The minimum inhibitory concentration was determined by the microbroth dilution technique. MIC determination of the tested agents and for both S. epidermidis strains (9142 and 1457) was performed by the serial two fold dilution method at concentrations ranging from 0.007μgml−1 to 16μgml−1, and 0.007mgml−1 to 16mgml−1, for linezolid and NAC, respectively. The controls were cells not exposed to the antimicrobial agents tested. All experiments were carried out in triplicate and repeated three times.Biofilm formation and susceptibility/synergy testsBiofilms were formed in 96 well tissue culture plates (Sarstedt, Newton, NC, USA) containing 200μl of S. epidermidis cell suspension (1×106CFUml−1) in TSB supplemented with 0.25% of glucose per well to promote biofilm formation. Plates were incubated at 37°C with orbital shaking at 130rpm for 24h. At the end, the supernatant was carefully removed and the biofilm was washed twice with 200μl of saline solution [0.9% NaCl (Merck)]. The biofilm was then incubated for 24h, in fresh nutrient medium (TSB) containing linezolid (at MIC 1μgml−1 and PS 18μgml−1) or NAC (MIC 4000μgml−1 and 10×MIC 40,000μgml−1) or a combination of both. Enumeration of colony forming units (CFU), crystal violet staining (CV) and XTT reduction assays was performed after 24h of exposure to the antimicrobial agents tested, either alone or in combination. At time 0 (before exposure to antibiotics) the initial biofilm cellular concentration of (∼2×108CFUml−1) was determined.To assess the number of viable cells after antibiotic treatment, the planktonic cells were carefully removed and then the biofilms were washed with 250μl of saline solution. Every well was resuspended in 0.9% NaCl, thoroughly scraped and biofilm cells transferred to centrifuge tubes and centrifuged for 10min at 9500×g and 4°C. The pellet was resuspended in 0.9% NaCl and washed twice, followed by 20s of sonication at 22W to homogenize the suspension. Viable cells were determined by performing 10-fold serial dilutions in saline solution and plating in TSA. Colonies were counted after 24h incubation at 37°C.Crystal violet (CV) staining was used as an indicator of total biofilm biomass. After exposure to the treatment agents, biofilms were washed with 250μl of saline solution. Then, 250μl of methanol was added and allowed to act for 15min. Afterwards, methanol was removed and crystal violet 1% (v/v) was added (5min). The wells were washed with distilled water and finally, acetic acid 33% (v/v) was added and the absorbance of the solution was measured at 570nm.Another colorimetric method, based on the reduction of XTT ({2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide}), was applied to assess cellular activity (XTT is converted to a colored formazan salt in the presence of metabolic activity).<a class="elsevierStyleCrossRef" href="#bib0135">27</a> After exposure to antimicrobial agents, biofilms were washed with 250μl of saline solution, followed by the addition of 250μl of a solution containing 200mgl−1 of XTT and 20mgl−1 of PMS (phenazine methosulfate) (Sigma–Aldrich, St. Louis, MO) to each well. The microtiter plates were incubated for 3h at 37°C in the dark. The absorbance was measured at 490nm.A control was performed with biofilm cells not exposed to the antibiotics tested. All experiments were carried out in triplicate and repeated three times.Statistical analysisData were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey method for pairwise multiple comparisons using SPSS software (Statistical Package for the Social Sciences). p<0.05 was considered statistically significant.Results<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> presents the MIC values of linezolid and NAC (for planktonic cells) determined in this study and peak serum concentrations obtained from the literature for linezolid<a class="elsevierStyleCrossRef" href="#bib0140">28</a> and NAC after oral<a class="elsevierStyleCrossRef" href="#bib0145">29</a> and intravenous dose.<a class="elsevierStyleCrossRef" href="#bib0150">30</a><elsevierMultimedia ident="tbl0005"></elsevierMultimedia>Overall, linezolid showed the best activity against both S. epidermidis planktonic strains, with MIC=1μgml−1 (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>) and both agents were equally effective against the two strains assayed.<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a> presents the effect of linezolid and NAC (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A) and the association linezolid+NAC (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>B) against S. epidermidis biofilm cells.<elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia>A bactericidal effect was defined as a reduction of ≥3log10 of the initial bacterial inoculum. A lesser reduction in bacterial count (<3log10) was defined as a bacteriostatic effect.<a class="elsevierStyleCrossRefs" href="#bib0155">31,32</a>Regarding the effect of each agent alone, NAC at 10×MIC showed the highest bactericidal activity against biofilm cells with a significant inhibitory effect (p<0.05), causing a reduction of approximately 4log in CFU retrieved from the biofilm. Although linezolid at peak serum concentration cannot be considered an effective bactericidal agent against S. epidermidis biofilms, it demonstrated a significant inhibitory and bacteriostatic effect, promoting a CFU reduction of ∼2log (p<0.05). However, linezolid at MIC combined with NAC either at MIC or 10×MIC displayed a synergistic effect against both strains in biofilm form. Linezolid at peak serum concentration plus NAC at MIC also had a synergistic effect against the S. epidermidis strain 9142. Overall, the most effective combination was linezolid (MIC)+NAC (10×MIC) causing a CFU reduction of approximately 5log. There was no synergistic or additive effect when linezolid at peak serum concentration was combined with NAC at 10×MIC.The results expressing the decrease in metabolic activity measured by the XTT reduction assay after treatment with linezolid or NAC alone and in combination are presented in <a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>. These results are in accordance with the results obtained by CFU enumeration confirming that linezolid (MIC)+NAC (10×MIC) promoted a very significant reduction in cellular metabolic activity. It should be stressed that the results obtained with the XTT reduction assay are more qualitative than quantitative because it is very difficult to find a direct relation between the number of cells and their metabolic activity in a biofilm, where the upper cell layer is in environmental conditions much different from more inner layers, that can be deprived of some nutrients.The variation in total biofilm biomass, assessed by CV staining, also confirms the effect of the antimicrobial agents tested. As it can be seen in <a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>, linezolid (MIC)+NAC (10×MIC) caused a significant reduction in biofilm biomass of S. epidermidis (p<0.05).<elsevierMultimedia ident="fig0015"></elsevierMultimedia>DiscussionThe results obtained demonstrate that linezolid is a very active agent against S. epidermidis planktonic cells being the minimal concentration necessary to inhibit the staphylococci growth (MIC) 4000-fold inferior than that of NAC (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). However, these two drugs have an opposite behavior when applied to biofilms, which can be related, partly, to diffusional limitations imposed by the biofilm matrix, since the molecular weight of linezolid (337.35) is approximately twice that of NAC (163.19) but mostly due to the lower metabolic activity displayed by biofilm cells. Linezolid works by inhibiting the bacterial protein synthesis at the initiation step, by binding to the 23S portion of the 50S subunit (the center of peptidyl transferase activity),<a class="elsevierStyleCrossRef" href="#bib0165">33</a> and for that it has to be up taken by the cells and this process is hindered in biofilm cells on account of their low metabolic activity or even dormancy.<a class="elsevierStyleCrossRef" href="#bib0170">34</a> On the other hand, NAC, well recognized as a mucolytic agent, acts by disrupting the biofilm structure through the splitting of disulfide bonds linking the proteins present in the extracellular matrix.<a class="elsevierStyleCrossRefs" href="#bib0090">18,21</a> Other authors also suggest that the mechanism for the antibacterial effect of NAC may be that it acts by competitively inhibiting amino acid (cysteine) utilization or, by virtue of possessing a sulfhydryl group, may react with bacterial cell proteins.<a class="elsevierStyleCrossRef" href="#bib0175">35</a>Previous studies have shown that NAC could decrease biofilm formation by a variety of bacteria such as Escherichia coli, S. epidermidis, Pseudomonas aeruginosa, etc.<a class="elsevierStyleCrossRefs" href="#bib0090">18–20,35</a> Zhao and Liu<a class="elsevierStyleCrossRef" href="#bib0175">35</a> concluded that NAC has antibacterial properties against P. aeruginosa, dispersing and detaching P. aeruginosa biofilms. NAC also inhibited bacterial adherence, reduced the production of extracellular polysaccharide matrix,<a class="elsevierStyleCrossRef" href="#bib0105">21</a> while promoting the disruption of mature biofilms, and reduced sessile cell viability.<a class="elsevierStyleCrossRefs" href="#bib0090">18–21</a>Therefore, this detachment and dispersive action of NAC in biofilms may make the biofilm-associated bacteria more susceptible to other antimicrobial agents,<a class="elsevierStyleCrossRef" href="#bib0110">22</a> as in the present association with linezolid. NAC alone, in this case at a concentration of 40mgml−1, also presented a very high inhibitory effect, promoting a 4log reduction in cultivable cells, and a significant reduction of total biofilm biomass and cellular metabolic activity. However, to the author's knowledge, NAC bactericidal mechanism of action is still not known, more probably consisting of a physical cleavage of the bacterial cell envelope. Our results are in accordance with those obtained in previous studies<a class="elsevierStyleCrossRefs" href="#bib0100">20–23,35</a>: NAC decreased sessile cell viability translated by the reduction of CFU; promoted biofilm detachment and dispersion translated by the reduction of biofilm biomass.Therapy almost always fails to eradicate the bacteria in biofilms. So, the search of effective drugs able to inhibit biofilm formation and/or disrupt/eradicate established biofilms or that may work in synergy with other antimicrobial agents, is essential for treating biofilm associated-diseases.Overall, a general promotion of biofilm weakness may be a potential help to human immune system in the combat of S. epidermidis biofilm associated-infections. However, the combination of linezolid (MIC=1μgml−1) with NAC (10×MIC=40mgml−1) demonstrated a synergistic effect, and is therefore more effective than NAC (10×MIC) alone. This combination was able to reduce the number of CFU from 8log to ∼3log, a nearly 5log reduction. Apart from being more active, the combination linezolid (MIC)+NAC (10×MIC) can reduce the probability of the onset of resistance. Other authors have already demonstrated a synergistic effect of NAC with tigecycline, another recent antibiotic, against S. aureus and S. epidermidis.<a class="elsevierStyleCrossRef" href="#bib0110">22</a> However, they have used higher concentrations of both agents, 1mgml−1 of tigecycline (1000×MIC) and 80mgml−1 of NAC, to obtain a similar 5log reduction in viable cells of S. epidermidis biofilm, although at half of the contact time, 12h against the 24h that we have used.NAC is widely used in medical practice via inhalation and oral and intravenous routes and it has an excellent safety profile.<a class="elsevierStyleCrossRef" href="#bib0180">36</a>Due to intrinsic differences between strains, further studies will be necessary using more clinical and reference strains with the objective of data confirmation. In conclusion, the combination of linezolid with NAC can be a possible therapeutic strategy to eradicate focus of S. epidermidis infections and, even more promisingly, can be further tested as a catheter lock solution.FundingBruna Leite acknowledges the financial support of ISAC/Program Erasmus Mundus External Cooperation and the IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus of Gualtar. Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support of Fundação para a Ciência e Tecnologia (FCT) through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively.Conflict of interestThe authors have no conflict of interest to declare." "textoCompletoSecciones" => array:1 [ "secciones" => array:12 [ 0 => array:2 [ "identificador" => "xres296297" "titulo" => array:5 [ 0 => "Abstract" 1 => "Introduction" 2 => "Methods" 3 => "Results" 4 => "Conclusion" ] ] 1 => array:2 [ "identificador" => "xpalclavsec279938" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xres296298" "titulo" => array:5 [ 0 => "Resumen" 1 => "Introducción" 2 => "Métodos" 3 => "Resultados" 4 => "Conclusión" ] ] 3 => array:2 [ "identificador" => "xpalclavsec279937" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Methods" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Bacterial strains and growth conditions" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Minimum inhibitory concentration (MIC)" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Biofilm formation and susceptibility/synergy tests" ] ] ] 6 => array:2 [ "identificador" => "sec0030" "titulo" => "Statistical analysis" ] 7 => array:2 [ "identificador" => "sec0035" "titulo" => "Results" ] 8 => array:2 [ "identificador" => "sec0040" "titulo" => "Discussion" ] 9 => array:2 [ "identificador" => "sec0045" "titulo" => "Funding" ] 10 => array:2 [ "identificador" => "sec0050" "titulo" => "Conflict of interest" ] 11 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2012-05-11" "fechaAceptado" => "2012-11-23" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec279938" "palabras" => array:5 [ 0 => "Antimicrobial agents" 1 => "Biofilm" 2 => "Linezolid" 3 => "N-acetylcysteine" 4 => "Staphylococcus epidermidis" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec279937" "palabras" => array:5 [ 0 => "Agentes antimicrobianos" 1 => "Biofilm" 2 => "Linezolid" 3 => "N-acetilcisteína" 4 => "Staphylococcus epidermidis" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "IntroductionStaphylococcus epidermidis is an organism commonly associated with infections caused by biofilms. Biofilms are less sensible to antibiotics and therefore are more difficult to eradicate. Linezolid and N-acetylcysteine (NAC), have demonstrated to be active against gram-positive microorganisms. Therefore and since linezolid and NAC have different modes of action, the main objective of this work was to investigate the single and synergistic effect of linezolid and NAC against S. epidermidis biofilms. MethodsThis work reports the in vitro effect of linezolid and NAC against S. epidermidis biofilms, treated with MIC (4mgml−1) and 10×MIC of NAC, and MIC (1μgml−1) and peak serum concentration (PS=18μgml−1) of linezolid alone and in combination. After exposure of S. epidermidis biofilms to linezolid and/or NAC for 24h, several biofilm parameters were evaluated, namely the number of cultivable cells [colony forming unit (CFU) enumeration], total biofilm biomass and cellular activity. ResultsWhen tested alone, NAC at 10×MIC was the most effective agent against S. epidermidis biofilms. However, the combination linezolid (MIC)+NAC (10×MIC) showed a synergistic effect and was the most biocidal treatment tested, promoting a 5log reduction in the number of biofilm viable cells. ConclusionThis combination seems to be a potential candidate to combat infections caused by S. epidermidis biofilms, namely as a catheter lock solution therapy." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "IntroducciónStaphylococcus epidermidis es un organismo comúnmente asociado con infecciones causadas por biofilms. Los biofilms son menos sensibles a los antibióticos y, por lo tanto, más difíciles de erradicar. El linezolid y la N-acetilcisteína (NAC) han demostrado ser activos contra los microorganismos grampositivos. Por lo tanto, y puesto que el linezolid y la NAC tienen diferentes modos de acción, el objetivo principal de este trabajo fue investigar el efecto individual y sinérgico del linezolid y la NAC frente a biofilms de S. epidermidis. MétodosEste trabajo reporta el efecto in vitro del linezolid y la NAC solos y en combinación contra biofilms de S. epidermidis, tratados con NAC en las concentraciones de MIC (4mg·ml–1) y 10×MIC, y linezolid en las concentraciones MIC (1mg·ml–1) y concentración sérica máxima (PS=18μg ml–1). Después de la exposición de los biofilms de S. epidermidis al linezolid y/o la NAC durante 24h, fueron evaluados varios parámetros del biofilm, a saber, el número de células cultivables (recuento de unidades formadoras de colonias [UFC]), la biomasa total del biofilm y la actividad celular. ResultadosDurante el ensayo solo, la NAC en la concentración de 10×MIC fue el agente más eficaz contra biofilms de S. epidermidis. Sin embargo, la combinación linezolid (MIC)+NAC (10×MIC) mostró un efecto sinérgico y fue el tratamiento con mayor efecto biocida, promoviendo una reducción logarítmica de 5 en el número de células viables del biofilm. ConclusiónEsta combinación parece ser un potencial candidato en el combate de las infecciones causadas por biofilms de S. epidermidis, es decir, como una terapia de solución de bloqueo del catéter." ] ] "multimedia" => array:4 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 662 "Ancho" => 3208 "Tamanyo" => 156111 ] ] "descripcion" => array:1 [ "en" => "Effect of linezolid (LIN) and/or NAC on biofilm cells of S. epidermidis 9142 and 1457, after 24h of contact with linezolid (MIC and PS) and NAC (4mgml−1 and 40mgml−1), alone (A) and in combination (B) assessed by CFU enumeration. Error bars represent standard deviation." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 799 "Ancho" => 3079 "Tamanyo" => 132388 ] ] "descripcion" => array:1 [ "en" => "Effect of linezolid (LIN) and/or NAC on the metabolic activity of biofilm cells of S. epidermidis 9142 and 1457, after 24h of contact with linezolid (MIC and PS) and NAC (4mgml−1 and 40mgml−1), alone (A) and in combination (B), after 24h of treatment expressed as XTT absorbance (cellular activity). Error bars represent standard deviation." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 788 "Ancho" => 3074 "Tamanyo" => 135645 ] ] "descripcion" => array:1 [ "en" => "Effect of linezolid (LIN) and/or NAC on total biofilm biomass of S. epidermidis 9142 and 1457, after 24h of contact with linezolid (MIC and PS) and NAC (4mgml−1 and 40mgml−1), alone (A) and in combination (B), after 24h of treatment expressed as CV absorbance (total biofilm biomass). Error bars represent standard deviation." ] ] 3 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:3 [ "leyenda" => "NAC, N-acetylcysteine." "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Antimicrobial agent \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">S. epidermidis 9142, MIC (μgml−1) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">S. epidermidis 1457, MIC (μgml−1) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Peak serum (μgml−1) \t\t\t\t\t\t\n \t\t\t\t</td></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Linezolid \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">18 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">NAC \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">4000 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="char" valign="\n \t\t\t\t\ttop\n \t\t\t\t">4000 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.35–4<a class="elsevierStyleCrossRef" href="#tblfn0005">a</a>or550<a class="elsevierStyleCrossRef" href="#tblfn0010">b</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab432069.png" ] ] ] "notaPie" => array:2 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "a" "nota" => "After oral dose." ] 1 => array:3 [ "identificador" => "tblfn0010" "etiqueta" => "b" "nota" => "After intravenous dose." ] ] ] "descripcion" => array:1 [ "en" => "MIC and peak serum concentrations of linezolid and NAC." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:36 [ 0 => array:3 [ "identificador" => "bib0005" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Success through diversity—how Staphylococcus epidermidis establishes as a nosocomial pathogen" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "S.M. 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Combined effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms

Efecto combinado de linezolid y N-acetilcisteína contra Staphylococcus epidermidis biofilms

Bruna Leitea,b, Fernanda Gomesa, Pilar Teixeiraa, Clovis Souzab, Elisabeth Pizzolittob,c, Rosário Oliveiraa,

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roliveira@deb.uminho.pt

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a IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal

b Department of Biotechnology, Federal University of São Carlos, São Carlos, São Paulo, Brazil

c Faculty of Pharmaceutical Sciences, São Paulo State University, Araraquara, Brazil

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epidermidis</span> infections and is a major clinical problem&#44; mainly due to the high level of biofilm tolerance&#47;resistance to antibiotics&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> In fact&#44; resistance to antibiotics by pathogenic <span class="elsevierStyleItalic">S&#46; epidermidis</span> isolates is an increasing trait&#46;<a class="elsevierStyleCrossRefs" href="#bib0020"><span class="elsevierStyleSup">4&#44;5</span></a> When in biofilms&#44; cells can be up to 1000 times more tolerant to antibiotics than their planktonic counterparts&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> This coagulase negative staphylococci is the cause of a great number of infections&#44;<a class="elsevierStyleCrossRefs" href="#bib0035"><span class="elsevierStyleSup">7&#44;8</span></a> namely endocarditis and infections of any type of indwelling medical devices&#44; such as peripheral or central intravenous catheters &#40;CVCs&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">In the beginning of this decade&#44; 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epidermidis</span> biofilm cells to a new antibiotic &#40;linezolid&#41; in combination with a non-antibiotic drug &#40;NAC&#41;&#44; both with different modes of action&#44; searching for possible synergistic interactions&#46; For that&#44; we assessed the inhibitory effect of these antimicrobial agents through the enumeration of biofilm viable cells&#44; metabolic activity and total biofilm biomass&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Bacterial strains and growth conditions</span><p id="par0025" class="elsevierStylePara elsevierViewall">Two clinical isolate strains of <span class="elsevierStyleItalic">S&#46; epidermidis</span>&#44; 9142 and 1457 were used in this study because they produce very thick biofilms with an abundant amount of extracellular matrix&#46;<a class="elsevierStyleCrossRefs" href="#bib0125"><span class="elsevierStyleSup">25&#44;26</span></a> Both strains were provided by Dr&#46; 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for linezolid and NAC&#44; respectively&#46; The controls were cells not exposed to the antimicrobial agents tested&#46; All experiments were carried out in triplicate and repeated three times&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Biofilm formation and susceptibility&#47;synergy tests</span><p id="par0045" class="elsevierStylePara elsevierViewall">Biofilms were formed in 96 well tissue culture plates &#40;Sarstedt&#44; Newton&#44; NC&#44; USA&#41; containing 200<span class="elsevierStyleHsp" style=""></span>&#956;l of <span class="elsevierStyleItalic">S&#46; epidermidis</span> cell suspension &#40;1<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>CFU<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; in TSB supplemented with 0&#46;25&#37; of glucose per well to promote biofilm formation&#46; Plates were incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C with orbital shaking at 130<span class="elsevierStyleHsp" style=""></span>rpm for 24<span class="elsevierStyleHsp" style=""></span>h&#46; At the end&#44; the supernatant was carefully removed and the biofilm was washed twice with 200<span class="elsevierStyleHsp" style=""></span>&#956;l of saline solution &#91;0&#46;9&#37; NaCl &#40;Merck&#41;&#93;&#46; The biofilm was then incubated for 24<span class="elsevierStyleHsp" style=""></span>h&#44; in fresh nutrient medium &#40;TSB&#41; containing linezolid &#40;at MIC 1<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> and PS 18<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; or NAC &#40;MIC 4000<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> and 10&#215;MIC 40&#44;000<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; or a combination of both&#46; Enumeration of colony forming units &#40;CFU&#41;&#44; crystal violet staining &#40;CV&#41; and XTT reduction assays was performed after 24<span class="elsevierStyleHsp" style=""></span>h of exposure to the antimicrobial agents tested&#44; either alone or in combination&#46; At time 0 &#40;before exposure to antibiotics&#41; the initial biofilm cellular concentration of &#40;&#8764;2<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">8</span><span class="elsevierStyleHsp" style=""></span>CFU<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; was determined&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">To assess the number of viable cells after antibiotic treatment&#44; the planktonic cells were carefully removed and then the biofilms were washed with 250<span class="elsevierStyleHsp" style=""></span>&#956;l of saline solution&#46; Every well was resuspended in 0&#46;9&#37; NaCl&#44; thoroughly scraped and biofilm cells transferred to centrifuge tubes and centrifuged for 10<span class="elsevierStyleHsp" style=""></span>min at 9500<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> and 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The pellet was resuspended in 0&#46;9&#37; NaCl and washed twice&#44; followed by 20<span class="elsevierStyleHsp" style=""></span>s of sonication at 22<span class="elsevierStyleHsp" style=""></span>W to homogenize the suspension&#46; Viable cells were determined by performing 10-fold serial dilutions in saline solution and plating in TSA&#46; Colonies were counted after 24<span class="elsevierStyleHsp" style=""></span>h incubation at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall">Crystal violet &#40;CV&#41; staining was used as an indicator of total biofilm biomass&#46; After exposure to the treatment agents&#44; biofilms were washed with 250<span class="elsevierStyleHsp" style=""></span>&#956;l of saline solution&#46; Then&#44; 250<span class="elsevierStyleHsp" style=""></span>&#956;l of methanol was added and allowed to act for 15<span class="elsevierStyleHsp" style=""></span>min&#46; Afterwards&#44; methanol was removed and crystal violet 1&#37; &#40;v&#47;v&#41; was added &#40;5<span class="elsevierStyleHsp" style=""></span>min&#41;&#46; The wells were washed with distilled water and finally&#44; acetic acid 33&#37; &#40;v&#47;v&#41; was added and the absorbance of the solution was measured at 570<span class="elsevierStyleHsp" style=""></span>nm&#46;</p><p id="par0060" class="elsevierStylePara elsevierViewall">Another colorimetric method&#44; based on the reduction of XTT &#40;&#123;2&#44;3-bis&#40;2-methoxy-4-nitro-5-sulfophenyl&#41;-5-&#91;&#40;phenylamino&#41;carbonyl&#93;-2H-tetrazolium hydroxide&#125;&#41;&#44; was applied to assess cellular activity &#40;XTT is converted to a colored formazan salt in the presence of metabolic activity&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a> After exposure to antimicrobial agents&#44; biofilms were washed with 250<span class="elsevierStyleHsp" style=""></span>&#956;l of saline solution&#44; followed by the addition of 250<span class="elsevierStyleHsp" style=""></span>&#956;l of a solution containing 200<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>l<span class="elsevierStyleSup">&#8722;1</span> of XTT and 20<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>l<span class="elsevierStyleSup">&#8722;1</span> of PMS &#40;phenazine methosulfate&#41; &#40;Sigma&#8211;Aldrich&#44; St&#46; Louis&#44; MO&#41; to each well&#46; The microtiter plates were incubated for 3<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in the dark&#46; The absorbance was measured at 490<span class="elsevierStyleHsp" style=""></span>nm&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">A control was performed with biofilm cells not exposed to the antibiotics tested&#46; All experiments were carried out in triplicate and repeated three times&#46;</p></span></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Statistical analysis</span><p id="par0070" class="elsevierStylePara elsevierViewall">Data were analyzed by one-way analysis of variance &#40;ANOVA&#41; followed by the Tukey method for pairwise multiple comparisons using SPSS software &#40;Statistical Package for the Social Sciences&#41;&#46; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered statistically significant&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Results</span><p id="par0075" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> presents the MIC values of linezolid and NAC &#40;for planktonic cells&#41; determined in this study and peak serum concentrations obtained from the literature for linezolid<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">28</span></a> and NAC after oral<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> and intravenous dose&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">30</span></a></p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0080" class="elsevierStylePara elsevierViewall">Overall&#44; linezolid showed the best activity against both <span class="elsevierStyleItalic">S&#46; epidermidis</span> planktonic strains&#44; with MIC<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>1<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41; and both agents were equally effective against the two strains assayed&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a> presents the effect of linezolid and NAC &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41; and the association linezolid<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>B&#41; against <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilm cells&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0090" class="elsevierStylePara elsevierViewall">A bactericidal effect was defined as a reduction of &#8805;3<span class="elsevierStyleHsp" style=""></span>log<span class="elsevierStyleHsp" style=""></span>10 of the initial bacterial inoculum&#46; A lesser reduction in bacterial count &#40;&#60;3<span class="elsevierStyleHsp" style=""></span>log<span class="elsevierStyleHsp" style=""></span>10&#41; was defined as a bacteriostatic effect&#46;<a class="elsevierStyleCrossRefs" href="#bib0155"><span class="elsevierStyleSup">31&#44;32</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Regarding the effect of each agent alone&#44; NAC at 10&#215;MIC showed the highest bactericidal activity against biofilm cells with a significant inhibitory effect &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#44; causing a reduction of approximately 4<span class="elsevierStyleHsp" style=""></span>log in CFU retrieved from the biofilm&#46; Although linezolid at peak serum concentration cannot be considered an effective bactericidal agent against <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms&#44; it demonstrated a significant inhibitory and bacteriostatic effect&#44; promoting a CFU reduction of &#8764;2<span class="elsevierStyleHsp" style=""></span>log &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; However&#44; linezolid at MIC combined with NAC either at MIC or 10&#215;MIC displayed a synergistic effect against both strains in biofilm form&#46; Linezolid at peak serum concentration plus NAC at MIC also had a synergistic effect against the <span class="elsevierStyleItalic">S&#46; epidermidis</span> strain 9142&#46; Overall&#44; the most effective combination was linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; causing a CFU reduction of approximately 5<span class="elsevierStyleHsp" style=""></span>log&#46; There was no synergistic or additive effect when linezolid at peak serum concentration was combined with NAC at 10&#215;MIC&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">The results expressing the decrease in metabolic activity measured by the XTT reduction assay after treatment with linezolid or NAC alone and in combination are presented in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#46; These results are in accordance with the results obtained by CFU enumeration confirming that linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; promoted a very significant reduction in cellular metabolic activity&#46; It should be stressed that the results obtained with the XTT reduction assay are more qualitative than quantitative because it is very difficult to find a direct relation between the number of cells and their metabolic activity in a biofilm&#44; where the upper cell layer is in environmental conditions much different from more inner layers&#44; that can be deprived of some nutrients&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">The variation in total biofilm biomass&#44; assessed by CV staining&#44; also confirms the effect of the antimicrobial agents tested&#46; As it can be seen in <a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#44; linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; caused a significant reduction in biofilm biomass of <span class="elsevierStyleItalic">S&#46; epidermidis</span> &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Discussion</span><p id="par0110" class="elsevierStylePara elsevierViewall">The results obtained demonstrate that linezolid is a very active agent against <span class="elsevierStyleItalic">S&#46; epidermidis</span> planktonic cells being the minimal concentration necessary to inhibit the staphylococci growth &#40;MIC&#41; 4000-fold inferior than that of NAC &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; However&#44; these two drugs have an opposite behavior when applied to biofilms&#44; which can be related&#44; partly&#44; to diffusional limitations imposed by the biofilm matrix&#44; since the molecular weight of linezolid &#40;337&#46;35&#41; is approximately twice that of NAC &#40;163&#46;19&#41; but mostly due to the lower metabolic activity displayed by biofilm cells&#46; Linezolid works by inhibiting the bacterial protein synthesis at the initiation step&#44; by binding to the 23S portion of the 50S subunit &#40;the center of peptidyl transferase activity&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">33</span></a> and for that it has to be up taken by the cells and this process is hindered in biofilm cells on account of their low metabolic activity or even dormancy&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">34</span></a> On the other hand&#44; NAC&#44; well recognized as a mucolytic agent&#44; acts by disrupting the biofilm structure through the splitting of disulfide bonds linking the proteins present in the extracellular matrix&#46;<a class="elsevierStyleCrossRefs" href="#bib0090"><span class="elsevierStyleSup">18&#44;21</span></a> Other authors also suggest that the mechanism for the antibacterial effect of NAC may be that it acts by competitively inhibiting amino acid &#40;cysteine&#41; utilization or&#44; by virtue of possessing a sulfhydryl group&#44; may react with bacterial cell proteins&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">35</span></a></p><p id="par0115" class="elsevierStylePara elsevierViewall">Previous studies have shown that NAC could decrease biofilm formation by a variety of bacteria such as <span class="elsevierStyleItalic">Escherichia coli</span>&#44; <span class="elsevierStyleItalic">S&#46; epidermidis</span>&#44; <span class="elsevierStyleItalic">Pseudomonas aeruginosa</span>&#44; etc&#46;<a class="elsevierStyleCrossRefs" href="#bib0090"><span class="elsevierStyleSup">18&#8211;20&#44;35</span></a> Zhao and Liu<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">35</span></a> concluded that NAC has antibacterial properties against <span class="elsevierStyleItalic">P&#46; aeruginosa</span>&#44; dispersing and detaching <span class="elsevierStyleItalic">P&#46; aeruginosa</span> biofilms&#46; NAC also inhibited bacterial adherence&#44; reduced the production of extracellular polysaccharide matrix&#44;<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">21</span></a> while promoting the disruption of mature biofilms&#44; and reduced sessile cell viability&#46;<a class="elsevierStyleCrossRefs" href="#bib0090"><span class="elsevierStyleSup">18&#8211;21</span></a></p><p id="par0120" class="elsevierStylePara elsevierViewall">Therefore&#44; this detachment and dispersive action of NAC in biofilms may make the biofilm-associated bacteria more susceptible to other antimicrobial agents&#44;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> as in the present association with linezolid&#46; NAC alone&#44; in this case at a concentration of 40<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#44; also presented a very high inhibitory effect&#44; promoting a 4<span class="elsevierStyleHsp" style=""></span>log reduction in cultivable cells&#44; and a significant reduction of total biofilm biomass and cellular metabolic activity&#46; However&#44; to the author&#39;s knowledge&#44; NAC bactericidal mechanism of action is still not known&#44; more probably consisting of a physical cleavage of the bacterial cell envelope&#46; Our results are in accordance with those obtained in previous studies<a class="elsevierStyleCrossRefs" href="#bib0100"><span class="elsevierStyleSup">20&#8211;23&#44;35</span></a>&#58; NAC decreased sessile cell viability translated by the reduction of CFU&#59; promoted biofilm detachment and dispersion translated by the reduction of biofilm biomass&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Therapy almost always fails to eradicate the bacteria in biofilms&#46; So&#44; the search of effective drugs able to inhibit biofilm formation and&#47;or disrupt&#47;eradicate established biofilms or that may work in synergy with other antimicrobial agents&#44; is essential for treating biofilm associated-diseases&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">Overall&#44; a general promotion of biofilm weakness may be a potential help to human immune system in the combat of <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilm associated-infections&#46; However&#44; the combination of linezolid &#40;MIC<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>1<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; with NAC &#40;10&#215;MIC<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>40<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; demonstrated a synergistic effect&#44; and is therefore more effective than NAC &#40;10&#215;MIC&#41; alone&#46; This combination was able to reduce the number of CFU from 8<span class="elsevierStyleHsp" style=""></span>log to &#8764;3<span class="elsevierStyleHsp" style=""></span>log&#44; a nearly 5<span class="elsevierStyleHsp" style=""></span>log reduction&#46; Apart from being more active&#44; the combination linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; can reduce the probability of the onset of resistance&#46; Other authors have already demonstrated a synergistic effect of NAC with tigecycline&#44; another recent antibiotic&#44; against <span class="elsevierStyleItalic">S&#46; aureus</span> and <span class="elsevierStyleItalic">S&#46; epidermidis</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> However&#44; they have used higher concentrations of both agents&#44; 1<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> of tigecycline &#40;1000&#215;MIC&#41; and 80<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> of NAC&#44; to obtain a similar 5<span class="elsevierStyleHsp" style=""></span>log reduction in viable cells of <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilm&#44; although at half of the contact time&#44; 12<span class="elsevierStyleHsp" style=""></span>h against the 24<span class="elsevierStyleHsp" style=""></span>h that we have used&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">NAC is widely used in medical practice via inhalation and oral and intravenous routes and it has an excellent safety profile&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">36</span></a></p><p id="par0140" class="elsevierStylePara elsevierViewall">Due to intrinsic differences between strains&#44; further studies will be necessary using more clinical and reference strains with the objective of data confirmation&#46; In conclusion&#44; the combination of linezolid with NAC can be a possible therapeutic strategy to eradicate focus of <span class="elsevierStyleItalic">S&#46; epidermidis</span> infections and&#44; even more promisingly&#44; can be further tested as a catheter lock solution&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Funding</span><p id="par0150" class="elsevierStylePara elsevierViewall">Bruna Leite acknowledges the financial support of <span class="elsevierStyleGrantSponsor">ISAC&#47;Program Erasmus Mundus External Cooperation</span> and the <span class="elsevierStyleGrantSponsor">IBB-Institute for Biotechnology and Bioengineering&#44; Centre of Biological Engineering&#44; University of Minho&#44; Campus of Gualtar</span>&#46; Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support of <span class="elsevierStyleGrantSponsor" id="gs0005">Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia &#40;FCT&#41;</span> through the grants <span class="elsevierStyleGrantNumber" refid="gs0005">SFRH&#47;BD&#47;32126&#47;2006</span> and <span class="elsevierStyleGrantNumber" refid="gs0005">SFRH&#47;BPD&#47;26803&#47;2006</span>&#44; respectively&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle">Conflict of interest</span><p id="par0155" class="elsevierStylePara elsevierViewall">The authors have no conflict of interest to declare&#46;</p></span></span>"
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        "resumen" => "<span class="elsevierStyleSectionTitle">Introduction</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Staphylococcus epidermidis</span> is an organism commonly associated with infections caused by biofilms&#46; Biofilms are less sensible to antibiotics and therefore are more difficult to eradicate&#46; Linezolid and <span class="elsevierStyleItalic">N</span>-acetylcysteine &#40;NAC&#41;&#44; have demonstrated to be active against gram-positive microorganisms&#46; Therefore and since linezolid and NAC have different modes of action&#44; the main objective of this work was to investigate the single and synergistic effect of linezolid and NAC against <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms&#46;</p> <span class="elsevierStyleSectionTitle">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">This work reports the in vitro effect of linezolid and NAC against <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms&#44; treated with MIC &#40;4<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; and 10&#215;MIC of NAC&#44; and MIC &#40;1<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; and peak serum concentration &#40;PS<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>18<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41; of linezolid alone and in combination&#46; After exposure of <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms to linezolid and&#47;or NAC for 24<span class="elsevierStyleHsp" style=""></span>h&#44; several biofilm parameters were evaluated&#44; namely the number of cultivable cells &#91;colony forming unit &#40;CFU&#41; enumeration&#93;&#44; total biofilm biomass and cellular activity&#46;</p> <span class="elsevierStyleSectionTitle">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">When tested alone&#44; NAC at 10&#215;MIC was the most effective agent against <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms&#46; However&#44; the combination linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; showed a synergistic effect and was the most biocidal treatment tested&#44; promoting a 5<span class="elsevierStyleHsp" style=""></span>log reduction in the number of biofilm viable cells&#46;</p> <span class="elsevierStyleSectionTitle">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">This combination seems to be a potential candidate to combat infections caused by <span class="elsevierStyleItalic">S&#46; epidermidis</span> biofilms&#44; namely as a catheter lock solution therapy&#46;</p>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<span class="elsevierStyleSectionTitle">Introducci&#243;n</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Staphylococcus epidermidis</span> es un organismo com&#250;nmente asociado con infecciones causadas por biofilms&#46; Los biofilms son menos sensibles a los antibi&#243;ticos y&#44; por lo tanto&#44; m&#225;s dif&#237;ciles de erradicar&#46; El linezolid y la N-acetilciste&#237;na &#40;NAC&#41; han demostrado ser activos contra los microorganismos grampositivos&#46; Por lo tanto&#44; y puesto que el linezolid y la NAC tienen diferentes modos de acci&#243;n&#44; el objetivo principal de este trabajo fue investigar el efecto individual y sin&#233;rgico del linezolid y la NAC frente a biofilms de <span class="elsevierStyleItalic">S&#46; epidermidis&#46;</span></p> <span class="elsevierStyleSectionTitle">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Este trabajo reporta el efecto in vitro del linezolid y la NAC solos y en combinaci&#243;n contra biofilms de <span class="elsevierStyleItalic">S&#46; epidermidis</span>&#44; tratados con NAC en las concentraciones de MIC &#40;4<span class="elsevierStyleHsp" style=""></span>mg&#183;ml<span class="elsevierStyleSup">&#8211;1</span>&#41; y 10&#215;MIC&#44; y linezolid en las concentraciones MIC &#40;1<span class="elsevierStyleHsp" style=""></span>mg&#183;ml<span class="elsevierStyleSup">&#8211;1</span>&#41; y concentraci&#243;n s&#233;rica m&#225;xima &#40;PS<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>18<span class="elsevierStyleHsp" style=""></span>&#956;g ml<span class="elsevierStyleSup">&#8211;1</span>&#41;&#46; Despu&#233;s de la exposici&#243;n de los biofilms de <span class="elsevierStyleItalic">S&#46; epidermidis</span> al linezolid y&#47;o la NAC durante 24<span class="elsevierStyleHsp" style=""></span>h&#44; fueron evaluados varios par&#225;metros del biofilm&#44; a saber&#44; el n&#250;mero de c&#233;lulas cultivables &#40;recuento de unidades formadoras de colonias &#91;UFC&#93;&#41;&#44; la biomasa total del biofilm y la actividad celular&#46;</p> <span class="elsevierStyleSectionTitle">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Durante el ensayo solo&#44; la NAC en la concentraci&#243;n de 10&#215;MIC fue el agente m&#225;s eficaz contra biofilms de <span class="elsevierStyleItalic">S&#46; epidermidis&#46;</span> Sin embargo&#44; la combinaci&#243;n linezolid &#40;MIC&#41;<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>NAC &#40;10&#215;MIC&#41; mostr&#243; un efecto sin&#233;rgico y fue el tratamiento con mayor efecto biocida&#44; promoviendo una reducci&#243;n logar&#237;tmica de 5 en el n&#250;mero de c&#233;lulas viables del biofilm&#46;</p> <span class="elsevierStyleSectionTitle">Conclusi&#243;n</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Esta combinaci&#243;n parece ser un potencial candidato en el combate de las infecciones causadas por biofilms de <span class="elsevierStyleItalic">S&#46; epidermidis</span>&#44; es decir&#44; como una terapia de soluci&#243;n de bloqueo del cat&#233;ter&#46;</p>"
      ]
    ]
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Effect of linezolid &#40;LIN&#41; and&#47;or NAC on biofilm cells of <span class="elsevierStyleItalic">S&#46; epidermidis</span> 9142 and 1457&#44; after 24<span class="elsevierStyleHsp" style=""></span>h of contact with linezolid &#40;MIC and PS&#41; and NAC &#40;4<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> and 40<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; alone &#40;A&#41; and in combination &#40;B&#41; assessed by CFU enumeration&#46; Error bars represent standard deviation&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Effect of linezolid &#40;LIN&#41; and&#47;or NAC on the metabolic activity of biofilm cells of <span class="elsevierStyleItalic">S&#46; epidermidis</span> 9142 and 1457&#44; after 24<span class="elsevierStyleHsp" style=""></span>h of contact with linezolid &#40;MIC and PS&#41; and NAC &#40;4<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> and 40<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; alone &#40;A&#41; and in combination &#40;B&#41;&#44; after 24<span class="elsevierStyleHsp" style=""></span>h of treatment expressed as XTT absorbance &#40;cellular activity&#41;&#46; Error bars represent standard deviation&#46;</p>"
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        "etiqueta" => "Fig&#46; 3"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Effect of linezolid &#40;LIN&#41; and&#47;or NAC on total biofilm biomass of <span class="elsevierStyleItalic">S&#46; epidermidis</span> 9142 and 1457&#44; after 24<span class="elsevierStyleHsp" style=""></span>h of contact with linezolid &#40;MIC and PS&#41; and NAC &#40;4<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span> and 40<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; alone &#40;A&#41; and in combination &#40;B&#41;&#44; after 24<span class="elsevierStyleHsp" style=""></span>h of treatment expressed as CV absorbance &#40;total biofilm biomass&#41;&#46; Error bars represent standard deviation&#46;</p>"
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          "leyenda" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">NAC&#44; <span class="elsevierStyleItalic">N</span>-acetylcysteine&#46;</p>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t" style="border-bottom: 2px solid black">Antimicrobial agent&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">S&#46; epidermidis</span> 9142&#44; MIC &#40;&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">S&#46; epidermidis</span> 1457&#44; MIC &#40;&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" style="border-bottom: 2px solid black">Peak serum &#40;&#956;g<span class="elsevierStyleHsp" style=""></span>ml<span class="elsevierStyleSup">&#8722;1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">Linezolid&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">18&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">NAC&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
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                  \t\t\t\t">4000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
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                  \t\t\t\t">4000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;35&#8211;4<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>or550<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">b</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
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              "identificador" => "tblfn0010"
              "etiqueta" => "b"
              "nota" => "<p class="elsevierStyleNotepara">After intravenous dose&#46;</p>"
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        "descripcion" => array:1 [
          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">MIC and peak serum concentrations of linezolid and NAC&#46;</p>"
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0005"
          "bibliografiaReferencia" => array:36 [
            0 => array:3 [
              "identificador" => "bib0005"
              "etiqueta" => "1"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Success through diversity&#8212;how <span class="elsevierStyleItalic">Staphylococcus epidermidis</span> establishes as a nosocomial pathogen"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "S&#46;M&#46; Schoenfelder"
                            1 => "C&#46; Lange"
                            2 => "M&#46; Eckart"
                            3 => "S&#46; Hennig"
                            4 => "S&#46; Kozytska"
                            5 => "W&#46; Ziebuhr"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.ijmm.2010.04.011"
                      "Revista" => array:6 [
                        "tituloSerie" => "Int J Med Microbiol"
                        "fecha" => "2010"
                        "volumen" => "300"
                        "paginaInicial" => "380"
                        "paginaFinal" => "386"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/20451447"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bib0010"
              "etiqueta" => "2"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Intravascular device infections&#58; epidemiology&#44; diagnosis&#44; and management"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "G&#46; Gandelman"
                            1 => "W&#46;H&#46; Frishman"
                            2 => "C&#46; Wiese"
                            3 => "V&#46; Green-Gastwirth"
                            4 => "S&#46; Hong"
                            5 => "W&#46;S&#46; Aronow"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1097/01.crd.0000197966.53529.67"
                      "Revista" => array:6 [
                        "tituloSerie" => "Cardiol Rev"
                        "fecha" => "2007"
                        "volumen" => "15"
                        "paginaInicial" => "13"
                        "paginaFinal" => "23"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/17172879"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            2 => array:3 [
              "identificador" => "bib0015"
              "etiqueta" => "3"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on <span class="elsevierStyleItalic">Staphylococcus epidermidis</span> and <span class="elsevierStyleItalic">Staphylococcus haemolyticus</span> biofilms"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "N&#46; Cerca"
                            1 => "S&#46; Martins"
                            2 => "S&#46; Sillankorva"
                            3 => "K&#46;K&#46; Jefferson"
                            4 => "G&#46;B&#46; Pier"
                            5 => "R&#46; Oliveira"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1128/AEM.71.12.8677-8682.2005"
                      "Revista" => array:6 [
                        "tituloSerie" => "Appl Environ Microbiol"
                        "fecha" => "2005"
                        "volumen" => "71"
                        "paginaInicial" => "8677"
                        "paginaFinal" => "8682"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/16332862"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            3 => array:3 [
              "identificador" => "bib0020"
              "etiqueta" => "4"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Antimicrobial resistance of coagulase-negative staphylococci from bovine subclinical mastitis with particular reference to macrolide-lincosamide resistance phenotypes and genotypes"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "P&#46; Luthje"
                            1 => "S&#46; Schwarz"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1093/jac/dkl061"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Antimicrob Chemother"
                        "fecha" => "2006"
                        "volumen" => "57"
                        "paginaInicial" => "966"
                        "paginaFinal" => "969"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/16524893"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            4 => array:3 [
              "identificador" => "bib0025"
              "etiqueta" => "5"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of <span class="elsevierStyleItalic">Staphylococcus aureus</span> and <span class="elsevierStyleItalic">Staphylococcus epidermidis</span>"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "F&#46; Martineau"
                            1 => "F&#46;J&#46; Picard"
                            2 => "N&#46; Lansac"
                            3 => "C&#46; M&#233;nard"
                            4 => "P&#46;H&#46; Roy"
                            5 => "M&#46; Ouellette"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "Antimicrob Agents Chemother"
                        "fecha" => "2000"
                        "volumen" => "44"
                        "paginaInicial" => "231"
                        "paginaFinal" => "238"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/10639342"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            5 => array:3 [
              "identificador" => "bib0030"
              "etiqueta" => "6"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Mechanisms of biofilm resistance to antimicrobial agents"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "T&#46;C&#46; Mah"
                            1 => "G&#46;A&#46; O&#8217;Toole"
                          ]
                        ]
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Article information

ISSN: 0213005X
Original language: English

DOI:10.1016/j.eimc.2012.11.011

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	5	0	5
2024 October	36	6	42
2024 September	43	7	50
2024 August	43	4	47
2024 July	24	5	29
2024 June	20	1	21
2024 May	27	9	36
2024 April	24	5	29
2024 March	40	4	44
2024 February	35	4	39
2024 January	30	3	33
2023 December	52	6	58
2023 November	48	4	52
2023 October	62	8	70
2023 September	49	5	54
2023 August	44	1	45
2023 July	37	3	40
2023 June	55	2	57
2023 May	78	2	80
2023 April	92	2	94
2023 March	62	7	69
2023 February	25	3	28
2023 January	31	6	37
2022 December	49	6	55
2022 November	57	6	63
2022 October	58	20	78
2022 September	35	20	55
2022 August	36	23	59
2022 July	23	15	38
2022 June	22	9	31
2022 May	26	6	32
2022 April	34	10	44
2022 March	42	12	54
2022 February	24	12	36
2022 January	28	8	36
2021 December	16	14	30
2021 November	25	13	38
2021 October	40	10	50
2021 September	18	10	28
2021 August	25	9	34
2021 July	35	10	45
2021 June	27	10	37
2021 May	19	7	26
2021 April	54	7	61
2021 March	42	4	46
2021 February	31	12	43
2021 January	28	14	42
2020 December	17	4	21
2020 November	16	6	22
2020 October	10	6	16
2020 September	36	8	44
2020 August	27	8	35
2020 July	16	10	26
2020 June	21	13	34
2020 May	31	13	44
2020 April	25	5	30
2020 March	22	8	30
2020 February	25	10	35
2020 January	27	5	32
2019 December	39	14	53
2019 November	19	5	24
2019 October	28	6	34
2019 September	43	11	54
2019 August	11	9	20
2019 July	21	14	35
2019 June	38	21	59
2019 May	94	23	117
2019 April	44	11	55
2019 March	21	4	25
2019 February	19	6	25
2019 January	11	6	17
2018 December	10	6	16
2018 November	18	12	30
2018 October	29	5	34
2018 September	36	4	40
2018 August	12	5	17
2018 July	17	4	21
2018 June	20	5	25
2018 May	8	3	11
2018 April	15	4	19
2018 March	4	0	4
2018 February	7	2	9
2018 January	19	2	21
2017 December	5	2	7
2017 November	17	10	27
2017 October	16	2	18
2017 September	20	9	29
2017 August	18	9	27
2017 July	18	7	25
2017 June	42	12	54
2017 May	32	14	46
2017 April	28	18	46
2017 March	34	15	49
2017 February	39	3	42
2017 January	35	0	35
2016 December	40	6	46
2016 November	36	1	37
2016 October	89	7	96
2016 September	53	6	59
2016 August	46	9	55
2016 July	37	4	41
2016 June	52	6	58
2016 May	26	8	34
2016 April	23	25	48
2016 March	37	13	50
2016 February	33	17	50
2016 January	44	12	56
2015 December	32	10	42
2015 November	25	2	27
2015 October	34	4	38
2015 September	35	5	40
2015 August	38	3	41
2015 July	31	8	39
2015 June	17	2	19
2015 May	20	8	28
2015 April	25	5	30
2015 March	24	11	35
2015 February	24	3	27
2015 January	14	3	17
2014 December	26	5	31
2014 November	13	4	17
2014 October	16	3	19
2014 September	17	3	20
2014 August	11	3	14
2014 July	12	5	17
2014 June	6	1	7
2014 May	9	12	21
2014 April	6	7	13
2014 March	39	25	64
2014 February	47	22	69
2014 January	56	61	117
2013 December	51	118	169

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