The reduction of GLUT-1 expression as a consequence of CF administration was up to 70% in U937 cells. Figure 6 Western Blotting analysis of GLUT-1 receptor in Jurkat, U937, and K562 leukemia cell lines after 72 h of incubation with CF (5 μl/ml) as compared to untreated cells (control). Results are representative of three independent experiments. Other than GLUT-1 up-regulation, the activation of HIF-1 also contributes to the conversion of glucose to lactate. In

fact, when stabilized, HIF-1α is directly GF120918 clinical trial involved in the overexpression of many glycolytic enzymes as well as LDH, the NADH-dependent enzyme that catalyzes the conversion of pyruvate to lactate [38]. Based on the observed strong LDH dependency for tumor proliferation

from both in vitro and in vivo studies [39, 40], inhibition of LDH may represent an alternative strategy toward the development of anti-glycolytic-based therapeutic strategies for the treatment of cancer. Noteworthy, our data revealed that CF induced a significant decrease in LDH activity after 72 hours from its administration (up to 28%) (Figure 7A). At the same time, the amount of lactate released in the extracellular environment was also reduced in CF treated cells as compared to untreated cells (up Tariquidar chemical structure to 37%) (Figure 7B). Figure 7 LDH activity (A) and lactate release in the culture medium (B) in leukemia cells after 72 h of incubation with CF (5 μl/ml) in comparison with untreated cells (control). Data are expressed as mean ± SD of at least three independent experiments. *p < 0.05 vs.

untreated cells. The reversion of the glycolytic phenotype is known to render tumor cells susceptible to apoptosis and decrease their growth rate [15–17]. In this context, our findings are in accord with recent observations indicating that the in vitro inhibition of tumor cell survival (T cell lymphoma) by compounds targeting tumor metabolism was accompanied Arachidonate 15-lipoxygenase by a modulation of lactate concentration in the tumor-conditioned medium, by altered expression of HIF-1α and by an alteration in the expression of apoptotic (such as caspase-3) and cell survival regulatory Selleck CA4P molecules (such as GLUT-1) [17]. Another important control point might be the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [41]. If the oxygen supply is normal, NADH reducing equivalents that are generated by GAPDH are transported inside the mitochondria in order to reach the respiratory chain. In hypoxic conditions, the above reducing equivalents are used by LDH to convert pyruvate into lactate and no ATP can be produced into the mitochondria. This reaction is prominent in tumor cells, thus the evaluation of CF effect on GAPDH activity could also be of great interest.

Figure 8a presents the 10-nm-thick Ag film deposited on glass, whereas Figure 8b shows an image of the uncoated substrate. Two-dimensional histograms containing surface height check details values determined from the respective topographies are also shown. The obtained Ag

film exhibited a root-mean-square (RMS) roughness of 0.177 nm. The images (1 μm × 1 μm or 512 × 512 pixels) were automatically plane-fitted (to compensate for any sample tilts), and a color scale was used to represent the height distribution. The Z axes of the height histograms were scaled relative to the peak height. In addition, the surface of the evaporated Ag/glass film usually had an RMS roughness above 5 nm [13], which is an order of magnitude greater than that for the optical monitored ion etching treated E-beam coating with IAD films. Figure 8 AFM topography images of (a) an ultra-smooth, thin Ag film on glass (B270) and (b) an uncoated glass substrate (B270). (c,d) Histograms (2D surface XMU-MP-1 manufacturer height values) obtained from the respective topography images. Electrical Selleck C59 wnt properties The ideal work function of Ag is 4.4 eV, which is smaller than that of TiO2 (4 to 6 eV) [14] and higher than that of SKh (3.03 to 3.41 eV) [15]. When two layers are in contact with each other, the Fermi levels align in equilibrium by the transfer of electrons from

Ag to SiO2 and TiO2. The electrical properties of the system improve under GBA3 these conditions. In this case, there is no barrier for the electron flow

between Ag and SiO2, which means that the electrons can easily move from the Ag layer to the SiO2 layer. According to Schottky’s theory, we expect high carrier concentrations in multilayer TAS films. X-ray photoelectron spectroscopy Figures 9 and 10 show the XPS spectra of a TAS sample in the Si 2p, Ti 2p, O 1s, and Ag 3d regions. The same TiO2, SiO2, and silver peaks have also been clearly identified for other bimetallic clusters, revealing that our multilayer samples are composed of stable titanium oxide and silicon oxide films and contain pure Ag atoms. The observed peak positions are very close to those reported for ideal vacuum-evaporated TiO2, SiO2, and silver films, with the differences (including those between the 3d5/2 and 3d3/2 peaks for silver, 6.0 eV) also being exactly the same as the handbook values reported for zero-valent silver [16]. This observation suggests that most of the silver atoms in the TAS multilayers are in the zero-valent state. One would expect that a significant amount of the outer metal atoms is oxidized from Ag0 to Ag+1 upon thiolate formation, with a shift of the Ag 3d5/2 peak to higher binding energies (by 0.7 to 0.9 eV). Figure 9 Relationship between atomic percentage and etching depth, determined by XPS analysis. Figure 10 XPS analysis of the bonds. (a) The oxide bond. (b) The Si-O bond of SiO2.

donovani infection in

hamsters and BALB/c mice when administered through the intraperitoneal route [4, 5]. However, immunization via the subcutaneous route with the same liposomal vaccine failed to elicit protection [6]. This low Ku-0059436 in vitro efficacy following subcutaneous injection represents a critical barrier that currently limits the clinical applicability of a liposomal LAg subunit vaccine. Whilst many adjuvants which are routinely used in laboratory animals are often incompatible for human use, alum has been licensed for human vaccines for decades and is still widely incorporated into new vaccine formulations currently in development [7]. In relation to leishmaniasis, alum has been used in combination with IL-12 and killed promastigotes, resulting in effective protection click here in a primate model of CL [8]. Furthermore, an alum-absorbed preparation of autoclaved L. major (alum-ALM) mixed with Bacillus Calmette-Guerin (BCG) protected Langur monkeys against VL [9]. Indeed, alum-ALM was found to be tolerable in healthy volunteers, whilst imparting minimal side-effects and conferring improved immunogenicity compared to preparations lacking the alum component [10]. These observations led to the use of this vaccine as an immunological stimulus for the treatment

selleck products of patients with persistent post kala-azar dermal leishmaniasis (PKDL), where vaccine administration was shown to significantly improve C1GALT1 the clinical outcome of PKDL lesions [11]. Saponin consists of natural glycosides of steroid or triterpene, which can activate the mammalian immune system, leading to significant interest in developing saponin as a vaccine adjuvant. Saponin has already been included as an adjuvant in clinical vaccine

formulations against HIV and cancer [12]. Combined administration of saponin and fucose manose ligand (FML) antigen from L. donovani was additionally found to be protective against VL in both mice and dogs [13, 14], and moreover the FML-vaccine was also effective in an immunotherapeutic context against the same disease [15, 16]. Similarly the Leishmune® vaccine, composed of FML antigen with an increased concentration of saponin exhibited immunotherapeutic potential in dogs, reducing clinical symptoms following L. chagasi challenge [17]. There is therefore much hope for a saponin-adjuvanted leishmanial vaccine in veterinary and clinical research. Alum and saponin are both approved for human use and have been widely applied in numerous clinical vaccine trials [7, 12]. Therefore, in the present study we investigated the protective efficacy of LAg against L. donovani challenge in isolation, or in combination with either alum or saponin adjuvants administered through a subcutaneous route, as compared to the highly efficacious intraperitoneal route of lip + LAg administration in BALB/c mice.

We are also very grateful to Professor Zhou Q. L.,

Professor Xie J. H., and their group for providing solution of benzene thiol in ethanol. References 1. Nie S, Remory S: Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 1997, 275:1102–1106.BAY 63-2521 price CrossRef 2. Kneipp K, Wang Y, Kneipp H, Perelman LT, Itzkan I, Dasari RR, Field MS: Field single molecule detection using surface-enhanced Raman scattering. Phys Rev Lett 1997, 78:1667–1670.CrossRef 3. Li JF, Huang YF, Ding Y, Yang ZL, Li SB, Zhou XS, Fan FR, Zhang W, Zhou ZY, Wu DY, Ren B, Wang ZL, Tian ZQ: Shell-isolated nanoparticle-enhanced Raman spectroscopy. Nature 2010, 464:392–395.CrossRef 4. Liang HY, Li ZP, Wang WZ, Wu YS, Xu HX: Highly surface-roughened see more “”flower-like”" silver nanoparticles for extremely sensitive substrates of surface-enhanced https://www.selleckchem.com/products/px-478-2hcl.html Raman scattering. Adv Mater 2009, 21:4614–4618.CrossRef 5. Liu FX, Cao ZS, Tang CJ, Chen L, Wang ZL: Ultrathin diamond-like carbon film coated silver nanoparticles-based substrates for surface-enhanced Raman spectroscopy. ACS Nano 2010, 4:2643–2648.CrossRef 6. Ryckman JD, Liscidini M, Sipe JE, Weiss SM: Direct imprinting of

porous substrates: a rapid and low-cost approach for patterning porous nanomaterials. Nano Lett 2011, 11:1857–1862.CrossRef 7. Schmidt MS, Hubner J, Boisen A: Large area fabrication of leaning silicon nanopillars for surface enhanced Raman spectroscopy. Adv Mater 2012, 24:11–18. 8. Caldwell JD, Glembocki O, Bezares FJ, Bassim ND, Rendell RW, Feygelson

M, Ukaegbu M, Kasica R, Shirey L, Hosten C: Plasmonic nanopillar arrays for large-area, high-enhancement surface-enhanced Raman scattering sensors. ACS Nano 2011, 5:4046–4055.CrossRef 9. Zhang L, Lang X, Hirata A, Chen M: Wrinkled nanoporous gold films with ultrahigh surface-enhanced Raman scattering enhancement. ACS Nano 2011, 5:4407–4413.CrossRef 10. Duan H, Hu H, Kumar K, Shen Z, Yang JKW: Direct and reliable patterning of plasmonic Staurosporine molecular weight nanostructures with sub-10-nm gaps. ACS Nano 2011, 5:7593–7600.CrossRef 11. Wang HH, Liu CY, Wu SB, Liu NW, Peng CY, Chan TH, Hsu CF, Wang JK, Wang YL: Highly Raman-enhancing substrates based on silver nanoparticle arrays with tunable sub-10 nm gaps. Adv Mater 2006, 18:491.CrossRef 12. Siegfried T, Ekinci Y, Solak HH, Martin OJF, Sigg H: Fabrication of sub-10 nm gap arrays over large areas for plasmonic sensors. Appl Phys Lett 2011, 99:263302.CrossRef 13. Cho WJ, Kim Y, Kim JK: Ultrahigh-density array of silver nanoclusters for SERS substrate with high sensitivity and excellent reproducibility. ACS Nano 2012, 6:249–255.CrossRef 14. Hu X, Meng G, Huang Q, Xu W, Han F, Sun K, Xu Q, Wang Z: Large-scale homogeneously distributed Ag-NPs with sub-10 nm gaps assembled on a two-layered honeycomb-like TiO2 film as sensitive and reproducible SERS substrates. Nanotechnology 2012, 23:385705.CrossRef 15.

J Mater Chem 2011, 21:5938–5943.CrossRef 21. Wu Y, Li Y, Ong BS: A simple and efficient

approach to a printable silver conductor for printed electronics. J Am Chem Soc 2007, 129:1862–1863.CrossRef 22. Osch THJ, Perelaer J, de Laat AWM, Schubert US: Inkjet printing of narrow conductive tracks on untreated polymeric substrates. Adv Mater 2008, 20:343–350.CrossRef 23. Kim TY, Kim YW, Lee HS, Hyeongkeun K, Yang WS, Suh KS: Uniformly interconnected silver-nanowire networks for transparent film heaters. Adv Funct Mater 2013, 23:1250–1255.CrossRef 24. Russo A, Ahn BY, Adams JJ, Duoss EB, Bernhard JT, Lewis JA: Pen-on-paper flexible electronics. Adv Mater 2011, 23:3426–3431.CrossRef SCH727965 order 25. Korte KE, Skrabalak SE, Xia YJ: Rapid synthesis of silver nanowires Pictilisib clinical trial through a CuCl-or CuCl 2 -mediated polyol process. Mater Chem 2008, 18:437–442.CrossRef 26. Liu CH, Yu X: Silver nanowire-based transparent, flexible, and conductive thin film. Nanoscale Res Lett 2011, 6:75–83.CrossRef Competing MLN8237 ic50 interests The authors declare that they have no competing

interests. Authors’ contributions YT synthesized the silver nanowire and prepared the SNW ink. Y-LT fabricated the conductive pattern and investigated the conductive properties. L-YW, Y-XT, B-BW, and Z-GY gave many advices and took part in writing the whole manuscript. All authors read and approved the final manuscript.”
“Background One of the most commonly used approaches to tune the fluorescence properties of fluorophores is to couple them to plasmonic excitations in metallic nanoparticles [1]. Large variations of shapes and sizes of metallic nanostructures provide almost infinite space for spectral engineering of optical properties of emitters, ranging from control of the fluorescence intensity, fluorescence decay dynamics, as well as the emission spectrum itself. Remarkable effects of plasmon coupling have been demonstrated on a single-molecule level, where a fluorophore was approached in a controllable way by a spherical metallic nanoparticle [2]. For large distances, the emission remained unaffected;

however, Thymidylate synthase as the separation decreased, a strong enhancement of the fluorescence emission has been measured. Upon further reduction of the separation between the fluorophore and metallic nanoparticle, the intensity of the fluorescence emission decreased rapidly. This result demonstrates allimportant effects of plasmon coupling in such experimental configuration, and they are associated with modifications of fluorescence quantum yield of the fluorophore, enhancement of its excitation rate, and quenching due to nonradiative energy transfer to the metallic nanoparticle. As these processes compete against each other, in order to achieve strong enhancement of the fluorescence intensity, it is crucial to put attention to the geometry of the hybrid plasmonic nanostructure, in particular to the control of the separation between fluorophores and metallic nanoparticles.

For clarity, each selleck chemical spectrum of Δ and Ψ are shifted by 200° and 50°, respectively. The fitted parameters of the TM-doped TiO2 films determined by the SE spectra are listed in Table 1.

From the table, the film thickness of undoped TiO2 film is the largest and that of Co-doped TiO2 films is the smallest. Compared with the undoped TiO2 film, the addition of dopant decreases A 0 and increases Γ, which suggests that the Urbach tail absorption characteristics were formed. Note that it is common to observe the development of an Urbach tail on doping transition metal oxides [45, 46]. Table 1 The fitted parameters of the TM-doped TiO 2 films determined by the SE spectra   Г (eV) E OBG(eV) ϵ ∞ A 0(eV3/2) df (nm) ds (nm) C TM(%) selleck chemicals llc Undoped 0.02 ± 0.01 3.58 ± 0.01 0.11 ± 0.03 136.6 ± 10 355 ± 10 5 ± 2 S6 Kinase inhibitor   Dopant content                 Fe 0.01

0.030 ± 0.01 3.56 ± 0.02 0.260 ± 0.02 132.31 ± 12 288 ± 8 3 ± 1 0.8 0.03 0.085 ± 0.06 3.54 ± 0.02 0.087 ± 0.02 126.23 ± 20 265 ± 6 4 ± 2 2.7   Ni 0.01 0.035 ± 0.02 3.53 ± 0.01 0.1 ± 0.04 134.48 ± 13 233 ± 7 3 ± 1 0.9 0.03 0.036 ± 0.03 3.50 ± 0.01 0.517 ± 0.11 128.18 ± 14 219 ± 6 3 ± 1 2.9   Co 0.01 0.042 ± 0.01 3.48 ± 0.02 0.528 ± 0.10 125.11 ± 11 215 ± 5 3 ± 2 0.8 0.03 0.106 ± 0.04 3.43 ± 0.01 0.353 ± 0.15 118.9 ± 6 206 ± 5 4 ± 2 2.8 The film thickness (df), the thicknesses of the surface rough layer (ds), and the parameter value of Adachi’s model (A 0) for TM-doped TiO2 films with dopant content extracted from the simulation of SE in Figure 7. The 90% reliability of the fitted parameters is shown with ± sign. The TM atom composition C TM derived by the XPS spectra is also listed. Figure 8 depicts the variation in dielectric function of the TM-doped TiO2 films with photon Paclitaxel mouse energy. In general, in all samples, we found that the real part

ϵ r of the dielectric function increases and gradually nears the maximum, and then decreases due to the Van Hove singularities. This is the typical optical response of dielectric or semiconductor materials [44]. The imaginary part ϵ i of the dielectric function nears zero in the transparent region (E OBG > E) and sharply increases further with increasing photon energy in the absorption region (E OBG < E). Figure 8 Imaginary part ϵ i and real part ϵ r of the complex dielectric functions of the undoped and TM-doped TiO 2 films. For clarity, the ϵ i and part ϵ r of the films are shifted by 2 and 5, respectively. The dopant content dependence of the E OBG of the TM-doped TiO2 films is presented in Figure 6c. It is can be seen that the E OBG of the TM-doped TiO2 films decreases with increasing dopant content.

subset3 ITS3-4B_3mis ITS3-4B_0mis Agaricales 361 269 (74.5) 118 (32.7) Boletales 18 17 (94.4) 15 (83.3) Cantharellales 33 31 (93.9) 0 Hymenochaetales 10 7 (70) 0 Polyporales 28 8 (28.6) 0 Russulales 97 64 (66.0) 0 Thelephorales 6 4 (66.7) 0 Dacrymycetes 1 0 0 Tremellomycetes 38 13 (34.2) 0 Pucciniomycotina 8 0 0 Ustilaginomycotina 21 0 0 Other categories * 71 21 (29.6) 3 (4.2) * ‘Other categories’ represent smaller orders including Agaricomycetidae. see more Our in silico analyses further indicate that most of the primers will introduce a taxonomic bias due to higher levels of mismatches in certain taxonomic groups.

When allowing one mismatch (corresponding to rather stringent PCR conditions) we found that the primer pairs ITS1-F, ITS1 and ITS5 preferentially amplified basidiomycetes whereas the primer pairs ITS2, ITS3 and ITS4 preferentially amplified ascomycetes. This type of bias must also be considered before selecting primer pairs for a given study. Also in molecular surveys of protistan and prokaryotic diversity, it has been documented that different 16S primers target different parts of the diversity [32–34]. In addition,

our results clearly demonstrate that basidiomycetes, on average, have significantly longer amplicon sequences than ascomycetes both for the whole ITS region, and the ITS2 region. This fact probably also introduces Selleckchem NSC23766 taxonomic bias during PCR Emricasan clinical trial amplification of environmental samples, since shorter fragments are more readily amplified compared to longer ones. In several studies, it has been demonstrated that a greater proportion of the diversity can be detected with short target sequences compared to longer ones [35, 36]. Hence, using the ITS2 region or the whole ITS region, a higher number of the ascomycetes will probably be targeted compared

to basidiomycetes. This bias could be avoided by using primers amplifying ITS1 only, but this would imply a preferential amplification of the ‘non-dikarya’ fungi. Conclusion The in silico method used here allowed for the assessment of different parameters for commonly used ITS primers, including the length amplicons generated, taxonomic heptaminol biases, and the consequences of primer mismatches. The results provide novel insights into the relative performance of commonly used ITS primer pairs. Our analyses suggest that studies using these ITS primers to retrieve the entire fungal diversity from environmental samples including mixed templates should use lower annealing temperatures than the recommended Tm to allow for primer mismatches. A high Tm has been used in most studies, which likely biases the inferred taxonomic composition and diversity. However, one has to find a balance between allowing some mismatches and avoiding non-specific binding in other genomic regions, which can also be a problem.

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“Background Renal cell carcinoma (RCC) is the most common type of kidney cancer (8.

The unloaded ZnO and ZnO NPs loaded with Au (1.00 mol%) were produced by a single-step FSP technique. The particle analyses using XRD, HR-TEM, MCC-950 and BET indicated that ZnO NPs were highly crystalline with a typical hexagonal structure of ZnO, and ultrafine Au NPs with 1 to 2 nm in diameter were formed around ZnO NPs. Composite P3HT:1.00 mol% Au/ZnO NPs films with different compositions were prepared by solution mixing and casting. Film characterizations by XRD and FE-SEM confirmed the presence of P3HT/ZnO phases and HDAC inhibition porous nanoparticle structures in the composite

thick film. The gas sensing results showed that the inclusion of 1.00 mol% Au/ZnO NPs at a low content provided significant NH3 sensing enhancement. In particular, the P3HT:1.00 mol% Au/ZnO NPs composite film with the ratio of 4:1 exhibited the best NH3 sensing performances with a high sensor response of approximately 32 and short response time within a minute to 1,000 ppm of NH3 at a room temperature.

In addition, the optimal composite film exhibited higher NH3 selectivity against C2H5OH, CO, H2S, NO2, and H2O than other composites as well as P3HT and 1.00 mol% Au/ZnO NPs. The observed composite gas sensing behaviors were explained based on the increased specific surface area by porous blended nanoparticle structure and catalytic effect of Au/ZnO NPs. From overall results, the P3HT:1.00 mol% Au/ZnO NPs composite sensor is a highly promising candidate for the efficient detection of NH3 at room temperature. C188-9 ic50 Acknowledgements The authors gratefully

acknowledge the financial support from the Thailand Research Fund (TRF), the Office of the Higher Education Commission and Maejo University, Thailand (MRG5580067); Program in Materials Science, Faculty of Science, Maejo University, Thailand; the National Research Council of Thailand; the National Research University under the Office of Higher Education Commission; Materials Science Research Urocanase Center, Faculty of Science, Chiang Mai University, Thailand; and National Electronics and Computer Technology Center (NECTEC), Pathumthani, Thailand. References 1. Narasimhan LR, Goodman W, Kumar C, Patel N: Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis. Proc Natl Acad Sci U S A 2001, 98:4617–4621.CrossRef 2. de la Hoz RE, Schueter DP, Rom WN: Chronic lung disease secondary to ammonia inhalation injury: a report on three cases. Am J Ind Med 1996, 29:209–214.CrossRef 3. Leung CM, Foo CL: Mass ammonia inhalation burns-experience in the management of patients. Ann Acad Med Singapore 1992, 21:624–629. 4. Michaels RA: Emergency planning and acute toxic potency of inhaled ammonia. Environ Health Perspect 1999, 107:617–627.CrossRef 5. Close LG, Catlin FI, Cohn AM: Acute and chronic effects of ammonia burns on the respiratory track. Arch Otolaryngol 1980, 106:151–158.CrossRef 6.

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drug efflux systems. J Mol Microbiol Biotechnol 2001, 3:145–150.PubMed 33. Johnson JM, Church GM: Alignment and structure prediction of divergent protein families: periplasmic and outer membrane Selleck RG7420 proteins of bacterial efflux pumps. J Mol Biol 1999, 287:695–715.PubMedCrossRef 34. Delcour AH: Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta 2009, 1794:808–816.PubMedCrossRef 35. Vaara M: Agents that increase the permeability of the outer membrane. Microbiol Rev 1992, 56:395–411.PubMed 36. Savage PB: Multidrug-resistant bacteria: overcoming antibiotic permeability barriers of gram-negative bacteria. Ann Med 2001, 33:167–171.PubMedCrossRef 37. Mahachai V, Sirimontaporn N, Tumwasorn S, Thong-Ngam D, Vilaichone RK: Sequential therapy in clarithromycin-sensitive and –resistant Helicobacter pylori based on polymerase

chain reaction molecular test. J Gastroenterol Hepatol 2011, 26:825–828.PubMedCrossRef 38. Bina JE, Alm RA, Uria-Nickelsen M, Thomas SR, Trust TJ, Hancock RE: Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro. Antimicrob Agents Chemother 2000, 44:248–254.PubMedCrossRef 39. Nikaido H: Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 2003, 67:593–656.PubMedCrossRef Competing interests The authors declare that they have no competing interests. This work was supported in part by Over Italia, S.r.l., Sora (Frosinone) (Contract of research between Over and University of Siena Janus kinase (JAK) N. 52514/III-17) Italy. Over s.r.l. is the owner of the patent PCT/IT2011/000175. Authors’ contribution NF: substantial contributions to conception and design, bacterial culture, susceptibility tests and manuscript writing. EM: substantial contributions to conception and design electron microscopy and manuscript writing. RM and GC substantial contributions to conception and design. GC: electron microscopy, revision of the manuscript. AS and AS: contribution of interpretation of the data. All the authors revised the manuscript and gave their final approval.