mallei SR1 ATCC 23344 sucrose-resistant

derivative [40] DDA0742 SR1 derivative harboring a deletion of the 156 bp NarI–SfuI fragment internal to hcp1; Δhcp1 [25] B. thailandensis DW503 E264 derivative; Δ(amrR-oprA) (Gms) rpsL (Smr) [41] DDII0868 DW503::pGSV3-0868; Gmr; hcp1 – This study Plasmids pCR2.1-TOPO 3,931-bp TA vector; pMB1 oriR; Kmr Invitrogen pCR2.1-0868 pCR2.1-TOPO containing 342-bp PCR product generated with II0868-up and II0868-dn This study pGSV3 Mobilizabile Gmr suicide Ralimetinib vector [42] pGSV3-0868 pGSV3 derivative containing EcoRI insert from pCR2.1-0868 This study a r, resistant; s, susceptible. PCR The two deoxyribonucleotide primers used for PCR amplification of an internal gene fragment of B. thailandensis BTH_II0868 (hcp1) were purchased from Invitrogen (Frederick, MD) and designated II0868-up (5’-AGGGCAAGATTCTCGTCCAG-3’) and II0868-dn (5’-TCTCGTACGTGAACGATACG-3’).

The PCR product was sized and isolated using agarose gel electrophoresis, cloned using the pCR2.1-TOPO TA Cloning Kit (Invitrogen), and transformed into chemically competent E. coli TOP10. PCR amplification was performed in a final reaction volume of 100 μl containing 1X Taq PCR Master Mix (Qiagen), 1 μM oligodeoxyribonucleotide ATM Kinase Inhibitor research buy primers, and approximately 200 ng of B. thailandensis DW503 genomic

DNA. PCR cycling was performed using a PTC-150 MiniCycler with a Hot Bonnet accessory (MJ Research, Inc.) and heated Tau-protein kinase to 97°C for 5 min. This was followed by 30 cycles of a three-temperature cycling protocol (97°C for 30 s, 55°C for 30 s, and 72°C for 1 min) and one cycle at 72°C for 10 min. DNA manipulation and plasmid conjugation Restriction enzymes, Antarctic MCC950 ic50 phosphatase, and T4 DNA ligase were purchased from Roche Molecular Biochemicals and were used according to the manufacturer’s instructions. DNA fragments used in cloning procedures were excised from agarose gels and purified with a GeneClean III kit (Q · BIOgene). Bacterial genomic DNA was prepared by a previously described protocol [29]. Plasmids were purified from overnight cultures by using Wizard Plus SV Minipreps (Promega). Plasmid pGSV3-0868 (Table 2) was electroporated into E. coli S17-1 (12.25 kV/cm) and conjugated with B. thailandensis for 8 h, as described elsewhere [30]. Pm was used to counterselect E. coli S17-1 (pGSV3-0868).

Rectal examination was guaiac-negative, and a complete blood count indicated leukocytosis with left shift. CT scan of abdomen showed a gastric dilatation, marked thickening of the anterior

wall and necrotic areas within. An exploratory upper laparotomy confirmed acute gastric dilatation and necrosis of the anterior surface of the stomach. A “sleeve” gastrectomy to ablate the necrotic area was performed and a feeding jejunostomy. The gastric wall appeared very thin and totally necrotic upon macroscopic examination by the pathologist. No layers or structures were identifiable on histological examination, but numerous fungal yeasts were identified inside the necrotic areas with PAS and Gomori Silvermthenamina stains (Figure 1). Figure 1 Histological section. A) Very thin and totally necrotic gastric wall. B, C) Numerous fungal yeasts were present. PAS stain (A) ×100; (B) ×200; (C) TH-302 research buy ×400. Culture of the intra-operative surgical

specimen confirmed the presence of Candida albicans. Yeast isolates were identified to the species level by conventional morphological and biochemical methods, as previously reported [3, 7, 8]. The yeast isolate was susceptible to fluconazole and echinocandin, according to CLSI cut off values [9, 10]. It is noteworthy that blood cultures were negative. Echinocandin check details (70 mg on the first day, i.e., day 103, followed by 50 mg/day) was administered parenterally for a total of 14 days, followed by maintenance therapy with 400 mg of oral fluconazole per day. The patient was discharged in stable condition and antifungal therapy was continued in an outpatient setting. She has been doing well since then. Second case In January 2013, a 62 year-old woman of Italian origin and nationality with BMI of 35 kg/m2, presented to the general surgery and emergency unit of the “P. Giaccone” Teaching Hospital in Palermo, Italy, with complicated clonidine midline incisional hernia,

nausea, vomiting and abdominal distension. Her initial vital signs were notable for a temperature of 38°C, respiratory rate of 22 breaths per minute, heart rate of 110 beats per minute and blood pressure of 90/60 mmHg. She was suffering from severe abdominal pain and breathing difficulties. On clinical examination, she presented a tender abdomen, ulcerated skin with associated necrosis and dry skin. Her past medical history showed three caesarean sections, treatment for arterial hypertension, COPD and a diagnosis of type II diabetes mellitus (DM) about 15 years previously, treated with insulin. Emergency surgery was required, and surgical exploration showed a congested, edematous and necrotic strangulated intestinal tract. The section of necrotic intestine was removed and ileo-ileostomy was performed. The surgery was successful, without additional complications, and an abdominal subcutaneous drain was inserted. The surgical specimen was sent to the Pathology Selleckchem BAY 1895344 Laboratory for histological examination.

coli 803 strain. Mating assays were performed by mixing equal volumes of overnight cultures

of donor and recipient strains. Briefly, the cells were harvested by centrifugation and resuspended in a 1/20 volume of LB broth. Cell suspensions were poured onto LB agar plates and incubated at 37°C for 6 h. The cells were then resuspended in 1 ml of LB medium, and serial dilutions were plated onto appropriate selective media to determine the numbers of donors, recipients, and exconjugants. Frequency of transfer was expressed as the number of exconjugant Go6983 cells per donor cells in the mating mixture at the time of plating. V. cholerae O139 MO10 [14], V. cholerae E4:ICEVchInd1 [21], V. cholerae O1 VC20 [22], V. cholerae N16961 [23], V. cholerae O1 CO840 [22], V. cholerae www.selleckchem.com/products/azd6738.html O1 VC7452, VC15699, and VC9258 isolated in India (Maharashtra) [16], and E. coli AB1157:R391 [24] were appropriately used as negative or positive controls for class 1 integrons, ICE, tcpA, and rstR detection, CTXΦ array and ribotype analysis. Molecular biology procedures Bacterial

DNA for PCR analysis was prepared with a Wizard Genomic DNA Purification kit (Promega). Amplicons to be sequenced were directly purified from PCR or extracted from agarose gel by Wizard SV Gel and PCR Clean-up System (Promega) according to the manufacturer’s instructions. DNA sequences were determined by BMR Genomics (Padova, Italy). Class 1 integron detection was performed by PCR amplification with specific primer pairs as previously AZD4547 supplier described [11]. ICEs of the SXT/R391 family were screened by PCR analysis, using 17 specific primer pairs previously described by our group [25, 26]. int SXT, prfC/SXTMO10 right junction, floR, strA, strB, sul2, dfrA18, dfrA1, rumAB operon, traI, traC, setR,

and Hotspots or Variable Regions s026/traI, s043/traL, traA/s054, s073/traF Ixazomib datasheet and traG/eex were screened. A second set of 15 primer pairs designed on the specific sequences of ICEVchInd5 [16] were used to detect ICEVchInd5 and ICEVchAng3 specific Hotspots and Variable Regions. All PCR reactions were set in a 50-μl volume of reaction buffer containing 1 U of Taq polymerase as directed by the manufacturer (Promega). Ribotype analysis Ribotyping of V. cholerae strains was performed by BglI restriction of chromosomal DNA with fluorescent-labeled 16S and 23S DNA (Gene Images 3540 RPn3510, Amersham) generated by reverse transcriptase polymerase chain reaction of ribosomal RNAs, as already described [25]. CTX array analysis and ctxB, tcpA, rstR biotype characterization The structure of CTX array was determined by multiple PCR analysis (Table 2) and by Southern Blot hybridization. The genetic structure of the two CTX prophage arrays described in Figure 1 was determined using the primers described in Table 2.

Table 3 Distribution of the proteins identified by CMAT and 2D-PAGE across phage genomes Gene Other Stx phages carrying the proteins in the study (identity) Accession number Other phages Accession number CM1 Stx2 converting phage II (99%) YP_003828920.1       phage Selleck Thiazovivin VT2-Sakai (99%) NP_050557.1       phage 933 W (99%) NP_049519.1       Stx1 converting phage (99%) YP_003848832       phage BP-933 W (99%) YP_003848832.1       phage VT2phi_272 (99%) ADU03741.1       phage Min27(100%) ADU03741     CM2 Stx2 converting phage II (100%) BAC78116       phage VT2-Sakai (100%) NP_050531.1       phage Min27

(100%) YP_001648926       phage HK97 (99%) AAF31137       phage Lahn2 (99%) CAJ26400       phage Lahn3 (98%) CAC95062.1       phage 2851 (99%) CAQ82016       phage CP-1639(99%) https://www.selleckchem.com/products/mek162.html CAC83142       prophage CP-933 V(99%) AAG57233       Phage Nil2 (99%)(99%) CAC95095       Stx1

converting phage (99%) YP_003848889.1       Phage CP-1639 (99%) CAC83142.1       Phage YYZ-2088 (99%) YP_002274170.1       Stx2-converting phage 1717 (99%) YP_002274244.1     CM5 phage Min27 (100%) YP_001648966.1       Stx2 converting phage II(100%) YP_00388933.1       Stx2 converting phage I(100%) NP_612929.1       phage VT2-Sakai (100%) NP_050570.1       phage 933 W (100%) NP_049532.1       phage VT2phi_272 (100%) ADU03756     CM7 phage VT2-Sakai (99%) NP_050570       Stx1 converting phage (99%) BAC77866.1       Phage VT2phi_272 (97%) ADU03756.1       Phage 933 W (97%) NP_049532.1       Stx2 converting phage I (97%) NP_612929.1       Stx2 converting phage II(97%) BAC78032.1       Phage BP-933 W (97%) AAG55616.1       Stx2 converting phage 86 (91%) YP_794082.1       Phage Min27 4EGI-1 (97%) YP_001648966.1     CM18 phage VT2-Sakai (100%) NP_050564.1       Stx1 converting phage Celecoxib (100%) YP_003848839.1

      Phage 933 W (100%) NP_049526.1       Stx2 converting phage I (100%) ZP_02785836.1       Stx2 converting phage II (100%) YP_003828926.1       Phage BP-933 W (100%) NP_286999.1       Stx2 converting phage 86 (97%) YP_794076.1       Phage Min27 (100%) YP_001648959.1     P1 Stx2 converting phage II (99%) YP_003828937.1 Phage phiV10 (78%) YP_512303.1   Stx2 converting phage I (99%) NP_612952.1       Phage 933 W (99%) NP_049538.1       Phage BP-933 W (99%) AAG55619.1       phage VT2-Sakai (99%) NP_050575.1       Phage Min27 (96%) YP_001648901.1       Stx2-converting phage 86 (96%) YP_794094.1       Phage BP-4795 (96%) YP_001449244.1       phage CP-1639 (74%) CAC83133.1     P2 Stx2 converting phage I (100%) NP_612997.1 Salmonella enteric YP_002455860.1   Phage 933 W (100%) NP_049484.1 bacteriophage SE1 (86%)     Phage BP-933 W (100%) AAG55573.1 Salmonella phage ST160 (86%) YP_004123782.1   Phage Min27 (100%) ABY49878.1       Stx2-converting phage 86 (100%) YP_794109.1     P3 Stx2 converting phage I (100%) NP_612995.1       Phage 933 W (100%) NP_049483.1       Stx2-converting phage 86 (100%) YP_794108.1       Phage Min27 (100%) YP_001648915.

Due to differences in the amount of sequence reads obtained from individual samples,

the relative distribution of sequences was calculated on the basis of the total number of reads from the sample. OTUs that accounted for > 1% of the total number of sequences were considered as dominant species. Table 2 The distribution of sequence reads, OTU’s in absolute numbers and the ratio between Firmicutes and Bacteroides in pooled caecal samples   Conventional Evofosfamide mw cage Furnished cage Aviary   Before inoculation 4 weeks PI Before inoculation 4 weeks PI Before inoculation 4 weeks PI Number of reads 51,863 21,714 42,885 42,520 51,715 40,410 Number of OTU/total number of OTU 185/197 178/197 196/197 193/197 195/197 193/197   93.9% 90.4% 99.5% 98.0% 99.0% 98.0% Firmicutes/Bacteroides ratioa 0.81 0.61 0.87 0.74 0.69 0.68 a The ratio was calculated by dividing all OTU that could be affiliated to Firmicutes (Clostridia and Bacilli)

by the number of OTU’s from Bacteroides. In total, 197 different OTUs were identified, and 196 and 195, respectively, out of these were found in non-inoculated samples from AV and FC, however, for CC a progressive decrease in numbers of OTUs was observed in both samples before and after inoculation with Salmonella. In these cages, 185 OTUs were identified before inoculation and 178 OTUs four weeks after inoculation, while in the other cages 193 OTUs were detected at the end of the experiment. Due to a different number of reads obtained Staurosporine clinical trial from each sample, normalized prevalence values

of each OTU were calculated. Using a cut-off value of 0.01%, the difference in diversity between cages was still observed where the BIBW2992 mouse dominating genera in CC constituted a larger proportion Phosphatidylinositol diacylglycerol-lyase of the microbiota at the expense of fewer OTU’s, compared to the two other cages (Figure 2). Figure 2 The distribution of OTU’s according to the prevalence in the microbiota. The number and prevalence of OTU based on the relative prevalence in each sample (cut off < 0.01%). The number of different OTU’s in the group of less abundant genera was highest in furnished and aviary cage, in contrast to conventional cage where we observed fewer but more dominating genera. The consensus sequence from each OTU was compared against the Ribosomal Database (RDP server) to find the most related species or genus. Though many of the bacterial species in the caecal microbiota still remain to be characterized, it was possible to classify 92% of all OTUs to phylum level, and out of these were 86% classified to class level and 55% to genus level. Although variation was observed in the relative presence that colonized the caecum, it was the same group of genera that were dominating in all cages before and after inoculation, accounting for more than 74% of the total amount of reads (Table 3).

The data indicated that the MIC for nitrofurantoin STI571 was approximately 3:g/ml for all strains tested (data not shown). When plates were incubated for an additional 24 hrs, a small number of colonies arose, and these were presumptive nitrofurantoin-resistant mutants. By comparing the number of colonies found on an agar

plate after 48 hours incubation in the presence of nitrofurantoin to the number of colonies obtained when a similar aliquot was inoculated onto media lacking the antimicrobial agent, we were able to calculate the spontaneous buy GSI-IX mutation frequency to resistance to this agent. The data (Fig. 1) indicate that the mutation frequency associated with this antimicrobial agent varied about 10 fold among strains, with FA1090 being the least mutable among the strains tested, and MS11 being the most mutable. However, since it was possible to isolate mutants that readily grew on media containing levels of nitrofurantoin above the MIC, we hypothesized that the mutation responsible for this phenotype was in the coding sequence for the putative gonococcal nitroreductase gene. Figure 1 Spontaneous mutation frequency of various lab strains of N. gonorrhoeae. Mutation frequencies were determined BKM120 mouse by counting the number of colonies arising on the GCK +

Nitrofurantoin (3 μg/mL) plates after 48 hours of incubation at 37°C, 6% CO2, and dividing this number by the number of colonies arising on the GCK plates after 48 hours of incubation at 37°C, 6% CO2. Data represents

experiments done in triplicate, with error bars representing standard error. Identification of potential nitroreductase genes E. coli possesses two nitroreductases that can reduce these nitro-aromatic compounds; nfsA and nfsB, plus a nitroreductase activity encoded by a gene that has yet to be identified [18, 24]. Therefore, it is possible that GC may possess additional cAMP genes that confer nitroreductase activity. In E. coli, resistance to these nitro-aromatic antimicrobial agents occurs in a step-wise manner. A mutation that knocks out the function of NfsA raises the MIC about three fold. A second mutation that knocks out the function of NfsB increases resistance to about 10 times the MIC of wild-type strains [18, 24]. All attempts to isolate second-step mutants in N. gonorrhoeae were unsuccessful, indicating that this species only contains a single functional nitroreductase, or that the additional nitroreductases were insensitive to nitrofurantoin. Since two nitroreductases have been identified in E. coli, nfsA and nfsB [30, 31], we used the amino acid sequence for these two gene products to search the gonococcal translated genomic DNA sequence database. No significant similarity was found to nfsA. However, an ORF encoding a protein with some similarity to nfsB was found.

Figure  3a reveals that the imperfect internal quantum process caused by the surface recombination and other carrier loss mechanisms results in a great degradation on the electrical properties of the top (a-Si:H) cell, which is reflected

as a much discrepancy between P a-Si:H and EQEa-Si:H https://www.selleckchem.com/products/cbl0137-cbl-0137.html especially at short-wavelength region. However, for the bottom junction, P μc-Si:H ~ EQEμc-Si:H is always observed since the material defects are much less and the bottom junction is far from the top surface where the surface recombination is strong. Spectral integrations to the EQE spectra indicate that under TE (TM) illumination, J aSi can be risen by 2.11 (2.35) mA/cm2, resulting in the rise of 2.23 mA/cm2 in the top junction under an learn more unpolarized injection. However, the raise of photocurrent in

bottom junction is especially dramatic (4.63 mA/cm2), which has been actually expected from the multi-peaked absorption spectra. Therefore, although significant improvement on the absorption and light-conversion capability has been realized by two-dimensionally nanopatterning a-Si:H. The performance gain has not been evenly distributed to the top and bottom junctions, leading to a photocurrent Kinase Inhibitor Library supplier mismatch high up to 2 mA/cm2. It is found that the incorporation of a ZnO intermediate layer between the junctions can increase the absorption and photocurrent of the top junction through light reflection from the a-Si:H/ZnO/μc-Si:H interfaces [13]. However, a too thick ZnO layer leads to rapidly degraded total photocurrent; therefore, its thickness has to be designed carefully.

According to our calculation, a ZnO layer with thickness of 18 nm is an optimal design for realizing the best photocurrent match without degrading J tot noticeably. EQE spectra of a-Si:H and μc-Si:H junctions incorporating Urease the intermediate ZnO layer are given in Figure  3b. Comparing to Figure  3a, it can be seen that for wavelength between 500 and 700 nm, the EQEa-Si:H has been increased for a higher J aSi. Since less light is coupled into μc-Si:H layer, J μcSi is slightly lowered for better current match. By integrating 2D nanopattern and ZnO intermediate designs into the a-Si:H/μc-Si:H tandem TFSCs, J sc can be up to 12.83 mA/cm2 under an unpolarized solar illumination, which has been enhanced by 35.34% compared to the planar system (i.e., increases by 3.35 mA/cm2 from 9.48 mA/cm2). Finally, based on the previously calculated J sc and the dark current densities in top and bottom junctions under continuously increasing forward electric biases (V), the current–voltage characteristics of the proposed a-Si:H/μc-Si tandem TFSCs obtained are explored and illustrated in Figure  4. For an accurate prediction of the electrical performance, series and shunt resistances (R s and R sh) of the solar devices have been taken into account.

2004). Consequently, recent studies have trying to understand what are the possible adaptation concepts and technologies of biological UV dosimetry, when developed for

applications under climates like space and Mars surface. In this context, characteristics as a high resistance of bacterial spores to extreme conditions under extraterrestrial environments are required (Nicholson et al. 2000). A biosensor GSK2118436 based in the spore inactivation doses (SID) of Bacillus subtilis strain TKJ6312 has been applied in the monitoring of the UV and the results compared with UV data obtained by Brewer Spectrophotometers at the INPE’s Southern Space Observatory (SSO, 29.4° S, 53.8° W), South of Brazil. Due to the deficiency in both DNA repair mechanisms, Nucleotide Excision Repair (NER) and Spore Photoproduct Lyase (SP lyase), this strain is sensible to UVR and maintain the resistant for others environment conditions (Munakata

et al. 2000). The biological dosimetry fulfills the criterions established by BIODOS project from the European Commission to be applied as UV-biosensor including its simplicity, facility of use and transport, long term storage and action spectrum with a good resolution (Schuch et. al. 2006). The high correlation index around 0.9 of the continuous monthly exposition of the biosensor, which began in 2000 at the SSO, when compared with Brewer’s UV measurements, demonstrates its application check details Etofibrate for long-term monitoring of the UV biologically-effective solar radiation. Furthermore, spore’s data analyses from other sites around the world agree with the UV seasonal variation data cited by the literature in terms of different and adverse environmental conditions from equatorial to higher latitudes sites (Munakata et. al. 2006). Considering the expectations of international exobiology groups to study the spatial solar radiation under different planetary environments using biological

systems the application of the Bacillus subtilis TKJ 6312 seems to be a very nice biosensor tool. Munakata, N., Kazadzis, S., Bais, A. F., Hieda, K., Rontó, G., Rettberg, P., and selleck chemicals Horneck, G. (2000). Comparisons of spore dosimetry and spectral photometry of solar UV radiation at four sites in Japan and Europe. Photochemistry and Photobiology, 72: 739–745. Munakata, N., Cornain, S., Kanoko, M., Mulyadi, K., Lestari, S., Wirohadidjojo, W., Bolsee, D., Kazadzis, S., Schuch, N. J., Casiccia, C., Kaneko, M., Liu, C. M., Jimbow, K., Saida, T., Nishigori, C., Ogata, K., Nonaka, S., Hieda, K., and Ichihashi, M. (2006). Biological monitoring of solar-UV radiation at 17 sites in Asia, Europe and South America from 1999 to 2004. Photochemistry and Photobiology, 82: 689–694. Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J., and Setlow, P. (2000).

The procedure of experiment is composed of the steps of spin coating, preexposure baking, exposing, post-exposure baking, developing, and hard baking in sequence. The obtained nanostructures are measured, characterized, and analyzed with an atomic force microscopy (AFM, Veeco Dimension 3100 AFM system, Veeco Instruments Inc., Plainview, NY, USA). To obtain the LY333531 nanopatterns with high precision and consistency, the focal sphere

should be accurately focused onto the surface of the photoresist. Furthermore, the motion of the scanning stage is required to be synchronized with laser exposure for fast fabricating nanopatterns. Results and discussion Experimental results Figure  2 is a typical image of a nanopillar array fabricated in the experiments. The top surface pattern of the overall topography is displayed

as Figure  2a. The scan range is about 10 μm × 10 μm. Each nanopillar Ipatasertib nmr is located in a circular pit whose external diameter is around 950 nm. The average diameter of the nanopillar is 65 nm, which is much smaller than the size of Abbe’s limit. Figure  2b is an AFM 3D image of the nanopillar array. Figure  2c represents the cross-sectional topography along the dark line which is shown in Figure  2a, and it illustrates the flatness of the coating surface. Figure 2d, e shows more details about the typical nanopillar in the array. Figure  2d is the top view of the nanopillar which is marked by selleck compound the arrow in the nanopillar array of Figure  2a. A dark line in Figure  2d acts as the symmetry axis of the pattern. It passes through RVX-208 the apex of the nanopillar, and its corresponding cross-sectional image is illustrated in Figure  2e. With careful calibration and analysis, it is found that the diameter of the pillar is around 48 nm, which is about λ/11, much smaller than the diffraction limit

λ/2, where λ is the incident laser wavelength at about 532 nm. Figure  2 demonstrates that the nanopillar array can be manufactured to sub-diffraction limit size with our donut-shaped CW visible laser system. Figure 2 Typical image of a nanopillar array fabricated in the experiments. (a) AFM image of nanopillar array fabricated with 532-nm CW laser and (b) its corresponding 3D image. (c) Roughness of coating along the dark line in (a). (d) Enlargement of one unit and (e) its cross section marked in (a). Figure  3 shows the typical nanopillars fabricated in our experiments. The AFM images of Figure  3a, b, c show the three different nanopillars which are fabricated with the same laser power. Figure  3d,e,f is the corresponding cross-sectional information along the black lines in Figure  3a, b, c, respectively. These black lines are drawn as symmetry axis of the patterns in Figure  3a, b, c.

1 M buy AZD5582 phosphate buffer pH 7.0 (PB). The pellet was resuspended in 2 ml PB with addition of 100 μg/ml lysozyme and 1 mM EDTA pH 8.0 and incubated at room temperature for 10 minutes. Cells were disintegrated using a French Press and centrifuged as above to remove unbroken cells. The low-speed centrifugation supernatant was then centrifuged at 30,000 × g for 30 High Content Screening minutes at 4°C to separate the cytoplasm (supernatant) and the membrane fraction (pellet). The pellet was resuspended in 1 ml of PB. Protein concentrations were determined and 25 μg of total

proteins was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Bands of interest were excised from the gel and the corresponding proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of the peptide generated by in-gel trypsin digestion ([35]; performed by CEINGE, University of Naples, Italy http://​www.​ceinge.​unina.​it/​). Measurement of gene expression by Real Time-PCR Gene expression determination was performed using Real Time-PCR as previously described [29]. RNA was extracted from bacterial cultures grown as for membrane protein extraction. Production

of cDNAs was obtained by reverse transcription using 1 μg total RNA, along with negative control samples incubated without reverse transcriptase. Primer sequences for genes of interest were designed based on the available genome sequences for A. baumannii and were tested in PCR experiments on A. baumannii SMAL genomic DNA to verify the 4EGI-1 nmr presence of the gene and the correctness of the expected products. Primer sequences were as follows: fchR_for: 5′-ACGTCAAGCGGTTGCTCCAT-3′, fchR_rev: 5′-CCTGTAATCGGGTCTGTTGG-3′, tonB_for: 5′-ATGGCAAGATACCGATGCCC-3′, tonB_rev: Gemcitabine clinical trial 5′-CCGATATCTTCGCTTGAGCG-3′, csuC_for: 5′-GCCCGCCTGTAGCCAAAATT-3′, csuC_rev: 5′-GAAGCATCTTGCTCGTTGCC-3′, csuE_for: 5′-TAGCGGGCCTGATGGCAATT-3′, csuE_rev: 5′-ACCCAGGGCTCTCAAAGAAG-3′, 16S_for: 5′-TGTCGTCAGCTCGTGTCGTGA-3′, 16S_rev: 5′-TGATGACTTGACGTCGTCCCC-3′.

Each Real Time PCR experiment was performed in triplicate and included negative control samples, which never showed significant threshold cycles. The relative transcript amounts were determined using 16S rRNA as the reference gene ([CtGene of interest-Ct16S] = ΔCt value). The results are the average of at least three independent experiments showing standard deviations ≤10%. Other methods Resistance to desiccation was performed as described in [29]. Sensitivity to oxidative stress was determined by treatment with hydrogen peroxide (H2O2), as described previously [50]. Transmission electron microscopy analysis was performed as described [51]. Acknowledgements We would like to thank M. Spalla for her excellent technical collaboration and L. Dolzani for providing A. baumannii strains RUH134 and RUH875.