Aerial hyphae scant, short, erect, loosely disposed, simple, becoming fertile. Autolytic activity absent or inconspicuous. No coilings noted. No diffusing pigment seen; odour indistinct or slightly mushroomy.

Chlamydospores rare. Conidiation noted after 4–6 days on scant short solitary conidiophores with minute wet conidial heads 10–40(–50) μm diam, and mostly dry in shrubs LY2109761 chemical structure becoming visible as white floccules, growing to circular or oblong pustules 1–2.5 mm diam, confluent to 5–7 mm length, spreading across the plate; after 6–11 days turning light green, 27DE4–6, 28CE5–7(–8), often with white margin; pustule surface appearing granular due to condensed whorls of phialides. Conidiation sometimes also within the agar in aged cultures. selleck screening library Conidiophores (after 10–12 days) usually on short stipes with mostly asymmetrical branching,

with two to several primary branches often dichotomously branched at several levels. Stipe and primary branches 6–10 μm wide, thick-walled (to 1.5 μm), with coarsely wavy outer wall; further branches thin-walled and 2.5–5 μm wide; origin of phialides often thickened, sometimes globose, to 7 μm wide. Branches often curved or sinuous. Peripheral conidiophores short (30–100 μm), variable, either with long sterile stretches and short irregular terminal heads, or regularly symmetrical with densely arranged, paired, 1–2 celled branches at right angles or slightly inclined upwards; often branches of similar length on all levels. Production of conidia starting within the pustule. Phialides solitary along terminal branches in short intervals and in whorls of 3–5(–6). Phialides (4–)5–10(–20) × (2.8–)3.0–4.0(–4.8)

μm, l/w 1.3–3.0(–6.3), (1.5–)2.3–3.2(–4.0) μm wide at the base (n = 70), variable, ampulliform or lageniform, with short necks, widest mostly below the middle; straight or curved upwards and inequilateral, sometimes sigmoid, typically narrowly lageniform on younger Gefitinib supplier and more simple conidiophores; terminal phialides in extension of main axes often appearing longer, but separated from the origin of the whorl by an additional cell. Conidia (2.5–)3.0–5.0(–6.8) × (2.0–)2.5–3.0(–3.7) μm, l/w (1.1–)1.2–1.6(–2.0) (n = 80), pale greenish, variable, ellipsoidal or subglobose, sometimes oblong, smooth, with 1–2 guttules; scar indistinct or narrowly projecting; aggregating in chains in age. At 15°C conidiation abundant in large green, 27–28CD4–7 to 27E4–8, pustules aggregating to 10 mm length. At 30°C either hyphae dying after a few days or colony dense, downy, with growth slowing down after 1 weeks; autolytic activity conspicuous, excretions yellow; conidiation effuse, colourless. On PDA after 72 h 5–8 mm at 15°C, 8–9 mm at 25°C, 1–3 mm at 30°C. Growth limited, typically stopping before covering the plate.

6 ± 2.6, 20.7 ± 2.5, 21.6 ± 2.7 min for raisin, chews and water respectively). While RPE was not different, HR was higher for both CHO treatments compared Pexidartinib manufacturer to water only during the 5-km TT. Figure 5 Time of completion and average rate of

perceived exertion (RPE) and heart rate (HR) (value/10) during the 5-km time trial. Values are means ± SD for 11 men. *, significantly different from water (p ≤ 0.05). Questionnaires There were no differences due to treatment in the whole body soreness and fatigue questionnaires (Table 4), but all values increased over pre-exercise and remained higher 5-hr post-exercise. GI disturbance was very low for all categories (Figure 6). Values were averaged over the entire exercise trial including both sub-maximal exercise and the time trial. GI disturbance was in the mild range for all treatments. Belching was higher with both CHO treatments compared to water only. Table 4 Data from Questionnaires Variable Pre-Exercise Post-Exercise   2-Hr Post   5-Hr Post   Whole Body Muscle Soreness

(out of 100 mm)  Water 15.4 ± 3.7 31.8 ± 5.2 + 34.5 ± 4.1 + Selleckchem GSK3 inhibitor 29.8 ± 3.7 +  Raisin 16.5 ± 4.2 35.3 ± 5.5 + 35.4 ± 5.2 + 34.0 ± 5.2 +  Chews 15.2 ± 3.8 37.4 ± 4.6 + 40.6 ± 4.9 + 40.6 ± 5.6 + Whole Body Fatigue (out of 100 mm)  Water 19.6 ± 4.8 50.4 ± 6.9 + 43.1 ± 4.2 + 42.9 ± 6.2 +  Raisin 23.7 ± 5.0 47.0 ± 6.2 + 43.2 ± 5.1 + 42.4 ± 3.9 +  Chews 21.4 ± 4.6 49.0 ± 6.9 + 43.6 ± 6.4 + 39.6 ± 7.1 + Values are means ± SD for 11 men. +, significantly different from pre-exercise. Figure 6 Gastrointestinal disturbance by category over the entire exercise bout on a scale from 0–6 with 1

being mild and 6 being unbearable. Values are means ± SD for 11 men. *, significantly different from CYTH4 water (p ≤ 0.05). Discussion Our results indicate that ingestion of a natural food product, raisins, had similar performance effects as a commercial sports product in chews and both products improved running time trial performance over water only. Raisins and chews maintained a higher % of non-protein macronutrient oxidation derived from CHO over the 80-min running bout at 75% VO2max than water only. The commercial product did cause slightly higher insulin levels and CHO oxidation rates during exercise than raisins. Raisins had a greater increase in creatine kinase during exercise than both chews and water only. Our data suggests that consuming a natural, relatively fiber-rich CHO source (raisins) had similar GI effects as a commercial product. All treatments maintained blood glucose levels at pre-exercise values during the 80-min sub-maximal trials. However, the glucose levels during exercise were higher with the commercial product compared to water only. Similar glucose responses between carbohydrate forms is in agreement with a study examining the metabolic effects of raisins (glycemic index (GcI) = 62) versus sport gels (GcI = 88) in cyclists [10].

Biodivers Conserv 14:251–259CrossRef Lodé T, Cornier JP, Le Jacques D (2001) Decline in endangered species as an indication of anthropic pressures: the case of European mink Mustela lutreola

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(1980) Sexual dimorphism in the body size of mustelids (Carnivora): the roles of food habits and breeding systems. Oikos 34:147–158CrossRef Mortelliti A, Amori G, Boitani L (2010) The role of habitat quality in fragmented landscapes: a conceptual overview and prospectus for future research. Oecologia 163:535–547PubMedCrossRef Navarro C (1980) Contribución al estudio de la

flora y vegetación del Duranguesado y la Busturia. Universidad Complutense de Madrid, Master thesis O’Connell M, Wright JM, Farid A (1996) Development of PCR primers for nine polymorphic American mink Mustela vison microsatellite loci. Mol Ecol 5:311–312PubMed Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295CrossRef Peakall R, Ruibal M, Lindenmayer DB (2003) Spatial autocorrelation analysis offers new insights into gene flow in the Australian bush rat, Rattus fuscipes. Evolution 57:1182–1195PubMed MycoClean Mycoplasma Removal Kit Petts GE (1984) Impounded rivers: perspectives for ecological management. Wiley, Chichester Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMed Raymond M, Rousset F (1995) Genepop (version-1.2)—population-genetics software for exact tests and ecumenicism. J Heredity 86:248–249 Rollins LA, Woolnough AP, Wilton AN, Sinclair R, Sherwin WB (2009) Invasive species can’t cover their tracks: using microsatellites to assist management of starling (Sturnus vulgaris) populations in Western Australia.

Thus, while our results support the role of CsrA as a major regulator of pgaABCD expression, they also suggest that the current model for pgaABCD post-transcriptional regulation, which is based on data obtained in E. coli K-12, PS-341 molecular weight may not readily apply to E. coli C. The additive effect observed upon combining Δpnp 751 with deletions targeting different sRNAs suggest that PNPase and the sRNAs may act independently on pgaABCD regulation. Figure 5 pgaABCD expression in mutants defective for CsrA-dependent regulation elements and/or PNPase. See Table 1 for the complete

list of strains used in these experiments. A Δpnp ΔcsrA double mutant could not be obtained. A. pgaABCD mRNA expression. RNA was extracted from cultures grown in M9Glu/sup to OD600 = 0.8 and analyzed by quantitative RT-PCR as described in Methods. White bars, pnp + strains; dark grey, Δpnp strains. The “Relative expression” values indicated in the graph are the average of three independent experiments, each performed in duplicate, and standard deviations

are shown. The overall p-value obtained by ANOVA is indicated in the graph. Letters provide the representation for posthoc comparisons. According to posthoc analysis (Tukey’s HSD, p < 0.05), means sharing the same letter are not significantly different from each other. B. PNAG production. Crude extracts from overnight cultures were filtered onto RGFP966 chemical structure a nitrocellulose membrane, and PNAG detection was carried out using polyclonal PNAG specific antibodies as detailed in Materials and Methods. PNAG determination was repeated at least four times on three independent EPS extractions with comparable results; data shown are from a typical experiment. Discussion In this report, we have shown that PNPase negatively regulates the production

of the adhesion factor PNAG, thus maintaining the bacterial cells in a planktonic state (Figures 1 3) when grown at 37°C in supplemented minimal medium. Our results are in line with previous Neratinib in vitro works by other groups connecting PNPase to regulation of outer membrane proteins in E. coli[59] and curli production in Salmonella [60]. Thus, PNPase seems to play a pivotal role in regulating the composition of cell envelope and the production of adhesion surface determinants. PNPase-dependent regulation of PNAG production requires its ribonuclease activity, as suggested by the observation that overexpression of RNase II can compensate for lack of PNPase (Figure 1B). Cell aggregation in the absence of PNPase is suppressed by RNase II, but not by RNase R. This reminds what previously showed for cold sensitivity in pnp mutants, which is also solely suppressed by RNase II [61] and reinforces the notion that, albeit partially redundant, RNA degradation pathways possess a certain degree of specificity and are not fully interchangeable [62].

Bars indicate the standard error of the mean. Student’s t-test was performed. ns = not significant. Three independent experiments were performed. The expression of p21 (also known as Cip1 and WAF1) in response to genotoxic stress is tightly regulated by p53 (reviewed in [45]), and we therefore measured it as an additional indicator of p53 activity. The fraction of p21-positive cells was approximately doubled by selenite treatment (Figure 2F–J). Although these changes are statistically significant, the positive fraction Trametinib was very small even after selenite treatment. As a positive control, epithelioid cells were treated

with 2 μM doxorubicin and showed a 22% positive fraction (not shown). Cells of either phenotype treated with the p53 inhibitor Pifithrin did not show a decreased apoptosis frequency as judged by Annexin-PI (Figure 1), nor a smaller loss of δΦm (Table 2). This is particularly interesting since p53 inhibition decreased the baseline apoptosis in untreated cells (Figure

1, Additional file 1). Consequently, p53 was active in the control cells but was inactivated by selenite. Apoptosis was still induced by selenite, implicating p53-independent pathways in this process. To find the mechanism of inhibition, we considered the complex regulation of p53 activity. The central DNA-binding Selleck BIBW2992 core domain of p53 contains one zinc atom. Zinc chelators have been shown to cause accumulation of wild-type p53 in a structurally aberrant form with inhibited DNA-binding activity [46]. Selenium is a known chelator of zinc and when applied in vivo as selenite or Benzatropine its reduced form selenide, it forms nanocrystals

of zinc-selenium with free or loosely bound zinc [47]. Another possibility is that selenite as an oxidizing agent may act directly upon regulatory cysteines on the p53 molecule, leading to an accumulation of oxidized p53 incapable of DNA-binding [48]. Also, secondary mediated redox regulation needs to be considered. The multifunctional protein Redox Effector Factor 1 (Ref-1) is involved in the redox regulation of stress inducible transcription factors such as Activating Protein-1, Nuclear Factor-κB, Hypoxia Inducible Factor-1 and p53, and may play an important role in this system. Ref-1 depends on thioredoxin (Trx) to maintain its active reduced state [49–51]. In a yeast experimental system, it has been shown that deletion of thioredoxin reductase (TrxR) downregulates p53 activity by keeping it in its oxidized form [52, 53]. Trx overexpression on the other hand has been shown to increase p53 transactivation of reporter genes in human cell lines [49]. Protein levels of Trx were reduced by selenite treatment in sarcomatoid cells, from 175 ng/mg to 100 ng/mg. The epithelioid cells had a baseline expression of 57 ng/mg, decreasing slightly to 52 ng/mg after selenite treatment (Figure 3).

5 16.8 VGII 34.4 17.9 −16.5 non-VGIII 40.0 13.8 −26.2 non-VGIV VGII B7466 VGIIc 30.8 20.8 −10.0 non-VGI 22.4 33.6 11.2 VGII 37.4 23.7 −13.7 non-VGIII 40.0 19.5 −20.5 non-VGIV VGII B7491 VGIIc 26.9 17.3 −9.6 non-VGI 19.2 33.0 13.8 VGII 0.0 16.8 16.8 non-VGIII 40.0 16.7 −23.3 non-VGIV VGII B7493 VGIIc

27.1 17.4 −9.7 non-VGI 18.6 33.6 15.1 VGII 36.6 20.7 −15.8 non-VGIII 40.0 16.1 −23.9 non-VGIV find protocol VGII B7641 VGIIc 26.0 17.3 −8.7 non-VGI 18.7 32.3 13.7 VGII 34.3 20.0 −14.3 non-VGIII 40.0 15.6 −24.4 non-VGIV VGII B7737 VGIIc 28.0 18.5 −9.6 non-VGI 20.1 34.3 14.2 VGII 37.0 23.0 −14.0 non-VGIII 40.0 18.0 −22.0 non-VGIV VGII B7765 VGIIc 22.5 13.0 −9.5 non-VGI 14.5 34.1 19.6 VGII 33.1 23.4 −9.7 non-VGIII 40.0 12.9 −27.1 non-VGIV VGII B8210 VGIIc 27.8 18.1 −9.7 non-VGI 19.6 33.3 13.7 VGII 33.0 19.4 −13.5 non-VGIII 40.0 16.8 −23.2 non-VGIV VGII B8214 VGIIc 27.1 17.7 −9.5 non-VGI 19.8 34.9 15.1 VGII 34.1 20.1 −14.0 non-VGIII 40.0 16.1 −23.9 non-VGIV VGII B8510 VGIIc 26.8 17.6 −9.2 non-VGI 18.8 33.2 14.5 VGII 35.2 19.1 −16.1 non-VGIII 40.0 15.6 −24.4 non-VGIV VGII B8549 VGIIc 26.8 16.2 −10.6 non-VGI 18.7 33.5 14.8 VGII 37.4 20.5 −16.9

non-VGIII 40.0 29.6 −10.4 non-VGIV VGII B8552 VGIIc 27.1 17.0 −10.1 non-VGI 18.6 33.2 14.6 VGII 34.3 19.7 −14.6 non-VGIII 40.0 16.6 −23.4 non-VGIV VGII B8571 VGIIc 28.8 19.4 −9.4 non-VGI 21.5 33.4 11.9 VGII 34.5 22.8 −11.8 non-VGIII 40.0 19.5 −20.5 non-VGIV VGII B8788 VGIIc 26.0 16.0 −10.0 non-VGI 18.5 29.5 11.0 VGII 38.0 20.4 −17.6 non-VGIII 40.0

16.6 selleck inhibitor −23.4 non-VGIV VGII B8798 VGIIc 36.0 24.7 −11.4 non-VGI 26.5 33.3 6.8 VGII 37.2 19.2 −18.0 non-VGIII 40.0 22.5 −17.5 non-VGIV VGII B8821 VGIIc 30.5 20.5 −10.0 non-VGI 22.3 33.0 10.7 VGII 37.0 29.0 −8.0 non-VGIII 40.0 18.7 −21.3 non-VGIV VGII B8825 VGIIc 27.4 17.8 −9.6 non-VGI 19.6 33.7 14.1 VGII 36.0 20.5 −15.5 non-VGIII 40.0 17.5 −22.5 non-VGIV VGII B8833 VGIIc 29.2 20.7 −8.6 non-VGI 19.5 33.4 13.9 VGII 35.4 19.6 −15.8 non-VGIII 40.0 15.5 −24.5 non-VGIV VGII B8838 VGIIc 29.2 19.1 −10.1 non-VGI 21.5 32.8 11.3 VGII 32.9 22.3 −10.6 non-VGIII 40.0 18.5 −21.5 non-VGIV VGII B8843 VGIIc 29.5 19.4 −10.1 non-VGI 21.5 33.7 12.2 VGII 37.5 22.1 −15.4 non-VGIII 40.0 19.1 −20.9 non-VGIV VGII B8853 VGIIc 33.3 23.1 −10.2 non-VGI 24.8 33.7 8.9 VGII 34.2 27.8 −6.4 non-VGIII 40.0 21.5 −18.5 non-VGIV VGII B9159 VGIIc 29.6 17.5 −12.1 Methocarbamol non-VGI 19.1 29.9 10.7 VGII 40.0 26.0 −14.0 non-VGIII 40.0 18.0 −22.0 non-VGIV VGII B9227 VGIIc 24.4 15.3 −9.1 non-VGI 15.5 28.1 12.6 VGII 27.9 16.1 −11.9 non-VGIII 31.0 16.3 −14.7 non-VGIV VGII B9235 VGIIc 24.6 15.1 −9.5 non-VGI 15.3 28.9 13.7 VGII 29.2 16.4 −12.7 non-VGIII 31.2 15.9 −15.3 non-VGIV VGII B9244 VGIIc 27.3 18.4 −8.9 non-VGI 18.5 31.8 13.3 VGII 28.2 21.0 −7.2 non-VGIII 30.6 18.8 −11.8 non-VGIV VGII B9245 VGIIc 26.8 17.9 −8.9 non-VGI 18.0 33.5 15.5 VGII 31.2 19.3 −11.9 non-VGIII 34.2 18.5 −15.6 non-VGIV VGII B9295 VGIIc 28.6 19.5 −9.1 non-VGI 19.9 40.0 20.1 VGII 33.6 25.5 −8.1 non-VGIII 34.4 20.3 −14.2 non-VGIV VGII B9302 VGIIc 24.6 14.1 −10.5 non-VGI 16.

Moreover a clear separation between above-ground (stem and leaves) and below-ground environments (soil and nodules) was detected. An analysis of the clone libraries, prepared from above-ground and below-ground pooled samples, revealed an uneven distribution of bacterial classes, with a marked pattern highlighting the class of Alphaproteobacteria as the more abundant in plant tissues (this class represented

half of the clones in the stem + leaf library). The same uneven pattern PD 332991 was then observed, at lower taxonomic ranks, within the Alphaproteobacteria, with sequences of clones belonging to members of the Methylobacteriaceae and Sphingomonadaceae families being more abundant in stem than in soil and nodules. Methylobacteria and Sphingomonadaceae have been found as endophytes in a number of plants [8, 12, 31, 33, 42–45] and it is believed that this group of bacteria may take advantage from living as plant-associated, thanks to its ability to utilize the one-carbon alcohol methanol discharged by wall-associated pectin metabolism of growing plant cells. Concerning root nodule bacterial communities, obtained

data indicated that very diverse see more bacterial taxa are associated with nodules, the most represented being the specific rhizobial host of M. sativa, the alphaproteobacterium S. meliloti. However, additional taxa have been found, including members of Actinobacteria Flavobacteria Gammaproteobacteria and Betaproteobacteria, which may have some additional plant growth-promoting activities (see for

instance [46, 47]). In soil, Amrubicin Acidobacteria was one of the most important divisions (in terms of number of clones in the library) and was present exclusively in the soil clone library, in agreement with many previous observations [48, 49]. A relatively high presence of Archaea (Thermoprotei) was also found. Checking the 16 S rRNA gene sequences present in the Ribosomal Database for 799f/pHr primer annealing, we found that PCR amplification from Thermoprotei was theoretically possible with this primer pair (data not shown). The presence of Archaea in the soil is not unexpected [50] and could be linked also to the anoxic or nearly anoxic conditions present in the bottom of the pot. However, since the low coverage of soil clone library, the presence of many other additional taxa, as well of different proportions of those found here cannot be excluded. In addition, it should be mentioned that differences between soil and plant-tissues bacterial communities could also be ascribed to the different DNA extraction protocols we were obliged to use, since a unique protocol (bead-beading protocol for both soil DNA and plant DNA) failed in a successful extraction of DNA from both soil and plant tissues (data not shown). A similar technical need was encountered by other authors also [33], which renders the study of the relationships between plant-associated and soil bacterial communities still at its beginning.

Amino-acids highlighted in grey indicate variations when compared to the nisin A (the first nisin variation

to be discovered) references. The complete amino-acid sequencesfrom the 9 wild strains have been deposited in GenBank (accession numbers KF146295 to KF146303, respectively). Table 3 shows the inhibitory activity of the nis positive Lactococcus isolates against several microbial targets. It can be observed that the isolates presented inhibitory activity mainly against the tested Gram positive bacteria, and lower frequencies of inhibition against Gram negative bacteria. These results indicate that the bacteriocins produced by the tested LAB isolates have interesting Alvelestat clinical trial antimicrobial activities, highlighting the relevance of raw goat milk as a source of bacteriocinogenic

strains [23]. In addition, the obtained results indicate that the PF-02341066 ic50 variations in nisin structure predicted in the present study (Figure 3) did not affect the antimicrobial activity of the isolates. Considering the main characteristics of bacteriocins, the inhibitory activity against the tested Gram negative bacteria must be due to non-specific antimicrobial substances produced by the LAB strains, such as organic acids or peroxide [24, 34]. Table 3 Inhibitory activity (diameters of inhibition halos, mm) of nis positive Lactococcus isolates obtained from raw goat milk against target microorganisms, identified by the spot-on-the-lawn methodology Target genus Species/serotype Origin* nispositive isolates       GLc04 GLc05 GLc08 GLc14 GLc18 GLc19 GLc20 GLc21 GLc03 Lactobacillus L. sakei ATCC 15521 11 13 9 9 5 11 0 0 5 Lactococcus L. lactis subsp. lactis ATCC 7962 11 9 8 7 0 7 0 0 0   L. lactis subsp. lactis

GLc18, wild strain, present study 13 11 11 11 0 12 0 0 7   L. lactis subsp. lactis GLc22, wild EGFR inhibitor strain, present study 13 11 11 7 7 10 7 7 7 Listeria L. monocytogenes ATCC 7644 11 11 11 9 15 13 7 7 9   L. monocytogenes ATCC 15313 9 9 7 7 0 7 7 5 10   L. monocytogenes 60, wild strain, beef origin 15 14 12 9 7 13 5 5 5   L. inoccua 76, wild strain, beef origin 5 5 5 5 5 5 5 5 9 Staphylococcus S. aureus ATCC 12598 9 7 7 7 7 5 7 7 7   S. aureus ATCC 14458 9 7 7 7 7 9 11 7 7   S. aureus ATCC 29213 8 7 7 7 7 7 9 0 7   S. aureus 27AF1, wild strain, cheese origin 9 9 9 7 5 11 7 0 9   S. aureus 27ST1, wild strain, cheese origin 9 9 9 7 5 7 11 7 9   S. aureus 26BP6, wild strain, cheese origin 13 13 14 7 7 13 7 0 7 Escherichia E. coli ATCC 11229 0 0 0 0 0 0 0 0 0   E. coli ATCC 00171 0 0 0 0 0 0 0 0 0 Pseudomonas P. aeruginosa ATCC 27853 5 5 5 5 0 0 5 0 0   P. fluorescens ATCC 10038 5 5 5 0 0 0 0 0 0 Salmonella S. Typhimurium ATCC 14028 7 7 5 5 0 0 0 0 0   S. Cholerasuis 38, wild strain, beef origin 0 0 0 0 0 0 0 0 0   S. Enteritidis 258, wild strain, poultry origin 7 7 7 5 5 5 5 5 0   S.

In this study we described six NDM-4-producing PS-341 E.coli isolates obtained from two patients admitted to an Italian hospital. We also present data on the localization and the genetic environment of the bla NDM-4 gene. Methods Bacterial strains Six E.coli isolated from urine samples of two inpatients at the San Martino-IST University Hospital (Genoa, Italy)

were studied. Isolates were taken as part of standard patient care and informed consent for the use of clinical data has been obtained by both patients. Strain identification, antibiotic susceptibility testing and phenotypic screening for MBL production Routine identification and antibiotic susceptibility testing were carried out using the Vitek-2 automated system (BioMérieux, Marcy-L’etoile, France). In vitro activity of carbapenems, aztreonam, fosfomycin and nitrofurantoin was further determined by the broth microdilution method and interpreted according to the of European Committee on Antimicrobial Susceptibility Testing (EUCAST ) guidelines (Version 4.0, 2014) [6]. To detect metallo-β-lactamase (MBL) production,

a synergy test using imipenem and EDTA discs was used [7]. Pulsed-field gel electrophoresis (PFGE) Genomic DNA was prepared, digested with XbaI (New England Biolabs Inc., MA, USA) and subjected to PFGE with the CHEF DRII device (Bio-rad, Milan, Italy), as described previously [8]. Fingerprinting pattern was interpreted Silmitasertib according to the method of Tenover et al. [9]. Multilocus sequence typing (MLST) MLST was carried out using protocols and conditions described on the E.coli MLST website (http://​mlst.​warwick.​ac.​uk/​mlst/​dbs/​Ecoli/​documents/​primersColi_​html).

Sequence types were assigned using the website interface. Molecular analysis techniques Polymerase chain reaction (PCR) amplification of the bla NDM gene and direct sequencing of the PCR products was performed as previously described [10]. Screening for resistance genes was carried out using primers and conditions previously described [11–13]. Phylogenetic analysis using multiplex PCR method as described previously [14] was used. PCR experiments were performed to identify the upstream- and downstream-located regions of the bla NDM-4 gene [15]. Mapping of the variable region of class 1 integron was performed by PCR as described previously [16]. The genetic environment of bla NDM-4 was studied by PCR mapping and sequencing Carnitine palmitoyltransferase II as described previously [13]. Conjugation assay and plasmid study Plasmid transfer was attempted by conjugation, using E.coli J53 as the recipient, as described previously [17]. Plasmid DNA, isolated from E.coli, was obtained by the alkaline lysis method and was used as a template in PCR analysis with primers that are specific for bla NDM and bla CTX-M[18]. To rule out chromosomal DNA contamination the template was used to amplify an internal fragment of the house-keeping recA gene. A PCR-based replicon typing method was used to identify the incompatibility group [19].

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