“The impact of four electron acceptors on hydrocarbon-indu

“The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons

may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or Selleckchem ACP-196 1-13C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogensis. The rates of methanogenesis were negatively affected by Palbociclib nmr sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy.

ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: ‘methyl coenzyme A’ changed to ‘methyl coenzyme M’ in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings. Roughly, one third of oil in reservoirs remains inaccessible (US Department of Energy, 2006). Since Zengler et al. (1999) reported the conversion of hexadecane to methane, it has been suggested that remaining energy can be recovered as methane gas (Anderson & Lovley, 2000; Head et al., 2003). Moreover, the conversion of hydrocarbons to carbon

dioxide (CO2) or methane represents a useful tool for Fludarabine bioremediation of oil-impacted ecosystems. The overall reaction kinetics of hydrocarbon biodegradation are controlled by the initial attack on hydrocarbons, where hydrocarbon biodegradation with oxygen as an electron acceptor is the energetically most favorable process. However, microbial methanogenesis usually requires anoxic conditions and methanogenesis, including the conversion of hexadecane to methane, is a slow process (Zengler et al., 1999; Feisthauer et al., 2010). The initial anaerobic activation of hexadecane may be irreversible and the removal of reaction products is unlikely to accelerate the initial steps or the overall degradation (Cravo-Laureau et al., 2005; Callaghan et al., 2006). However, β-oxidation and the release of electrons are essential steps in hydrocarbon biodegradation pathways (Fig. 1; Kniemeyer et al., 2003; Rabus, 2005; Callaghan et al., 2006).

In these experiments, the orientation discrimination task was sim

In these experiments, the orientation discrimination task was simply performed on the second stimulus without reference to the first one. This was done either by randomizing

the orientations of the composing lines in the first stimulus (Experiments III and IV; Figs 3A and 4A), or by entirely removing the first stimulus (Experiment V; Fig. 5A). The detailed designs of each of these experiments were as follows. In Experiment Ulixertinib manufacturer III, even though the first stimulus composed of randomly oriented lines was irrelevant to the orientation discrimination task performed on the second stimulus, the subjects were required to perform an additional luminance task on the first stimulus to ensure that some voluntary attention was still allocated to it. The luminance of randomly oriented lines in the first stimulus of a trial was either identical (0.36 cd/m2) or randomly interleaved (0.61 or 0.11 cd/m2; Fig. 3A). The subjects performed a dual task: first, to report whether the random lines in the first stimulus were iso-luminant, and then to judge whether the second stimulus (composed of iso-oriented and iso-luminant lines) was tilted clockwise or counterclockwise relative to its mean orientation (55° or 140°, i.e. the standard orientation used in Experiments I and II), which can be established internally by the

subjects within a block of trials (Vogels & Orban, 1985; Shiu & Pashler, 1992). This manipulation contrasts with Experiments IV NVP-AUY922 and V, in which less or no spatial attention was required to allocate to the first stimulus: in Experiment IV, where the first stimulus consisted of randomly oriented and iso-luminant lines (Fig. 4A), and in Experiment V, where the first stimulus was not displayed but the gaze shift was still required (Fig. 5A), the subjects were simply instructed

to perform the orientation discrimination task on the second stimulus around its mean orientation. The subjects were trained for 6 days, with eight blocks of trials (a total of 300–400 trials) per day. Auditory feedback was given on error responses. The trained (i.e. standard) orientation in all experiments was 55°, whereas both 55° and 140° orientations were examined in the post-training test for learning specificity. In Experiment I, two groups of N-acetylglucosamine-1-phosphate transferase subjects were trained in the congruent and incongruent conditions, respectively; in the other experiments, all subjects were trained in the congruent condition. To counterbalance eye movement training, every four blocks of training trials were interleaved with a block of 50 saccade-balanced trials. In these trials, no stimulus was displayed; the subjects made a gaze shift opposite to that in the training blocks, and performed an irrelevant task to discriminate a slight change in luminance of the shifted FP (brighter or dimmer than the initial FP in the screen center).

, CH

, Trichostatin A in vivo 2009). From this analysis, we found three additional sophorolipid-producing species of the Starmerella clade, one of which is a novel species, and have determined that two forms of sophorolipids are selectively synthesized by different species within the clade. The strains examined in this study were obtained from the ARS Culture Collection (NRRL), National Center for Agricultural Utilization Research, Peoria, IL, and maintained on YM agar (3 g L−1 yeast extract, 3 g L−1 malt extract, 5 g L−1 peptone, 10 g L−1 glucose and 20 g L−1 agar, in distilled water). The medium used for production of sophorolipids was termed SL

medium and composed of 100 g L−1 glucose, 87.5 g L−1 (100 mL L−1) oleic acid (Aldrich, technical grade), 1.5 g L−1 yeast extract, 4 g L−1 NH4Cl, 1 g L−1 KH2PO4·H2O, 0.1 g L−1 NaCl and 0.5 g L−1 MgSO4·7H2O, in distilled water. The initial Neratinib mw pH was adjusted to 4.5 with 6 N KOH. Unless specified otherwise, cultures were grown at 25 °C in 50-mL Erlenmeyer flasks with 10 mL of SL medium and shaken at 200 r.p.m. in an Innova 4335 shaker incubator. Incubation times were either 96 or 168 h and the time is given with

each reported experiment. The pH of the flask cultures was adjusted to 3.5 twice daily by the addition of 1 N NaOH. The 10 mL of spent SL medium from each shake flask was acidified with 0.4 mL 6 N HCl and extracted twice with 40 mL of ethyl acetate to remove sophorolipids and unmetabolized oleic acid. The ethyl acetate extract was reduced to dryness in a rotoevaporator,

redissolved in 2 mL chloroform, transferred to a glass vial and reduced to dryness under a nitrogen gas stream. Oleic acid was separated from sophorolipids in the mixture by three separate 3 mL hexane extractions. The hexane was evaporated and the concentration of oleic acid was quantified by weight, which was confirmed by gas–liquid chromatography (Price et al., Cobimetinib molecular weight 2009). The residue that remained after hexane extraction was the sophorolipid fraction and the amount was determined by weight following confirmation of the presence of sophorolipids by MALDI-TOF MS as described below. Yields of sophorolipids and consumption of oleic acid are reported as averages and were determined from duplicate cultures, which varied no more than 11%. MALDI-TOF MS screening was accomplished using a Bruker Omniflex instrument in reflectron mode with positive ion detection. The samples were dissolved in ethyl acetate and the matrix used was 2,5-dihydroxybenzoic acid. The instrument conditions were used as described previously (Price et al., 2009). Determinations were performed in duplicate. The methods used for DNA isolation, purification and sequencing were reported earlier (Kurtzman & Robnett, 1998). DNA characterization was initiated by PCR amplification of the D1/D2 domain of the LSU rRNA gene followed by sequencing reactions using the ABI Big Dye Terminator v3.0 Cycle Sequencing Kit.

Patients with a connective tissue disease (CTD) who met the modif

Patients with a connective tissue disease (CTD) who met the modified definition of Angiogenesis inhibitor the WHO group I pulmonary arterial hypertension (PAH) were enrolled. PAH was defined as a systolic pulmonary arterial pressure > 40 mmHg

by echocardiography or mean pulmonary arterial pressure > 25 mmHg by right heart catheterization. Hemodynamic parameters and clinical data such as demographics, functional class, underlying disease, organ involvement, laboratory tests and current treatment were recorded. A total of 321 patients were enrolled during the 2-year study period from 2008 to 2010. The mean age of the patients at registration was 51.9 years and 87.5% were female. Most patients were diagnosed by echocardiography and only 24 patients (7.5%) underwent cardiac catheterization. Exertional dyspnea was present in 63.6% of patients and 31.8% were New York Heart Association class III or IV. Among the patients,

systemic lupus erythematosus accounted for 35.3%, systemic sclerosis 28.3%, rheumatoid arthritis 7.8%, overlap syndrome 9.0%, and mixed connective tissue disease 5.9%. There were no significant differences in hemodynamics, functional class, diffusing capacity and N-terminal pro-brain natriuretic peptide levels between the disease subgroups. Treatments consisted of calcium antagonists (57.0%), endothelin antagonists (32.7%), prostanoids (27.1%), phosphodiesterase-5 inhibitors (14.3%) and combinations (37.4%). ABT-263 research buy Compared with previous studies, the results showed some differences: underlying diseases, functional status and treatments. This may be due to differences in ethnic background and Farnesyltransferase diagnostic methods of our study. “
“Knee joint replacement is an effective and cost-effective intervention for severe symptomatic osteoarthritis of the knee joint. However, utilisation rates vary hugely, there are no indications, it is difficult to know when (in the course of arthritis) it is best to operate, and some 10–20% of people who have this surgery are unhappy with the outcome, and have persistent pain. In this article

we briefly discuss the variations in utilization of knee joint replacement, and then outline four different approaches to the selection and prioritisation of patients for this procedure. Consensus criteria, including appropriateness criteria are available, but if produced by professionals alone, they may conflict with the views of patients and the public. Databases and cohort studies can be used to attempt relating outcomes to baseline characteristics, but at present we can only account for a small percentage of the variance with this technique. Finally, we propose use of the ‘capacity to benefit framework’ to attempt providing guidance to both patients and healthcare professionals. “
“Psoriatic arthritis is an inflammatory rheumatic disorder of unknown etiology occurring in patients with psoriasis.

These glycoproteins, which include Flo1p, Flo5p, Flo9p, Flo10p an

These glycoproteins, which include Flo1p, Flo5p, Flo9p, Flo10p and Flo11p, are termed flocculins or adhesins (reviewed in Verstrepen & Klis, 2006; Dranginis et al., 2007; Bauer et al., 2010). On the basis of their sensitivity to sugar inhibition, three distinct flocculation phenotypes have been characterized, which include Flo1-type

[mannose-sensitive (MS)], NewFlo-type [glucose- and mannose-sensitive (GMS)] and a mannose-insensitive (MI)-type (Masy et al., 1992). Both MS- and GMS-types are Ca2+-dependent flocculation phenotypes that can be attributed to FLO1-, FLO5- and FLO9-overexpression in Saccharomyces cerevisiae strains (Guo et al., 2000; Liu et al., 2007; Govender et al., 2008, 2010; Van Mulders et al., 2009). It should also be noted that Flo11p is required for strong Flo1-type flocculation

in Saccharomyces diastaticus strains (Bayly et al., 2005). In contrast, the MI phenotype displays Ca2+-independent flocculation and is yet to beta-catenin signaling be ascribed to a particular FLO gene. To meet the demands of a consumer-driven market, wine processing currently involves fining and clarification procedures to produce clear and physicochemical stable wines. Wine fining entails the purposeful addition of an adsorptive compound (bentonite, gelatin or albumin), followed by the settling or precipitation (cold stabilization) of partially soluble components from the wine. Further clarification is usually achieved by sedimentation, racking, centrifugation and filtration (Boulton et al., 1996; Rucaparib supplier Ribéreau-Gayon et al., 2000; Pretorius & Bauer, 2002). Moreover, studies have shown that filtration alters the aroma and colour of the wine and also removes molecules that would otherwise positively contribute to the impression of body and volume on the palate (Lubbers et al., 1994; Boulton et al., 1996; Moreno & Azpilicueta, 2004; Moreno et al., 2007). Thus, it may be concluded that the fining and clarification of wine are expensive and time-consuming procedures that ultimately negatively impact on

the cost of the finished product. Efficient wine yeast flocculation after primary alcoholic fermentation leads to the formation of compacted sediments (Lahtchev & Pesheva, 1991) or ‘caked’ lees, thereby reducing the handling of wines and minimizing problems however associated with wine clarification (Pretorius & Bauer, 2002). As such, this ultimately contributes to lower volume loss of the finished wine products. The fact that the natural flocculent ability of certain commercial wine yeast strains is advertised by retailers of active dry wine yeasts further highlights the significance and attractiveness of this trait to the wine industry (http://www.maurivinyeast.com/media/51.pdf, 18 January 2010). Being mindful of this, we showed in a recent study that by placing the native chromosomal copies of two dominant flocculation genes, FLO1 and FLO5 in two nonflocculent commercial S.

In summary, the primer sets are not always the best in terms of s

In summary, the primer sets are not always the best in terms of sequence differences or software score, but are often a compromise between the results of sequence alignment and software design. This could explain why A. flavus/A. oryzae and A. parasiticus/A. sojae cannot be differentiated

with our real-time method. The validation on 11 species of this section demonstrated that identification results are more precise than those obtained by the single gene sequencing method. From a taxonomic point of view, it is worth noting that the section Flavi is still a matter of debate. Indeed, although a lot of genetic approaches failed to identify interspecific differences between A. flavus and A. oryzae, or between A. parasiticus DMXAA supplier and A. sojae (Egel et al., 1994; Geiser et al., 1998a, b), other studies confirmed that A. flavus and A. oryzae are almost genetically identical, but show some slight differences

at the level of the genes involved in the aflatoxin this website biosynthetic pathway (Watson et al., 1999; Geiser et al., 2000; Tominaga et al., 2006). Regrettably, these differences are minimal and do not allow researchers to design correct real-time primers assuring good PCR efficacy. Our tests on aflT and aflR genes to differentiate those two species were laborious and unsuccessful. Up to now, only genetic analyses based on the total DNA can differentiate these two pairs of species because they take genome differences into account. However, A. oryzae can be separated from A. flavus by SmaI digestion of total DNA (Klich & Mullaney, 1987), whereas A. parasiticus and A. sojae can be differentiated from each other only by RAPD analysis of the total DNA (Yuan et al., 1995). Furthermore, A. oryzae and A. sojae are considered to be domesticated forms of A. flavus and A. parasiticus, respectively (Kurtzman et al., 1986; Klich & Pitt, 1988; Geiser et al., 1998a, b; Kumeda & Asao, 2001). According to several authors, the absence of interspecific variability

provided no justification for maintaining the industrial species A. oryzae and A. sojae as individual species (Klich & Pitt, 1988; Kumeda & Asao, 2001). However, from a mycotoxigenic point of Thalidomide view, the proposition to meld taxonomically species used in the food-processing industry and aflatoxin-producing species was not received enthusiastically by food mycologists (Geiser et al., 2000). From an ecological point of view, A. flavus and A. parasiticus are commonly found in the environment, whereas A. oryzae and A. sojae, used for industrial applications, would not live in the same niches as A. flavus and A. parasiticus (Yuan et al., 1995; Pitt & Hocking, 1999; Cruz & Buttner, 2008; Gonzalez-Salgado et al., 2008). Nevertheless, the necessary discrimination of A. flavus from A. oryzae and A. parasiticus from A.

Similarly, dideoxynucleosides cause peripheral neuropathy [106],

Similarly, dideoxynucleosides cause peripheral neuropathy [106], a common toxicity of taxanes and vinca alkaloids, so co-prescribing should be avoided. Both ZDV and dideoxynucleosides are no longer recommended for

initiation of ART but some treatment-experienced patients may still be receiving these drugs and alternatives should be considered. With Ponatinib supplier the widespread use of effective combination ART, the incidence of severe HIV-associated cerebral disease has declined dramatically [107]; however, more subtle forms of brain disease, known as HIV-associated NC disorders are reported to remain prevalent [108]. This NC deficit may present with a wide spectrum of clinical symptoms, but typically includes patterns involving ineffective learning and problems with executive function, rather than pure difficulties in formulating new memory (the cortical defect typical of Alzheimer’s disease [109]). Given the changing picture of this disease, a revised nomenclature system has been proposed classifying subjects with abnormal neuropsychological testing

results in to three categories based on patient’s symptoms, measured via the activities Hydroxychloroquine molecular weight of daily living scale [108]. Subjects with abnormal neuropsychiatric testing results, who are otherwise asymptomatic, are classified as having HIV-associated asymptomatic NC impairment; those who are mildly symptomatic are classified as having HIV-associated mild NC disorder; and those who are severely symptomatic are classified as having HIV-associated dementia. The clinical relevance of asymptomatic NC impairment, namely asymptomatic subjects with abnormal results on neuropsychological testing, remains unclear. Reports describing rates of NC impairment vary with some groups describing that up to 50% of HIV-positive subjects meet the above diagnostic criteria [110]. However, such reports should be interpreted with caution as asymptomatic subjects are C1GALT1 often included and not all reports correct

for effective ARV use. A Swiss cohort has reported 19% of aviraemic HIV-positive subjects meet the classification for mild NC disorder or above [111]. Risk factors for the development of NC disorders are poorly understood and are likely to be multifactorial, including both HIV disease factors [112] and concomitant diseases [113]. Although it is possible the choice of combination ART a subject receives may influence NC function, this is a controversial area without definitive evidence. The following recommendations apply to patients with symptomatic HIV-associated NC disorders. We recommend patients with symptomatic HIV-associated NC disorders start ART irrespective of CD4 lymphocyte count (1C). Proportion of patients with symptomatic HIV-associated NC disorders on ART. Current evidence suggests NC function improves after commencing ART for the first time [114] in both cognitively symptomatic [115] and asymptomatic [116] subjects.

Some drugs are likely to be available in the near future that mig

Some drugs are likely to be available in the near future that might be sequenced in the same class (e.g. dolutegravir) although others with novel sites of action (e.g. maturation inhibitors, CD4 receptor antagonists, etc.) are still in earlier phases of

development and some years off randomized trials. Drugs developed for, and Ibrutinib solubility dmso used in, other settings such as pegylated interferon that have been incidentally demonstrated to decrease VL should not be used without discussion with an experienced HIV physician as data are either too limited or contradictory. Several studies and an early meta-analysis suggested that CCR5 receptor antagonists were associated with significant gains in CD4 cell counts even in the presence of C-X-C chemokine receptor type 4 tropic virus. However, a more recent meta-analysis refuted this finding (P=0.22) when comparing with other new drugs [53]. A priority Ribociclib in vitro question that the Writing Group addressed was whether 3TC/FTC should be used in maintaining an RT mutation at codon 184 in patients with limited or no therapeutic options. Although the M184V mutation is associated with resistance to 3TC/FTC, the mutation has a broad influence on the RT enzyme. In vitro studies have shown that M184V-possessing enzymes have lower processivity and higher fidelity and replicate more slowly than WT enzymes [70]. These observations have led to the hypothesis that maintaining this mutation

using 3TC/FTC would provide clinical benefit through the replication deficit provided by the M184V mutation combined with the residual antiviral activity of 3TC/FTC [71, 72]. It has been shown that patients harbouring M184V due to 3TC failure who continue on 3TC monotherapy maintain lower VLs than at baseline and rarely develop new RT or protease mutations [73]. Moreover, ceasing 3TC monotherapy has been demonstrated to result in replication capacity recovery and a reduction in CD4/CD8 ratio driven by the de-selection of the M184V mutation [74]. This strategy is supported by the E-184 study which was a small but randomized, open-label study of 3TC monotherapy vs. no therapy in patients failing ART [75].

Monotherapy was associated with Molecular motor significant smaller increases in VL, smaller declines in CD4 cell counts, and no selection of additional RT mutations. Finally, the presence of M184V mutation enhances in vitro susceptibility to TDF and this translated into a significant HIV RNA response in clinical trials of TDF intensification [76, 77]. “
“HIV-1 non-B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non-B clades and their association with demographic factors, over the entire course of the HIV-1 epidemic, have not been fully investigated in Italy. We carried out a phylogenetic analysis of HIV-1 pol sequences derived from 3670 patients followed at 50 Italian clinical centres over nearly three decades. Overall, 417 patients (11.

Bilirubin elevation of any grade occurred in 14 of 40 infants (35

Bilirubin elevation of any grade occurred in 14 of 40 infants (35%). No difference in the time course of bilirubin elevation occurred between infants whose mothers received the

300/100 mg and 400/100 mg doses of ATV/r in the third trimester. Among infants who had elevated bilirubin in the first 14 days, none was elevated to grade 1 because thresholds for grade 1 bilirubins are higher in the first 2 weeks of life to account for normal, physiological bilirubin elevations [21]. Six infants underwent phototherapy (at ages ranging from 3 to 6 days); four infants had 2 days of phototherapy, one had 3 days, and one had 4 days; bilirubin levels associated with these selleck chemicals events ranged from 8.1 to 13 mg/dL. The total bilirubin level was <4 mg/dL in all infants by week 12.

All grade 3–4 bilirubins occurred after day 14; however, levels were decreasing by day 14 in all cases. The infants’ total bilirubin levels on delivery day correlated weakly with the mothers’ total bilirubin levels at delivery (Fig. 3a). When the infants’ total bilirubin levels were compared with the mothers’ bilirubin levels over the last 4 weeks of pregnancy, an Alectinib datasheet even weaker correlation was observed (Fig. 3b). ATV/r 300/100 mg qd dosing had a lower AUCτ and Cmax in the third trimester of pregnancy compared with the AUCτ and Cmax in nonpregnant adults; however, Cmin values were similar. Increasing the dose of ATV/r to 400/100 mg achieved AUCτ and Cmax values similar to those in nonpregnant adults

and higher Cmin values. ATV concentrations were elevated in the postpartum period. This observation has been made for other protease inhibitors [22–24]. ATV concentrations appear to normalize by week 7 postpartum [18]. The ratio of maternal to cord blood ATV indicates dipyridamole that, as with other protease inhibitors [25], ATV does not freely cross the placenta; however, in this study, plasma protein binding in cord blood was lower than in maternal blood, indicating that free drug concentrations in the fetus were approximately twice as high as in the mothers at a similar total (bound and unbound or ‘free’) ATV plasma concentration [26], suggesting that the levels achieved in the cord blood may provide some antiviral protection to the fetus [18]. A theoretical concern with ATV use during pregnancy is an increase in bilirubin in newborns [1]. This could occur either by passive back-diffusion of infant bilirubin across the placenta, as a result of saturation of protein binding of bilirubin in mothers with elevated maternal bilirubin, or theoretically through the effect of UGT1A1 inhibition in the fetus as a result of ATV crossing the placenta. The placenta normally only allows unidirectional flow of bilirubin from the fetus to the mother, and not from the mother to the fetus.

Five microlitres of purified ligated DNA were used as a template

Five microlitres of purified ligated DNA were used as a template in PCR experiments carried out with the divergent primers IF505 (5′-CGT GAA GTA TCT TCC TAC AGT-3′) and IF452 (5′-ACT CAT TCT AAT AGC CCA TTC-3′) or with IF433 (5′-GGT GGA ACT TAT CAA TCC CAT-3′) and IF506 (5′-GGA TAA ATC GTC GTA TCA AAG-3′). 5-Fluoracil in vitro DNA sequence analysis including coding sequence identification was carried out using the software artemis ver.

11 available for download at http://www.sanger.ac.uk/Software/Artemis/website. Manual gene annotation was carried out by conducting blast homology searches of the databases available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/sites/gquery) Ceritinib and at the S. pneumoniae Sybil website (http://strepneumo-sybil.igs.umaryland.edu/). Protein domains were identified by searching the protein family database

Pfam available at the Wellcome Trust Sanger Institute (http://pfam.sanger.ac.uk). Multiple sequence alignments were performed using the clustalw2 tool at the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw2/). Plate mating experiments were performed essentially as already described (Iannelli & Pozzi, 2007). Donor and recipient cells were grown separately in TSB in the presence of appropriate antibiotics at 37 °C, until the end of the exponential phase (OD590 nm=0.5). Cells were mixed at a 1 : 10 ratio, harvested by centrifugation for 15 min at 3000 g, resuspended in 0.1 mL of TSB and plated on TSA enriched with 5% horse blood. Following 4 h of incubation in 5% CO2 at 37 °C, cells were harvested by scraping the plates with a sterile plain swab and resuspended

in 1 mL of TSB containing 10% glycerol. Selection of transconjugants was carried out with the multilayer clonidine plating. Briefly, 2 mL of TSB/10% horse blood containing the appropriately diluted mating reactions were combined with 6 mL of melted TSA and poured into a Petri dish containing a base layer of TSA. After 90 min of incubation at 37 °C for phenotypic expression, an 8 mL TSA layer containing the appropriate antibiotics, for the resistance marker of the donor genetic element and for the chromosomal resistance marker of recipient strain (where available), was added. The antibiotic concentrations were as follows: chloramphenicol 5 μg mL−1, fusidic acid 25 μg mL−1, novobiocin 10 μg mL−1, rifampicin 25 μg mL−1, spectinomycin 400 μg mL−1, streptomycin 1000 μg mL−1 and tetracycline 5 μg mL−1. Conjugation frequencies were determined by plating each parent strain alone. At this stage, we carefully performed genetic analysis of the transconjugants in order to exclude isolation of spontaneous mutants or colonies that might grow even in the absence of any genotype conferring resistance.