0045). Factors associated with a higher risk of pneumonia at admission in the univariate analysis, other than being HIV-negative, were: older age (mean 45 years in those with pneumonia

vs. 39 years in those without; P=0.0066), headache (31%vs. 13%, respectively; P=0.0009), tiredness (27%vs. 5%, respectively; P=0.0006), dyspnoea (35%vs. 17%, P=0.0099), longer time from the onset of symptoms to hospital admission (mean 5 vs. 2.6 days, respectively; P=0.0001), and delayed influenza A H1N1 diagnosis (56%vs. 17%, respectively; Selleckchem LY2157299 P=0.0001). In the multivariate analysis, being HIV-positive was not an independent risk factor for pneumonia at admission. We identified time from the onset of symptoms to hospital admission [odds ratio (OR) 1.82 per extra day; 95% confidence interval (CI) 1.50–2.22; P<0.0001] and tiredness (OR 4.40; 95% CI 1.19–16.23; P=0.0260) as independent factors associated Selleckchem EMD 1214063 with pneumonia at admission. Among HIV-positive patients, those with pneumonia at admission were more commonly active smokers (100%vs. 49% for those with and without pneumonia, respectively; P=0.0545) and former/current injecting drug users (100%vs. 31%, respectively; P=0.0053), and more frequently had dyspnoea (60%vs. 14%, respectively; P=0.0351), respiratory

failure (60%vs. 4%, respectively; P=0.0034), and concomitant bacterial infections (60%vs. 2%, respectively; P=0.0014) compared with those without pneumonia. Among HIV-positive patients, presenting with pneumonia was not associated with gender, comorbidities, travel/contacts, age, time from HIV diagnosis, CD4 cell count nadir, log10 HIV-1 RNA zenith, prior/current C events, delayed influenza A H1N1 diagnosis, time between the onset of symptoms and

hospital admission, temperature at admission, or laboratory parameters, including most recent CD4 cell count, CD8 cell count and HIV-1 RNA measurement. Because of the low number of HIV-positive patients with pneumonia, multivariate analyses assessing independent risk factors could not be performed. Most recent CD4 and CD8 cell ounts and HIV-1 RNA measurement this website prior to influenza A H1N1 diagnosis were available for all patients (n=56) within 4 months preceding influenza A H1N1 diagnosis (median 7 weeks; interquartile range 2–13 weeks). CD4 and CD8 cell counts and HIV-1 RNA were determined 4–6 weeks after discharge in 51 patients. Compared with values obtained before diagnosis, there were slight decreases in CD4 count (median −15 cells/μL; interquartile range −44 to 39 cells/μL), CD4 percentage (median −0.4%; interquartile range −0.8 to 2.3%), CD8 count (median −14 cells/μL; interquartile range −122 to 77 cells/μL) and CD8 percentage (median −0.7%; interquartile range −2.8 to 1.5%), but none of these changes was statistically significant (P>0.05 for all comparisons). Plasma HIV-1 RNA and the number of patients with plasma HIV-1 RNA below the detection limit remained unchanged.

The addition of 1 mM nitrate or 10 mM sulfate almost completely inhibited methanogenesis in Eckernförde Bay microcosms

(Fig. 3a). Hexadecane-dependent methanogenesis (46.5±3.5 nmol methane cm−3 day−1) was higher than naturally occurring methanogenesis without hexadecane of no more than 10 nmol methane cm−3 day−1 in the sediment layer of the highest methanogenesis (Fig. 3a; Treude et al., 2005). While hexadecane-dependent methanogenesis occurred without additional electron acceptors at a rate of 24.5±1.7 nmol methane cm−3 day−1, selleck monoclonal antibody the process was significantly slower than that in incubations with 2 mM sulfate concentrations 46.5±3.5 nmol methane cm−3 day−1 (Fig. 3b). Also, the addition of ethylbenzene significantly increased methanogenesis in microcosms containing Zeebrugge sediment (Fig. 2b). Compared with 2 mM sulfate, the addition of ferrihydrite or manganese dioxide reduced methanogenesis from 58.1±0.6 to 39.6±0.9

or 28.2±12.1 nmol methane cm−3 day−1, respectively (Fig. 2b). www.selleckchem.com/products/Metformin-hydrochloride(Glucophage).html Like in hexadecane incubations, an increase of sulfate concentrations to 22 mM decreased the methanogenesis rate to 10.0±0.5 nmol methane cm−3 day−1. Nitrate inhibited methanogenesis completely. The addition of ethylbenzene inhibited CO2 release (Fig. 2b) compared with unamended controls. The lowest CO2 production rate was detected with nitrate (19.5±0.6 nmol CO2 cm−3 day−1), while 22 mM sulfate led to an increase in CO2 release to 45.9±0.3 nmol CO2 cm−3 day−1. Methanogenesis depending on 1-13C-naphthalene commenced between days 124 and 235 in 2 mM sulfate Baricitinib incubations, with maximum rates of 12.5±0.3 pmol methane cm−3 day−1 (Table 1). At the same time, the was −37.1±1.6‰ (unamended control: =−43.2±1.1‰; Fig. 4d). At day 435, 1-13C-naphthalene-derived 13CH4

formation was also detected as indicated by the elevated values compared with unamended controls. Methanogenesis rates were, however, within the same order of magnitude in all microcosms (Table 1). Furthermore, a strong enrichment in 13CO2 was observed already after 42 days of incubation in all setups amended with 1-13C-naphthalene (Fig. 4e–h). The values ranged from +34.9±2.6‰ (nitrate addition) to +68.4±23.5‰ (iron addition), which was significantly different from the values produced in microcosms amended with unlabelled naphthalene (total mean −26.6±0.2‰). In the 1-13C–naphthalene-degrading cultures, the values further increased to a maximum at day 235 (total mean +419±21‰; Fig. 4e–h). The CO2 release rates were at least 200 times higher than the methane formation rates (Table 1). Ferrihydrite addition resulted in relatively low CO2 formation rates from 1-13C-naphthalene of 236.7±3.4 pmol CO2 cm−3 day−1, while the highest rate was observed with nitrate (499.4±0.5 pmol CO2 cm−3 day−1). In parallel experiments, anaerobic oxidation of methane (AOM) was observed in Zeebrugge microcosms. Incubations with 22 mM sulfate showed the highest AOM rates (1216.0±135.

Earlier studies have focused on cell counts and the activity of bacteria in the reed rhizosphere using cultivation-based techniques (Borsodi et al., 2003). Others have focused

on the community structure and diversity of buy Fulvestrant bacteria associated with the reed rhizosphere in freshwaters using molecular methods (Borsodi et al., 2007; Ravit et al., 2007; Rusznyak et al., 2007; Vladar et al., 2008), but no study has examined the endophytic bacteria associated with reed roots and their possible roles in phytoremediation mediated by reed wetland. This paper describes the diversity and community structure of endophytic bacteria in reed roots growing in a constructed wetland. We used the 16S rRNA library technique, a culture-independent method, with the goal of understanding the role of bacteria within reed roots in enhancing the phytoremediation of eutrophic water mediated by reed-constructed wetland. Reed roots were obtained see more from the common reed (P. australis Cav. Trin.) zone of Beijing CuiHu Wetland, China, in July 2008. The wetland was used to treat a mixture of domestic wastewater from the surrounding area and water from Shangzhuang reservoir. In this study, one treatment region with marshy

plants (mainly reed) and one control region (without any plants) were chosen to measure the water quality, in order to determine the effect of reed on the water body. The control region shared the same water source with the reed planted region, but was 50 m away from it. The physicochemical characteristics

of the water in the treatment region were as follows: pH 7.34, 1.37 mg L−1 total nitrogen (N), 0.13 mg L−1 total phosphorus (P), and 27.85 mg L−1 organic matter. In the control region, the water quality indexes were as follows: pH 7.56, 3.11 mg L−1 total nitrogen, 0.25 mg L−1 total phosphorus, and 31.90 mg L−1 organic matter. The observations and sampling GPX6 took place in July 2008. The reed roots were sampled from 15 cm below the water surface within the treatment region. Three samples of 1 g fibrous roots were taken from three different locations with a distance of about 10 m. They were immediately mixed and transported to the laboratory. Reed roots were first washed three times with tap water to remove attached soil. Subsequently, the roots were immersed in 70% ethanol for 3 min, washed with a fresh sodium hypochlorite solution for 5 min, rinsed three times with 70% ethanol for 30 s, and finally washed five times with sterile-distilled water as described in Sun et al. (2008). To confirm that the disinfection process was successful, aliquots of the sterile-distilled water used in the final rinse were set on Luria–Bertani (LB) medium plates. The plates were examined for bacterial growth after incubation at 30 °C for 3 days.

Flanking regions of genomic DNA in the rescued plasmids were mapped to genes

by sequencing. The regions Selleckchem STA-9090 rescued are shown in Fig. 2. When AF210.1 was retransformed with p11, p56 and p101, the phenotype of the original REMI-11, REMI-56 and REMI-101 strains was reconstituted. Frequency of reconstruction of REMI insertions was 1 in 380 transformants for p11, 2 in 870 for p56 and 4 in 40 for p101. These transformants had the same azole susceptibilities as the original REMI strains. A Southern hybridisation carried out on genomic DNA isolated from these transformants confirmed that they had the same hybridisation pattern as the original transformants, indicating that the insertion had occurred at the same parts in the genome (Fig. 3). We note that for retransformation of AF210 with p56, aberrant band sizes were obtained on a Southern blot after XhoI digestion, although the expected sizes were obtained after ClaI digestion. We are unable to explain this banding pattern, and REMI-56 was therefore included in the complementation experiments described later. The plasmids used for the other reconstruction experiments lacked long flanking sequences on one side of the insertion and it is possible that the double recombination event required for reconstitution of the REMI was inefficient. learn more A high number of transformants was tested in these cases (670 for

p102, 3200 for p85, 540 for p14D and 420 for p103). To determine whether phenotype and insertion were linked, it was decided to attempt to complement the mutations using genes amplified by PCR from Af293.

Plasmids p5G07550, p1G05010, p2G11840, p2G11020, p4G10880 and p6G12570 were cotransformed into REMI-85, REMI-56 REMI-102, REMI-14D, REMI-103 and REMI-116, respectively. Wild-type (AF210) or parental (AF210.1) levels of azole resistance were obtained from all transformations except that of REMI-116 5-Fluoracil datasheet (Table 3). Primers flanking the original insertion site were used to confirm that intact copies of complementing genes were present in strains where wild-type phenotypes had been restored. In all cases, restoration of parental AF210.1 phenotype as assessed by MICITR corresponded to integration of an intact genomic copy of the appropriate gene. In this study, we aimed to discover new genes and mechanisms involved in ITR resistance in A. fumigatus. Several insertional mutants were isolated from a REMI screen and characterised. Eight of 4000 mutants tested displayed altered azole sensitivity with four mutants showing increased sensitivity and four showing decreased sensitivity. Two putative transporter genes were isolated in the screen as being involved in resistance to azoles (i.e. the insertions were more sensitive to azoles). One gene identified is an ABC transporter and is probably an orthologue of the A. nidulans AtrG and Pmr1 of Penicillium digitatum (Nakaune et al., 1998).

05. Hypoxic cultures (standing) were established by dispensing 200 μL culture aliquots into 96-well black, clear-bottom microtitre plates and incubating the plates at 37 °C. The aerobic promoter activity was measured in cultures that were simultaneously grown in 50-mL tubes (5 mL of culture). Culture aliquots of 200 μL were sampled at 48 h and the GFP fluorescence was measured in a spectrofluorimeter (Molecular Devices, Sunnyvale, CA) with an excitation wavelength of 483 nm and an emission wavelength of 515 nm. The 178-bp narK2 promoter region was amplified Hydroxychloroquine supplier by PCR using NarK2R1 and NarK2F

primers (Fig. 1, Table 2) and genomic DNA of the various standard or clinical strains. The PCR conditions were a 10-min initial denaturation phase at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C and, finally, 7 min at 72 °C. CHIR-99021 mw A 10-μL aliquot of

the PCR product was digested with NheI for 90 min, electrophoresed on a 6% nondenaturing polyacrylamide gel and visualized using ethidium bromide. Mycobacterium bovis AN5 was complemented with the integrating plasmid pNarG-GM1 expressing the M. tb narGHJI operon (Sohaskey & Modesti, 2009) or the pNarK2X plasmid expressing the M. tb narK2X operon, (see Table 1) or both pNarG-GM1 and pNarK2X. To construct pNarK2X, the region encompassing the coding regions of narK2 and narX along with a 280-bp upstream promoter was amplified by PCR using Fusion DNA polymerase (NEB, UK) and M. tb H37Rv DNA and cloned in the EcoR1 and Digestive enzyme HindIII sites of pFPV27 mycobacterial shuttle vector. The resultant plasmid was electroporated into M. bovis or M. bovis-harbouring pNarG-GM1. Nitrate reductase assay was performed with aerobic

shaking and 48-h standing cultures (hypoxic). Briefly, the cultures were grown aerobically as described above in the presence of 5 mM nitrate and standing cultures (starting OD595 nm, 0.05) were maintained for 48 h in 96-well microtitre plates as described previously (Chauhan & Tyagi, 2008b). The nitrite concentration was determined using the Griess reaction as described (Wayne & Doubek, 1965). Briefly, 50 μL of sulfanilamide was added to 50 μL of cultures (both aerobic and standing) and incubated at room temperature for 5–10 min. Next, 50 μL of N-1-napthylethylenediamine dihydrochloride was added and the A595 nm was measured in a plate reader (Biorad). To test the hypothesis that the lack of hypoxic induction of narK2 and narX in M. bovis/BCG is because of a −6T/C SNP in the narK2X promoter region, we mutated the M. tb narK2 promoter by changing thymine at the −6 position to cytosine (−6TC) in the narK2 promoter plasmid, pnarK2, to mimic the observed mutation at this site in M. bovis/BCG. The effect of this mutation on promoter activity was assessed in M. tb H37Rv under hypoxic conditions using the GFP reporter assay. The −6TC mutation completely abolished the hypoxic induction of pnarK2 (Fig.

An increased risk of life-threatening infection was also observed when the results of three Phase II studies were pooled, combining rituximab with the infusional CDE chemotherapy regimen in 74 patients with ARL [50]. However, subsequent Phase II studies of R-CHOP (without maintenance rituximab) from Europe did not show an increased risk of infectious deaths, instead showing that rituximab was beneficial [14,17]. The AMC went on to perform a randomized

study of DA-EPOCH with either concurrent or sequential rituximab [19]. Concurrent administration was superior, with no increase in infectious deaths, but outcome in both groups was excellent, supporting the efficacy and tolerability of concurrent rituximab. A recent Selleckchem MK-8669 meta-analysis of prospective studies has confirmed the benefit in response rate and overall survival (OS) of the addition of rituximab to chemotherapy [20]. A pooled analysis of both AMC studies mentioned above suggested that R-EPOCH resulted in superior response rates and survival compared to R-CHOP [18], although these regimens have not been compared

in any randomized study. Importantly, the R-EPOCH study was performed during a later time period (2002–2006) than the R-CHOP study (1998–2002), suggesting other variables, including supportive http://www.selleckchem.com/products/PD-98059.html care and antiretroviral drug options, may have differed. Consistent with this, the patients treated with R-EPOCH routinely received concurrent antifungal and antibacterial prophylaxis, which was omitted from those treated earlier with R-CHOP. The AMC have recently reported the results of a prospective, multicentre Phase II trial of R-CHOP, but with pegylated, liposomal doxorubicin in order to limit toxicity. Of note, HAART was continued during chemotherapy. The treatment was well tolerated without any deaths from infection, even in those with a low CD4 cell count, thus supporting the inclusion of rituximab in treatment regimens. However, the response rate was inferior to that reported in prior studies (overall response 76.5%, CR 47.5%) [51]. Thus, the addition of rituximab to chemotherapy is now recommended (-)-p-Bromotetramisole Oxalate for DLBCL in HIV-positive patients. Although the use of rituximab is contentious in patients with a CD4 count <50 cells/μL

[27], with appropriate antimicrobial prophylaxis (cotrimoxazole, fluconazole, aciclovir, azithromycin), pre-emptive G-CSF and prompt treatment of opportunistic infection, rituximab is recommended for all patients with DLBCL. The rate of overall response (CR and partial remission; PR) and CR to R-CHOP chemotherapy is reported to be around 66–87% and 58–77%, respectively [14,17,27,29]. In one study with long follow-up, the 8-year OS was 46% [52]. (See Table 4.5 for summary of R+ chemotherapy studies in HIV-positive patients.) R-EPOCH (Concurrent rituximab only) As mentioned, the IPI score at diagnosis is prognostic of outcome, such that those patients with high-risk disease (IPI score 3–5) have a lower response rate and overall survival to standard chemotherapy [17,27].

Bachiller

Luque (Hospital Universitario del Río Hortera, Valladolid); A. Castro Iglesias, S. López (Hospital Universitario Juan Canalejo, A Coruña); J. R. Arribas, J. González García, I. Pérez Valero (Hospital Universitario La Paz, Madrid); J. Sanz Sanz, I. Santos (Hospital Universitario La Princesa, Madrid); J. Sanz Moreno, A. Arranz Caso (Hospital Universitario Príncipe de Asturias, Alcalá de Henares); J. Antonio Girón (Hospital Universitario Puerta de Mar, Cádiz); M. A. López Ruz, M. López, J. Pasquau Liaño, C. García (Hospital Universitario Virgen selleck compound de las Nieves, Granada); M. Crespo Casal (Hospital Vall d’Hebrón, Barcelona); C. Galera Peñaranda (Hospital Virgen de la Arrixaca, Murcia); A. Chocarro Martínez, I. García (Hospital Virgen de la Concha, Zamora); Pompeyo Viciana (Hospital Virgen del Rocío, Sevilla); J. Rodríguez Baños, C. Machado (Hospital

Virgen Macarena, Sevilla). Representatives of Abbott Laboratories Medical Department participating in this study were: B. Tribis-Arrospe, J. A. García, M. J. Fuentes, N. García, X. Gómez and L. Griffa. “
“The aims of the study were to describe the sociodemographic profile of men who have sex with men (MSM) who have never been tested for HIV and to analyse factors associated with never having been tested. The European MSM Internet Survey (EMIS) was implemented in 2010 in 38 European countries Selleckchem AZD1208 on websites for MSM and collected data on sociodemographics, sexual behaviour, and other sexual health variables. A logistic regression analysis was conducted to assess variables associated with never having been tested for HIV. Of the 13 111 respondents living in Spain, 26% had never been tested for HIV. Those who had never Verteporfin manufacturer been tested were significantly more likely to live in a settlement with fewer than 100 000 inhabitants, be

younger than 25 years old, have a lower education level, be a student, and identify themselves as bisexual. In the multivariate analysis, to have never been tested for HIV was associated with being born in Spain [odds ratio (OR) 1.35; 95% confidence interval (CI) 1.192–1.539], living outside large settlements (OR 1.37; 95% CI 1.216–1.534), being younger than 25 years old (OR 2.94; 95% CI 2.510–3.441), being out to no one or only a few people (OR 2.16; 95% CI 1.938–2.399), having had no nonsteady partners in the last 12 months (OR 1.26; 95% CI 1.109–1.422), and being not at all confident to access HIV testing (OR 3.66; 95% CI 2.676–5.003), among others factors. The profile of the MSM who had never been tested for HIV indicates that most of them were men who were hard to reach (young, bisexual men, in the closet). Interventions should aim to improve access to and the convenience of testing. In Spain, an increase in the prevalence of sexually transmitted infections (STIs), including HIV infection, as well as high-risk sexual behaviour among men who have sex with men (MSM) has been reported in recent years.

Here, we use an Escherichia coliΔnanT strain to characterize

the function of known and proposed bacterial sialic acid transporters. We discover that the STM1128 gene from Salmonella enterica serovar Typhimurium, which encodes a member of the sodium solute symporter family, is able to restore growth on sialic acid to the ΔnanT strain and is Dorsomorphin able to transport [14C]-sialic acid. Using the ΔnanT genetic background, we performed a direct in vivo comparison of the transport properties of the STM1128 protein with those of sialic acid transporters of the major facilitator superfamily and tripartite ATP-independent periplasmic families, E. coli NanT and Haemophilus influenzae SiaPQM, respectively. This revealed that both STM1128 and SiaPQM are sodium-dependent and, unlike SiaPQM, both STM1128 and NanT are reversible secondary carriers, demonstrating qualitative functional differences in the properties of sialic acid transporters

used by bacteria that colonize humans. Sialic acids are a family of related nine carbon sugar acids that play important roles in the biology this website of a wide range of both eukaryotic and prokaryotic organisms (Schauer, 2004; Vimr et al., 2004). In mammals, sialic acids are a predominant feature on the surface of many cell types, and bacteria have evolved multiple mechanisms to exploit these host-derived sugars (Vimr et al., 2004; Severi et al., 2007). For example, Escherichia coli is able to grow on the most common sialic acid N-acetylneuraminic

acid (Neu5Ac) as a sole carbon and nitrogen source (Vimr & Troy, 1985), which is important for successful colonization of the mouse gut (Chang et al., 2004). Other bacteria such as Haemophilus influenzae use host-derived Neu5Ac in an immune evasion mechanism by adding it as a terminal component of their lipopolysaccharide (Bouchet et al., 2003). While some pathogens have evolved de Clomifene novo biosynthesis pathways for Neu5Ac (Vimr et al., 2004), many bacteria rely on the acquisition of Neu5Ac from their environment and hence require high-affinity transport systems (Bouchet et al., 2003). The pioneering work of Vimr and colleagues led to the first molecular characterization of a bacterial Neu5Ac transporter, which was the NanT protein from E. coli (Vimr & Troy, 1985). This is a secondary transporter and a member of the major facilitator superfamily (MFS) (Martinez et al., 1995). Very recently, another MFS family member, distinct from NanT, has been implicated in sialic acid uptake in Bacteriodes fragilis (Brigham et al., 2009) and Tannerella forsythia (Thompson et al., 2009). We and others have characterized a tripartite ATP-independent periplasmic (TRAP) transporter for Neu5Ac from H. influenzae, SiaPQM, that is important for virulence (Allen et al., 2005; Severi et al., 2005; Mulligan et al., 2009).

That is, a short exposure of 6 h with ofloxacin (Hansen et al., 2008) selleck may only

identify mutants with a minor persistence phenotype. In addition, an important difference in the study by Hansen et al. (2008) from ours is that the persister mutant identified in their study had a weak phenotype of only a 10-fold drop in persisters compared with the wild-type strain, which is likely an indication of short antibiotic exposure and ‘of shallow or intermediate persister’ phenotype identified in that screen. In contrast, in our study we used a longer exposure of 24 h and 5 days with ampicillin and identified only two genes ubiF and sucB as being involved in persister survival. The persister phenotype was more obvious with large differences in the number of persisters between the sucB and ubiF mutants and the parent strain in persister and stress assays (Tables 2–6). ubiF encodes 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol oxygenase, an important enzyme in ubiquinone biosynthesis (Kwon et al., 2000). Ubiquinone is an acceptor of electrons from many cellular dehydrogenases involved in oxidative metabolism and is responsible for transporting electrons from complexes I and II to complex III of the respiratory electron transport chain (Moat & Foster,

1995). The ubiquinone forms hydroquinone upon receiving 2e− and 2H+ from the cytosol, which plays a critical role in the generation of ATP and in the maintenance of membrane potential (Moat & Foster, 1995). The increased susceptibility of buy PD98059 the ubiF mutant to antibiotics and stresses is presumably Methocarbamol due to its impaired ability to provide energy source for the persister bacteria under antibiotic and stress conditions, thus leading to reduced persister survival. sucB encodes dihydrolipoamide succinyltransferase (SucB), which is a subunit (E2) of the 2-oxoglutarate dehydrogenase complex together with 2-oxoglutarate decarboxylase (E1) and lipoamide dehydrogenase (E3, Lpd). The 2-oxoglutarate dehydrogenase complex catalyzes the reaction 2-ketoglutarate +coenzyme A+NAD+succinyl-CoA+CO2+NADH, and is a key enzyme

of the TCA cycle (Moat & Foster, 1995). In Mycobacterium tuberculosis, SucB together with Lpd, AhpC and AhpD form a complex, which functions as NAD-dependent peroxidase and peroxynitrite reductase for antioxidant defense (Bryk et al., 2002). Consistent with this finding, in this study, we found that the E. coli sucB mutant showed higher sensitivity to peroxide than the parent strain did. However, sucB has not previously been shown to be involved in susceptibility to antibiotics and stresses and persister survival. We have found that the sucB mutant, besides being more susceptible to peroxide, had higher susceptibility to acid pH and weak acid salicylate and also various antibiotics for both log phase and stationary phase cultures.

The TOL plasmid, originally isolated from P. putida strain mt-2, is one of the best-studied catabolic plasmids belonging to the IncP-9 group, encoding biodegradation pathways for toluenes and xylenes (Williams & Murray, 1974). The TOL plasmid can be transferred to other pseudomonads and has earlier been reported as derepressed for transfer (Benson & Shapiro, 1978; Bradley & Willams, 1982; Ramos-Gonzalez et al., 1991). During earlier studies, we had observed a stimulatory effect of TOL plasmid carriage on biofilm formation in Metformin solubility dmso P. putida

KT2440 at the air–water interface (Arango Pinedo et al., 2003). Here, we provide quantitative support for this biofilm enhancement at both the air–liquid and the liquid–solid interface and show that extracellular DNA (eDNA) may be responsible for the plasmid-stimulated interfacial growth. Pseudomonas putida KT2440 is a plasmid-free, restriction-deficient derivative of P. putida mt-2 (Bagdasarian et al., 1981). TOL is the archetypical catabolic plasmid pWWO (Williams & Murray, 1974). The TOL-free strain was chromosomally tagged with a miniTn5-Plac-gfpmut3b-kanR cassette (Normander et al., 1998), the TOL plasmid was tagged with a miniTn5-Plac-gfpmut3b-tetR cassette as per Christensen et al. (1996), and carried in a

wild-type KT2440 host. Strains were cultured in AB medium [15.1 mM (NH4)2SO4, 42.2 mM Na2HPO4, 22 mM KH2PO4, 51.3 mM NaCl, selleck chemicals llc 100 μM MgCl2, 10 μM CaCl2, 1 μM FeCl3, Clark & Maaløe, 1967] supplemented with 1 mM (flow cells) or 40 mM (static cultures) sodium citrate. Solid media were prepared by adding 20 g L−1 agar to AB medium. All cultivations were at 25 °C, unless observed otherwise. Antibiotics were added to all precultures to a final concentration of 50 μg mL−1. Static cultures were performed in replicate 500-mL Erlenmeyer flasks (70 mL medium) for bulk measurements, EPS extractions, and viscosity measurements or in 20 mL test tubes (5 mL medium) for microscopy, flow cytometry, and β-glucosidase assays. To test DNase effect, duplicate cultures were supplemented with DNaseI (Qiagen, 20 U mL−1), magnesium chloride (250 μM), and calcium Ergoloid chloride (4 μM). Pellicles were sampled with a

10-μL inoculation loop, or tweezers for very eDNA-rich pellicles, applied to a microscope slide, and stained with PI (15 μL, 10 μg mL−1) or Cytox Orange before viewing. Flow cell biofilms were established in three-channel flow cell setups, as described before (Møller et al., 1996). Flow cells were inoculated by adjusting the OD600 nm of precultures to 1.0, washing and resuspending the cells in 0.9% NaCl, and injecting 300 μL of this suspension into each channel with an insulin syringe. AB medium was continuously supplied by a peristaltic pump (Watson & Marlow 205S) to each channel at a rate of 2.7 mL h−1. After 2 and 7 days of incubation, three-dimensional image stacks of the biofilms (3 z-stacks from three replicate channels in duplicate flow cells for each strain) were recorded by confocal laser scanning microscopy (CLSM).