1 Motamedi SM, Posadas-Calleja

J, Straus S, Bates DW et 

1. Motamedi SM, Posadas-Calleja

J, Straus S, Bates DW et al. The efficacy of computer-enabled discharge interventions: a systematic review. BMJ Qual Saf 2011; 20: 403–415. 2. Callen J, McIntosh J, Li J. Accuracy of medication documentation in hospital discharge summaries: A retrospective analysis of medication transcription errors in manual and electronic discharge summaries. International Journal of Medical Informatics 2010; 79: 58–64. “
“K. Sonnexa,b, H. Alleemuddera, Sunitinib L-C. Chena aUniversity of Nottingham, Nottingham, UK, bNottingham University Hospitals, Nottingham, UK ICS have been shown to reduce the decline in lung function in COPD. However, it is thought that smoking causes resistance to the effects of ICS. Never-smokers, ex-smokers and light smokers had a better improvement in lung function1 after six months ICS use than current and heavy smokers. ‘Steroid resistance’ due to smoking may cause lower efficacy selleck products of ICS in this patient group but further work is required. It has been shown that smoking accelerates the loss in lung function (as measured by forced

expiratory volume in 1 second; FEV1) and increases mortality in Chronic Obstructive Pulmonary Disease (COPD) patients.1 Later stages of COPD may require regular treatment with inhaled corticosteroids (ICS) in addition to bronchodilators. One of the mechanisms by which ICS exerts its effect is by acting on enzyme histone deacetylase 2 (HDAC2) to suppress the release of inflammatory mediators.2 ICS have been shown

to reduce exacerbation rates and possibly reduce the decline in FEV1 in comparison to placebo.1 However, there is some evidence that smoking inactivates HDAC2, resulting in smokers being resistant to the effects of ICS.2 The aim of this research was to conduct a systematic review of the evidence that smoking affects efficacy of ICS in COPD. An electronic database search of PubMed, Ovid Medline, Ovid Embase and Cochrane Library (2000–2014) was performed using appropriate free text and MeSH terms. The reference lists of the retrieved papers were searched for retrieving further relevant studies. Fully published RCTs evaluating the use of ICS Vasopressin Receptor in COPD adults and stratifying the participants by smoking status were included. Review articles, abstracts, papers which are not fully published or published in languages other than English were not included. Retrieved trials that include COPD patients with asthma, lung cancer and pneumonia were excluded. Ethics approval was not required. A total of 44 studies were identified, 40 were excluded because participants were not stratified according to smoking status or the population did not meet the inclusion criteria. Of the remaining four trials, one was not randomised and was thus excluded. COPD severity varied across the studies ranging from mild to very severe. Two types of ICS were studied as monotherapy or in combination with salbutamol or salmeterol: fluticasone and budesonide.

Because a decrease in the transcription of fliC in the fliC-lux r

Because a decrease in the transcription of fliC in the fliC-lux reporter correlates with a decrease in the luminescent signal, our data support the hypothesis that growth in the presence of PMs results in a reduction of fliC expression. As may be seen in Fig. 1a–c, the fliC gene is maximally expressed at 1 h after inoculation, which

agrees with reports from other groups (Lane et al., 2007a, b). To compare the effect of the different PMs on fliC expression, the maximal normalized luminescence of each treatment Etoposide was divided by the control conditions (Max. normalized luminescencetreatment/Max. normalized luminescencecontrol). This calculation revealed that PG, PGP, and PGRE, all at a concentration of 10%, reduced the normalized luminescence signal to 12%, 30%, and 8% of that of the control, respectively. Hence, we concluded that the strongest inhibitor find protocol of fliC expression in this study is the PGRE at a

concentration of 10%. To further assess the conditions under which PMs reduce fliC transcription, CFT073 PfliC-lux bacteria were grown in LB, harvested, resuspended in fresh media, and spiked with PGRE at different concentrations (0–10% v/v). Luminescence and OD600 were measured as described above and the normalized luminescence calculated. The maximum normalized luminescence, which was observed at 15 min after the PGRE addition, was plotted vs. the PGRE concentration and may be seen in Fig. 1d. The results obtained show that, relative to the control, a spike of PGRE reduces the normalized luminescence in a concentration-dependent manner. Based on

this data, we concluded that growth in PGRE is not necessary to achieve a reduction in the level of expression of the flagellin gene. To confirm that the reduced expression of fliC that results from growth in the presence of PMs decreases the production of flagellin, we conducted a Western blot analysis using H1 flagellin antiserum (Fig. 2a). This analysis confirmed that flagellin production declined when CFT073 was grown in LB supplemented with PMs because flagellin bands were observed only when the bacterium was grown under control conditions or when supplemented with 1% PGRE. For all other conditions tested, no bands were observed. Furthermore, as expected, no flagellin bands were observed on the blot in the lane also corresponding to the negative control, CFT073 ∆fliC. Additional validation of the observed decrease in flagellin production upon exposure to PGRE was obtained by imaging bacteria grown in LB with and without 10% PGRE using SEM. As shown in Fig. 2b–e, the bacteria grown in LB (Fig. 2b and c) had several flagella, whereas those grown in the presence of PGRE (Fig. 2d and e) exhibited few or no flagella. Next, we set out to evaluate whether the downregulation of fliC expression and corresponding drop in flagellin production that result from growth or exposure to PMs would impair bacterial motility.

, 2000) The epidemiological relationship was studied by REP-PCR

, 2000). The epidemiological relationship was studied by REP-PCR (Vila et al., 1996). Nalidixic acid susceptibility was tested using the disk-diffusion method following CLSI recommendations (CLSI, 2008). Data were statistically analyzed using the Fisher exact test due to the small size of the sample. We studied 331 vaginal samples (114 from pregnant and 217 from nonpregnant women from 16 to

50 years old) and 317 endocervical samples (271 and 46, respectively). Eighty-six (86/648, 13%) samples were positive for E. coli: 48 (15%) from pregnant and 38 (12%) from nonpregnant women. REP-PCR did not show any epidemiological relationship between isolates (data not shown). Table 2 summarizes the different virulence factors and the phylogenetic characteristics among E. coli strains in general Ceritinib purchase and stratified by pregnancy status. Phylogenetic group B2 was the most frequent among the strains (51%), followed

by groups D (34%), A (12%) and B1 (3%). Sixty percent of the strains from pregnant women were phylogenetic group B2 vs. 39% of those from nonpregnant women (P=0.043). The iroN, fyu, pap and iutA genes were the virulence factors found most frequently (57%, 53%, 51% and 41%, respectively). However, only the hly, cnf, pap and iroN genes occurred significantly 5-FU concentration more frequently when comparing the strains from pregnant women (48) with those from nonpregnant women (38) (Table 2). In contrast, the adhesin iha occurred more frequently among strains from nonpregnant women (17% vs. 39%, P=0.017). The iucD and iutA genes tended to be more frequent among strains from nonpregnant women (Table 2), but the differences were not statistically significant. No statistically significant differences were found in nalidixic acid susceptibility between E. coli strains collected from pregnant and nonpregnant women, although the strains from pregnant women presented a lower resistance to this antimicrobial agent than those from nonpregnant women. The comparison Phloretin between nalidixic acid-susceptible (67) and -resistant (19) strains showed

that those that were resistant presented hly, cnf1 and focG less frequently (Table 3). It is also of note that among nalidixic acid-susceptible strains, phylogenetic group B2 was significantly more frequent, confirming greater virulence. On the other hand, phylogenetic group D was the most frequent among nalidixic acid-resistant strains (Table 3). The predominant flora in the vagina consists of Lactobacillus and Streptococcus species; however, the presence of other bacteria such as E. coli may be very important, albeit not necessarily synonymous with infection. Vaginal E. coli may cause symptomatic infections and is associated with neonatal sepsis (Percival-Smith et al., 1983). These strains possess several virulence factors allowing vaginal and/or endocervical colonization. We analyzed the prevalence of E.

, 2008) Experiments performed with viable bacteria yielded the e

, 2008). Experiments performed with viable bacteria yielded the equivalent of 5 × 108S. aureus Cowan I from a suspension with an optical density (OD600 nm) of 1.0. Staphylococci were adjusted to an estimated concentration of 2 × 108 CFU mL−1 cell culture medium and kept at +4 °C until use.

Three FACS experiments were performed as previously described, on different days in duplicates, Selleckchem Dasatinib and up to 5000 invasion events were counted, unless described elsewhere. Staphylococcus aureus Cowan I and S. carnosus TM 300 were measured in the same experiment as a positive control and a negative control, respectively. The arbitrary value of FITC-stained bacteria, used as a surrogate for invasion of cells, was normalized to the positive control S. aureus Cowan I to display the relative invasiveness of the tested strains to the strongly invasive S. aureus Cowan I. Purified fibrinogen (plasminogen, von Willebrand-factor and fibronectin depleted; Enzyme Research Laboratories, Crizotinib manufacturer South Bend, IL) was coated to a 96-well microtiter as previously described (Szabados et al., 2011). For the fibronectin binding, a precoated microtiter plate was used (BD Biocoat™ Cellware Human Fibronectin; BD, Bedford, MA). The binding experiments were performed as previously described (Szabados et al., 2011). An OD550 nm

value of 0–0.06 was interpreted as negative, 0.07–0.15 as intermediately positive (+), 0.15–0.3 as positive (++), and > 0.3 as strongly positive (+++). Staphylococcus aureus Cowan I was used as positive control for fibrinogen and fibronectin binding. A sample without bacteria Protein kinase N1 and the S. carnosus TM 300 were used as negative controls. Bacteria (1 × 108) were washed with PBS and suspended in an estimated 1 μg mL−1 FITC and incubated for 30 min. Bacteria were washed three times with ice-cold PBS. Sulfo-NHS-LC-biotin (Pierce Biotechnology, Rockford, IL) was solved at a final concentration of 0.3 mg mL−1

in PBS as previously described (Agerer et al., 2004). Samples were washed three times with PBS, mounted with embedding medium ProLong® Gold (Invitrogen) in glass slides and sealed with nail polish. The glass slides were examined using confocal microscope Leica DM IRE2 (Leica, Solms, Germany). A suspension of human urinary bladder carcinoma cells 5637 from the FACS assay was used. The lysis step was omitted and cells were centrifuged gently (1000 g) for 60 s and transferred into 500 μL D-PBS (PAA) and fixed with 500 μL glutaraldehyde 2.5% as previously described. Only three of eight strains (Stlu 12, Stlu 50, and Stlu 108) showed binding to solid-phase fibrinogen (Fig. 1a)- as seen in previous results (Szabados et al., 2011). Four of eight strains (Stlu 30, Stlu 33, Stlu 36 and Stlu 108) showed binding to solid-phase fibronectin (Fig. 1b). One strain (Stlu 108) showed binding to immobilized fibrinogen and also to immobilized fibronectin.

[3] Few data exist on the use of JE-VC vaccine to boost immunity<

[3] Few data exist on the use of JE-VC vaccine to boost immunity

following a primary course of JE-MB. Both are derived from different viral strains, and in this case, follow-up serology indicated protective immunity after one dose of JE-VC. Previously, a primary series of JE-VC was recommended to all travelers regardless of prior vaccination history, but a recent study has demonstrated the efficacy of a single dose of JE-VC in JE-MB-primed travelers.[10] This would suggest that the viral strains Nakayama and SA14-14-2 are immunologically similar and elicit cross-reactive immune responses. This may underlie the allergic Selleckchem Oligomycin A reaction in this case. The similar nature of the reactions to both JE-MB/rabies and later JE-VC lead us to hypothesize

that the JE vaccine precipitated the allergic reaction in both vaccination schedules. Decline in antibody levels occurs with both vaccines after 1 year and booster doses may be needed in travelers with continued risk.[2] In the case we have reported, we will repeat serology, and if the risk benefit analysis favors a further booster dose we may consider experimental boosting intradermally at one-fifth of the normal dose with anti-histamine cover. This approach has been successful with egg-allergic yellow fever vaccine recipients.[11] Pirfenidone molecular weight JE-VC vaccine is associated with a lower risk of adverse events than JE-MC vaccines. We describe a case in which a similar allergic response occurred to both JE-MB and JE-VC vaccines. In the absence of identifiable allergogenic excipients, this may represent an allergy to the JE virus antigen. Cross-reactivity between the

JE-MB and the JE-VC vaccines remains poorly understood. While JE-VC undergoes post-marketing surveillance, we recommend vigilance and reporting of adverse reactions to improve the characterization of the safety profile of this new vaccine. The authors state that they have no conflicts of interest. “
“Background. Cebiche is a common dish in Latin America, Interleukin-3 receptor prepared using raw fish mixed with vegetables and marinated with lime juice. The acidity of the lime juice is commonly believed to destroy bacteria and render cebiche as safe to eat. Little data exist concerning rates of cebiche-associated gastroenteritis outbreaks, although these may be high given the popularity of the dish. Methods. We inoculated raw fish with Aeromonas hydrophila, Vibrio parahaemolyticus, and enterotoxigenic Escherichia coli to determine the effect of the cebiche preparation process on bacterial viability. Raw fish were exposed to a suspension of 1.0 × 108 colony-forming units (CFUs) of each organism in a 50-mL solution, prior to the addition of cebiche ingredients. A typical Peruvian cebiche recipe was used combining limes, onions, sweet potatoes, cilantro, and hot peppers marinated together for 30 minutes.

TraB is a hexameric pore-forming ATPase that resembles the chromo

TraB is a hexameric pore-forming ATPase that resembles the chromosome segregator protein FtsK ABT-199 order and translocates DNA by recognizing specific 8-bp repeats present in the plasmid clt locus. Mobilization of chromosomal genes does not require integration of the plasmid, because TraB also recognizes clt-like sequences distributed all over the chromosome. Mycelium-forming actinomycetes

do not divide by binary fission but grow by apical tip extension and undergo a complex life cycle ending in sporulation (Flardh & Buttner, 2009). They are well known for the production of antibiotics, a feature probably developed to inhibit competitors in the soil community (Allen et al., 2010). During evolution of the antibiotic biosynthetic gene clusters, they also evolved specific resistance

genes as a part of the cluster to protect themselves from their own compounds. Because a typical Streptomyces strain contains 10–20 different gene clusters for the production of antibiotics and other bioactive secondary metabolites (Bentley et al., 2002; Medema et al., 2011), streptomycetes form a huge reservoir of antibiotic resistance genes in the soil, which can be passed to other bacteria by horizontal gene transfer (D’Costa et al., 2006; Allen et al., 2010). Therefore, the antibiotic KU-57788 ic50 producers not only compete with other organisms by the production of antimicrobial compounds but they also provide resistance genes that can help others to survive. In Streptomyces and related actinomycetes, even small multi-copy plasmids of < 10 kb in size are normally self-transmissible and able to mobilize chromosomal resistance genes and auxotrophic markers (Kieser et al., 1982; Kataoka et al., 1991; Servin-Gonzalez et al., 1995). These plasmids are normally cryptic and do not confer phenotypic traits (Hopwood

& Kieser, 1993; Vogelmann et al., 2011a). Efficiency of transfer reaches nearly 100% and between 0.1% and 1% of MG-132 solubility dmso the transconjugants obtain chromosomal fragments during mating (Kieser et al., 1982). DNA transfer takes place only on solid surfaces in the early growth phase of the life cycle, when Streptomyces grows as substrate mycelium (Pettis & Cohen, 1996; Possoz et al., 2001). The transfer determinants of many Streptomyces plasmids were initially identified as killing functions (kilA, traB), which could only be subcloned in the presence of the corresponding killing override (korA, traR) region (Kendall & Cohen, 1987; Hagege et al., 1993; Reuther et al., 2006a). Probably due to the toxic effects of the transfer determinants, plasmid transfer is associated with the formation of so-called pock structures having a diameter of 1–3 mm. Pocks are formed when donor spores germinate on a lawn of a plasmid-free recipient. Pocks represent temporally retarded growth inhibition zones and indicate the area, where the recipient mycelium has obtained a plasmid by conjugation (Fig. 1).

TraB is a hexameric pore-forming ATPase that resembles the chromo

TraB is a hexameric pore-forming ATPase that resembles the chromosome segregator protein FtsK Belnacasan mw and translocates DNA by recognizing specific 8-bp repeats present in the plasmid clt locus. Mobilization of chromosomal genes does not require integration of the plasmid, because TraB also recognizes clt-like sequences distributed all over the chromosome. Mycelium-forming actinomycetes

do not divide by binary fission but grow by apical tip extension and undergo a complex life cycle ending in sporulation (Flardh & Buttner, 2009). They are well known for the production of antibiotics, a feature probably developed to inhibit competitors in the soil community (Allen et al., 2010). During evolution of the antibiotic biosynthetic gene clusters, they also evolved specific resistance

genes as a part of the cluster to protect themselves from their own compounds. Because a typical Streptomyces strain contains 10–20 different gene clusters for the production of antibiotics and other bioactive secondary metabolites (Bentley et al., 2002; Medema et al., 2011), streptomycetes form a huge reservoir of antibiotic resistance genes in the soil, which can be passed to other bacteria by horizontal gene transfer (D’Costa et al., 2006; Allen et al., 2010). Therefore, the antibiotic buy AZD2014 producers not only compete with other organisms by the production of antimicrobial compounds but they also provide resistance genes that can help others to survive. In Streptomyces and related actinomycetes, even small multi-copy plasmids of < 10 kb in size are normally self-transmissible and able to mobilize chromosomal resistance genes and auxotrophic markers (Kieser et al., 1982; Kataoka et al., 1991; Servin-Gonzalez et al., 1995). These plasmids are normally cryptic and do not confer phenotypic traits (Hopwood

& Kieser, 1993; Vogelmann et al., 2011a). Efficiency of transfer reaches nearly 100% and between 0.1% and 1% of Florfenicol the transconjugants obtain chromosomal fragments during mating (Kieser et al., 1982). DNA transfer takes place only on solid surfaces in the early growth phase of the life cycle, when Streptomyces grows as substrate mycelium (Pettis & Cohen, 1996; Possoz et al., 2001). The transfer determinants of many Streptomyces plasmids were initially identified as killing functions (kilA, traB), which could only be subcloned in the presence of the corresponding killing override (korA, traR) region (Kendall & Cohen, 1987; Hagege et al., 1993; Reuther et al., 2006a). Probably due to the toxic effects of the transfer determinants, plasmid transfer is associated with the formation of so-called pock structures having a diameter of 1–3 mm. Pocks are formed when donor spores germinate on a lawn of a plasmid-free recipient. Pocks represent temporally retarded growth inhibition zones and indicate the area, where the recipient mycelium has obtained a plasmid by conjugation (Fig. 1).

, 2001) This appearance has been well studied in higher organism

, 2001). This appearance has been well studied in higher organisms particularly in Insecta (Ghiradella, 1991; Vukusic et al., 2004;

Seago et al., 2009), Aves (Greenewalt et al., 1960; Prum & Torres, 2003; Doucet et al., 2006), and in fishes (Land, 1972; Lythgoe & Shand, 1989). Iridescence is also encountered in viruses (Williams & Smith, 1958) and in marine organisms such as ctenophore (Welch et al., 2006) and diatoms (Noyes et al., 2008). Iridescence ABT-888 in vivo has been poorly studied in the prokaryote kingdom. Both direct illumination and trans-illumination have been used to observe colonies’ iridescence on solid media (Pijper, 1923; Nogrady & Guérault, 1964; Zierdt, 1971). Recently (Kientz et al., 2012), a comparison of a wide range of bacterial strains

permitted to defined four classes of iridescence: rainbow-diffuse and rainbow-edge iridescences under trans-illumination and, metallic appearance and intense glitter-like iridescence under direct illumination. Cellulophaga lytica was the unique bacterium belonging to the latter class. As this type of iridescence occurred under direct natural light exposure, it was described ZD1839 as a more natural coloration effect. The visual appearance corresponds to sub-millimeter-sized centers of color of varying brightness distributed across the biofilm giving a glitter-like character. Iridescent green is the dominant color, but red and blue-violet are also observed at the colonies’ edges on classical marine

media. Though the physiology of C. lytica has never been thoroughly characterized, some microbiological features (Johansen et al., 1999) and genomic data (Pati et al., 2011) suggest that the bacterium is well adapted to extreme conditions. Moreover, C. lytica is frequently isolated from coastal shore. In this biotope, high variations of temperature, salinity, or light exposure are common. It is still unknown whether C. lytica’s iridescence can occur under such conditions, in vitro or in natural habitats. In the present work, we examine the effect of key abiotic factors on C. lytica’s iridescence. Several stress conditions that mimic the natural Flavopiridol (Alvocidib) biotope of the bacterium were preferentially employed. Unless otherwise specified, agar concentration was 1.5%. Ready-to-use media marine agar (MA), nutrient agar (NA), tryptic soy agar (TSA), and Luria–Bertani (LB) were purchased from Dutscher (France). Cytophaga agar (CYT ASW) and low nutrient (LN ASW) media were made with artificial seawater (ASW) Instant Ocean© (30 g L−1 in pure water). CYT ASW medium contained 1 g tryptone, 0.5 g yeast extract, 0.5 g CaCl2·2H2O, 0.5 g MgSO4·7H2O, and 15 g agar in 1 L of ASW (Johansen et al., 1999). Casein was replaced by tryptone because C. lytica does not degrade casein (Kientz et al., 2012). LN ASW medium only contained agar (15 g) in 1 L of ASW (Jensen et al., 1996).

These liver-specific cDNAs were ligated into the pMD18-T vector (

These liver-specific cDNAs were ligated into the pMD18-T vector (TaKaRa Biotech) and amplified by PCR. A total of 278 cDNA clones obtained after SCOTS were selected for Southern dot blot analysis. Thirty-six clones that did not hybridize with the probes from the normal culture, but had highly positive signals with the probes from rabbit livers after P. multocida infection (Fig. 1), were subjected to further sequence analysis. Selectively captured sequences RO4929097 were designated scs-L. All 36 cDNA clones corresponded to regions within the P. multocida

Pm70 genome (May et al., 2001), and 31 genes were identified. These 31 genes were divided into five functional groups: (1) 15 genes were involved in metabolism, including peptide and amino acid catabolism, pyrimidine biosynthesis, and energy metabolism; (2) four genes were involved in the construction of the cell wall, among them lpxB and pfhaB1/pfhaB2; (3) three genes were involved in the production

of transporters; (4) 6 transcriptional regulators were identified; (5) the remaining three genes had identity with genes of unknown function in P. multocida (Table 2). To investigate the SCOTS genes expressed by P. multocida C51-17 in infected rabbit livers, five genes (purF, lon, dnaB, ftsQ, and glpT) were selected randomly and validated by qRT-PCR. The expression levels of all five genes were upregulated in infected rabbit livers when compared with in vitro cultures, and 16S rRNA gene as internal control, changes ranging from 1.61-fold to 13.55-fold, respectively (Fig. 2). According to the functional Navitoclax clinical trial annotation, only very few scs-L clones were associated with the genes identified by STM in mice and/or chickens and in P. multocida recovered from the blood and liver tissue of chickens using the DNA microarray method. In the current study, most of the differentially expressed genes from P. multocida C51-17 identified by SCOTS analysis were involved in amino acid and

carbohydrate metabolism. This is because bacteria regulate and adapt their biosynthetic and metabolic pathways in the host to acquire necessary nutrients, including necessary amino acids and carbohydrates. In contrast, genes involved in certain biosynthetic pathways, such as pathways for the synthesis Dimethyl sulfoxide of aromatic amino acids and nucleotides, are generally attenuated. Such genes include an amidophosphoribosyltransferase encoded by scs-L6 which is involved in purine biosynthesis. Several of the genes involved in purine biosynthesis in P. multocida were upregulated in bacteria recovered from the blood of infected chickens, for example, purD, purF, purH, and purN (Boyce et al., 2002). In addition, purF and purN mutants of P. multocida have been identified by STM to be attenuated in mice and/or chickens because of the reduced ability of the mutants to replicate in vivo (Fuller et al., 2000; Harper et al., 2003).

Cat Cmpd) including ETBR (Son et al, 2003) Overexpression of t

Cat. Cmpd) including ETBR (Son et al., 2003). Overexpression of the Enterobacter cloacae sugE homolog

in E. coli generated cells with increased resistance to several QACs and ETBR (He et al., 2011). Overexpression of the Aeromonas molluscorum sugE homolog in E. coli generated resistance to ETBR, but not the QAC cetylpyridinium RG 7204 chloride (Cruz et al., 2013). Also, when the E. coli SugE protein was assembled in membrane mimics, it bound to ETBR with a Kd in the low micromolar range, which is consistent with a role in ETBR transport (Sikora & Turner, 2005). Thus, in this work, we directly tested the model that Dcm influences sugE expression and thereby affects SugE-mediated resistance to antibacterial compounds. The bacterial strains used in this study are shown in Table 1 (Baba et al., 2006; Militello et al., 2012); plasmids were a gift from Ashok VEGFR inhibitor Bhagwat (Sohail et al., 1990). The lack of 5-methylcytosine in the dcm knockout strain JW1944-2 has been previously reported (Militello et al., 2012). The absence of the sugE gene in JW5738-1 and the rpoS gene in JW5437-1 was confirmed by PCR (data not shown). Liquid bacterial cultures were grown at 37 °C at 250 r.p.m. in either Luria Broth (LB) or M9 minimal media containing 0.4% glucose (Difco). Ampicillin was added to liquid cultures containing

dcm plasmids at 25 μg mL−1. Solid cultures were grown at 37 °C in the same media containing 15 grams of agar per liter, and when necessary ampicillin was added at 50 μg mL−1. All experiments to assess the sensitivity of strains to antibacterial compounds were performed in minimal media containing glucose as many QACs precipitate in LB. Bacteria were grown in LB at 37 °C at 250 r.p.m. to early logarithmic phase (A600 nm of c. 0.45) and early stationary phase (A600 nm c. 3.0). Total RNA was isolated from

3–4 mL of bacteria cultures using the MasterPure RNA Isolation Ketotifen Kit (Epicentre). For 5-azacytidine experiments, the drug (Sigma-Aldrich) was dissolved in 1X phosphate-buffered saline (PBS), and PBS was added to untreated samples as a control. RNA quality was assessed using bioanalysis at the University of Rochester Genomics Research Center. Prior to reverse transcription, RNA was treated with RQ1 RNase-free DNase (Promega). One microgram of total RNA was used for reverse transcription using the New England BioLabs Protoscript kit with random primers. cDNA was used as a template for qPCR reactions on a Stratagene MX3000p machine. All reactions were run in triplicate or quadruplicate (technical replicates), and each experiment was performed 3–4 times (biological replicates). Data were normalized to the levels of malate dehydrogenase (mdh) using the ΔΔCt method (Livak & Schmittgen, 2001). The primer sequences are listed in Table S1.