The cereulide-producing B. cereus strain NVH 1257 was used for positive control. The Bacillus spp. strains were tested for their ability to produce cereulide under standard conditions, essentially as described by Andersson et al. (2004), with minor modifications. The cereulide-producing strain NVH 1257 was used as a positive control. The virulence properties of the various strains were assessed by comparing the killing effect, by injection into the haemocoel and buy LEE011 by oral force feeding. The tests were performed with G. mellonella last-instar larvae weighing about 200 mg, reared at the

INRA laboratory by free feeding on pollen and beeswax at 25 °C. The general protocols have been described earlier (Bouillaut et al., 2005). Briefly, both oral and haemocoel infections were performed with exponential growth phase bacteria

(OD600 nm≈1–2). The needed volume (≈1–3 mL) of bacterial Luria–Bertani culture was centrifuged at 20 000 g. for 5 min, and pellets were suspended in phosphate-buffered saline (PBS), pH 7, either alone (for haemocoel) or in Cry1C toxin diluted in PBS (0.3 mg mL−1) http://www.selleckchem.com/products/ch5424802.html for oral infection. A total of 300 μL suspension was prepared for each dose in order to infect 20 larvae with 10 μL of this suspension. For haemocoel infections, tested doses were from 5.0 × 103 to 1.4 × 104 bacteria per larva and oral infection was performed with 3–7 × 106 bacteria per larva. Cry1C toxin is necessary for sacrifice by oral infection because neither the toxin nor bacteria alone confer high mortality (Salamitou et al., 2000); meanwhile, the exact role of the synergistic effect of Cry1C toxin is not yet elucidated. Bacterial suspensions used for infection

experiments were quantified by plate counting for every experiment, as confirmation of estimated dose from measurements of OD600 nm before infection. Tests were repeated at least three times. Control larvae were injected with PBS, pH 7.4, or PBS and Cry1C for oral infection. Infected larvae were kept at 15 and 37 °C [five larvae per Petri-dish (5 cm diameter) without food] and mortality was recorded at 24, 48, 66, 96 and 120 h postinfection. Mortality this website analyses comparing temperature, time, strains and route of infection were carried out using regression analysis. The dataset consisted of 505 observations from two species and seven strains (four B. weihenstephanensis and three B. cereus strains). Linear regression was performed with mortality as the response variable and categorical factors: temperature (low=15 °C, high=37 °C), species (B. cereus, B. weihenstephanensis), hours after infection (numerical) and infection route (haemocoel=haemocoel injection, oral=oral force feeding) as predictor variables. To account for the inherent time aspect of mortality, two interaction terms were added to model interconnectivity between hours after infection and both infection route and temperature.

, 2008). Microcystis aeruginosa is a species of freshwater cyanobacteria that can form harmful algal blooms that are of economic and ecological importance. Cells of this species usually are organized into colonies as a biofilm-like sheath. As QS has proven to be an

important factor in the control of biofilm architecture (Davies et al., 1998; Huber et al., 2001; Lynch et al., 2002), is it possible that M. aeruginosa Compound Library has a QS regulation system? This study aims at detecting whether M. aeruginosa has a QS phenomenon by bioreporter assay and liquid chromatography–mass spectrometry (LC-MS) analysis. The ecological role of QS in M. aeruginosa has been discussed. The understanding of the role of QS in the regulation of M. aeruginosa growth and environmental adaptation may http://www.selleckchem.com/epigenetic-reader-domain.html be useful in developing new strategies to control bloom formation and outbreak in freshwater ecosystems. An axenic strain of M. aeruginosa PCC-7820, which was kindly supplied by Dr Pengfu Li at Nanjing University, China, was grown and maintained in a growth chamber at 28 °C day−1 and 22 °C night−1, with a 14 : 10 h light–dark regime under an illumination of 3000 lx (Mu et al., 2007).

For determination of growth rate and AHLs, a 200-mL culture of M. aeruginosa at the exponential phase was harvested under sterile conditions and centrifuged at 8000 g for 10 min. The pellet was resuspended in 500 mL of fresh sterile BG11 medium to a final cell concentration of 1 × 106 mL−1 in 1-L flasks. The flasks were incubated in a growth chamber as described above. The cultures were sampled at 10, 20, 30, and 40 days after inoculation for growth measurement at 680 nm with an ultraviolet/visible spectrophotometer (PGENERAL, China), bioreporter assay, and AHLs detection with LC-MS analysis. Each sample was replicated for three times. AHLs were extracted from the culture in accordance with reported literature CHIR-99021 research buy (Yates et al., 2002) with some modifications. Three hundred milliliter of algal culture was centrifuged at 8000 g for 10 min to remove cells, and then the supernatant was adjusted to pH 2 and stored at 4 °C for 16 h. After that, the sample was extracted three times

with 150 mL of dichloromethane. The combined dichloromethane extracts were dried by anhydrous MgSO4 and evaporated to dry. The resulting residue was dissolved in 1 mL of HPLC-grade methanol, sealed, and stored at −20 °C until they were required. Three bioreporters were used to test whether M. aeruginosa can produce a QS signal. Agrobacterium tumefaciens (AT) bioassay strain KYC55 (pJZ410) (pJZ372) (pJZ384), which was kindly supplied by Dr Jun Zhu at Nanjing Agricultural University and was cultivated in AT medium supplemented with appropriate antibiotics (Zhu et al., 2003). The dichloromethane extracts were added to AT medium containing the AHL monitor strain A. tumefaciens KYC55 and tested for β-galactosidase activity (Miller, 1972).

, 1996; Bearson et al., 1998). In addition, Bearson et al. (2006) have recently identified the phoP, rpoS, fur and pnp genes as being involved Selleck Natural Product Library in protecting serovar Typhimurium against

exposure to lactic acid. Our group has previously reported that, at the concentration present in Lactobacillus CFCSs, lactic acid plays no role in the anti-Salmonella activities of L. johnsonii NCC533, L. rhamnosus GG, Lactobacillus casei Shirota YT9029, L. casei DN-114 001, L. rhamnosus GR1 or Lactobacillus acidophilus LB strains (Bernet et al., 1994; Coconnier et al., 1997, 2000; Hudault et al., 1997; Lievin-Le Moal et al., 2002; Fayol-Messaoudi et al., 2005). Here, we found that at the concentration present in Lactobacillus CFCS lactic acid alone plays no role in the killing effect of L. johnsonii NCC533 or vaginal L. gasseri KS120.1 against two other pathogens: UPEC CFT073 and G. vaginalis DSM 4944 strains. The observation that the killing activity of lactic acid develops at high concentrations is consistent with Makras et al. (2006), who

have shown that activities of lactic acid started at 100 mM. In contrast, based on the fact that the activity of L. rhamnosus GG CFCS against the growth and survival of serovar Typhimurium disappears after dialysis eliminating lactic acid, whereas it is still present after dialysis against a lactic acid solution, De Keersmaecker et al. (2006) have concluded that lactic acid is responsible for the activity of L. rhamnosus GG. However, eliminating Ivacaftor mw lactic acid could have an effect on some other molecule(s) secreted by Lactobacillus that kill pathogens in co-operation with lactic acid. Consistent with this hypothesis, Niku-Paavola et al. (1999) have proposed that compounds secreted by Lactobacillus plantarum act synergistically with lactic acid, and Makras et al. (2006) observed that L. johnsonii NCC533 CFCS was effective against serovar Typhimurium by unknown inhibitory substance(s) that are only active in the presence of lactic acid. These nonlactic acid, heat-resistant anti-Salmonella molecule(s) present in the CFCSs of probiotic Lactobacillus strains have not yet been identified (McGroarty & Reid, 1988; Bernet-Camard

et al., 1997; Coconnier et al., 1997; Hudault et al., 1997; Ocana et al., 1999; Aroutcheva et al., Protirelin 2001b; van de Guchte et al., 2001; Sgouras et al., 2004, 2005; Fayol-Messaoudi et al., 2005, 2007; Atassi et al., 2006a). It has already been suggested that pyroglutamic acid may be responsible for the antimicrobial activity of L. rhamnosus GG and L. casei strains LC-10 and LB1931 (Silva et al., 1987; Huttunen et al., 1995; Yang et al., 1997), but it has been found to be intrinsically present in MRS medium and it does not increase during bacterial growth (De Keersmaecker et al., 2006). Adding increasing concentrations of acetic or formic acid to MRS medium has no effect on the viability of serovar Typhimurium (De Keersmaecker et al., 2006).

In addition, we performed receiver operating characteristic (ROC) analysis to assess the accuracy of EBV DNA load as a predictive marker of lymphoma [as estimated by the area under the curve (AUC)]. The optimal cut-off value of EBV DNA load for differentiating patients at risk of lymphoma from other patients was determined as the point of the ROC curve with the shortest distance to the 100/100% sensitivity/specificity angle (upper left corner) [i.e. lowest value for the term (1 – sensitivity)2 + (1 – specificity)2, assuming equal costs of false positive and false negative results]. The sensitivity, specificity and OR for developing lymphoma were then provided for the identified

cut-off point. All statistical analyses were performed using sas 9.2 (SAS Institute Inc., Selleck Tacrolimus Cary, NC). EBV DNA was positive in PBMC samples from all lymphoma cases collected over the 3 years preceding the PI3K Inhibitor Library mw diagnosis, while it was positive in 78 to 81% of samples from controls collected during the same period of time (Fig. 1a). Interestingly, eight of the 37 controls had undetectable EBV loads in PBMC1 while none of the 20 cases had undetectable EBV loads in PBMC1 (P = 0.04) (Fig. 1a). EBV load in PBMCs measured a median of 10 months before diagnosis was associated with an increased risk of B lymphoma [OR 2.48 (95% CI 1.16; 5.32) per increase in EBV

load of 1 log copies/106 PBMCs] (Table 2). Similar results were obtained when the OR was adjusted for CD4 cell count nadir instead of CD4 cell count at sample date (OR 2.33; 95% CI 1.12; 4.81). The OR associated with EBV load quantified in a sample collected earlier (median of 24 months before diagnosis) was of borderline significance, probably because of a smaller number of PBMC samples available for that period. When we restricted the analysis to the patients with a CD4 cell count > 300 cells/μL, the median EBV load was still lower in controls (median 2.69) than in cases (median 3.63), mainly because four out of 14 controls had undetectable EBV load vs. none of

seven cases. EBV DNA was more often detectable (> to EBV PCR threshold value or detectable but < to EBV PCR threshold value) in sera from cases than in sera from controls (with 24 to 25% positive detection in the last 3 years for cases vs. 8 to 10.5% for Flucloronide controls) (Fig. 1b); however, this difference was significant only for serum 2 samples (collected a median of 15.3 months before the diagnosis of lymphoma) (Table 2). EBV DNA was positive in PBMC samples from all tested cases during the 3 years preceding the diagnosis of cerebral lymphoma, but it was also positive in 87 to 94% of controls during the same period (Fig. 2a). EBV DNA was not more often detectable (> threshold or detectable < threshold) in sera from cases than in sera from controls (with 0 to 23.1% positive detection in the last 3 years for cases vs. 4.8 to 12% for controls) (Fig. 2b).

Figure 3 shows that there was a gradual decrease in the ThyA level during the stationary growth phase to 40% of that in the LDK378 in vitro late-exponential phase cells in LB medium (Fig. 3a and c). Conversely, ThyX was maintained at the same

level in both the late-exponential and stationary phase cells (Fig. 3b and c), indicating that the levels of ThyA and ThyX were regulated by different mechanisms and that ThyX could play a role in the stationary growth phase of C. glutamicum. The thyX gene is located on an operon with dapB and dapA, and these genes are transcribed as a single unit, dapB-thyX-dapA (Park et al., 2010). Two putative promoter regions of dapB were identified by primer extension analyses (Pátek et al., 1996), and one of the promoters or both (p1-dapB and/or p2-dapB) might be recognized by SigB. SigB was shown to be induced during the transition from the exponential to the stationary growth phase (Larisch et al., 2007; Pátek & Nešvera, 2011).

To examine whether the level of ThyX was regulated by SigB, a ΔsigB strain was constructed by allelic replacement using a sucrose counter-selectable suicide plasmid. Deletion of sigB was confirmed Z-VAD-FMK nmr by PCR amplification of the sigB region, with primers binding upstream and downstream of sigB. A 1329-bp fragment containing intact sigB was seen in the wild-type strain, and a 324-bp fragment was seen in the mutant strain (Fig. 1b). The transcriptional activity of the dapB-thyX promoter region was quantified in the wild-type and ΔsigB strain KH4 after the

introduction of plasmid pMTXL1. The thyX promoter in the ΔsigB strain revealed about 25% of the activity shown in the parental wild-type strain (Fig. 4a). Thus, SigB was shown to be necessary for the induction of thyX. The levels of ThyA or ThyX in the wild-type, KH4, and KH5 strains of C. glutamicum were analyzed by immunoblotting using antiserum against ThyA or ThyX, respectively. Whereas the level of ThyA in the ΔsigB strain was comparable to that of the parental wild-type, the level of ThyX was diminished significantly in the deletion mutant (Fig. 4b). Complementation of the ΔsigB mutation was performed with a plasmid containing wild-type sigB, including its putative promoter region. Western blotting analysis revealed that expression Rho of functional sigB in the complemented strain restored the accumulation of ThyX to nearly wild-type levels (Fig. 4b). This result confirmed that SigB is necessary for maintenance of the level of ThyX during transition into the stationary growth phase. To investigate the role of the sigma factor SigB on sensitivity to a DHFR inhibitor, WR99210-HCl, wild-type, KH4, and KH5 strains grown to log-phase were inoculated into MCGC minimal medium containing isocitrate and glucose with 3 µM WR99210-HCl. Growth was monitored for 36 h, and the KH4 strain appeared to be sensitive to WR99210-HCl.

To describe the dental characteristics of parents of children with non-syndromic cleft lip ± palate. Unaffected parents of Australian children with a cleft of the lip ± palate underwent dental examination including radiographs, photographs, and impressions.

Dental anomalies were identified. Data were available on 101 parents (49 males, 52 females). Fifty-one participants had at least one dental anomaly. Twelve (11.8%) individuals had congenital absence of teeth, with seven missing multiple teeth. The tooth most commonly missing was the upper right lateral incisor. Five subjects (4.9%) had microdontia (upper lateral incisor most commonly affected). PARP inhibitor Four subjects (4.0%) had supernumerary teeth. Enamel defects were present in 27 (26.7%) cases with the incisors

(46.8%) followed by premolars (24.2%) most affected. This study supports previous work suggesting that ‘unaffected’ parents of children with clefts of the lip ± palate may present with dental anomalies. “
“International Journal of Paediatric Dentistry 2013; 23: 56–63 Objective.  To compare clinical and radiographic outcomes of pulpotomy treatment using calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA) in carious-exposed vital immature permanent first molars. Design.  Fifty-one immature molars with clinical carious exposure with symptomatic/asymptomatic pulpitis met the inclusion criteria and randomly assigned to one of the treatment groups (CEM [26 teeth; 59 roots], MTA [25 teeth; 59 roots]). After performing pulpotomy and covering the radicular pulps this website with the biomaterials, all teeth were permanently

restored. Blinded clinical and radiographic evaluations were performed at 6 and 12 months after operation for signs of success or failure. Radiographs were evaluated for complete/partial apical closure. The data were analysed using chi-square test and generalized estimating equation (GEE) model. Results.  There was no significant difference at the baseline between the two experimental groups. fantofarone All available cases (49 teeth) showed pulp survival and signs of continuous root development after 12 months. Overall, complete apical closure (apexogenesis) occurred in 76.8% and 73.8% of radiographically interpreted roots in CEM cement and MTA groups, respectively. There was no statistical difference in terms of radiographic outcomes between two groups. Conclusions.  Calcium-enriched mixture cement and MTA showed similar performance in pulpotomy of immature caries-exposed permanent molars. “
“International Journal of Paediatric Dentistry 2012; 22: 342–348 Background.  Streptococcus mutans and Streptococcus sobrinus are known to be associated with dental caries in humans. Aim.  We used a polymerase chain reaction method to detect S. mutans and S. sobrinus in 128 Japanese schoolchildren and then compared their presence with the dental caries experience. Design.

While current

initiatives involve roles and practice developments of rural healthcare providers, there is potential to further enhance medication services in rural areas. The review has also shown the value of pharmacy along the medication pathway, and there is potential to better involve pharmacy to provide support mechanisms and/or medication consultation services. The Authors declare that they have no conflicts of interest to disclose. This work was supported by the Pharmacists Board of Queensland Pharmacy College Trust (grant number 2010000973). The authors EGFR inhibitor gratefully acknowledge technical assistance from Victoria Jarvis, BPharm MPS. “
“Objectives  To identify the type and frequency of services provided through community pharmacies in the United Arab Emirates (UAE). Methods  A survey was conducted using an anonymous questionnaire distributed by hand to 700 community pharmacies. Items included information about the pharmacists and pharmacies, type of products sold, type and extent of enhanced Apoptosis Compound Library nmr services provided and perceived barriers to providing these services.

Key findings  Most pharmacies provided a wide range of medicinal and non-medicinal products. The frequency with which services were provided was assessed on a scale of 1 (never) to 5 (always). Enhanced professional services were not provided to a large extent in most pharmacies. Fewer than one-third (29%) reported they always supplied printed information to patients (mean = 3.37, 95% confidence interval = 3.23–3.52); fewer than one-third (28%) counselled patients on a regular basis STK38 (3.25, 3.09–3.40); nearly two-thirds (62%) reported monitoring patients’ adherence to therapy at least sometimes (2.96, 2.81–3.10). Most pharmacies (92%) in the UAE did not routinely keep patient records (2.09, 1.96–2.32). While just over a quarter of respondents claimed that they always reported medication errors (27%) and adverse drug reactions (28%), these activities were not often performed in around 40% of pharmacies. Conclusions  This is the first study to explore the type and extent of professional services

provided through community pharmacies in the UAE and provides baseline data critical to inform the development of strategies to improve the quality of community pharmacy services. “
“J. Waterfield De Montfort University, Leicester, UK To determine how ‘pharmacy knowledge’ is viewed by pharmacy educators There is a distinct contrast in how knowledge is defined between pharmaceutical scientists and pharmacy practitioners Theoretical insights into how pharmacy knowledge is utilised is vital for the ongoing development of the MPharm curriculum With the increasing emphasis on a more practice-based, integrated MPharm curriculum it is important to determine how pharmacy knowledge is viewed by different educators within the pharmacy education field.

3% of all missed doses were not being recorded as per hospital policy and therefore identified as an area for improvement.2 A vital element of service improvement is to have reliable and accurate data,

therefore lack of good data for omitted doses made service MDX-1106 improvement in this area more challenging. The Trust strategy to improve data is the introduction of the EPMA system, where the recording of the reasons for missed doses is mandatory and patients’ allergy must be entered onto the system before a drug can be prescribed. Forty paper drug charts (pre EPMA, November 2012) and 40 electronic drug charts (post EPMA, January 2013) on the Paediatric wards were randomly selected for data collection. Prior to EPMA, data were collected from the drug charts of patients whom had been receiving medication for over 24 hours. Medication prescribed regularly and medical and nursing notes were screened for comments indicating a reason for a medication omission. A blank recording panel on the paper drug chart in place of nurse initials indicated an omitted medicine. The data post EPMA implementation was obtained as an electronic spread sheet identifying patients’ allergy status and omitted doses of medication with reasons for omissions. The number of recorded medication omissions and allergy status were collected by a Pre-registration pharmacist

and collated onto an Excel spread BIRB 796 sheet to compare the pre and post implementation data. This audit was viewed as service improvement performed to meet specific local needs and ethics approval was not sought. Following the implementation of the EPMA system 100% (40/40) patients had their allergy recorded prior to having any medication prescribed, this compares to 90% (36/40) prior to EPMA implementation.

A marked reduction in the number of blank administration boxes were seen post EPMA Morin Hydrate implementation. Prior to EPMA system, 64.6% of medication administration boxes were left blank and post implementation only 3.4% of administration boxes were left blank. The introduction of EPMA system into Paediatrics has improved two important patient safety issues. With the wider roll out the EPMA system it is expected that other patient safety incidents regarding the prescribing and administration of medicines will decrease. For those ward areas that are live with EPMA a webpage has been developed that highlights all missed doses over the last 7 days so the appropriate staff can identify missed dosing issues in a timely manner. One limitation of this audit is the small sample size due mainly to the exclusion of patients who had been in hospital for less than 24 hours. 1. National Patient Safety Agency. Safety in doses; medication safety incidents in the NHS. 2007. 2. Royal Cornwall Hospital Trust. Delayed and Omitted Doses of Medicines Procedure. June 2011.

Trials containing voluntary electromyographic activity were excluded from further analysis. The effect of the attention locus (baseline, attention to hand, visual attention) on MEP amplitudes (series 1) was examined using repeated-measures anova with factors of Condition and ISI. For SICI and ICF, separate anova s with Condition and ISI as a repeat factor were analysed. More detailed information is given

in the Results. If appropriate, correction for multiple comparisons was used. For all experiments significance was set at P < 0.05. The behavioural results showed similar values for the visual and the attention-to-hand tasks (correct answers: visual attention 87.77 ± 6.5%; this website attention to hand, cutaneous stimulation above

the FDI muscle area 92.48 ± 1.7%; attention to hand, cutaneous stimulation above ADM area 93.32 ± 2.0%), indicating similar difficulty SB203580 purchase for the two tasks and suggestive of similar levels of attentional demand. Figure 3(A) shows the MEP amplitude in the three blocks of trials (no attention, attention to hand, visual attention) as the difference between the two attention blocks and the no-attention block. An anova on the MEP amplitudes (no attention, 1.2 ± 0.1 mV; attention to hand, 0.87 ± 0.3 mV; visual attention, 1.87 ± 0.2 mV) revealed a significant effect of Condition (F2,22 = 23.67, P < 0.001). Post-hoc analysis confirmed that visual attention significantly increased the MEP size compared with baseline

(P < 0.001) and compared with attention to the hand (P < 0.005). Attention to the hand (at this location of the stimulus, i.e. the dorsum manum) did not significantly change the MEP size compared with baseline, although there was a trend (P = 0.06) towards suppression (Fig. 3). There was no difference in any condition between SICI measured at an ISI of 2 or 3 ms. Figure 3(B) shows the mean SICI in the three conditions as the difference between the two attention tasks and the baseline task, Figure 4 shows corresponding Methane monooxygenase absolute values. Two-way anova on the amount of SICI (in % unconditioned test MEP) (no attention: 2 ms, 54.1 ± 8.6; 3 ms, 62.9 ± 13.8; attention to hand: 2 ms, 62.1 ± 15.2; 3 ms, 59.5 ± 12.4; visual attention: 2 ms, 76.5 ± 14.3; 3 ms, 78.1 ± 12.4) revealed a significant main effect of Condition (F2,22 = 4.24, P < 0.05), no significant effect of ISI (F1,11 = 0.06, P > 0.5) and no significant interaction of both (F2,22 = 0.43, P > 0.5). Post-hoc analysis showed that SICI was less effective during visual attention both when compared with the baseline task (P < 0.05) and the attention-to-hand task (P < 0.05). There was no difference in the amounts of ICF (in % unconditioned test MEP) at the two ISIs (no attention: 12 ms, 167.5 ± 23.5; 15 ms, 163.2 ± 20.8; attention to hand: 12 ms, 149.0 ± 14.1; 15 ms, 146.2 ± 18.4; visual attention: 12 ms, 159.1 ± 24.1; 15 ms, 137.5 ± 22.1).

, 2011). The phylogenetic composition of the extracted DNA in relation

to the original bacterial community has however received less attention. One major problem in assessing this is the fact that it is very difficult to know the true community composition. In two recent studies, it was shown that a mechanical bead-beating step during cell lysis resulted in increased complexity of extracted DNA as evidenced by an increased number of distinct bands in PCR-DGGE profiles (Ariefdjohan et al., 2010; Smith et al., 2011). The fact that different extraction procedures performed on the same fecal sample may lead to different estimations of the bacterial community composition is not surprising, but may well be disturbing for comparisons between separate studies. Within a study, it is most probable that the same DNA Pembrolizumab price extraction method be used throughout; however, other parameters that may affect extraction, such as storage conditions of fecal samples, may vary. It is for instance common practice, mainly for practical reasons, to freeze fecal samples immediately after sampling and then collectively extract the DNA and perform downstream analysis such as sequencing or qPCR, at some later stage (Mariat et al., 2009; Santacruz et al., 2009). In this study, the effect of freezing fecal samples prior to DNA extraction was evaluated for alterations in DNA

recovery and bacterial community composition as determined by downstream quantitative PCR analysis. Fecal samples were obtained from three healthy adult volunteers (two women, one man), homogenized thoroughly in four volumes diluent (0.85% NaCl, 0.1% peptone), selleck compound centrifuged at 300 g for 2 min to remove large debris, and finally 0.5 mL of aliquots (average 8 mg dry weight) were pelleted at 10 000 g for 5 min (Fig. 1). Extraction of DNA was performed immediately with three different extraction methods (five replicates), or samples were frozen at −20 °C for 53 ± 5 days (F) prior to extraction.

Methods used for DNA extraction were M: PowerSoil® DNA Isolation kit (MO BIO Laboratories, Carlsbad), Q: QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), and B: Modified QIAamp DNA Stool Mini Kit extraction procedure with the incorporation of a bead-beating step to potentially improve Anacetrapib cell lysis (Leser et al., 2000). Briefly, bead-beating was performed by adding 500 μL autoclaved 0.1 mm zirconia silica beads (Biospec Products Inc., Bartlesville, OK) and 30 μL of 10% sodium dodecyl sulfate and processing for 4 min. at 30 cycles per second on a Mixer Mill MM 300 (Retsch GmbH, Haan, Germany). Extractions were performed as directed by the suppliers with minor modifications, including standardized initial sample size and elution in 200 μL, 10 mM Tris, to allow better comparison of the methods. For extractions with method M, bacterial cells were treated in a Mixer Mill MM 300 (4 min at 30 cycles per second) and not the suggested Vortex adaptor.