, 2010). To date, Fe(II)-dependent or -enhanced growth has been shown only for a handful of freshwater isolates including species from the genera Gallionella and Sideroxydans (Hallbeck & Pederson, 1991; Emerson & Moyer, 1997; Weiss et al., 2007). Since the known FeOB are phylogenetically and physiologically diverse and the functional genes unique to Fe(II) oxidation are unknown, the use of nonculture-based, molecular methods to study FeOB ecology and distribution can be problematic. It therefore remains critical to further our knowledge of FeOB using enrichment and isolation techniques. The genus Dechlorospirillum has been primarily described in the literature

as a perchlorate and nitrate reducer (Coates, 1999; Bender et al., 2004; Bardiya & Bae, 2008), and Fe(II)-oxidation-dependent growth of this genus has not been demonstrated previously. The objective Selleck Tanespimycin of our studies was to determine whether a Dechlorospirillum sp. isolated from an Fe(II)-oxidizing, microbial mat is involved in and benefits from microaerophilic Fe(II) oxidation in gradient cultures. The inoculum consisted of sediment and microbial mat samples collected in June 2007 from a

portion of Jackson Creek (Bloomington, IN) fed by an iron-rich groundwater spring. In addition to irregular mats several centimeter thick on the creek bed, the creek also contained orange, bulbous, and filamentous formations of up to 20 cm diameter. Apoptosis inhibitor Under microscopic examination, we found that these formations primarily consist of both iron (oxy)hydroxide precipitates and mostly empty, Leptothrix-like sheaths. In addition to the sheaths, large numbers of other bacteria were observed including occasional spiral stalks characteristic of Gallionella. The pH of the site water was 6.6 on the day

of inoculum collection and typically ranges from 5.8 to 6.8. During the period that samples were obtained, the spring water typically contained 0.36–1.8 mM Fe2+, 0.02–0.18 mM NO3−, and approximately 2 mg L−1 dissolved organic carbon. Samples of the flocculent mat and sediment were collected in sterile bottles, returned to the laboratory, and used to inoculate gradient-culture bottles on the day of collection. Opposing-gradient-culture Protein kinase N1 systems, inoculation procedures, and enrichment transfers were similar to those described elsewhere (Emerson & Moyer, 1997; Sobolev & Roden, 2001). Initially, we used 250- or 40-mL screw-cap bottles containing a lower layer of 50 mM FeCl2, stabilized by 2% Difco noble agar (Becton, Dickinson and Company, MD) and buffered at pH 7 by 20 mM 1,4-piperazinediethanesulfonic acid (PIPES). The upper layer consisted of 0.5% noble agar, 30 mM NaHCO3, 10 mM NH4Cl, 1 mM KH2PO4, 5 mL L−1 vitamin solution (Strąpoćet al., 2008), and 2.5 mL L−1 trace mineral solution (Strąpoćet al., 2008).

5). These results suggested that the cell death process may not be associated with activation of inflammasomes, but rather that IL-1β and ATP are released from damaged cells. Alternatively, oxidative stress may contribute to the cell death, because ROS inhibitor reduced the cell death of macrophages (Fig. 5). ROS generated from damaged mitochondoria are known to induce cell death in various ways (Ott et al.,

2007). In this regard, several oral streptococcal species including S. sanguinis are known to produce hydrogen peroxide (Chen et al., 2011). This bacterial product is a possible candidate for click here the virulence factor that mediates cellular damage in macrophages, because Streptococcus gordonii, another oral streptococcus, is reported to induce cell death of endothelial cells by peroxidogenesis (Stinson

et al., 2003). Our preliminary study suggested that the concentrations of hydrogen peroxide in the culture supernatants of S. sanguinis were <5 μM under the conditions of the infection assay, although its effect on macrophages was unknown. The involvement of hydrogen peroxide produced by S. sanguinis in the cell death of infected macrophages should be investigated further. To evaluate the molecular mechanisms underlying S. sanguinis-induced cell death, further study on the mitochondorial dysfunction induced by this microorganism will be required. This work was supported in part by Grants-in-Aid for Scientific Research (A) (#19209063), (B) (#20390465, #20390531) and (C) (#20592398), and Grants-in-Aid for Young Scientists (B) (#21792069,

#21791786) from the Japan Gefitinib in vitro Society for the Promotion of Science. We thank Dr M. Killian for providing 4-Aminobutyrate aminotransferase S. sanguinis strain SK36. “
“Ophiobolins are sesterterpene-type phytotoxins produced by fungi belonging mainly to the genus Bipolaris. In this study, the antifungal effect of ophiobolins A and B on different zygomycetes has been examined. Depending on the zygomycete tested, MIC values of 3.175–50 μg mL–1 were found for ophiobolin A and 25–50 μg mL–1 for ophiobolin B. Ophiobolin A inhibited sporangiospore germination of Mucor circinelloides and caused morphological changes; the fungus formed degenerated, thick or swollen cells with septa. Cytoplasm effusions from the damaged cells were also observed. Fluorescence microscopy after annexin and propidium iodide staining of the treated cells suggested that the drug induced an apoptosis-like cell death process in the fungus. Ophiobolins are secondary metabolites of certain fungi belonging to the genera Bipolaris, Drechslera, Cephalosporium and Aspergillus (Au et al., 2000a). These sesterterpene-type compounds (C25) are characterized by a unique tricyclic chemical structure (Fig. 1). More than 25 ophiobolin analogues have been described (Au et al., 2000a; Wei et al., 2004; Evidente et al., 2006) and various biological actions have been attributed to them, such as phytotoxic (Au et al.

In general, ITPs complained about their heavy workload, long working hours and lack of support from their employers. Specifically, EEA pharmacists in most cases felt excluded from the professional network and sensed colleagues saw them as ‘foreigners’ while some non-EEA pharmacists had to deal with a level of hostility from patients. This novel research provides a foundation for future work on ITPs in GB and could assist employers to better target Tacrolimus manufacturer their efforts in development of standards to support the working experiences of ITPs in GB. “
“Objectives The aim of the study was to assess and improve first-year student pharmacists’

satisfaction and learning experience in a Student-Run Free Medical Clinic

Obeticholic Acid datasheet Project (SFMCP) providing medical care to an underserved population. Methods Two consecutive classes of first-year student pharmacists at the University of California San Diego (UCSD) Skaggs School of Pharmacy and Pharmaceutical Sciences participated in an Introductory Pharmacy Practice Experience (IPPE) at the UCSD SFMCP. This IPPE involved two inter-professional evening free clinics which provide medical care to an underserved population and opportunities for healthcare professional training and service. Year 1 students completed a self-assessment survey instrument and year 2 students completed the survey instrument plus a new competency checklist tool. Average scores from the self-assessment survey instrument were compared between years 1 and 2. Key findings Initial survey results showed that students felt the SFMCP was worthwhile; however, they did not experience enough involvement in the patient assistance programme or non-pharmacy-related clinic activities. After the competency checklist tool implementation, overall student Aldehyde dehydrogenase pharmacist satisfaction of the SFMCP IPPE remained high (88%), participation in identified weak areas improved and students agreed that the tool helped focus

their clinic experience. Conclusions Areas of improvement were identified with the survey instrument and the competency checklist tool increased achievement of learning objectives. Overall, student pharmacists felt the SFMCP IPPE was a good learning experience. Practising pharmacists can employ these or similar tools in specific practice settings, to evaluate and help ensure that student pharmacists or interns are achieving applicable learning objectives. “
“To explore older people’s opinions of current community pharmacy provision and identify potential areas for improvement. A pilot focus group was conducted to finalise the topic areas for discussion. Three focus groups and three small group interviews were held with a total of 25 people aged over 65 years. A purposive sampling approach was used to maximise variation in likely responses. All focus group discussions were transcribed and analysed for emerging themes.

, 1987; Haug & Eggers, 1991). It is now believed that cell numbers in the frontal cortex are preserved through aging in humans (Haug et al., 1981, 1984; Freeman et al., 2008). Similar conclusions have been drawn for frontal areas in nonhuman primates (Peters et al., 1996, 1998a; Smith et al., 2004), with the exception of prefrontal area 8A, a region of the dorsolateral PFC, which was shown

to have a significant decline in Nissl-stained neurons (Smith et al., 2004). In rodents the cell counting results are conflicting. One group reports decreases in neuron numbers in the dorsal PFC areas but preservation in the ventral PFC areas (Stranahan et al., 2012), and another found the opposite, with cell loss in the ventral PFC and preservation in dorsal PFC (Yates et al., 2008). Because the same rat strain was utilized, Stranahan et al. (2012) suggest that different learn more delineation of brain structures high throughput screening compounds during counting could explain the disparate findings. Nonetheless, the current view is that the cell numbers in the PFC are reasonably well preserved during aging, although there may be focal points of cell loss in nonhuman primates and rodents. In line with the overall reduction in frontal lobe volume mentioned above, age-related decreases in gray matter volumes and cortical

thickness have been reported in humans (Haug & Eggers, 1991; Raz et al., 1997, 2005; Good et al., 2001; Tisserand et al., 2002; Salat et al., 2009; Bergfield et al., 2010; Giorgio et al., 2010; Thambisetty et al., 2010; Burzynska et al., 2012; Kalpouzos et al., 2012), nonhuman primates (Alexander et al., 2008; Shamy et al., 2011; Fig. 2B) and rats (Alexander et al., 2011). However, an earlier stereological study performed using Nissl-stained slices from monkeys reported a general preservation of area 46 (O’Donnell et al., 1999), which is in contrast with the findings from MRI studies presented above. These differences may be the result Fludarabine of the research method employed or may be caused by inter-individual variability of age effects on this part of the brain. Nonetheless,

the changes in volume of the dorsolateral PFC in nonhuman primates have also been shown to correlate with accuracy on a recognition memory task (Shamy et al., 2011). Specifically, aged monkeys with larger PFC volumes identified more correct nonmatch objects on the DNMS task than did monkeys with smaller PFC volumes (Shamy et al., 2011; Fig. 2D). This correlation held even when the analysis was restricted to PFC gray matter or white matter volumes separately. Rather than cell loss, the gray matter volume decrease in the PFC is in part caused by age-related changes in neuron morphology, particularly the loss of synapses and the regression of apical dendrites (reviewed in Peters et al., 1996; Markham & Juraska, 2002; Dickstein et al., 2007; Luebke et al., 2010; Pannese, 2011; Morrison & Baxter, 2012). Decreases in spine numbers and density, and changes in spine morphology, have been reported in humans (Jacobs et al.

All efforts should be made to involve the woman’s GP and health visitor. It may be necessary to involve some of the following: patient advocates, social workers, legal advocacy, clinical psychologists, Selleck NU7441 psychiatrists, counsellors, health advisors, Citizens Advice Bureau workers, interpreters, community midwives, clinical nurse specialists and health visitors [4]. In settings with relatively few HIV-positive pregnant women, it is still important to develop robust pathways of care with identified members of an MDT. Regular links, formal or informal, can also be established with a larger unit to provide advice and support as

necessary. Good communication is vital in view of the complexity of the issues involved. An early assessment of the social circumstances of a newly diagnosed HIV-positive woman is important. Patients who initially refuse interventions or default from follow-up need to be identified and actively followed-up. Support by trained peer-support workers is a valuable component of the management of HIV-positive pregnant women. Many newly diagnosed selleck screening library HIV-positive pregnant women are initially reluctant to engage with peer support; however, the great majority of women who do engage

with it find that it becomes one of the most highly valued of all the interventions that they undertake [5]. The importance of informing appropriate healthcare workers should be emphasized. This includes midwives, general practitioners, health visitors and paediatricians. The process of in-patient care should be explained clearly, so that the women can be helped

to inform ward staff explicitly about levels of disclosure to visitors. Depending on the setting, levels of disclosure of newly diagnosed pregnant women about their HIV status vary, and there are cultural factors that influence the patterns of self-disclosure to partners and other social network members [4, 6]. Disclosure should be encouraged in all cases but Myosin may be viewed as a process that may take some time [7, 8]. There are situations where a newly diagnosed HIV-positive woman refuses to disclose to a current sexual partner, or appears to want to delay disclosure indefinitely. This can give rise to very complex professional, ethical, moral and, potentially, legal situations. There is a conflict between the duty of confidentiality to the index patient and a duty to prevent harm to others. Breaking confidentiality to inform a sexual partner of the index patient’s positive HIV status is sanctioned as a ‘last resort’ by the WHO [9] and General Medical Council [10]. However, it is not to be taken lightly as it could have the negative impact of deterring others from testing because of the fear of forced disclosure and loss of trust by patients in the confidential doctor–patient relationship.

The

EMG raw signals were amplified (1000 ×) and band-pass filtered (20 Hz–2 kHz) by a Digitimer D360 amplifier (Digitimer, Welwyn Garden City, Hertfordshire, UK), digitized at a sampling rate of 4 kHz by an analogue-to-digital interface (Micro 1401; Cambridge Electronic Design, UK) and stored on a laboratory computer for off-line analyses. The EMG traces were analysed using customized Signal® version 4.00 (Cambridge Electronic Design, UK) and matlab® version 7.1 (The MathWorks, Natick, USA) selleck screening library software. Participants were comfortably seated in a chair with the arms slightly abducted from the trunk (~45–50 °), the elbow flexed (~90 °) and both forearms in prone position. The right forearm and wrist were tightly attached on the armrest with straps. The right wrist was kept in a neutral position. The right BIBW2992 nmr thumb was slightly abducted, and fingers 2–5 adducted extended at the inter-phalangeal and flexed at the metacarpo-phalangeal joints (~70–80 °). The motor training

task was adopted from previous studies (Agostino et al., 2007, 2008). Participants were first asked to keep their dominant index finger extended and in line with the forearm. Participants were then instructed to produce ballistic finger abductions of their dominant index finger, so as to achieve the highest initial acceleration possible, in response (but not to react immediately) to a ‘go’ signal, given randomly at ~0.2 Hz, and to return to the neutral position. While performing fast abductions with their dominant index finger, participants were instructed to pinch with the 1st and 2nd finger a cylindrical body in order to isometrically recruit at ~5–10% of the maximal voluntary contraction in the contralateral FDIMIRROR (Fig. 2A; Giovannelli et al., 2006; Hübers et al., 2008).

The maintenance of a constant level of isometric contraction in the FDIMIRROR was monitored online by displaying the continuous EMG activity on a PC Hydroxychloroquine clinical trial screen in front of the participants. In each training session 100 movements were collected; 10 consecutive movements were considered as a trial and averaged (Fig. 2A). A rest interval of 10 s was left between trials to avoid fatigue (Fig. 2A). Before starting the motor training, one practice trial was permitted for the participants to become familiar with the experimental setup. In the present study we adopted a simple ballistic motor task with no real requirements for accuracy, just acceleration, as it fitted in well with the possibility to explore the effects of motor practice on the EMG mirroring activity related to fast finger movements. Moreover, although the after-effect of a simple ballistic motor task has been clearly described in terms of changes of corticospinal excitability, i.e. cortical plasticity (Classen et al., 1998; Muellbacher et al., 2001, 2002; Agostino et al.

043) and at follow-up 6–12 months later (P=0.017). There were weaker nonsignificant associations with VL in treated patients (Table 2). Higher scores on ‘medical decision’ were also related to higher CD4 cell count at baseline

(P=0.034). The relationship between concordance and CD4 cell count at baseline remained significant after controlling for treatment status (on treatment/stopped treatment) (P=0.019) as did the relationship between concordance IDO inhibitor and CD4 cell count 6–12 months later after controlling for treatment status and baseline CD4 cell count (P=0.043) (Table 4). Higher concordance was associated with on average a CD4 count that was 84 cells/μL higher at questionnaire completion, and 51 cells/μL higher at 6–12 months, after adjusting for treatment status (and baseline CD4 count for the latter). The relationship between ‘medical decision’ and CD4 cell count at baseline remained significant after controlling for ethnicity (P=0.011) (see Table 4). The relationship between concordance and CD4 cell count at baseline remained significant when quality of life-anxiety/depression

was added to the regression model [unstandardized coefficient (B) (standard error (SE))=86.21 (36.46), P=0.019, n=138] as did the relationship between concordance and CD4 cell count 6–12 months later [B (SE)=53.64 (25.91), P=0.040, n=130]. To test whether adherence (defined as doses missed last week) mediated the relationship between concordance and CD4 cell count, a subgroup analysis Hydroxychloroquine mouse was run on only patients on treatment. The relationship between concordance and CD4 cell count at baseline was similar [B (SE)=70.61 (37.61), P=0.063, n=127] and this trend remained after we added adherence to the linear regression model [B (SE)=67.93 (37.79), P=0.075, n=126]. The relationship between concordance Demeclocycline and CD4 cell count at 6–12 months was significant after controlling for baseline CD4 cell count [B (SE)=58.15 (26.85), P=0.032, n=121] and was not changed after we added adherence to the linear regression model [B (SE)=59.39 (27.12), P=0.030, n=120]. In this study, high levels of concordance, with

positive implications for patient wellbeing, were found during HIV treatment switch decision-making. Observed concordance was higher than that reported by Elwyn et al. [11] in consultations in GP. Given the pivotal role of adherence for maximum success of HAART, doctors might be more likely to take patients’ experiences into account when treatment decisions involve switching antiretrovirals. In the United Kingdom, HIV care is ‘open access’ and patients can move freely from one clinic to another to find services that are felt to be a ‘good fit’, which may result in higher concordance. Alternatively, the difference may be a reflection of the self-reported nature of our data rather than third-party observer reports.

In P. putida KT2440, the cfaB gene is transcribed divergently with respect to the lpd3 gene encoding a dihydrolipoamide dehydrogenase and convergently with the cls (cardiolipin synthase) gene (Fig. 2a), suggesting that the cfaB gene is a monocistronic unit. In order to identify the promoter of the cfaB gene, we first determined the transcriptional start point (tsp) of the KT2440 cfaB by primer extension analysis. The tsp was found to be identical to that of the P. putida

DOT-T1E strain (Pini et al., 2009) and located 53 nucleotides upstream of the proposed ATG codon of the CFA sequence (Fig. 2b). Putative consensus sequences for the Shine–Dalgarno box and for the −35 and −10 boxes of an selleck chemicals RpoS-dependent promoter were found upstream from the transcription E7080 in vitro initiation point (Fig. 2b). To confirm that the expression from the cfaB promoter in this strain was RpoS-dependent, the cfaB promoter was fused to the ‘lacZ gene in plasmid pMP220 and β-galactosidase activity was measured in P. putida KT2440 and in its isogenic RpoS mutant (Ramos-González & Molin, 1998). As can be seen in Fig. 2c, expression of the cfaB promoter in

P. putida KT2440 was fully dependent on the growth phase and no expression was detected in the RpoS knockout mutant strain. As expected, real-time PCR assays showed that the expression of rpoS and cfaB was almost nonexistent in the exponential growth phase, while both genes were expressed at a relatively high level during the stationary phase (Fig. 2d). cfaB expression started to decrease slightly before the expression of the rpoS gene. In the cfaB promoter, the proposed consensus sequence for RpoS recognition differs only in one position from the E. coli consensus (Fig. 3a) and it covers mafosfamide from the bases from −8 to −14 rather than −7 to −13. To analyze the importance of each nucleotide in the putative RpoS recognition site of the cfaB promoter, we generated transverse

point mutations in each of the seven nucleotides between positions −8 and −14 (Fig. 3b). The mutant promoters were cloned into the pMP220 plasmid and β-galactosidase expression was followed throughout the growth curve. Expression from wild-type and mutant promoters during the exponential phase of growth was low (never higher than 100 Miller Units) (not shown). However, the expression increased when the culture reached a turbidity at 660 nm of approximately 1.5 and high levels (1300 Miller Units) were detected when the cultures had reached a turbidity of 3 (Fig. 3b). Mutations at positions −14, −13, −12 and −9 completely abolished the cfaB promoter activity.

Computer-aided analysis of the affected genes also revealed the presence of inverted repeats highly similar

to the conserved Rex-binding site, -TTGTGAAW4TTCACAA-, in the promoter regions of most, but not all genes identified by microarray (Schau et al., 2004; Gyan et al., 2006; Pagels et al., 2010). Efforts to investigate whether Rex can bind to the promoter of the targeted genes and how NAD+/NADH balances affect Rex-regulated gene expression are ongoing It is apparent that Rex-deficiency did not have any significant effect on the morphology and growth rate of the deficient mutant when grown planktonically under the conditions studied (Fig. 1a). However, the deficient Alectinib mutant did show a decreased ability to develop biofilms on a surface, and it formed biofilms with an altered structure (Figs 2 and 3). These defects could be in part attributed to the altered expression of genes central to carbohydrate fermentation and energy metabolism (e.g. pflC and pdhAB), NAD+/NADH recycling (e.g. adhE, adhAB and frdC) and oxidative homeostasis (mleSP and gshR) (Table 2 and Table S1). One particularly interesting observation of the Rex-deficient mutant is that while it had a decreased ability to form biofilms, it also appeared to generate more glucans (Figs 2 and 3). Streptococcus mutans possesses at least three glucosyltransferases (GtfB, -C and -D) and one fructosyltransferase click here (Ftf).

The enzymes use sucrose as the primary substrate, assembling glucans and fructans from the glucose- and fructose-moiety of sucrose, respectively (Burne, 1998). At a significant level of P<0.01, gtfC was also identified by DNA microarray analysis to be Erastin upregulated by 1.56-fold in TW239, but not gtfB, gtfD and ftf (data not shown). When analyzed by RealTime-PCR, the expression of gtfC was found to be increased by >13-fold in TW239 (Table 2), but again no significant differences were detected in the expression of either gtfB, gtfD or ftf. Similar observations were also made recently in S. mutans grown with aeration (Ahn et al., 2007). Consistent with the severely impaired ability to form biofilms,

S. mutans grown in the presence of oxygen showed major changes in the amount and localization of the Gtf enzymes. In particular, the cell surface-associated GtfC was found by Western blotting to be dramatically increased in cells grown aerobically, as compared with those prepared under anaerobic conditions. However, it remains to be investigated whether the localization of any of the Gtf enzymes were altered in S. mutans as a result of Rex-deficiency. Glucosyltransferase GtfB is known to produce α1,3-linked, water-insoluble glucans that play a central role in S. mutans adherence and accumulation on surfaces, whereas the glucan products of GtfC contain α1,3-linked, water-insoluble and a substantial amount of α1,6-linked water-soluble glucans (Bowen & Koo, 2011).

coli TOP10. The resulting pCR4-16S rDNA vectors were analyzed by DNA sequencing (GATC Biotech, Germany) and the blast program was used to compare the sequences with the data in GenBank. All the colonies formed on 2YT or YPD agar were scraped from the plates into 5 mL 2YT (2YT plates) or YPD medium (YPD plates)

and centrifuged (3381 g for 15 min). DNA was extracted according to Matson et al. (2007), with some modifications. The pellet of bacteria was suspended in 1 × TE buffer (1 mM Tris-HCL, 0.1 mM EDTA pH 8.0, Sigma-Aldrich) containing 1% w/v polyvinylpolypyrrolidone (PVPP), 2% sodium dodecyl sulfate, 30% phenol, and 500 mg Zirconia/Silica Beads Compound Library (Stratech Scientif Unit). The cells were broken by three cycles of homogenization at 376 g for 30 s (Mikro-Dismembrator U, Sartorius) and chilling on ice for 30 s. The lysate was purified with the Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions (for the method described for crude lysate purification). Genomic DNA was partially digested with Sau3AI see more and fragments ranging

in size from 5 to 10 kb were purified from the agarose gel with the QIAEX II Gel Extraction Kit (Qiagen). The digested DNA fragments were ligated into the BamHI-linearized vector YEp356 (Myers et al., 1986) and the ligation products introduced into E. coli Max Efficiency DH5α competent cells (Invitrogen). Each colony was collected into a well of a 96-well plate containing 2YT

medium/50 μg mL−1 ampicillin and incubated at 37 °C for 16 h before replication and long-term storage at −80 °C. The quality of the library was determined on 96 randomly selected clones. The plasmids were isolated and then digested with EcoRI and HindIII. The digestion products were analyzed by agarose gel electrophoresis. CYTH4 The colonies in 96-well plates were replicated on the following media for enzyme detection. α-Amylase activity was detected after incubation for 24 h at 37 °C on 2YT agar containing 0.5% soluble starch. The medium was then covered with Gram’s iodine solution (Sigma-Aldrich), and α-amylase-producing colonies were identified by the absence of dark blue stain (due to the starch–iodine complex) in the zone surrounding them. Xylanase and endoglucanase were detected on 2YT agar containing an appropriate chromogenic substrate (respectively, AZCL-xylan or AZCL-HE-cellulose, Megazyme). Colonies producing these activities were identified by the blue color produced. β-Glucosidase was detected on 2YT agar containing 0.5% esculin (Sigma-Aldrich) and 0.1% ammonium iron (III) citrate (Sigma-Aldrich) after incubation at 37 °C for 24 h. Bacteria with β-glucosidase activity hydrolyze the substrate esculin to glucose and esculetin, which combines with ferric ions to yield a dark-brown color in the agar around the bacteria. The plasmid of the positive clone (named P11-6B) was isolated and analyzed by DNA sequencing (GATC Biotech).