Streptomyces sp. JAJ06 is a seawater-dependent antibiotic producer, previously isolated and characterised from an Indian coastal solar saltern. This paper reports replacement of seawater with a defined salt formulation in production medium and subsequent statistical media optimization to ensure consistent as well as improved antibiotic production by Streptomyces sp. JAJ06. This strain was observed to be proficient to produce antibiotic compound with incorporation of chemically defined sodium-chloride-based salt formulation instead of seawater into the production medium. Plackett-Burman design experiment was applied, and three media constituents, starch, KBr, and CaCO3, were recognised to have significant effect on the antibiotic production of Streptomyces JAJ06 at their individual levels. Subsequently, Response surface methodology with Box-Behnken design was employed to optimize these influencing medium constituents for the improved antibiotic production of Streptomyces sp. JAJ06. A total of 17 experiments were conducted towards the construction of a quadratic model and a second-order polynomial equation. Optimum levels of medium constituents were obtained by analysis of the model and numerical optimization method. When the strain JAJ06 was cultivated in the optimized medium, the antibiotic activity was increased to 173.3 U/mL, 26.8% increase as compared to the original (136.7 U/mL). This study found a useful way to cultivate Streptomyces sp. JAJ06 for enhanced production of antibiotic compound. Streptomyces are the prime producers of bioactive compounds for biotechnology industry. Several clinically significant antibiotics as well as widely used drugs against common diseases have been derived from this unique genus affiliated with the order Actinomycetales [Streptomyces have been isolated from previously unexplored natural habitats to nourish the current microbial antibiotic searching programs [Streptomyces sp. JAJ06 is a Gram-positive, moderately halophilic Streptomyces strain, which has previously been isolated from hypersaline coastal solar saltern at Tuticorin, India [ycetales . Diverseprograms , 3. Stren, India . This stn, India . ProductMedia components and their optimum levels are critical to the secondary metabolites produced by microorganisms. In the field of antibiotics, much effort was directed toward optimizing production rates and directing the product spectrum . ProductIn present work, efforts taken to expel the seawater from the production medium of JAJ06, with incorporation of chemically defined salt formulations. Further, in order to enhance the antibiotic production by JAJ06, production medium was optimized using a successive optimization strategy, selection of media components that significantly influence the antibiotic production using Plackett-Burman design and optimization of these media components using RSM with Box-Behnken design.Streptomyces sp. JAJ06 was previously isolated from a coastal solar saltern soil and extensively studied for their antibacterial secondary metabolite [The antibacterial compound producing strain, tabolite . The str4, 2 g of CaCO3, 0.04 g of FeSO4·7H2O, and 0.1 g of KBr in 1 L of sterile seawater was taken as basal production medium for further modification and statistical optimization.A nutrient medium contained 10 g of starch, 4 g of yeast extract, 2 g of peptone, 1 g of MgSO2·2H2O, 10.7 mg of H3BO3, 7.3 mg of SrCl2, 1.3 mg of NaF, and 26 μg of CoCl2·6H2O in 1 liter of deionized water; salt formulation II [2·2H2O, 10.7 mg of H3BO3, 7.5 mg of SrCl2, 1.3 mg of NaF, and 26 μg of CoCl2·6H2O in 1 liter of deionized water.In order to replace the chemically nondefined seawater, two defined salt formulations were screened and compared with seawater to support antibiotic compound in JAJ06. According to previous reports, two salt formulations were prepared with some modifications: salt formulation I [ation II  containe8 spores in 50 mL liquid medium. Cultures were incubated on a shaker at 120 rpm at 30°C for 3 days and used as seed stocks. For antibiotic production, strain JAJ06 was inoculated into production medium and incubated on a shaker at 120 rpm for 8 days at 30°C.Spore suspension of JAJ06 was prepared in distilled water from cultures grown on modified ISP-4 medium supplemented with 3% of sea salt (w/v) at 30°C for 7 days. The suspension was then added to ISP-2 broth in 250 mL Erlenmeyer flask at a rate of 10Cell-free supernatant of fermentation broth was recovered by centrifuging it at 10000 rpm for 10 min. Ethyl acetate was added to the supernatant in 1 : 1 proportion and the mixture was agitated for 10 min. The solvent layer containing antibiotic substance was separated from broth and it was further centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth. The antimicrobial crude extract was concentrated tenfold using a rotational vacuum concentrator and used for antibacterial assay.Bacillus subtilis MTCC 441 by agar diffusion plate assay [B. subtilis suspension adjusted to a McFarland standard of 0.5, which is equivalent to 1.5 × 108 CFU/mL. A sterile disc impregnated with ethyl acetate was used as control. The plates were incubated at 37°C for 24 h and the inhibition zone formed around the disc was measured with transparent ruler in millimeter. Maxwell et al. [The extracted crude substance was assayed in triplicate for their antimicrobial activity against te assay , 20. TheStreptomyces sp. JAJ06. Minitab 15.0  was used for the experimental designs and subsequent analysis of the experimental data. In the experimental design, 7 medium components (independent variables) were screened by representing them at two levels, low (−) and high (+) in 12 trials. B. subtitles was taken as the response. The variables with confidence levels above 90% were considered to have significant effect on antimicrobial compound production and thus used for further optimization.Plackett-Burman design (PBD) was employed for screening the most significant medium components for growth and antimicrobial compound production by 3 for enhanced antibiotic production in JAJ06. The three medium components (independent variables) were studied at three different levels: (−), (0), and (+) for low, intermediate, and high concentrations, respectively  was used with Box-Behnken design to optimize the selected media constituents: starch, KBr, and CaCOectively . The expectively  with fivY is the predicted response, β0 is the intercept term, βi is the linear coefficient, βij is the quadratic coefficient, βii is the interaction coefficient, and XiXj represent the independent variables.For predicting the optimal point, a second-order model was fitted to correlate the relationship between independent variables and response. The behaviour of the system was explained by the following quadratic equation:F-test and its associated probability P(F). The lack of fit was also applied to estimate the model. Lack of fit values lower than 0.05 indicates that there might be a contribution to the variables-response relationship that the model does not take into account. The quality of the polynomial model equation was judged statistically by coefficient of determination (R2) and adjusted R2. The fitted polynomial equation was then expressed in the form of three-dimensional response surface plots, to illustrate the relationship between the responses and the experimental levels of each independent variable. The Design Expert's numerical optimization method was employed to optimize the level of each variable for maximum response.The Design Expert trial package (Version 7.0) was used for the experimental design and the regression analysis of the data obtained. The statistical significance of the model was verified by applying the analysis of variance (ANOVA). Overall model significance was determined using Fisher's The combination of different optimized variables, which yielded the maximum response, was experimentally validated by culturing JAJ06 in optimized and unoptimized production medium. The cell-free culture broths were collected and extracted with equal volume of ethyl acetate and the top organic layer was dried for further analysis. The dried ethyl acetate extracts were resuspended in methanol and assayed as above for antibiotic activity.Streptomyces sp. JAJ06 in production media containing various salt compositions are given in The maximal antibiotic activity of JAJ06 in three different media prepared separately with nondefined sterile seawater and two chemically defined salt formulations was examined in shake flask cultures. Growth and antibiotic activity exerted by Streptomyces sp. JAJ06. t-value, P value, and confidence level of each component from the result of antibiotic assay given in X1), KBr (X5), and CaCO3 (X7) had apparent influences in antibiotic production by JAJ06. Confidence levels of these three variables were higher than 90% which indicates their significant contributions than those of other media components. The same was confirmed from the Pareto graph  can be expressed in terms of the following regression equation:Y represented the response of antibiotic activity, and A, B, and C were the coded values of starch, KBr, and CaCO3, respectively.Based on the results of the Plackett-Burman design, starch, KBr, and CaCOP value (<0.0001) of the Fisher's F-test. Significance of model was also supported by statistically insignificant lack of fit, as was evident from the lower calculated F value (4.01). The model R2 of 0.9831 implied that model equation could explain 98.31% of the total variation which suggested a good agreement between predicated values and experimental data.The ANOVA was performed to inspect the second-order response surface model and results are given in Diagnostic plots were drawn to judge the model adequacy and clarify the signs of any problems in the experimental data. Plot of observed response (antibiotic activity) versus predicted reponse is shown in 3 on antibiotic production by JAJ06  response surface plots were drawn to illustrate the individual and interactive effects of starch, KBr, and CaCOby JAJ06 . Each 3D Figures , respect Figures .3 was just beyond the middle level , the antibiotic activity increased with increasing concentration of starch from 2 to 15 g/L  and further increase of starch to its high level resulted mild decrease in antibiotic production  and further slightly increased at a moderate level of CaCO3 . Lower concentration of CaCO3 was found to be supportive for antibiotic activity; however the activity was suppressed when the concentration of CaCO3 was higher in production medium.With the increase of the concentration of KBr from 0.02 to 1.1 g/L , the antibiotic activity significantly increased to certain level at a low concentration of CaCO3 = −0.20 (3, respectively. The validation of the statistical results using the optimized medium was accomplished by carrying out shake-flask experiments in triplicate. The maximum antibiotic activity unit obtained experimentally was found to be 173.3 U/mL, (Streptomyces sp. JAJ06. The final optimized medium contained 7.4 g of starch, 4 g of yeast extract, 2 g of peptone, 1 g of MgSO4, 0.8 g of CaCO3, 0.04 g of FeSO4·7H2O, and 0.048 g of KBr in 1 L of sodium chloride based salt formulation I.On the basis of numerical optimization, the quadratic model predicted that the maximum antibiotic activity was 169.07 U/mL, when the optimal values of test factors in the coded units were starch = 0.37, KBr = 0.24, and CaCO = −0.20 , which w.3 U/mL,  which isStreptomyces are potential producers of secondary metabolites with antibiotic properties [Streptomyces sp. JAJ06 as a function of levels of constituents in production medium. The Streptomyces sp. JAJ06 has previously been identified as seawater dependent antibiotic producing actinomycete [Salinispora tropica strain NPS21184 [operties –23. Theioperties , 25. PreXenorhabdus bovienii and recorded 37.8% increase in antibiotic activity. Recently, Chen et al. [Bacillus sp. ZJUIBE-076 using RSM approach.Small manipulations in the culture medium composition can exert significant effect on secondary metabolites biosynthesis in microorganisms , 26. Sevn et al.  reportedStreptomyces sp. JAJ06 and recorded 26.8% increase in antibiotic activity. The results of PBD revealed that the crucial media components related to the antibiotic production by JAJ06 were starch, KBr, and CaCO3. Raytapadar and Paul [Streptomyces aburaviensis 1DA-28. Similarly, CaCO3 has been identified as a crucial ingredients related to the production of cyclic hexapeptide antibiotic by Streptomyces alboflavus [R2 value 0.9831 which was comparable with the earlier reports [In the present study, PBD and RSM were applied for medium optimization for antibiotic production by and Paul  reportedboflavus . RSM was reports , 27.Streptomyces sp. JAJ06 as a function of various salt compositions and levels of ingredients in production medium. PBD and RSM were found to be very effective in selecting and optimizing the medium components in manageable number of experimental trials with overall 26.8% increase in antibiotic activity. Moreover, the optimum culture medium obtained in this experiment will be useful for further study with large scale fermentation in a fermenter for the efficient production of antibiotic from this Streptomyces sp. JAJ06.Present investigation focused primarily on improved production of antibiotic by


This is an excellent starting point for research on antibiotics. The site contains a large list of antibiotics that is arranged into different structural classes.This review describes a book that provides an excellent background on the types of antibiotics, their synthesis and their mechanisms of action.This timeline shows the dates of introduction of new antibiotics. It should be noted that the major classes of antibiotics were largely discovered more than 40 years ago and that many recent antibiotics are elaborations on existing antibiotic structures.This article for a general audience lays out the idea that the discovery of new types of antibiotics has fallen off in recent years and that antibiotic resistance is increasingly a problem in the practice of medicine.Long-term, sub-therapeutic doses of antibiotics can increase weight gain for a given dietary intake. This has positive implications for purposeful increase in the weight of farm animals but negative consequences for humans where obesity is a major current health concern.Antibiotics.This link is for the open access journal Silver compounds have long been known to have antimicrobial activity, but new research suggests that silver acts synergistically with many common organic antibiotics and thus could help against antibiotic resistance issues.The Pew Charitable Trusts has invested in research and societal issues related to antibiotic resistance and new antibiotic development.A recent report suggests that antibiotics may damage the mitochondria in animal cells. This is interesting in the context of human health and evolution because mitochondria are thought to derive from prokaryotic cells.These pages from Ken Todar's online microbiology textbook give an excellent overview of antibiotics.This catalogue page provides links to lists of antibiotics, information on chemical classes and the mechanism of action of antibiotics.This popular science article discusses some of the controversies concerning the mechanisms of action of antibiotics.There is a common perception that combining antibiotics for medical treatment is the best way to protect health and prevent antibiotic resistance. The present paper uses computer simulations and experiments to suggest otherwise.Streptomyces.The major antibiotic-producing genus is the bacterial genus Streptomyces and the antibiotics they produce.This Microbe Wiki site discusses This report describes a discovery in which an antibiotic provides a regulatory signal for a bacterium to produce more antibiotics. This could be used as a strategy to increase the industrial production of antibiotics.This popular news article describes research in which bacteria can use antibiotics as a carbon and energy source. The danger is that high-level antibiotic degradation activity might spread among microbial populations.This article deals with the business aspects of antibiotics and antibiotic resistance.There are rising concerns that businesses do not have suitable incentives to develop new antibiotics and programmes are emerging to stimulate new research on antibiotics.


The binary effect of pulverized fuel ash (PFA) and palm oil fuel ash (POFA) on heat of hydration of aerated concrete was studied. Three aerated concrete mixes were prepared, namely, concrete containing 100% ordinary Portland cement (control sample or Type I), binary concrete made from 50% POFA (Type II), and ternary concrete containing 30% POFA and 20% PFA (Type III). It is found that the temperature increases due to heat of hydration through all the concrete specimens especially in the control sample. However, the total temperature rises caused by the heat of hydration through both of the new binary and ternary concrete were significantly lower than the control sample. The obtained results reveal that the replacement of Portland cement with binary and ternary materials is beneficial, particularly for mass concrete where thermal cracking due to extreme heat rise is of great concern. The hydration of cement compounds is exothermic where heat is liberated to the surrounding. In other words, when cement is hydrated, the compounds react with water to acquire stable low-energy states, and the process is accompanied by the release of energy in the form of heat . The quaRegarding the durability of concrete structures, modern concrete practices stipulate special measures to reduce peak and differential temperatures using materials with lower release of heat in order to minimise or prevent thermal cracking thereby avoiding the corrosion of embedded steel reinforcement , 6. LikeEffort towards sustainability and sustainable environment as made possible the use of pozzolanic material in concrete and other construction related materials. One of the latest additions of pozzolanic material is palm oil fuel ash (POFA). A waste material obtained from burning of palm oil husk, shell and fibre as fuel for palm oil mill boilers that has been identified as a good pozzolanic material –16. ThisThe name of materials that are used through this research and their characteristics are listed in this section.Cement. Ordinary portland cement (OPC) was prepared from the Tasek Corporation Berhad-Cement Industry of Malaysia. The OPC used complies with the Type I of the portland cement as stated in the ASTM C 150-05 [C 150-05 .POFA. Palm oil fuel ash (POFA) used was a by-product obtained from burning of palm oil shell and husk at temperature of 940°C from a Kahang mill, Kluang Johor, southern state of Malaysia. Afterwards, as suggested by Hussin and Awal [and Awal , the POFPulverized Fuel Ash (PFA). Pulverized fuel Ash (PFA) used for this research was obtained from the silos of Kapar Power Station, located Selangor, Malaysia. The fineness of PFA complies with the specifications of ASTM C 618-05 (2005).Fine Aggregate. In the case of preparing all the aerated concrete specimens, the aggregate used only comprises bottom Ash from Tanjung Bin power plant in Pontian, Johor. The Bottom ash is transported from the bottom of the boiler to the ash pond as liquid slurry by 200–250 mm diameter pipes. Initially the bottom ash was dried in an oven at the temperature of 105°C ± 5°C for 24 hours. Then, the oven-dried bottom ash is sieved passing 1.18 mm sieve before stored in the airtight container.Water. In this experiment, tap water was used for the manufacture of the lightweight concrete.Superplasticizer. The trade name of the superplasticizer used during this study was SIKAMENT NN. This superplasticizer was used as a chemical admixture. According to ASTM C494/C494 M-05 [494 M-05  that is Aluminum Powder. The type of the aluminum powder used through this study was Y250. It was the gaseous agent producing the porosity within the mass of aerated concrete.Thermocouple. Thermocouple used in this investigation was VITAR heater and thermosensor from the Vitar Group Heat and Temperature Division, Malaysia.2) within the minimum allowable that was mentioned by the ASTM C 618-12A [The chemical composition and physical properties of OPC, POFA, and PFA were shown in Tables  618-12A . The matIn the present investigation three types of the aerated concrete were made; first type (I) was with the OPC alone, used as a control sample, In the second type (Type II) the OPC was replaced by weight of 50% POFA, and in the third type (Type III), the OPC was replaced by weight of 30% POFA and 20% PFA. The mixtures  were selected according to the literatures that aimed only to improve the compressive strength of concrete –26. The A cubical plywood with the size of 280 mm was used as the exterior mould. Then, after installing the interior concrete mould, it was internally packed with 76 mm thick expanded polystyrene acting as the insulator . Each aeWhen aerated concrete was poured into the box, heat was liberated by the hydration process that subsequently increased the temperature of the aerated concrete mass. This increase in temperature and subsequent drop was monitored with close intervals during the first 24 hours. However measuring the heat of hydration was later monitored with lesser frequents until the temperature dropped close to its initial reading (first measurement when the test is just started). Recording the temperature was continued up to 5 days for the whole three aerated concrete types.The results of variation of the temperature versus time were recorded for the different aerated concrete types and presented in Although the initial temperature of the whole aerated concrete types was approximately same, more heat was considerably obtained from Type I aerated concrete during the first day of experiment, particularly in the first 8 hours after casting. A peak temperatures of 73.3°C was observed for Type I aerated concrete at 8 hours, while the peak temperature of 67.2 and 62.3°C were observed for Type II and Type III aerated concrete at 5: 30 hours, respectively .It is indeed well established that the fineness of cement influences the rate of heat development to some extent . In spitPOFA is relatively a new pozzolanic material and until now it does not have any specification compared to PFA. It has been characterized to be a unique supplementary cementing material for the concrete. The obtained results in this study demonstrate that the partial replacement of cement by POFA and PFA is advantageous and has very good potential to control the heat of hydration in concrete particularly in the first days of concrete casting. It can be concluded that is by incorporating 50% ashes  as a replacement to OPC (Type I aerated concrete), the heat of hydration significantly reduced through the aerated concrete. However there is limit on the usage of ashes because higher amount of ashes can cause strength reduction in the aerated concrete. To design an appropriate mix proportion for the aerated concrete, particularly in places that the thermal cracking (due to excessive heat rise) is of great concern, exploring the optimized amount of ashes that satisfy both the strength and heat of hydration factors is a key matter.


Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation.Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC In mammals, four profilin isoforms have been identified, each encoded by a distinct gene. Profilin-1 (PFN1) and profilin-2a (PFN2a), the classic “somatic” isoforms, are 65% identical in amino acid sequence, and highly conserved in 3D structure Profilins are small actin-binding proteins that are essential for all eukaryotic cells. They play a role in many cellular processes including cell motility, cytokinesis, gene transcription, endocytosis and neuronal plasticity Recent studies have linked profilins, in particular PFN1, to several human diseases. Our prior work identified PFN1 as an inhibitor of huntingtin aggregation, suggesting a role in the pathogenesis of Huntington Disease (HD). The anti-aggregation activity of PFN1 depends on its binding to both G-actin and a polyproline tract within huntingtin in vivobona fide phosphorylation site for the Rho-associated kinase ROCK Despite our detailed knowledge of profilin's structure and functions, we know little about how its cellular activities are regulated. PFN1 is a phospho-protein We previously generated a polyclonal phospho-specific antibody against pSer-137 of PFN1 (P3490). Using this antibody, we previously determined by Western blot (WB) that pS137-PFN1 levels in cultured cells are regulated by RhoA/ROCK signaling. The RhoA activator lysophosphatidic acid (LPA) mildly increased pS137-PFN1 levels in cultured cells, which was blocked by the ROCK inhibitor Y-27632 in situ by immunocytochemistry. P3490 heterogeneously stained several cell lines (predominantly the cytoplasm), i.e. not all cells were positive at the same time  markedly increased the number of P3490-positive cells, and this was blocked by ROCK inhibition with Y-27632  and PP2A (IC50∼0.1–0.3 nM) 50 for PP2A (0.1–0.3 nM), and more consistent with its known IC50 for PP1 (15–50 nM). We also used endothall, a structurally distinct phosphatase inhibitor that is also much more potent against PP2A (IC50 = 90 nM) than PP1 (IC50 = 5 µM) 50 for PP2A, but similar to that for PP1 , had no effect on P3490 staining, even at 1 µM (data not shown), a concentration expected to fully inhibit PP2A in a living cell To identify candidate phosphatases for pSer-137 of PFN1, we employed pharmacologic inhibitors of serine/threonine phosphatases in cultured cells. Okadaic acid (OA) inhibits the serine/threonine-specific phosphoprotein phosphatase (PPP) family, in particular its two most abundant members, PP1 and PP2A  for PP1 . Due to  for PP1 . FostrieThe above effects of phosphatase inhibitors indicated a role of PP1 in pS137-PFN1 dephosphorylation. To test this more definitively, we knocked down the alpha isoform of the PP1 catalytic subunit, PP1Cα, by transfecting HEK293 cells with siRNAs. In parallel, we also knocked down the alpha isoform of the PP2A catalytic subunit, PP2ACα. Based on Western blot analysis, endogenous PP1Cα and PP2ACα were both knocked down by 90% with no effect on cellular PFN1 levels . This waThe preceding experiments correlated PP1 activity with pS137-PFN1 dephosphorylation, but left uncertain whether the dephosphorylation is carried out directly by PP1, or by an unknown phosphatase whose activity is regulated by PP1. To test this, we transiently expressed Myc-PP1Cα and PFN1 in HEK293 cells, immunoprecipitated Myc-PP1Cα via an anti-Myc antibody, and tested for co-precipitation of PFN1. Myc-PP2ACα was separately co-expressed with PFN1 and immunoprecipitated as a control. We readily detected PFN1 in a complex with PP1Cα, but not PP2ACα . We nextin vitro. We immobilized recombinant 6His-PFN1  proteins on nickel beads, mixed them with bacterially expressed recombinant PP1Cα, and subsequently tested for their interaction via Western blot against PP1Cα. We did not detect any specific binding of PP1Cα to all three forms of 6His-PFN1, despite that all proteins were abundantly present . This is consistent with the fact that PFN1 lacks a typical PP1-docking motif ) that is found in most PIPs PP1 is a major eukaryotic serine/threonine phosphatase, and is thought to catalyze the majority of cellular protein dephosphorylation events As an essential protein for all eukaryotic cells, PFN1 has now been linked to several human diseases such as Huntington disease 5′-CCGGAATTCGCCGCCATGGCCTACCCATATGATGTTCCAGATTACGCTTCTTTGGGTGCCGGGTGGAACGCCTACATCGACAACCTCATG-3′ (upper primer containing an HA-tag); 5′-CCGCCGCTCGAGTCAGTACTGGGAACGCCGAAGGTGG-3′ (lower primer).Mammalian expression vectors encoding Myc-PP1Cα (wt or H125A) and Myc-PP2ACα were kindly provided by Dr. Hiroshi Shima  5′-GGAATTTAGCATGGATCTTCG-3′) was custom-designed to target 246-267nt of human PFN1 mRNA, and cloned as short hairpin RNA (shRNA) downstream of the U6 promoter in a lentiviral vector, pFLRu-FH (kind gift of Dr. Greg Longmore) 5′-CAACAAGATGAAGAGCACCAA-3′) adapted from the MISSION Non-Target shRNA control vector from Sigma. Lentiviral particles were purified by the Hope Center Viral Vectors Core at Washington University.Negative control siRNA (SI03650325) and human PP1Cα-specific siRNA (SI02225748) were purchased from Qiagen. A pool of three siRNAs targeting different regions of human PP2ACα were purchased from Santa Cruz (sc-43509). To silence PFN1, a 21nt sequence , mouse anti-Myc tag , rabbit anti-actin , rabbit anti-PP1Cα , rabbit anti-PP2ACα , mouse anti-HA tag . Custom-made antibodies include rabbit polyclonal antibody against the C-terminus of PFN1  and affinity purified rabbit polyclonal anti-pS137-PFN1 QY-OH) Alpha isoform of bacterially expressed recombinant PP1 catalytic subunit was purchase from Sigma (P7937). Nickel-NTA beads were purchased from Qiagen (30410). Protease inhibitor cocktail (11-836-170-001) was purchased from Roche Diagnostics. ECL Plus Western blotting detection kit (RPN2132) was purchased from GE Healthcare. Lysophosphatidic acid (L7260) was purchased from Sigma. Okadaic acid (495609), Endothall (324760), Fostriecin (344280) and Hydroxyfasudil (390602) were purchased from Calbiochem. Fetal bovine serum (SH3007103) was purchased from Hyclone. Gold anti-fade mounting media was purchased from Invitrogen (P36934).HEK293, HeLa and NIH 3T3 cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and penicillin/streptomycin. Lipofectamine 2000 (Invitrogen) was used for transfecting both DNAs and siRNAs using the protocol recommended by the manufacturer.in vitro pull-down experiment, 20 µl nickel NTA beads were pre-blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature, and subsequently mixed with 10 µg 6His-tagged PFN1 proteins previously purified from bacteria HEK293 cells over-expressing Myc-PP1Cα(wt or H125A) or Myc-PP2ACα were lysed with buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and protease inhibitors. Cleared lysate was mixed with agarose beads bound with anti-Myc antibody. After 2 hr of mixing at 4°C, beads were washed 4 times with lysis buffer, and analyzed by Western blot for co-immunoprecitation of untagged or HA-tagged PFN1 via antibodies against PFN1 (1∶1000) or the HA tag (1∶1000). For Cultured cells were fixed for 30 min with 4% paraformaldehyde, washed 3 times with PBS, followed by blocking with 2% BSA in PBS/0.1% Triton X-100 for 30 min. P3490 antibody was diluted at 1∶5000 (v/v) with blocking buffer and incubated with cells for 2 hr at room temperature or overnight at 4°C. Cells were washed 4 times with PBS/0.1% Triton X-100, incubated with 1∶500 dilution of Alexa Fluor® 488 goat anti-rabbit secondary antibody in blocking buffer for 2 hr at room temperature, washed 4 times with PBS/0.1% Triton X-100, and mounted. For total PFN1 staining, custom-made rabbit polyclonal antibody against the C-terminus of PFN1 (KCYEMASHLRRSQY) was used at 1∶100 and followed by secondary antibody incubation as for P3490. For double labeling, cells were simultaneously incubated with P3490 (1∶5000) and anti-Myc antibody (1∶1000) overnight at 4°C, followed by 2 hr incubation with 1∶500 dilution of Alexa Fluor® 488 goat anti-rabbit antibody and Alexa Fluor® 546 goat anti-mouse antibodies at room temperature. In all cases, DAPI was included in the secondary antibody solution at a concentration of 1 µg/ml. Images were acquired using the Zeiss LSM 5 PASCAL system equipped with the following lasers: 405 nm, 488 nm, 543 nm.For quantitative immunostaining within 96-well plates, HEK293 cells were grown to approximately 90% confluence, fixed, blocked, stained with P3490 (1∶5000) or anti-PFN1 antibody (1∶100) and Alexa Fluor® 488 goat anti-rabbit antibody (1∶500), and counterstained with DAPI as described above. Cells were washed 4 times with PBS/0.1% Triton X-100, left in 150 µl PBS, and quantified for fluorescence intensity (490/519 nm for Alexa Fluor® 488 and 365/439 nm for DAPI) using a fluorescence plate reader . Background fluorescence was acquired using cells stained only with the secondary antibody. Background subtracted fluorescence intensity for Alexa Fluor® 488, which represented P3490 reactivity, was then normalized against DAPI level to control for total cell number. For all experiments, at least 4 replicate wells were quantified for a given condition, and each experiment was repeated at least three times independently.P3490 was first diluted 1∶5000 in the blocking buffers for immunofluorescence (PBS/0.1% Triton X-100/2% BSA) or Western blot (TBS/0.05% Tween 20/5% skim milk), and then mixed at 1∶200 molar ratio (antibody/peptide) with PFN1 peptides containing (Ac-CMASHLRR(pS)QY-OH) or lacking a phosphate on Ser-137 (Ac-CMASHLRRSQY-OH) for 30 min at room temperature. A control antibody solution was set up by mixing an equivalent volume of water (instead of peptides) with P3490. All antibody solutions  were subsequently used to immunostain or immunoblot HEK293 cells as described above.


The presence of antibiotics in the environment and their subsequent impact on resistance development has raised concerns globally. Hospitals are a major source of antibiotics released into the environment. To reduce these residues, research to improve knowledge of the dynamics of antibiotic release from hospitals is essential. Therefore, we undertook a study to estimate seasonal and temporal variation in antibiotic release from two hospitals in India over a period of two years. For this, 6 sampling sessions of 24 hours each were conducted in the three prominent seasons of India, at all wastewater outlets of the two hospitals, using continuous and grab sampling methods. An in-house wastewater sampler was designed for continuous sampling. Eight antibiotics from four major antibiotic groups were selected for the study. To understand the temporal pattern of antibiotic release, each of the 24-hour sessions were divided in three sub-sampling sessions of 8 hours each. Solid phase extraction followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used to determine the antibiotic residues. Six of the eight antibiotics studied were detected in the wastewater samples. Both continuous and grab sampling methods indicated that the highest quantities of fluoroquinolones were released in winter followed by the rainy season and the summer. No temporal pattern in antibiotic release was detected. In general, in a common timeframe, continuous sampling showed less concentration of antibiotics in wastewater as compared to grab sampling. It is suggested that continuous sampling should be the method of choice as grab sampling gives erroneous results, it being indicative of the quantities of antibiotics present in wastewater only at the time of sampling. Based on our studies, calculations indicate that from hospitals in India, an estimated 89, 1 and 25 ng/L/day of fluroquinolones, metronidazole and sulfamethoxazole respectively, might be getting released into the environment per 100 hospital beds. One of the contributory factors implicated for the development of antibiotic resistance, is the exposure of bacteria to antibiotics in the environment, particularly the aquatic environment Therefore, we undertook a two-year study to estimate antibiotic residues in wastewater of two representative hospitals in India, with the aim of understanding seasonal and temporal variation in antibiotic release. Sampling was done using continuous and grab sampling methods to compare antibiotic release estimates given by the two methods. The long-term aim is to introduce interventions to reduce/remove antibiotic residues from hospital wastewater.The study was conducted in the central Indian province, Madhya Pradesh (MP). The sampling sites were two hospitals in Ujjain district. ‘Hospital 1’ is a non-teaching hospital with 350 patient beds located in Ujjain city catering mainly to city population, while ‘Hospital 2’, is a 570-bed teaching hospital attached to a medical college, located in a rural area 6 km from the city and catering predominantly to the rural population. Both hospitals have all major medical specialties.To understand the seasonal variation in antibiotic residues in hospital wastewater, the study was conducted during the three prominent seasons of India, i.e. summer, rainy season and winter. During these three seasons, weather conditions in the study setting remain as follows Summer sampling was done during the second week of May 2008 and the third week of May 2009. Rainy season sampling was done during the last week of July 2008 and the first week of August 2009. Winter sampling was done during the last week of January 2009 and the first week of February 2010. In total, six sampling sessions of 24 hours each were conducted in each hospital during the study . Furthermore, to understand the temporal pattern of antibiotic release from the hospitals, each of these 24-hour sessions was divided into three sub-sampling sessions of 8 hours each. The three sub-sampling sessions were, 0900–1700, 1700–0100 and 0100–0900 hours. The first sub-sampling session (0900–1700 hours) represented the hottest 8 hours of the day, when sunshine is relatively intense. The second sub-sampling session (1700–0100 hours) represented the cooling period and the third sub-sampling session (0100–0900 hours) represented the relatively cooler period of the 24 hours. This sequence of sampling was also convenient from the point of view of logistics of working.Wastewater sampling was done at all the wastewater outlets of the two hospital buildings at the points where water came out of the hospitals after use; four outlets in Hospital 1, and 6 in Hospital 2. Wastewater from each of the hospitals’ outlets was collected in a small wastewater chamber (2′×2′×2′) before it exited the hospitals’ premises. The wastewater in these chambers was sampled using (i) continuous, and (ii) grab methods.An in-house wastewater sampler was designed for continuous sampling, which consisted of an electrically-operated submersible pump , a polyvinyl chloride (PVC) tube (1/2 inch dia.) with flow regulator, a half turn PVC valve, a PVC Tee (T) joint (1/2 inch dia.), electric (on/off) switch and an indicator lamp to show power on/off. A representative diagram of the wastewater sampler’s assembly is given in On each sampling day, the 24 hours sampling session consisted of three consecutive continuous sub-sampling sessions of 8 hours each , in both the hospitals. At the end of every 8-hour session, all wastewater samples collected from all the outlets of each hospital were mixed together to make a composite sample representing that 8-hour duration for that hospital. From this sample, two liters of wastewater was sent for chemical analysis.Grab sampling consisted of taking wastewater samples instantly at one point in time. In this method, in each sampling session, a one-time separate grab sample (two liters each) was collected from all the wastewater chambers of both the hospitals, once in 8 hours. In ‘Hospital 1’, grab samples were collected at 1300, 2100 and 0500 hours, while in ‘Hospital 2’, they were collected at 1000, 1800 and 0200 hours. From a feasibility point-of-view, the sampling times and days for the two hospitals had to be kept different, but it was done on consecutive days. All samples from all the wastewater chambers of a hospital, taken at one time e.g. say at 1300 or 1000 hours were mixed to make one grab sample representing that sampling time. From this mixture of grab samples, a two-liter sample was sent for chemical analysis. In all, three grab samples were made from each hospital during each 24-hour sampling session.Both the hospitals received water from municipal as well as ground water sources. The water was received once daily in a water tank, located in the hospital area. Samples (2 L) representing the incoming water of each hospital were collected from the water tank at 0800 hours on the day of sampling as a one-time grab sample in each season and in each hospital. This was done to determine if the incoming water was already contaminated with antibiotics and also to ascertain that the quantities of antibiotics detected in the hospital wastewater represented the actual release by the hospitals.et al 2010 To prevent degradation of the antibiotics, all samples were placed in screw-capped amber bottles wrapped in silver foil. The samples were stored at <4°C immediately after collection until they reached the analytical laboratory (within 24 hours). Analysis was performed at the laboratories of Shriram Institute for Industrial Research, New Delhi, where the samples remained at −20°C until analyses. Details of sample storage and handling methods are described in Diwan Eight antibiotics from four major antibiotic groups - cephalosporins (ceftriaxone and cefoperazone), fluoroquinolones , sulfonamides (sulfamethoxazole) and imidazoles (metronidazole) - were selected for analyses. The selection was based on, (i) the prescription pattern in the inpatient wards of the hospitals, (ii) antibiotic residues found in the same setting in our previous study et al 2010 The details of the method of analyses can be found in Diwan Both the hospitals and R.D. Gardi Medical College are interlinked institutions and the study was approved for both the hospitals by the ethics committee of R.D. Gardi Medical College, Ujjain (No. 41/2007).The seasonal and temporal variation in antibiotic residues in the wastewater of the two hospitals obtained by continuous and grab sampling methods, is presented respectively in To our knowledge, this is the only study worldwide which has attempted to quantify antibiotic residues in hospital wastewater using concurrent continuous and grab sampling methods. Furthermore, the sampling in our study was done at locations closest to the hospitals’ wastewater outlets, whereas in most of the earlier published studies, sampling was done at the inlet and outlet points of wastewater treatment sites of hospitals, by the grab method . The water released from the hospitals contained antibiotics belonging to the fluoroquinolone, imidazole and sulphonamide groups. Although cephalosporins are prescribed in significant quantities in the hospitals No antibiotic residues were detected in the incoming water, indicating that the entire quantities of antibiotic residues detected in the hospital waste water were actually released by the hospitalsPseudomnas putida is 8,000 ng/L and for fish, daphnia and algae it is 3,000 ng/L Variation in the detection of antibiotic residues in the wastewater of the two hospitals was observed, between seasons, between sampling times and between the two years of sampling. Detecting variation in antibiotic residues in the wastewater of any two hospitals is always likely because of variation in patient admission with different maladies, preference and type of antibiotics prescribed and administration, variation in patient behavior and physicochemical behavior of antibiotics In the two hospitals, the number and concentration of antibiotics detected in wastewater varied from one season to another. There was also no similarity in the occurrence of antibiotic residues in the same season of the two years. When the continuous sampling method was used, concentrations of antibiotics detected in winter and rainy season were more than four times higher than in summer . For theet al reported a higher concentration of ciprofloxacin at 1300 and 1500 hours and of ofloxacin at 1300 and 1700 hours et al as we conducted our studies in tropical conditions in India, took samples in three seasons during three time frames over 24 hours and also employed two sampling methods. However, if a comparison had to be made under our conditions, the ambient temperatures in winter appear somewhat similar to summer temperatures in Sweden and during this season, using the grab sampling method , Lindberg It is suggested that temporal variation in antibiotic concentration in wastewater might arise due to variation in antibiotic administration during the course of the day and various pharmacokinetic factors such as metabolism, half-life and excretion, and environmental factors like flow and temperature The two different sampling methods we experimented on, indicated the release of different numbers and concentrations of antibiotics into hospital wastewater for the same time frame and even for the same hospital. In both the hospitals, the concentration of antibiotic residues detected by grab sampling was mostly on the higher side compared to continuous sampling. For a given time frame however, grab samples only give a ‘snap-shot’ picture, and these results are not representative of a whole timeframe. The continuous sampling method on the other hand, samples the wastewater continuously during the whole period and gives an estimation of the antibiotic residues for the whole timeframe in question. Low antibiotic levels observed in samples obtained by the continuous sampling method may be explained by a dilution effect; in continuous sampling, even when there is no antibiotic residue present in the wastewater, the sample is taken regardless and gets mixed with the total sample.To our knowledge, simultaneous sampling of hospital wastewater by grab and continuous methods has not been done earlier. It is evident from our studies however, that grab sampling gives an erroneous picture of the antibiotic residues present in wastewater, if the wastewater in question is a dynamic system. Grab sampling may be useful to some extent, to study antibiotic residues in stagnant water or when information on antibiotic residues is wanted for a particular point in time. For dynamic systems of water, we suggest use of continuous sampling over a time period, to obtain a more realistic picture of the status of antibiotic residues.If an attempt to generalize the quantities of antibiotics released into the environment from hospitals is made using information from et al showed that selection of resistant bacteria occurs at antibiotic concentrations several hundred-fold below the minimal inhibitory concentration (MIC) and de novo mutants get selected very rapidly at sub-MIC antibiotic concentrations and take over a susceptible population E. coli populations that have constituents that are ESBL-positive and are carrying resistance genes such as blaCTX-M, blaTEM,blaSHV and qnrA, qnrB, qnrS, aac (6′)-Ib-cr, qepAGullberg The aim of this study was to understand the seasonal and temporal dynamics of antibiotic residue levels in hospital-associated waters from the point-of-view of administering interventions to reduce/remove antibiotic residues from the wastewater. It appears that such interventions cannot be time and season specific, as antibiotics are released in hospital wastewater continuously, daily and all year round, with not much seasonal or temporal variation. Further based on our studies, calculations indicate that from hospitals in India, an estimated 89, 1 and 25 ng/L/day of fluroquinolones, metronidazole and sulfamethoxazole respectively, might be getting released into the environment per 100 hospital beds. More studies are needed in this area to confirm present findings. We think that if knowledge regarding realistic levels of antibiotic residues in hospital wastewater is to be obtained for the planning of interventions, then the continuous sampling method should be the method of choice.Table S1Seasonal and temporal variation in antibiotic residues (ng/L) in the wastewater of the two hospitals by continuous sampling.(DOC)Click here for additional data file.Table S2Seasonal and temporal variations in antibiotic residues (ng/L) in the wastewater of the two hospitals by grab sampling.(DOC)Click here for additional data file.


The risk of acquisition of antibiotic resistant-bacteria during or shortly after antibiotic therapy is still unclear and it is often confounded by scarce data on antibiotic usage.Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae in hospitalised patients, after starting antibiotic therapy.Primary objective of the study is to compare rates of acquisition of methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae will be obtained at hospital admission, discharge, at antibiotic start  and at the following intervals: day 3 (t1), 7 (t2), 15 (t3), and 30 (t4). Two nested case-control studies will be performed. The objective of the first study will be to define individual level of risk related to specific antibiotics. Patients acquiring methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Enterobacteriaceae (cases) will be compared with patients not acquiring antibiotic-resistant strains after starting antibiotic therapy . To define the impact of antibiotics on new acquisition of target antibiotic-resistant bacteria, a second nested case-control study will be done (ratio 1:4). Control group will be selected among patients not receiving antibiotics, admitted in the same ward on the day of the corresponding case, with negative cultures at admission. Epidemiological, clinical and microbiological data will be prospective collected.The study, running in three European hospitals, is a multicenter, prospective, longitudinal, observational cohort study funded from the European Community's Seventh Framework Programme [FP7/2007-2013] within the project 'Impact of Specific Antibiotic Therapies on the prevalence of hUman host ResistaNt bacteria' (acronym SATURN). Nasal and rectal screening for methicillin-resistant The rationale of this study is to better understand the impact of antibiotic use on acquisition, selection and transmission of antimicrobial resistant-bacteria in European hospitals.NCT01208519.ClinicalTrials.gov  Staphylococcus aureus (MRSA) was increased by 1.8-fold in patients who had taken antibiotics. Antibiotic exposure was determined in the 126 days preceding MRSA isolation [Despite many efforts, the control of nosocomial antibiotic-resistant infections is still a permanent and unresolved issue in healthcare institutions worldwide facing increased morbidity, mortality and hospital costs -3. Selecsolation . Such risolation .The likelihood of acquisition of nosocomial ARB during or shortly after therapy with different antibiotic agents is still unclear and is often confounded by scarce data on antibiotic usage at the individual patient level. Study design, selection bias, definition of control groups and failure to control for confounding variables might account for conflicting results . No prosWe designed a 2-year observational cohort study (WorkPackage 4 of the EU-funded SATURN project) in order to clarify and determine the role of antibiotic therapy in the acquisition of ARB in hospitalized patients. The study will run in three European hospitals, ranging from 700 to 3,500-bed capacity: Università Cattolica Sacro Cuore "Policlinico A. Gemelli" , Institute of Infectious Diseases "Matei Bals" , and Clinical Center of Serbia . Overall, 6 medical and 6 surgical wards will be included. All centers obtained ethical approval from the local institution review board.per os and/or intravenously will be sampled at antibiotic start  and at the following intervals: day 3 (t1), 7 (t2), 15 (t3), 30 (t4)  will be excluded from follow-up cultures and analysis. Patients will be followed during the hospitalisation and afterwards for a total of 30-day from the inclusion in the study. Screening will be performed in outpatient clinics after patients' discharge from the hospital, if possible.All adult patients > 18 years) starting intravenous and/or oral antibiotic treatments during hospitalisation will be included in the study. Written informed consent will be obtained by every patient before study inclusion. Exclusion criteria will be pregnancy and recent nose surgery. Patients starting antibiotic therapy 8 years sThe primary outcome will be the rate of acquisition of target antibiotic bacteria by 1,000 antibiotic-days in hospitalised patients. The rate will be also defined according to antibiotic class and single drug. Patients will be stratified by class of risk  and rate of acquisition will be defined. The secondary outcome will be the rate of nosocomial infections by 1,000 days of hospitalisation in newly colonised patients after undergoing antibiotic therapy.The following variables will be identified in cases and controls: age; gender; reason for admission; inter and intra-wards transfers; admission from home or other acute or long-term care institutions; previous hospitalisation; surgical procedures; Intensive Care Unit (ICU) stay (previous 12 months); residency in long-term care facility; transfer form another hospital; nursing and/or medical domiciliary assistance; urinary catheter; central venous catheter ; underlying conditions ; room sharing with a ESBL or MRSA patient; previous MRSA or ESBL carrier; consciousness at admission; antibiotic therapy (previous 30 days).Antibiotic therapy will be reported as for daily dosage, frequency of dosage(s), route of administration, type of antibiotic(s), reason for starting and stopping, duration of therapy. At every clinical follow-up the following variables will be also detected: presence of urinary catheters; CVC; surgery; room sharing with a ESBL or MRSA positive patient; MRSA or ESBL-producing gram-negative infections ; albumin, creatinine and blood urea nitrogen levels.S. aureus will be confirmed through the use of a classical DNase assay or automated phenotypic methods  [Dedicated personnel will be trained in performing nasal samples. Both nostrils will be sampled. Specimens obtained with a dry swab  will be incubated overnight in 2 ml of Mueller-Hinton broth supplemented with colistin 32 μg/mL and 2.5% NaCl (CS) at 35°C in room air. Then 10 μl of broth sample will be plated onto chromogenic medium   and willton, NC) . OxacillKlebsiella species, Escherichia coli and Proteus species will be detected by confirmatory double disk diffusion test (DDT). Ceftazidime (30 μg), cefotaxime (30 μg), or cefepime (30 μg) discs will be placed on Mueller-Hinton agar plates, 20 mm apart (centre to centre) from a clavulanate disc (clavulanic acid 10 μg). The test will be considered as positive when a decreased susceptibility to cefotaxime is combined with a clear-cut enhancement of the inhibition zone of cefotaxime in front of the clavulanate-containing disk, often resulting in a characteristic shape-zone referred to as 'champagne-cork' or 'keyhole' [Rectal specimens will be obtained with a dry swab  and will be inoculated onto plates of ESBL chromogenic medium . After the direct plating, 1 ml of trypticase soy broth (TSB) with 0.5% NaCl will be added to the transport tube and the selective broth will be incubated overnight at 37°C in air. The selective medium will be plated on the ESBL chromogenic medium following the same procedure as for direct plating. Colonies will be identified according to manufacturers' recommendations and subcultured for confirmatory testing. The ESBL phenotype of identified colonies of keyhole' ,13. Plat0) will be included in the longitudinal analysis. The length of exposure to antibiotic therapy for patients newly colonised by ARB will be defined by the number of days between the inclusion in the study and the date of the first positive sample. The length of exposure to antibiotic therapy for patients not colonised will be defined by the number of days between the inclusion in the study and the date of antibiotic discontinuation. The colonisation pressure will be calculated for each center and defined as the average point of prevalence for antibiotic-resistant bacteria for all days until acquisition or discharge. Time-to-event analysis (in days) between the inclusion in the study and the acquisition of colonisation will be done using Kaplan-Meier curves and Cox proportional hazard regression modeling in which the number of days until acquisition will be the dependent variable. Two nested case-control studies will be performed:The assessable population for each target organism will include all treated patients from whom baseline screening and at least one follow-up screening will be available. The incidence of colonisation will be defined by the number of new cases of colonisation for 1,000 days of antibiotic therapy. Only patients with a negative baseline . The control group will be selected among patients not receiving antibiotics, admitted in the same ward on the day of the corresponding case, with negative cultures at hospital admission. Matching criteria will include: age (± 5 years), sex, and total length of hospitalisation.To define the impact of antibiotics on new acquisition of MRSA and ESBL-producing Enterobacteriaceae will be compared with patients not acquiring ARB after starting antibiotic therapy (ratio 1:4). Previously known risk factors or clinically relevant significant variables from the univariate analysis will be considered for inclusion in multivariate logistic regression analysis. The Hosmer and Lemeshow goodness-of-fit test will be used to assess model fit.To define individual level of risk related to specific antibiotics, patients acquiring MRSA and ESBL-producing Enterobacteriaceae: 48/95, 121/201, and 88/147 for UCSC, IDMB, CCS, respectively.Since the rate of acquisition of ESBL-producing gram-negative bacteria in patients receiving antibiotic therapy has not been established in prospective cohort studies, prevalence proportion was derived from retrospective case-control studies and defined as 5%. Sample size calculation for the rate of new acquisition of MRSA has been derived by a previous study where the acquisition rate, after starting antibiotic therapy, was 3% . To be ahttp://www.saturn-project.eu provides the information and details concerning the different WPs and the entire project.SATURN project is a European multicentre study that aims to provide an in-depth knowledge of ARB acquisition through the analysis of antibiotic classes, duration of treatment and dosage in the nosocomial setting by a multidisciplinary approach. The SATURN project website Several outcomes will be obtained at the end of the study : the rate of acquisition of target ARB by 1,000 antibiotic-days according to different classes of antibiotics, duration of therapy, and antibiotic combinations (monotherapy versus combinations); risk factors associated with new colonisation by target ARB; and risks for nosocomial infections due to target ARB after a cycle of antibiotic therapy adjusted by length of hospitalisation and ward colonisation pressure.The WP4 study is currently recruiting patients in all clinical centers. The accomplishment of the study will be pursued thanks to the great strive of all clinicians, nurses and microbiology staff in every study step. An advertising campaign  was performed in the selected wards in order to thrill and sensitise the recruited staff.The results of the study should lead to the design of specific infection control measures for preventing spread of ARB in hospitalised non-ICU patients undergoing antibiotic therapy. A potential benefit for selective screening in high-risk patients during antibiotic therapy or shorter duration of antibiotics in such patients might be hypothesised. Readily available colonisation information could impact empiric therapy for nosocomial infections or, for example, the potential need of mupirocin prophylaxis for patients colonised by MRSA during hospitalisation. Assessment of the usefulness of the recommendations derived from the results of the SATURN study should be of valuable help for those in charge of infection control, for hospital administrators, and for those managing budgets of large healthcare organisations.We do expect that SATURN project results will have a direct impact on clinical practice, improving the appropriateness of antibiotic prescribing policy, the prevention of antibiotic-resistant infections and the heightener of patients' quality care.The authors declare that they have no competing interests.GDA participated in the elaboration of clinical protocol and drafted the manuscript. GR and SV participated in the design of the study. RC reviewed the manuscript. SMK, HG, and JS elaborated the microbiological protocol. ET conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/74/prepub


Though inconsistent, a number of studies have shown an association between vitamin D (25(OH)D) status, parathyroid hormone (PTH) and the metabolic syndrome (Met S). These have largely been carried out in Caucasians or black subjects living in high income countries. There no data on the relationship of 25(OH)D and PTH status with Met S in populations resident in Africa. The aims of this study were to evaluate if there was an association of 25(OH)D or PTH with Met S in non-Caucasian populations in South Africa, and whether these molecules explained ethnic differences in the prevalence of Met S and its individual components.We measured anthropometry, serum 25(OH)D and PTH levels and the components of Met S, plus related metabolic variables, in 374 African and 350 Asian Indian healthy adults from the greater Johannesburg metropolitan area.Met S was diagnosed in 29% of the African and 46% of the Asian Indian subjects (p<0.0001). Subjects with Met S had higher PTH than those without Met S, (p<0.0001), whilst 25(OH)D levels were not significantly different (p = 0.50). In multivariate analysis, 25(OH)D was not associated with any components of the Met S however PTH was shown to be positively associated with systolic (p = 0.018) and diastolic (p = 0.005) blood pressures and waist circumference (p<0.0001) and negatively associated with HOMA (p = 0.0008) levels. Logistic regression analysis showed that Asian Indian ethnicity  and raised PTH D) produced an increased risk of Met S but 25(OH)D did not .Plasma PTH but not 25(OH)D is an independent predictor of the Met S in African and Asian Indians in South Africa. The incidence of obesity is on the rise in many low and middle income countries (LMICs) The burden of disease related to CVD is expected to increase substantially in LMIC, such as South Africa, over the next decades and it is important to understand traditional and non-traditional risk factors that contribute to this disease burden. Some populations are more susceptible to CVD because of specific factors – e.g. familial hypercholesterolaemia in the South African Afrikaner population  2D). Hypovitaminosis D appears to exert its effects via reductions in intracellular calcium and through the effects of 1,25(OH)2D on the regulation of various target genes e.g. by lack of suppression of the renin gene leading to hypertension It has been suggested from several studies in various parts of the world that vitamin D deficiency or insufficiency may increase risk of the Met S and its sequelae, type 2 diabetes and CVD Elevated parathyroid hormone (PTH) levels have also emerged as a potential risk factor for Met S There are no studies from Africa that have investigated any associations between 25(OH)D, PTH and components of the Met S. Therefore, the aims of this study were to examine the association of 25(OH)D and PTH with Met S in African and Asian Indian population groups in South Africa and to determine whether ethnic differences in 25 (OH)D or PTH levels contribute to differences in the prevalence of Met S, its component parts and related metabolic variables, within these populations. The hypotheses to be tested were that 25(OH)D is lower and PTH is higher in Met S patients in both population groups and that ethnic differences in the prevalence of Met S and its component parts are related to ethnic differences in the blood level of 25(OH)D.This was a cross sectional study of African and Asian Indians from the greater Johannesburg-Soweto metropolitan area in South Africa. Participants were recruited via the Birth to Twenty study, which is a longitudinal analysis of more than 3200 children and their caregivers which began in 1990 and is representative of long-standing urban inhabitants After an overnight fast of at least 8 hours, peripheral venous blood was collected in EDTA tubes for 25(OH)D, PTH and glycated haemoglobin analysis (HbA1c), in fluoride tubes for glucose and in plain tubes for the rest of the analytes. Tubes were kept on ice and centrifuged after 30 minutes at 3500 g for 10 minutes. Glucose, HbA1c and lipid profile were assayed on the day of collection and aliquots for 25(OH)D, PTH, and insulin were stored at −80°C until analysis. Glucose, total cholesterol, HDL-C and triglycerides were measured enzymatically on an automated analyser . The HbA1c was measured on the ADVIA 1800, which was standardized according to the Diabetes Control and Complications Trial National Glycohemoglobin Standardization Program 2 and 25 (OH)D3 were detected at a wavelength of 264 nm. The chromatograms were integrated using peak height. Total vitamin D (25(OH)D) was taken as the sum of 25(OH)D2 and 25(OH)D3. The intra assay CV for 25(OH)D ranged from 0.9–4.9% and the inter-assay CV ranged from 3.0– 4.9%.The limit of detection was 2.5 nmol/l for 25(OH)D3 and 7.5 nmol/L for 25(OH)D2. Our laboratory participates in the vitamin D external quality assurance scheme (DEQAS).Plasma 25(OH)D was measured using the Clin Rep high performance liquid chromatography (HPLC) kit . In this analytical method, 25(OH)D was determined from plasma using HPLC with a photodiode array (PDA) detector. Prior to HPLC analysis a short liquid-liquid extraction was performed. Thereafter the samples were injected onto the HPLC system and the analytes were separated on the appropriate analytical column. The separated 25(OH)DMet S was determined using the harmonized definition A standardized questionnaire was used to collect information on age, sex, visit date, smoking , self-reported diabetes, hypertension, dyslipidaemia, coronary heart disease and medication use. Body weight and height were measured with subjects in light clothing and without shoes using an electronic scale and wall mounted stadiometer . The waist circumference was measured with a soft measuring tape to the nearest 0.5 cm at the level of the smallest girth above the umbilicus in the standing position. Blood pressure was measured thrice in the right arm, with the subject seated and after 5 minutes rest using an automated sphygmomanometer. The final reading was used.Stata 12  was used for all analyses. The distribution of variables was assessed by the Shapiro-Wilk’s W test and by examination of the normal probability plot. Logarithmic transformations were applied for all non-normally distributed variables. In descriptive analysis, normally distributed, continuous variables were reported as means ± SD whilst non-normally distributed data were expressed as median (interquartile range). Categorical data were reported as N (percent).Pearson’s correlation was used to assess the association of 25(OH)D and PTH with the selected variables. Multivariate regression analyses were conducted to assess the association of the Met S components with 25(OH)D and PTH after adjusting for potential confounders i.e. age, gender and body mass index (BMI). Ethnicity was also included as an independent variable to determine if ethnic differences in Met S components were mediated by 25(OH)D or PTH.In order to determine if the higher risk of Met S in the Indian population was mediated by 25(OH)D or PTH, logistic regression analysis was performed with the stepwise addition of 25(OH)D and PTH to a model that also included ethnicity, age, gender and BMI as independent variables. The attenuation of the p-value (from significant to non-significant) for the ethnicity odds ratio was used as the indicator of any effect.PTH was found to increase the risk of Met S and therefore we analysed which of the individual components of the Met S were influenced by PTH by separately adding each individual component to a logistic regression model for MetS risk which included PTH, age, gender, ethnicity, BMI and 25(OH)D as independent variables. As described above the attenuation of the p-value, in this case for the PTH odds ratio, was used as the effect indicator.In all regression models Africans were coded as 0 and Asian Indians as 1whilst females were coded as 0 and males as 1.Six participants were excluded from the analyses due to incomplete data collection and seven participants with serum calcium above the normal reference range (2.15–2.50 mmol/L) were also excluded. Characteristics of the participants (n = 717) are shown in In both ethnic groups females had lower 25(OH)D levels compared to their male counterparts (p = 0.0008 in Africans and p = 0.0002 in Asian Indians) and this was also observed for triglyceride levels (p = 0.03 in Africans and p<0.0001 in Asian Indians) . The PTHMet S was diagnosed in 38% of the participants, with a prevalence of 29% in the African and 46% in the Asian Indian group (p<0.0001). The data in In both ethnic groups, subjects with Met S had higher ages, HbA1c, HOMA, PTH, cholesterol and BMI measures than those without the Met S, whilst GFR was lower in those with Met S  see . CalciumIn a univariate analysis there was a significant negative correlation between PTH and 25(OH) D D and PTH with components of the Met S are presented in Logistic regression was used to determine whether the higher prevalence of Met S in the Indian population was related to PTH or 25(OH)D levels. The data in In order to determine which individual components of the Met S are influenced by PTH to increase the risk of Met S, logistic regression analysis was performed. The results in In this study we investigated whether 25(OH)D or PTH is associated with the Met S or its components. Our results show a significant association of PTH, but not 25(OH)D, with the Met S. Higher PTH levels were associated with increased systolic and diastolic blood pressure and reduced insulin resistance as assessed by the HOMA index and an increased odds ratio for the Met S. Furthermore our results suggest that neither 25(OH)D nor PTH contribute to ethnic differences in the prevalence of Met S between African and Asian Indian subjects. There are no previous studies from sub-Saharan Africa that have examined the relationship between 25(OH)D, PTH and the Met S. Our results also showed lower levels of 25(OH)D in Asian Indians compared to Africans. Studies from India have also shown that this population tends to have poor vitamin D status Our observations of a lack of association of 25(OH)D with Met S are in line with those of Scragg et al. who observed no association between 25(OH)D status and type 2 diabetes in non-Hispanic black subjects but did observe a negative associations between 25(OH)D and risk of diabetes in Mexican Americans and non-Hispanic white populations The results of studies on the association of 25(OH)D and PTH with the Met S, components of the Met S or related disorders have been inconsistent. In a population based cross sectional study of US men and women over 20 years-of-age  the prevalence of Met S and its components fell across increasing quartiles of 25(OH)D after adjustment for age, race, sex, income, lifestyle factors, calcium and energy intake. This study also showed that the odds ratio for Met S increased with increasing PTH in older men only A number of other studies have implicated PTH as being associated with the Met S rather than 25(OH)D Logistic regression models demonstrated that the increased risk of Met S in subjects with higher PTH levels is largely due to the positive and independent relationships of systolic blood pressure and waist circumference with PTH. Previous studies have also shown a positive association of PTH with obesity Our data show that Asian Indian ethnicity is associated with increased risk for Met S and most of its components. As a group, Asian Indians tend to be insulin resistant and at high risk for diabetes and premature coronary heart disease when compared to other ethnic groups This study is limited by the cross-sectional nature of the data, and as such we cannot draw any conclusions about causality. Another limitation is that blood samples were collected over several months, and vitamin D is known to display seasonal variation. Also, study subjects were not selected randomly and this may have introduced some level of selection bias. Finally, 25(OH)D levels were not measured using the reference method of liquid chromatography-tandem mass spectrometry (LC-MS/MS), however the technique used in this study (HPLC) has been shown to correlate well with the LC-MS/MS method In conclusion, PTH but not 25(OH)D is associated with the Met S in our African and Asian Indian populations. The PTH effect is largely via its impact on blood pressure and its relationship with waist circumference. The relationship between PTH and insulin resistance needs to be investigated further, as does the mechanism responsible for causing serum PTH levels to rise with increased adiposity.


We formulate a mathematical model for the cointeraction of schistosomiasis and HIV/AIDS in order to assess their synergistic relationship in the presence of therapeutic measures. Comprehensive mathematical techniques are used to analyze the model steady states. The disease-free equilibrium is shown to be locally asymptotically stable when the associated disease threshold parameter known as the basic reproduction number for the model is less than unity. Centre manifold theory is used to show that the schistosomiasis-only and HIV/AIDS-only endemic equilibria are locally asymptotically stable when the associated reproduction numbers are greater than unity. The impact of schistosomiasis and its treatment on the dynamics of HIV/AIDS is also investigated. To illustrate the analytical results, numerical simulations using a set of reasonable parameter values are provided, and the results suggest that schistosomiasis treatment will always have a positive impact on thecontrol of HIV/AIDS. Schistosoma or flat worms. The adult worms inhabit the blood vessels lining either the intestine or bladder, depending on the species of the worm , a modified Holling's type-II functional response , the response refers to the change in the density of susceptibles per unit time per pathogen as the schistosomiasis susceptible population density changes. From the functional response, we note that at low parasite density, contacts are directly proportional to host density, but a maximum rate of contact is reached at very high densities (saturation incidence). Individuals infected with schistosomiasis have an additional disease-induced death rate dB. Similarly, susceptible and infected snails have a natural death rate μS, and the infected snails have an additional disease-induced death rate dS. The total snail population is given by NS(t) = SS(t) + IS(t).NE of eggs leave the body through excretion (faeces and urine) and find their way into the fresh water supply where they hatch into free swimming ciliated miracidium at a rate γ for individuals without AIDS. Given the weakened immune system of AIDS individuals, they tend to excrete more often, thus releasing more eggs which will hatch into miracidia at a rate σγ, σ > 1. If the miracidium reaches a fresh water with snails of a suitable species, it penetrates at a rate λM, where Considering a schistosomiasis-infected individual, a number (portion) μM. The infected snails release a second form of free swimming larva called a cercariae which is capable of infecting humans at rate θ. Some cercariae also die naturally at a rate μP. Individuals infected with schistosomiasis are infected with HIV at a rate δλH with δ > 1 since infection by schistosomiasis creates wounds within the urethra as eggs are being released, which increases the likelihood of HIV infection per sexual contact. Individuals with HIV progress to the AIDS stage at a rate ρ. Individuals in the AIDS stage have an additional disease-induced death rate dA. We assume that antiretroviral therapy is given to AIDS individuals who are ill and have experienced AIDS-defining symptoms, or whose CD4+ T cell count is below 200/μL, which is the recommended AIDS defining stage T, wherea and b, say. After some little algebraic manipulations and rearrangements, it can be shown thatb may be expected in general for epidemic models because, in essence, using β as a bifurcation parameter often ensures b > 0 T, wherea and b yields𝒰2* is locally asymptotically stable for ℛB > 1.The unique endemic equilibrium a < 0, local stability of 𝒰2* implies its global stability.Since Model system  has evidWatmough , the repℛA and ℛB defined as earlier in 𝒰0 is locally asymptotically stable whenever ℛHB < 1 and unstable otherwise.The disease-free equilibrium In this section we investigate the effects of HIV/AIDS on schistosomiasis and vice versa, in the presence and absence of the aforementioned intervention strategies.α = 0) the reproductive number is denoted by ℛA0 and also in the absence of schistosomiasis treatment (ω = 0), ℛB = ℛB0. Thus, to express ℛB0 in terms of ℛA0, we solve for μH and obtainℛB0, we haveℛB0 partially with respect to ℛA0 yieldsℛB0 = 1 and expressing μH as the subject of formula, we haveθ1 = μMP0 and θ2 = μMP0βPNEγΛHℛS0. Consider θ3 − dBθ1)ℛB0 + θ4 > 0. Then, ℛA0 expressed in terms of ℛB0 readsℛA0 with respect to ℛB0 yieldsκ1h2 ≥ θ4h1,  treatment for model system  only, wiThe synergy between HIV and other diseases such as schistosomiasis provides more opportunities to combat HIV/AIDS by treating its coinfections with these other diseases.We shall use the following theorem of Castillo-Chavez et al.  in the sX ∈ ℝm denotes (its components) the number of uninfected individuals, Z ∈ ℝn denotes (its components) the number of infected individuals including latent, infectious, and so forth, U0 =  denotes the disease-free equilibrium of the system. Assume that (i) for dX/dt = F,X* is globally asymptotically stable, (ii) X, Z) ∈ 𝒟, where A = DZG is an M-matrix  and 𝒟 is the region where the model makes biological sense. Then the fixed point U0 =  is a globally asymptotic stable equilibrium of model system  , HIV on In this study, the impact of schistosomiasis and its treatment on the transmission dynamics of HIV/AIDS in the community is investigated by formulating a mathematical model that incorporates both key epidemiological parameters of both schistosomiasis and HIV/AIDS. Mathematical and numerical analysis of the model suggests that schistosomiasis may increase the prevalence of HIV/AIDS in the community. Analysis of the impact of schistosomiasis treatment has shown that the impact of this form of treatment depends on the sign of a certain threshold parameter Θ, and for Θ > 1, schistosomiasis treatment will have a positive impact, for Θ = 1, no impact, and for Θ < 1, a negative impact on controlling the co-interaction of the two diseases. We, however, note that from schistosomiasis and HIV/AIDS epidemiology, realistic parameter values always yield 1 < Θ. Consequently, schistosomiasis treatment will always have a positive impact on the control of both schistosomiasis and HIV/AIDS codynamics. Thus, schistosomiasis treatment can reduce the burden of schistosomiasis and HIV/AIDS coinfection in areas of extreme poverty, especially among the rural poor and some disadvantaged urban populations since it is less expensive and usually available in government clinics and hospitals. This outcome highlights the fact that global public health challenges require comprehensive and multipronged approaches to dealing with them. Current efforts that focus on a single infection at a time may be losing substantial rewards of dealing synergistically and concurrently with multiple infectious diseases .


Data from randomized trials on the development of anaemia after interruption of therapy is not well described. We aimed to describe the development of anaemia after interruption of cART and the relationship between the development of anaemia and clinical events  in the Strategic Management of Antiretroviral Therapy (SMART) randomised trial.2248 patients from the SMART study were included. We used Cox proportional hazards models to investigate development of new  or worsening (<8 mg/dl if anaemic at randomization) anaemia and poisson regression analyses to explore the relationship between anaemia and the development of AIDS, death or non-AIDS events.The change in haemoglobin and anaemia after randomisation to SMART is shown in Figure 759 patients developed new or worsening anaemia; 420/1106 (38.0%) in the drug conservation (DC) arm and 339/1127 (30.1%) in the virological suppression (VS) arm; p<0.0001. In the first 4 months following randomization, there was no difference in the risk of new or worsening anaemia when comparing the DC arm to the VS arm . After the initial 4 months, patients in the DC arm had a significantly increased risk of new or worsening anaemia . 56 patients died during 5811 person-years of follow-up (PYFU), 56 developed AIDS (5728 PYFU) and 100 developed a non-AIDS event (5664 PYFU). Currently anaemic patients had an increased incidence of AIDS , death  and non-AIDS events  compared to non-anaemic patients.Patients in SMART who interrupted cART had a higher risk of new or worsening anaemia. Patients with anaemia had a higher incidence of AIDS, non-AIDS defining events or deaths; whether this relationship is causal or a consequence of the disease is not clear but suggests that anaemia, or drop in haemoglobin, might be of use as a pre-clinical marker of disease. Further research is warranted to further understand the occurrence of anaemia, its consequences and underlying pathological mechanisms.