Previous studies have suggested the involvement of c-Met and/or its ligand, hepatocyte growth factor (HGF), in esophageal squamous cell carcinoma (ESCC), but the correlation between c-Met status and clinical outcome remains unclear. Furthermore, the identification of a novel molecular therapeutic target might potentially help improve the clinical outcome of ESCC patients.The expression of c-Met and HGF was immunohistochemically assessed in 104 surgically obtained tissue specimens. The correlation between c-Met/HGF expression and patients’ clinicopathological features, including survival, was evaluated. We also investigated changes in cell functions and protein expression of c-Met and its downstream signaling pathway components under treatments with HGF and/or c-Met inhibitor in ESCC cell lines.Elevated expression of c-Met was significantly correlated with tumor depth and pathological stage. Patients with high c-Met expression had significantly worse survival. In addition, multivariate analysis identified the high expression of c-Met as an independent prognostic factor. Treatment with c-Met inhibitor under HGF stimulation significantly inhibited the invasive capacity of an ESCC cell line with elevated c-Met mRNA expression. Moreover, c-Met and its downstream signaling inactivation was also detected after treatment with c-Met inhibitor.The results of our study identified c-Met expression as an independent prognostic factor in ESCC patients and demonstrated that c-Met could be a potential molecular therapeutic target for the treatment of ESCC with elevated c-Met expression.The online version of this article (doi:10.1186/s12885-015-1450-3) contains supplementary material, which is available to authorized users. Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy. Recently, several molecular markers have been identified as prognostic factors for ESCC, and the development of preoperative chemo-irradiation, surgical techniques, and postoperative chemotherapy has been reported . Howeverc-Met gene amplification and mutation in human malignancies have been reported to range from 1.4 % to 7.2 % for gene amplification [c-Met gene amplification has been reported in ESCC [c-Met, a transmembrane receptor tyrosine kinase, is composed of α- and β-chains connected by a disulfide linkage . It is afication –21 and 1fication , 21 in lfication , 22, 23,fication , 13.2 % fication , and 26.fication . Recentl in ESCC . However in ESCC , 30.in vitro experiments.Therefore, in this study, we examined the clinical and biological significance of c-Met in ESCC and evaluated the potential of c-Met as a molecular therapeutic target using We examined tissue samples from 104 primary ESCC patients who underwent surgery without neoadjuvant therapy from January 2000 to December 2006 at the Tohoku University Hospital, Sendai, Japan. The final diagnosis was made based on the 6th edition of the tumor-node-metastasis classification of malignant tumors by the Union for International Cancer Control . PatientImmunohistochemistry was performed using anti-c-Metβ polyclonal antibody  and anti-HGFα polyclonal antibody . All surgical pathology specimens, obtained from the sites of deepest invasion, were sectioned at 3-μm thickness. Antigen-retrieval was performed in 0.01 M citrate buffer (pH 6.0) by heating in a microwave. The slides were then washed with phosphate-buffered saline (PBS) and incubated with protein blocking solution  at room temperature. They were reacted with the primary antibodies overnight at 4 °C. Endogenous peroxidase activity was blocked by incubating the reacted slides in 0.3 % hydrogen peroxidase with methanol. Slides were then incubated with biotinylated goat anti-rabbit IgG (Nichirei) and horseradish peroxidase-conjugated streptavidin (Nichirei). The antigen-antibody complex was visualized with 3.3′-diaminobenzidine and counterstained with hematoxylin. Normal placenta was used as the positive control for c-Met and HGF immunoreactivity. The absorption test was performed using each antigen peptides .All immunostained slides were evaluated by two authors (YO and YN) without prior knowledge of any clinicopathological variables. Five different high-power fields were analyzed per slide, with each field containing more than 100 carcinoma cells. The H-score was determined using the percentage of immunopositive cells and their immunointensity. Immunointensity was evaluated and scored according to the following criteria: 0, completely negative; 1+, weakly positive; 2+, moderately positive, and 3+, markedly positive. The H-score was then calculated by multiplying the percentage of immunopositive cells to the immunointensity score (H-score ranging from 0 to 300). We also determined the optimal cut-off values using the receiver operating characteristic (ROC) curve method, which indicated that “40” was the optimal cut-off for patients’ survival outcome when analyzing c-Met and HGF immunohistochemistry results . It was also close to the optimal cut-offs for tumor depth  and lymph node metastasis . Cases with an H-score below the cut-off value were tentatively categorized as “low expression”, whereas those with an H-score greater than the cut-off value were considered as “high expression”.In order to evaluate the possible influence of inflammatory processes upon c-Met and HGF expression, the preoperative levels of percent vital capacity (%VC), forced expiratory volume in one second percentage (FEV 1.0 %), C-reactive protein (CRP), the retention rate of indocyanine green 15 min after administration (ICG-R15), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were evaluated because of the high frequency of alcohol consumption and smoking history in ESCC patients.2 incubator.Three human ESCC cell lines  were purchased from the Health Science Research Resources Bank . All cell lines were authenticated by using STR analysis  in July 2014. The three cell lines were maintained according to the manufacturer’s instruction. All cells were cultured at 37 °C in a 5 % COA small-molecule inhibitor for c-Met (PF-2341066) was purchased from Selleck Chemicals . PF-2341066 was reconstituted in dimethyl sulfoxide (DMSO). Recombinant human HGF (rHGF) was purchased from R&D Systems  and reconstituted in PBS with 0.1 % bovine serum albumin.TRIzol RNA Isolation Reagents  were used for total RNA extraction according to the manufacturer’s instruction. Reverse transcription was then performed in a thermal cycler using appropriate amounts of total RNA. The measurement of mRNA expression was performed using a Light Cycler equipment . The sequence of the primers used was as follows: c-Met forward: 5′-CACTTCTGAGAAATTCATCAGGCTGTGAAG-3′, reverse: 5′-AGAGGACTTCGCTGAATTGACCCATG-3′, and HGF forward: 5′-TGTGCCATTCCAAATCGTCCTGGT-3′, reverse: 5′-TCAACAAACATGACTCTCCAGTAGTTGTCT-3′. RPL13A was used as a housekeeping gene for mRNA quantification.For protein extraction, three ESCC cell lines  were lysed and scraped in Mammalian Protein Extraction Regent with 2 % protease inhibitor, 2 % phosphatase inhibitor, and 1 % ethylenediaminetetraacetic acid. Each protein sample was subsequently resolved in 10 % sodium dodecyl polyacrylamide gels and transferred onto nitrocellulose membranes using Amersham ECL semi-dry blotters . After blocking of nonspecific binding for 1.5 h in 5 % skimmed milk, the membranes were reacted with primary antibodies at 4 °C overnight. They were then washed with Tris-buffered saline and reacted with secondary antibody for 1 h. Protein expression was visualized using ChemiDoc™ XRS+ System . For quantification of protein expression by c-Met and components of its downstream signaling pathways, KYSE170 cells were cultured in fetal bovine serum (FBS)-free medium for 48 h prior to protein extraction. The quantitative determination was performed using the quantification tool of Image LabTM software (BIORAD). The primary antibodies used are summarized in Table 5 cells/chamber for KYSE170 and 1 × 105 cells/chamber for KYSE 150 and 180) with PF-2341066 treatment or vehicle control. These upper chambers were inserted in to a 24-well lower chamber containing FBS-free medium supplemented with 50 ng/ml of HGF or vehicle control. After the plate was cultured for 24 h at 37 °C with 5 % CO2, each upper chamber filter was fixed with methanol and counterstained with hematoxylin. The number of invasive tumor cells for each filter membrane was then counted in high-power fields.The invasive properties of ESCC cells were determined using Cell Culture Inserts with transparent PET membrane  and their companion plates . After culturing for 24 h in an FBS-free medium, three ESCC cell lines  were seeded in the upper chambers (3 × 104 cells/well). After the plate was cultured for 24 h in FBS-free medium, the medium was switched to one with or without 50 ng/ml of HGF and PF-2341066. Then, WST-8, a highly water-soluble tetrazolium salt, was added to each well at 24 h after PF-2341066 treatment, and absorbance at 450 nm was measured using a microplate reader (BIORAD). Equivalent volumes of DMSO were used as vehicle controls.ESCC cells were counted using the Cell Counting Kit-8 . Three ESCC cell lines  were seeded in a 96-well plate .Pearson’s chi-square test was used to assess the correlation of immunohistochemical protein expression with various clinicopathological features. Figure P = 0.013) and pathological stage (P = 0.010). On the other hand, HGF status was significantly correlated with tumor differentiation (P = 0.024), tumor depth (P = 0.028), lymph node metastasis (P = 0.014), and pathological stage (P = 0.022). However, no statistically significant correlation was detected between c-Met and HGF status of the patients. Results of the correlation analysis of ESCC patients’ clinicopathological variables and c-Met/HGF immunoreactivity in carcinoma cells are summarized in Table c-Met status was significantly correlated with tumor depth P = 0.03 and patP = 0.022)  , lymphatic invasion (P = 0.015), tumor depth (P = 0.015), lymph node metastasis (P < 0.001), pathological stage (P < 0.001), and c-Met expression (P = 0.017) (Table P = 0.033) and lymph node metastasis (P = 0.025) as independent prognostic factors  Fig. . This di15) Fig. . Univarirs Table . However2 Fig. . There were no significant correlations between c-Met/HGF expression and preoperative AST, ALT, ICG-R15, %VC and FEV 1.0 % in ESCC patients (data not shown).c-Met and HGF mRNA was detectable in all three cell lines examined Fig. . The amoP = 0.0019). However, the number of invaded cells decreased under PF-2341066 treatment to 54 ± 13.3 at 10 nM, 27.6 ± 4.5 at 100 nM, 36.2 ± 12.3 at 500 nM, and 25.2 ± 5.4 at 1 μM. In addition, treatment with 100 nM, 500 nM, and 1 μM of PF-2341066 significantly decreased cell invasiveness  ; that of KYSE170 decreased 11 % (P = 0.11), and KYSE180 cell proliferation decreased 16 % (P = 0.003)  under PF-2341066 treatment was evaluated as a decrease percentage compared to control (rHGF+/PF-2341066-). When 1 μM of PF-2341066 was added, the proliferation of KYSE150 cells decreased 16 %   prevalence of c-Met gene amplification in ESCC patients [c-Met gene mutation or amplification in ESCC is virtually unknown.The results of our nditions . The respatients , the staet al. reported the inhibition of MAPK and Akt signaling by PF-2341066 in ovarian cancer cells [et al. reported the inhibition of Akt signaling but not of the MAPK cascade by PF-2341066 in head and neck squamous cell carcinoma cells, possibly due to the high basal level of phosphorylated MAPK in these cells prior to HGF stimulation [The important downstream signaling of c-Met includes the MAPK cascade and PI3K/Akt signaling pathway, which mainly regulates cell proliferation and motility [er cells , which wmulation . Our resmulation , 38–41. PF-2341066 is also known to inhibit the protein activation processes resulted from gene rearrangement of anaplastic lymphoma kinase and ROS1 tyrosine kinase , 43 in aIn conclusion, the results of our study identified c-Met expression in carcinoma cells as an independent prognostic factor for ESCC and demonstrated the potential of c-Met as a molecular therapeutic target for the treatment of high c-Met expressing ESCC. However, further genetic and/or biological investigations are warranted.


Burnout is a psychosomatic syndrome characterized by three dimensions . We determined the prevalence of burnout and mental health status between HIV/AIDS healthcare workers and other healthcare workers, and determined the factors associated with burnout of HIV/AIDS healthcare workers.All participants were asked to complete a self-administered questionnaire. The participants were recruited from the departments of infectious diseases in four hospitals which treated HIV/AIDS. The questionnaire included demographics, the Maslach Burnout Inventory-General Survey (MBI-GS), the Symptom Checklist 90 (SCL-90), the Eysenck Personality Questionnaire (EPQ), and the Trait Coping Style Questionnaire (TCSQ).F = 6.350, p = 0.012) and depersonalization dimensions  were significantly higher than other healthcare workers. The HIV/AIDS healthcare workers had higher total scores and positive items on the Symptom Checklist 90 (SCL-90) compared with other healthcare workers. Low job satisfaction, serious somatization, interpersonal sensitivity, poor quality of sleep, high psychoticism scores, and use of negative coping styles were frequently associated with burnout.A total of 512 questionnaires were distributed; 501 questionnaires were completed and collected (the response rate was 97.9 %). After eliminating nine invalid questionnaires (1.80 %), 264 physicians and nurses caring for HIV/AIDS and 228 physicians and nurses caring for other infectious diseases provided valid responses (98.2 %). The HIV/AIDS healthcare workers’ scores on the emotional exhaustion (Burnout was shown to be highly prevalent in HIV/AIDS healthcare workers, 76.9 % of whom met the accepted criteria for burnout. In addition, compared with other healthcare workers, HIV/AIDS healthcare workers experienced lower levels of psychological health. Interventions should be targeted at reducing the occurrence of burnout and alleviating psychological pressure amongst HIV/AIDS healthcare workers. Burnout is a psychosomatic syndrome characterized by three dimensions  . The focIn contrast with other medical staff, HIV/AIDS healthcare workers have an excessive workload, higher levels of stress, and worse working conditions because of the particularity of HIV, which includes infectivity, stigma, and discrimination , 9. In COf particular importance are the consequences of burnout among HIV/AIDS healthcare workers, including poorer quality of care, decreased empathy, absenteeism, increased turnover, and decreased job performance , 15. BurDue to the adverse consequences of burnout among HIV/AIDS healthcare workers, determining the related risk factors is extremely important. On the basis of a previous study, burnout among healthcare workers is due to a combination of personal and workplace environment factors . These fIn the current study, we determined the prevalence of burnout among HIV/AIDS healthcare workers and determined the factors associated with burnout in HIV/AIDS healthcare workers. Our findings provide the theoretical basis for the effective promotion of physical and mental health of HIV/AIDS healthcare workers, for decreasing occupational burnout, and for improving work performance in HIV/AIDS treatment.The HIV/AIDS healthcare workers are concentrated in departments of infectious diseases in hospitals where HIV/AIDS is treated. Thus, the healthcare workers selected as the sample in this study were recruited from departments of infectious diseases. This cross-sectional study was conducted in Nanning, which is the capital of Guangxi province, and has one of the highest incidences of HIV/AIDS in China, including physicians and nurses from four hospitals that treat HIV/AIDS. We personally contacted the healthcare workers, invited them to participate, and clearly explained the aims and significance of this study, as well as the method by which to complete the questionnaires. Participation was completely voluntary, without any monetary motivation, and each participant signed a written informed consent. We ensured confidentiality and immediately provided an explanation without inducement for any unclear questionnaire items. Self-administered questionnaires were directly distributed to the participants who were allotted 15 min to complete the questionnaire. The questionnaires were collected immediately after completion. We checked the questionnaires to avoid errors and ensure quality. Five hundred twelve questionnaires were distributed and a total of 501 questionnaires were completed and collected (the response rate was 97.9 %), but there were 9 invalid questionnaires (1.80 %). Of the 264 physicians and nurses caring for HIV/AIDS and the 228 physicians and nurses caring for other infectious diseases in the departments of infectious diseases provided valid responses (98.2 %). This study was approved by the Ethics Committee of Harbin Medical University.The basic socio-demographic information included age, gender, education, marital status. The education was grouped into three types . The marital status was categorized as married or single.Burnout symptoms in this study were assessed by a Chinese version of the 15-item Maslach Burnout Inventory-General Survey (MBI-GS) . The MBIThe scale developed by Derogatis , 26 is aThis study used the Eysenck Personality Questionnaire to assess the personality traits of participants. The scale includes three 12-item indices , along with a 12-item lie (L) scale , 32. EacThe Trait Coping Style Questionnaire (TCSQ) consists of 20 items, rated on a 5-point Likert scale, ranging from 1 to 5  . The TCSThe job satisfaction was measured by asking the question, “Are you satisfied with current job” with the following response choices: “1 = satisfactory; 2 = moderately satisfactory; and 3 = unsatisfactory.”p < 0.05. HIV/AIDS healthcare workers and other healthcare workers were included in the analyses. Demographic data, including age, gender, education, marital status, and job satisfaction were recorded as numbers and percentages. Comparisons of burnout for demographics were tested by t-tests and ANOVA. Multivariate analyses of variance (MANOVA) were performed to compare the three dimensions of burnout and the 10 subscales of SCL-90 variables between two groups. The positive items of SCL-90 variables were compared between groups byχ2 tests. We used logistic regression to assess the factors associated with burnout symptoms.SPSS (version 18.0) was used for statistical analysis. All tests were two-tailed, with a statistical significance level set at a p > 0.05). The scores of the three dimensions of occupational burnout had no significant difference between physicians and nurses (p > 0.05).After excluding 9 invalid questionnaires, 492 participants completed the questionnaires during the survey. Of the 264 physicians and nurses caring for HIV/AIDS and the 228 physicians and nurses caring for other infectious diseases in the departments of infectious diseases provided valid responses. Of these physicians and nurses, 29.9 % were males and 70.1 % were females, and 68.2 % were married and 31.8 % were single. Of the participants, 21.3 % had attained a postgraduate qualification. The average age of the participants was 33.77 years (SD =8.63), with a range of 19–60 years. There was no significant association between burnout scores on age and gender  and DP  were significantly higher than other healthcare workers, but there were no significant differences noted in the dimension of reduced PA . As shown in Table F = 4.355, p = 0.038) and hostility  between HIV/AIDS healthcare workers and other healthcare workers.When compared with other healthcare workers, the HIV/AIDS healthcare workers scored higher in SCL-90 total scores. Chi-square tests showed that the number of positive items were significantly different between HIV/AIDS healthcare workers and other healthcare workers (Nearly one-fourth (24.6 %) of HIV/AIDS healthcare workers were satisfied with their work, 54.9 % were moderately satisfied, and 20.5 % were dissatisfied.On the basis of a bivariate logistic model, the possibility of having burnout symptoms was significantly higher in HIV/AIDS healthcare workers who were dissatisfied with their job, had serious somatization, interpersonal sensitivity, poor quality of sleep, high scores of psychoticism, and used negative coping styles frequently Table .Table 3FThe overall prevalence of burnout syndrome among the HIV/AIDS healthcare workers was 76.9 %. The prevalence of low EE, DP, and reduced PA was 41.7, 67, and 54.2 %, respectively. The prevalence of medium EE, DP, and reduced PA was 37.8, 25.4, and 28 %, respectively. The prevalence of high EE, DP, and reduced PA was 20.5, 7.6, and 17.8 %, respectively. Our study reported that HIV/AIDS healthcare workers exhibited a high level of EE, which is considered the core symptom of burnout. Gabbe et al. [The present results show that HIV/AIDS healthcare workers scored higher in total scores and positive items when compared with other healthcare workers. Comparing the 10 factors in SCL-90, we found that the scores of depression and hostility significantly differed between HIV/AIDS healthcare workers and other healthcare workers, which is similar to previous report . A previHIV/AIDS healthcare workers are a specific occupational group that often needs to deal with people living with HIV and AIDS patients, have more tedious work, long working hours, and irregular work and life time in contrast with other healthcare workers. Thus, HIV/AIDS healthcare workers are more likely to experience burnout because of their work and stress with such characteristics of professional background. Our study showed that burned-out HIV/AIDS healthcare workers have poor job satisfaction, serious somatization, interpersonal sensitivity, poor quality of sleep, high scores of psychoticism, use of negative coping styles frequently. Job satisfaction describes how people feel about their jobs—whether they like or dislike their jobs . In our In summary, occupational burnout and the mental health of HIV/AIDS healthcare workers should be taken seriously by authorities, in particular, the high AIDS epidemic province, such as Guangxi. Relevant departments should try to meet the reasonable material and spiritual needs of HIV/AIDS healthcare workers, improve welfare to stimulate their enthusiasm for work, and reduce the occurrence of burnout. Furthermore, enhancing education on reducing HIV-related stigma and discrimination, actively seeking countermeasures, and improving the level of mental health to alleviate psychological pressure is warranted. Moreover, departments should raise awareness of occupational protection and effective protective measures should be taken to reduce the occurrence of occupational exposure and improve workplace safety. The above measures can improve work efficiency of HIV/AIDS healthcare workers and enhance the prevention and treatment of AIDS.Despite these merits, there were several limitations in the present study. The first limitation is the potential for sampling bias, as the subjects in the present study were recruited from four hospitals that treated HIV/AIDS in Guangxi province, which might not be representative of HIV/AIDS healthcare workers throughout China. The second limitation involves the weak causal inference because this study had a cross-sectional design. Therefore, future studies should include a longitudinal follow-up to determine the relationships between burnout and other factors.Burnout is highly prevalent in HIV/AIDS healthcare workers, with 76.9 % of HIV/AIDS healthcare workers meeting the accepted criteria for burnout. Moreover, the HIV/AIDS healthcare workers’ scores of the dimensions of EE and DP were significantly higher than other healthcare workers, These findings indicate that some effective interventions for HIV/AIDS healthcare workers should be designed to address burnout. In addition, HIV/AIDS healthcare workers experience lower levels of psychological health, compared with other healthcare workers. In the present study, low job satisfaction, serious somatization, interpersonal sensitivity, poor quality of sleep, high psychoticism scores, and using a negative coping style frequently were associated with high burnout. Interventions should be targeted at reducing the occurrence of burnout and alleviating psychological pressure.


Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic’s ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens.Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as  Mycobacterium smegmatis and four evolutionarily divergent bacterial pathogens was blocked by conditions that worked to maintain intracellular pH homeostasis. Single-cell pH analysis demonstrated that antibiotics increased the cytosolic pH of M. smegmatis, while conditions that promoted proton entry into the cytosol prevented intracellular alkalization and antibiotic killing. These findings led to a hypothesis that posits antibiotic lethality occurs when antibiotics obstruct ATP-consuming biosynthetic processes while metabolically driven proton efflux is sustained despite the loss of proton influx via ATP synthase. Consequently, without a concomitant reduction in respiratory proton efflux, cell death occurs due to intracellular alkalization. Our findings indicate the effects of antibiotics on pH homeostasis should be considered a potential mechanism contributing to antibiotic lethality.Antibiotics target specific biosynthetic processes essential for bacterial growth. It is intriguing that several commonalities connect the bactericidal activity of seemingly disparate antibiotics, such as the numerous conditions that confer broad-spectrum antibiotic tolerance. Whether antibiotics kill in a manner unique to their specific targets or by a universal mechanism is a critical and contested subject. Herein, we demonstrate that the bactericidal activity of diverse antibiotics against IMPORTANCE Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic’s ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens. Antibiotics target biosynthetic processes essential for cell growth. Inhibition of nucleic acid, protein, or cell wall synthesis leads to cessation of growth and often cell death. However, why some drugs kill while others are bacteriostatic is not well understood. Additionally, the specific downstream effects of the primary drug-target interaction that ultimately cause bacterial death have yet to be elucidated. Multiple commonalities exist between the bactericidal activities of seemingly disparate antibiotic targets. These include the existence of generalized persister populations that are simultaneously tolerant to multiple antibiotics as well as specific metabolic conditions that confer broad-spectrum antibiotic tolerance, particularly those that inhibit respiration –8. TheAlthough the exact mechanism of antibiotic cell death is unclear, it appears that cellular metabolism plays a key role. Cellular processes involved in metabolism, growth, division, and maintaining energy balance are multifaceted and numerous. Cellular respiration in its simplest form can be described as a series of metabolic reactions used to break down nutrients in order to provide biochemical energy in the form of ATP. Electron carriers such as NADH feed electrons from catabolic reactions to electron transport systems, which translocate protons across the inner cytoplasmic membrane, forming a proton gradient. Energy from the proton gradient is harnessed via ATP synthase as protons flow back into the cell . Vast amIN), which if substantial enough could result in bacterial membrane disruption.We propose a model in which an imbalance in proton homeostasis, resulting in a decrease in intracellular proton concentration, is at least partially responsible for antibiotic-induced cell death. The majority of protons that reenter the cell do so through ATP synthase , 14, thuIN and was exacerbated under conditions in which pHIN increased. Using flow cytometry for single-cell analysis of Mycobacterium smegmatis, pHIN was observed to increase after antibiotic exposure but not in the presence of a protonophore, which decreased pHIN and rescued antibiotic killing. The observed decrease in antibiotic killing resulting from manipulation of pH homeostasis did not appear to be a result of a general disruption of metabolic processes or decreased antibiotic activity against specific antibiotic targets.We challenged Gram-positive, Gram-negative, and acid-fast bacteria with multiple classes of antibiotics to study the role of proton homeostasis in antibiotic-induced cell death. We demonstrated that antibiotic-induced death was abrogated by conditions that lowered pHM. smegmatis to determine a concentration that did not inhibit growth (m-chlorophenyl hydrazine (CCCP), also blocked antibiotic killing in a similar manner to DNP  . The funt growth . Use of t growth . A subinr to DNP . These fEX). We found an alkaline extracellular environment enhanced chlorpromazine killing, while an acidic environment blocked lethality  . In contrast, the protonophore CCCP was highly effective at dissipating the pH gradient and was used to calibrate the GFP ratiometric signal . pHIN of the overall population rose from 7.7 to 8.1 and 8.2 for kanamycin, norfloxacin, and chlorpromazine, but not rifampin (in (GFP) . Direct rifampin .M. smegmatis, rifampin was unique in having an extremely high MBC/MIC ratio. While the MIC for rifampin was 25 µg/ml (M. smegmatis (as defined by an MBC/MIC ratio of >4)  ; Fig. 1 o of >4) . In ordeIN (≥8.6) demonstrated that all antibiotics, including rifampin, increased the number of bacilli within this high-pHIN population. Addition of protonophore mitigated the alkalization caused by all antibiotics  (M. smegmatis (R2 = 0.999)  . The treM. smegmatis was exposed to phosphate citrate buffer at a pH near the pHIN, at 1 and 2 U higher or 1 U lower than the pHIN, and in the presence of the protonophores DNP and CCCP. Exposure of cells to a pH 1 U higher than the pHIN led to an ~10-fold greater drop in viability than exposure to a pHEX 1 U lower than the pHIN . A DNP concentration was determined for each species that did not affect growth and was used to ensure that growth arrest was not the basis of rescue . ATP levels in antibiotic-treated M. smegmatis cultures were the same as or higher than those in the negative control . Since the level of rifampin was 84 times higher than the MIC necessary to stop growth, it is likely that all rifampin RNA polymerase targets were saturated, resulting in maximum decrease in ATP utilization. In addition, the expected population-wide increase in pHIN from loss of ATP flux could have been largely offset from proton influx in exchange for rifampin efflux. However, the subpopulation increase with an alkaline pHIN at lethal levels of rifampin indicated that a fraction of the population lost the ability to maintain pH homeostasis, resulting in death. These differences between rifampin and the three bactericidal antibiotics may indicate fundamental differences in bacteriostatic and bactericidal antibiotics. It was recently reported that bactericidal antibiotics accelerate respiration, while bacteriostatic drugs either suppress or have no effect on respiration  (LB medium) were diluted 1:50 in LB medium at pH 6 (adjusted with hydrochloric acid), 7, or 8 (adjusted with sodium hydroxide) in 125-ml Erlenmeyer flasks. Cultures were allowed to grow while shaking for 1.5 h, and diluted to an optical density at 600 nm (OD600) of 0.1 with LB medium of the same pH. One-milliliter culture aliquots were added to 15-ml snap-cap tubes, antibiotics were added at concentrations listed in g for 1 min and suspended in LB medium, and survival was assayed by serial dilution and plating on LB agar plates. For M. smegmatis mc2155, overnight cultures grown in Dubos-Tween-albumin (DTA) medium  were diluted to an OD600 of 0.03 in DTA at pH 5.6 (adjusted with hydrochloric acid) or pH 6.6 or 7.6 (both adjusted with sodium hydroxide) and allowed to grow at 37°C in 125-ml Erlenmeyer flasks for 1.5 h while being stirred at 150 rpm. One-milliliter aliquots were added to 15-ml snap-cap tubes, and antibiotics and other drugs were added at the concentrations listed in g for 1 min and suspended in DTA medium, and survival was assayed by serial dilution and plating onto DTA agar plates containing 4 g/liter of activated charcoal. Mid-log-phase (OD600 of 0.2 to 0.4) cultures of M. tuberculosis H37Rv grown in DTA medium were diluted in DTA to an OD600 of 0.05 at pH 5.6, 6.6, or 7, and 1-ml aliquots were added to sterile glass tubes (20 by 125 mm) containing stir bars (12 mm by 4.5 mm). Cultures were outgrown 1 h at 37°C, antibiotics were added, and samples were stirred rapidly (200 rpm) using a rotary magnetic tumble stirrer . Samples were taken at intervals, serially diluted, and plated on DTA agar plates containing 4 g/liter of activated charcoal to determine survival.Overnight cultures of M. smegmatis cells were suspended in preincubated DTA containing antibiotic, and the killing assays were conducted as described for the experiments shown in To determine the relative stability of antibiotics at pH 5.6, 6.6, and 7.6, each antibiotic was preincubated in DTA medium for 24 h at each pH. The media containing drugs at pHs 5.6 and 7.6 were then adjusted to pH 6.6 followed by filter sterilization. Pelleted E. coli MG1655 single colonies were inoculated in 25-ml M9 minimal medium at pH 7 supplemented with 0.2% (wt/vol) succinate and 0.2% (wt/vol) d-lactate in 125-ml Erlenmeyer flasks and grown overnight at 37°C with shaking at 250 rpm. Cultures were diluted 1:50 into 10-ml anoxic M9 minimal medium supplemented with 0.2% (wt/vol) succinate, 0.2% (wt/vol) d-lactate, 400 mM sodium nitrate, and 0.1% (vol/vol) Oxyrase in 16-ml Hungate tubes with a tuberculin syringe through a butyl rubber septum stopper. Cultures were grown at 37°C for 16 h with shaking at 250 rpm, and diluted to an OD600 of 0.01 via the Hungate technique in fresh anoxic M9 medium at pH 6, 7, or 8 supplemented with 0.2% (wt/vol) succinate, 0.2% (wt/vol) d-lactate, 400 mM sodium nitrate and 0.1% (vol/vol) Oxyrase. Cultures were outgrown for 1.5 h, and antibiotics were added via syringe at concentrations listed in For anoxic growth, M. smegmatis were diluted to an OD600 of 0.05 and subjected to centrifugation, and pellets were suspended in phosphate-citrate buffer at pH 6.6, 7.6, 8.6, or 9.6. One-milliliter aliquots were added to 15-ml snap-cap tubes, and 25 µM CCCP or 1 mM DNP was added. Cultures were grown and survival determined as described above for M. smegmatis.To assay survival in phosphate citrate buffer of different pHs, overnight cultures of E. coli, S. aureus, P. aeruginosa, and M. smegmatis as described above. Bacteria were then exposed to 2-fold dilutions of antibiotics. Samples were plated at time zero and 3 h  or 24 h (for M. smegmatis). CFU were determined and recorded. The MIC was recorded as the minimum concentration at which the final CFU count was less than twice that at time zero. The values reported represent the mode of 3 biological replicates.Overnight cultures were started for M. smegmatis containing a plasmid encoding the ratiometric pH-sensitive green fluorescent protein (GFP) described elsewhere . An3H]norfloxacin  was prepared by diluting labeled drug with unlabeled norfloxacin to specific activities of 9.8 mCi/mg for E. coli and 3.6 µCi/mg for S. aureus, and M. smegmatis. Working concentrations of 10 µg/ml for E. coli experiments and 100 µg/ml for S. aureus and M. smegmatis experiments were used. Stock [3H]vancomycin  was prepared by diluting labeled drug with unlabeled vancomycin to a specific activity of 0.011 mCi/mg and a working concentration of 1.0 mg/ml. Stock [3H]tetracycline  was prepared by diluting labeled drug with unlabeled tetracycline to a specific activity of 0.51 mCi/mg and a working concentration of 10 mg/ml. Cultures were started as described above. Stock solutions of [3H]norfloxacin, [3H]vancomycin, or [3H]tetracycline were added to achieve a final antibiotic concentration shown in 3H-labeled antibiotic to filters was determined by adding labeled antibiotic to cell-free medium and subjecting it to the same vacuum filtration and washing as experimental samples, which served as a negative control. Internalized antibiotic was calculated by subtracting the negative control from each sample. The counts from this sample were divided from the counts obtained by adding pure labeled antibiotic of a known concentration to a membrane without washing to obtain a concentration of antibiotic associated with cells. This antibiotic concentration was divided by the total cellular protein concentration for each sample to obtain a measurement of antibiotic per total protein.Antibiotic uptake assays were performed similarly to those previously described . Stock [g for 1.5 min, and supernatants were carefully discarded. Pellets were suspended in equal volumes of 1× phosphate-buffered saline and subjected to lysis via sonication. Supernatants were clarified by centrifugation, and 150 µl was used for total protein analysis as per the manufacturer’s protocol (Bio-Rad QuickStart Bradford assay) in duplicate. The values from the duplicate samples were averaged per time point per biological replicate. Each reported value represents the mean ± standard deviation from 3 biological replicates for norfloxacin and tetracycline experiments and 6 biological replicates for vancomycin experiments.To determine cellular protein concentration, non-radiolabeled norfloxacin, vancomycin, or tetracycline was added to cultures as described above, and aliquots were removed for protein content determination with a Bio-Rad protein assay kit . Four hundred-fifty-microliter samples were taken at the same time points used for radioactive analysis and subjected to centrifugation at 16,000 × ATP measurements were taken as previously described . After 1t test was performed. For flow cytometry analyses, all data points were compared to the control sample from the same experiment. For survival studies, all data points were compared to the mid-pH sample from the same experiment unless otherwise noted.The Colorado Biostatistics Consortium at the Colorado School of Public Health was consulted to determine appropriate statistical analytical methods. For all statistical analyses, a paired, one-tailed


The Republic of Georgia lacks regulations regarding drug prescriptions. In pharmacies, all drugs except psychotropic medication are sold legally without prescription anti-, including anti-tuberculosis agents. Due to the lack of adequate policies and regulations, the big share of responsibility regarding antibiotic education lies with the general public. This study examines public knowledge and attitudes toward antibiotic use and resistance in the Republic of Georgia among personnel from government schools and other public institutions.This cross-sectional study was conducted in June 2011 using a quantitative questionnaire. Convenience sampling method was used. Participants included 250 individuals aged 21–80 years, from government schools and public institutions. Participants were from Tbilisi as well as the surrounding rural and urban areas. Respondents provided demographic data along with statements on knowledge and attitudes towards antibiotic use and resistance. Poisson and logistic regression models were used to study the relationship between knowledge, attitude outcomes and socio-demographic characteristics.The overall response rate was 75 % (n = 187), of which 80 % were female. Approximately 91 % of respondents had used antibiotics at least once and 55 % agreed that antibiotics speed up recovery from common colds. A number of respondents (55 %) reported having received antibiotics without previously consulting a doctor and 62 % reported having purchased antibiotics without a prescription. Respondents demonstrated some misunderstanding around the terms ‘bacteria’ and ‘virus.’ About 52 % of participants agreed that antibiotics are effective against bacteria; however, 55 % also agreed that antibiotics are effective against viruses. Trust in doctors was high at 80 %. More knowledge was associated with a lower probability of having purchased antibiotics without medical consultation.The study findings demonstrate that respondents have several misconceptions and lack knowledge on proper antibiotic use and resistance. High proportion of people use antibiotics without a medical prescription or consultation, while having high trust in the medical personnel. We believe that the high level of trust in doctors shown by our respondents should be acknowledged by the Georgian government, health care providers and public health policy professionals. Furthermore, the information should be utilized in future educational and antibiotic resistance awareness raising campaigns.The online version of this article (doi:10.1186/s13104-015-1477-1) contains supplementary material, which is available to authorized users. Antibiotic resistance can be defined as the ability of a microorganism to survive and resist exposure to antimicrobial drugs, threatening the effectiveness of successful treatment of infection. Resistance can be transferred genetically from one microorganism to another. Antibiotic resistance is a recognized public health issue at the local, national and global levels. Currently antibiotic resistance is a concern as it is no longer a predictor of maintaining health within populations but an increasing threat to future of health as antibiotics are being misused –5.Prevalence of antibiotic resistance varies among countries but in general it is positively correlated with prescribed outpatient antibiotic use at the national level , 7. HoweResistance is a severe problem in the treatment of tuberculosis (TB). In 2010, multi-drug-resistant TB (MDR-TB) was estimated at 3.4 % of all new TB cases worldwide. Georgia is one of the 18 high-priority countries with regards to TB with a 2008 prevalence rate of 135.5 cases/100,000 people, of which 6.4 % were TB cases in children under 15 years of age . While iGeorgia is a former Soviet Union Republic, which gained its independence on April 9th, 1991 . In 2011isoniazid and rifampin were widely available without prescription at pharmacies in Tbilisi [In Georgia, all drugs, except for psychotropic medication, are sold legally without prescription. Studies showed that even anti-TB drugs, including  Tbilisi .Due to the above-described situation, approximately 95 % of the Georgian population tends to avoid visiting a physician, which has resulted in the habit of self-treatment . Since mTo date, there is no study documenting knowledge and attitudes towards antibiotic use in Georgia. Thus, the aim of this study has been to examine the knowledge and attitudes toward antibiotic use and resistance among school and public institution personnel.In Georgia, people are often afraid to participate in research studies due to fear of exposure and confidentiality concerns. In order to have elicited participation in this study documenting knowledge and attitudes towards antibiotic use, considering the difficulty in accessing the public, we applied a convenience sampling method. We identified networks and contacts mostly in federally funded government institutions including schools, ministries and public service sectors in Tbilisi and the surrounding rural and urban areas. In 2011 eight schools and public institutions were approached. All individuals working at each school or institution, regardless of the type of their contract of employment  where invited to participate in this study. Participants were eligible if they were above 21 years of age. Medical personnel  were intentionally excluded due to possible response bias and more knowledge on the topic.The questionnaire  for analysis., awareness, resistance and general scores were created to assess knowledge. The awareness score was calculated as a sum of the correct answers to the questions under sections (2) (three questions on access to antibiotics) and (3) (14 questions on knowledge about antibiotic use and its effects). Resistance scores summed up the correct answers for the questions from section (4) (15 questions on side effects and resistance), whilst the general score represented the sum of correct answers on the questions in all of the above mentioned sections. The Cronbach’s alpha has been calculated for each score.During data analysis2 tests and correlation coefficients were used for bivariate analyses of the relationship between independent variables and the outcomes of interest.Descriptive statistics were calculated using median and standard deviation for numerical variables, and frequencies and percentages for categorical variables. T tests, χawareness,resistance, and general scores, stepwise Poisson regression models were used to study the relationship between scores and socio-demographic characteristics. Stepwise logistic regression models were performed to study the association between ‘having purchased Antibiotics without prescription’ and ‘having received Antibiotics without prior medical consultation’ (both binary variables), versus demographic characteristics  and knowledge scores . For all the regression models, variables with a p ≤ 0.25 in the bivariate analyses were considered in the stepwise models (to be more conservative in the selection of variables), and p < 0.05 were considered significant in the final models.For count outcomes such as, The Heads of the public institutions where the questionnaires were distributed were contacted prior to the study and were informed about the topic of the study. They gave their consent for their personnel to participate. Subsequently, potential participants were approached and given information about the study, and that results would only be presented at an aggregate level. Those who agreed gave their oral/written consent before completing the questionnaire. In Georgia, these kinds of studies do not require formal ethical permission.Two hundred and fifty participants were included in this study of which 187 completed the survey. The participants’ background characteristics are summarized in Table doctor–patient relationship. The majority of respondents (54 %) agreed with the statement that ‘pharmacy staff often tell you how antibiotics should be used’. Most reported trusting doctors’ decisions both for prescribing (76 %) and for not prescribing antibiotics (80 %).Table Knowledge scores were quite low. The median resistance score, based on 17 questions, equaled six, and median awareness score, based on 15 questions, did not exceed eight. According to the Cronbach’s alpha analyses the resistance, awareness and general scores have an acceptable level of reliability Table . MultiplLogistic regression analyses showed that a higher awareness score is associated with lower odds of having purchased antibiotics without a medical consultation (p = 0.007) Table . Age is This is the first study documenting knowledge and attitudes towards antibiotic use in Georgia. The topic has, however, been researched in similar studies in other countries such as Sweden, South Korea, Malaysia, the Denver metropolitan area, New Zealand, Netherlands and Tanzania –24. PoorThe participants in our study were not well-informed about antibiotics and their use, and the importance of antibiotic resistance and the spread of antibiotic-resistant bacteria. Our results show that more than half (55 %) of the respondents agreed that common colds are cured more quickly with antibiotics, which is more than double that reported in a similar Swedish study (19.1 %) ; in thisOne may argue that a possible reason for inadequate knowledge among this study’s participants could be due to the microbiological terms used (‘bacteria’ or ‘virus’). Respondents might lack sufficient knowledge to differentiate between the two groups of pathogens. It may perhaps be beneficial to explain the differences between these terms when prescribing or not prescribing antibiotics to patients.Our study revealed overlap between the results: more than half of participants received antibiotics without medical consultation and over 60 % purchased antibiotics without prescription. This can be explained by the fact that the questionnaire did not provide a clear differentiation between how study participants understood and perceived the word ‘prescription’. More specifically, whether a regular paper received from a doctor, with the name of a drug on it is perceived as a prescription, or whether they solely refer to special forms as prescriptions . It should also be noted that the questionnaire did not provide the opportunity for study participants to identify the sources from which they received antibiotics without medical consultation; therefore respondents could have received antibiotics from friends, neighbors, etc.Studies showed that broad-spectrum antibiotics use is common in Georgia, with co-amoxiclav accounting for 42.9 % of total antibiotic use in 2011 . RelatinStudies show that public knowledge of antibiotic treatment and antibiotic resistance awareness can influence patient demand for antibiotic prescriptions, as well as their behavior towards antibiotic use . It shouIt is interesting to note that almost 80 % of participants reported trusting doctors’ decisions both for prescribing (76 %) and for not prescribing an antibiotic (80 %). Similar findings have been seen in a Swedish report, with relatively high trust seen towards doctors who do not prescribe antibiotics . AlthougThe majority of participants had correct knowledge of the importance of completing a full course of antibiotics even when symptoms improved. However, our questionnaire did not cover questions on whether individuals continue antibiotic treatment when they start feeling better. In order to avoid selection of resistant organisms, public health campaigns have been encouraging people to finish antibiotic treatment as prescribed, an approach which is somewhat questioned today. Although limited research has been done in this area, it has been seen that prolonged treatment might result in higher resistance rates, whilst shorter treatments could be more appropriate .Notably, in government-funded institutions and public schools in Tbilisi, around 82 % of personnel are female , which egeneral score compared to those living in the Old Tbilisi region, which is considered one of the more affluent areas. Further studies are required for better understanding of the geographical differences. We also found that people older than 30 years of age were less likely to declare having received antibiotics without medical consultation. In addition, study participants with more knowledge on antibiotic resistance are more prone to purchase antibiotics without prescription. Further research would be useful to establish a clear motive and to better understand what is meant and understood by ‘prescription’.Didube-Chugureti and Gldani-Nadzaladevi regions, districts with lower economic status, had lower expected counts of the In this study convenience sampling method was used. We therefore acknowledge the possibility of selection bias and that the results cannot be generalized to the whole Georgian population.awareness, resistance and general scores, the scores were summed up without using any weighting, giving the same importance to each question.The lack of information on respondent’s income distribution limits our ability to determine whether socioeconomic status plays any role on behavior and attitudes towards antibiotic use. Respondents had the opportunity to take the questionnaires home. Consequently, they may have looked up the correct answers online or conferred with someone else before responding. If so, this may lead to some overestimation of knowledge and attitudes towards antibiotics compared to the general population. The consequence of this would be that the problems raised in this study may actually be more severe if the whole country is considered. Furthermore, the survey was conducted within school and governmental institutions, where almost all personnel had university-level education. In addition the study was based in the capital, Tbilisi, where the population’s socioeconomic status is relatively better in comparison to the rest of the country. We would like to note that while creating Tbilisi also offers relatively better access to healthcare facilities and higher possibilities of affording healthcare services. Thus, access to antibiotics without prescriptions and without medical consultation is expected to be more widespread among the general public. The knowledge scores were created summing up the number of correct answers without weighting the answers according the estimated importance. It should be noted that the study was based on self-reported information and depends on the honesty and recall ability of the study’s participants, as well as their understanding of the questionnaire.The findings demonstrate that respondents have several misconceptions and a lack of awareness on antibiotic use and resistance. Further qualitative and quantitative studies are needed to identify the determinants of attitudes, behavior, expectations and motivation that lead people to use and misuse antibiotics. Considering the complexity of infectious disease and antibiotic resistance management in the global healthcare arena, the Georgian government should commit to investing in public health education programmes for the public and healthcare professionals . In addition, the government should require its healthcare system to develop proper regulations and prescription policies as well as controls for prescription drugs and empower the pharmacists’ role in raising awareness around the use of antibiotics and the growing antibiotic resistance within populations. Finally, public health strategies—including educational programmes—should be developed, targeting specific areas of misconception, misuse of antibiotics, and identification of at-risk populations in terms of improper antibiotic consumption.


The disposal of ready mixed concrete truck wash water and returned plastic concrete is a growing concern for the ready mixed concrete industry. Recently, extended set-retarding admixtures, or stabilizers, which slow or stop the hydration of portland cement have been introduced to the market. Treating truck wash-water or returned plastic concrete with stabilizing admixtures delays its setting and hardening, thereby facilitating the incorporation of these typically wasted materials in subsequent concrete batches. In a statistically designed experiment, the properties of blended concrete containing stabilized plastic concrete were evaluated. The variables in the study included (1) concrete age when stabilized, (2) stabilizer dosage, (3) holding period of the treated (stabilized) concrete prior to blending with fresh ingredients, and (4) amount of treated concrete in the blended batch. The setting time, strength, and drying shrinkage of the blended concretes were evaluated.For the conditions tested, batching 5 % treated concrete with fresh material did not have a significant effect on the setting time, strength, or drying shrinkage of the resulting blended concrete. Batching 50 % treated concrete with fresh materials had a significant effect on the setting characteristics of the blended cocnrete, which in turn affected the water demand to maintain slump. The data suggests that for a known set of conditions, the stabilizer dosage can be optimized within a relatively narrow range to produce desired setting characteristics. The strength and drying shrinkage of the blended concretes were essentially a function of the water content at different sampling ages and the relationship followed the general trend of control concrete. In a ready mixed concrete plant, the waste water from several processes, including truck-mixer wash water, needs to meet certain criteria, established by environmental agencies, prior to discharge into a storm water drainage system or public waterway. In a plastic state, concrete is a perishable product and disposal of unused concrete left over from a job provides its own set of challenges. Increased environmental regulation requires industry to implement practices that will effectively reduce the quantity of byproduct material requiring disposal.A survey by the National Ready Mixed Concrete Association (NRMCA)  indicateMore recently, Environment Canada published the Environmental Code of Practice for the ready mixed concrete industry in British Columbia . BesidesPractices used for handling and disposal of truck wash water vary with job conditions, such as, restrictions on disposal at the construction site, space constraints at the plant, daily production amounts, process control, job schedules, concrete specifications, and local environmental regulations. The Specification for Ready Mixed Concrete, ASTM C 94, permits the use of water from mixer washout operations as mix water in subsequent batches. C 94 places certain criteria on the quality of wash water that can be used as concrete mix water. However, the reuse of wash water may not always be feasible, in which case it has to be disposed in an appropriate manner. Unused returned concrete can be routed to smaller jobs, when available, or used to make pre-cast concrete products at the plant. However, concrete that is past a certain stage in the hydration process will need to be disposed. Mechanical aggregate reclaimers are used to separate aggregates from the returned concrete. The cement slurry water is separated either for disposal or diluted for reuse as mixing water. While initial capital and operating costs of reclaiming units may be high for some of the smaller producers, cost savings from reduced need for settling basins and solids disposal can be significant.In some cases, blending returned plastic concrete with fresh materials may be a feasible alternative to disposal. Recent technological advances have introduced a chemical admixture ,4 that cThis study evaluates some of the processing conditions and the resulting properties of blended concrete containing plastic concrete treated with extended set-retarding admixtures. It should be noted at the outset that the response of a cement and admixture combination is relatively unique. Hence, the conclusions are specific, to a certain extent, to the brands of cement and admixture, as well as the operating conditions, used in this study. However, the results of this study should be qualitatively applicable. The nature and size of this experiment does not allow the determination of well founded uncertainty statements on the (interim) results obtained. Nonetheless the experimental results are valuable as relative, qualitative information for further study.the age of concrete when the admixture was added —stabilizer addition time (SAT)the stabilizer dose (SD) used to keep the treated concrete from setting for the desired periodthe duration for which the stabilized concrete was held prior to the addition of fresh material—concrete addition time (CAT), andthe percent (by mass) of treated concrete (PTC) in the blended batch.In addition to the normal process control factors that affect the uniform production of concrete, there are several additional processing factors that need to be considered when stabilized concrete is mixed with fresh materials to produce a blended concrete batch. Since it was not feasible to study all of these factors, the first step in planning the experiment was to identify the most important processing factors. The following factors were chosen as variables in this study:A schematic representation of the sequence of operational steps and the related factor settings is presented in Two levels of each factor were chosen to represent practical recycling scenarios of interest. The two levels are referred to as the “low” and “high” levels of the factor. The two settings for each factor are indicated in The low and high settings of the stabilizer addition time, SAT, were set at 45 min and 180 min. The low level, 45 min, represents the earliest time a producer might decide to use a stabilizer. The high level, 180 min, was selected to represent the latest time this particular concrete mixture could be stabilized, from a practical point of view. At 21 °C (70 °F), stabilizing the concrete after 180 min would have required excessive admixture and water addition.The low and high levels for the stabilizer dosage, SD, were selected so as to hold the treated concrete in a plastic state for the required period. The dosage was estimated based on preliminary tests, described in Sec. 3.2. The selected stabilizer dosage depended on the age of concrete at the time it was treated (SAT), and the duration for which the concrete had to be held prior to batching fresh material (CAT). In this sense these three factors were not mutually independent.The next processing factor was the percent of treated concrete, PTC, in a blended concrete batch. The levels of this factor were chosen to represent the cases of reuse of wash water and returned concrete, respectively. The mortar fraction, or “butter”, that sticks to the walls of a concrete truck mixer will generally constitute about 1 % of a full load of concrete, or about 270 kg (600 lb) of cement, fine aggregate, and water. The low level of the factor, PTC, was set at 5 % to represent a situation of recycling truck wash water. The high level of PTC was set at 50 % to represent an upper practical limit of recycling returned concrete.The last factor was the duration of holding treated concrete prior to adding fresh materials, or concrete addition time, CAT. The low level for this factor, 45 min, was chosen to represent recycling wash water or plastic concrete on the same day. The high level, 20 h, was chosen to test the case when stabilized concrete would be held in a truck overnight and batched with fresh materials the next day.It should be noted that the factor levels used for this experiment were chosen for their research significance and some of them may not be appropriate in ready mixed concrete production operations. For instance, batching fresh materials in a mixer containing 90 min old butter is regularly done and does not typically need the use of a stabilizer. Also, holding treated concrete overnight and combining it with 50 % fresh material is not recommended by the admixture suppliers. Without evidence that the use of recycling conditions more extreme than those recommended by the manufacturer gives satisfactory results, it would be premature to use those levels in production situations.the concrete temperature, which was maintained at (21±2) °C [(70±3) °F],the concrete ingredients, including cement and admixture brands and aggregates,the concrete mixture proportions for the original and the blended batches, andthe concrete slump, which was held relatively constant by retempering as required.The following factors, which have significant effects on the properties of the concrete, were held constant:the setting time of the blended concrete,the compressive strength at 28 d, andthe drying shrinkage of 28 d moist-cured specimens after 91 d in air.The response variables evaluated in this study were:As described above, four factors, each at two levels, were chosen for the experiment. It is usually possible to ensure that all effects of interest in a designed experiment can be estimated separately from extraneous effects, like batch-to-batch effects. However, in this experiment, it was not possible to do so, due to physical constraints on the operational procedures. Constraints included maintenance of minimum and maximum batch sizes in the available mixers, scheduling tests while the concrete was fresh, and the ability to hold fixed factors at the given levels over time.As a result of these constraints, operational sequences were scheduled so that 16 “batches” of concrete for different stabilization conditions  were made from four initial or original batches of concrete, each mixed on a different day. The 16 different stabilization conditions were divided into 4 groups, each of which was made from one of the four original batches. The groups of stabilization conditions to be derived from each original batch were carefully defined to minimize confounding of important effects with the (expected) batch-to-batch variability of the original batches. Nonetheless, the design did not allow for separation of the batch-to-batch effect from the effect of Concrete Addition Time (CAT). The experimental layout, as run,To  resolve the ambiguity arising from the use of this design, it was planned to assess batch-to-batch variability using samples from the original batches. Provided a low batch-to-batch variability of the original concrete was attainable, the effect of CAT on resulting properties of the blended concrete could be estimated. The test results for both the blended and original concretes are discussed in Sec. 4.An ASTM Type I portland cement was used. The composition of the cement is given in 3 (2.5 ft3) batch of concrete was mixed and split into four portions. Each portion was held, with mixing and retempering at regular intervals, until the desired ages of 8 min, 45 min, 90 min, and 180 min. At each age, incremental doses of the stabilizing admixture were mixed into the concrete batch and several set time samples, each containing different amounts of admixture, were obtained. The procedure for measuring the setting time is described in Sec. 3.4.1. This data provided dosage versus set time curves for concrete treated at different ages. The dosage versus set time curves for concrete treated at 45 min and 180 min are plotted in In order to predict the admixture dosage required to hold concrete without setting, the admixture had to be “calibrated” with the cement. A 0.07 mThe design mixture proportions of the concrete batches are given in Mixture ingredients required to produce about 180 kg (400 lb) of concrete were batched and mixed in mixer 1. At 45 min, approximately 55 kg (120 lb) of concrete was transferred to mixer 2 and stabilized with the admixture. The remaining concrete in mixer 1 was held until the concrete was 180 min old at which point it was stabilized with the appropriate admixture dose. Fresh material was batched with the treated concrete at either 45 min or 20 h (CAT) after the stabilizer was added. In the case of recycling on the same day (45 min), the concrete was retained in the mixer which was covered with a polyethylene sheet to prevent evaporation. Concrete that was treated to be recycled the next day was transferred to a metal container and covered with a polyethylene sheet for storage overnight.At the time of adding fresh material, the stabilized concrete was split into two portions to represent 5 % and 50 % of a blended batch. Fresh materials were batched with the stabilized concrete in mixers 3 and 4 to produce approximately 90 kg (200 lb) of blended concrete. Each original batch thus produced four batches of blended concrete.Samples taken at 8 min, 45 min, and 180 min, from the four original concrete batches were tested to serve as a benchmark or control measurements for the results of the blended concrete.2, in flake form, was added at the rate of 2 % by total weight of cement in the batch for all overnight blended concrete batches (CAT = 20 h). In order to make appropriate comparisons, calcium chloride was dosed on the basis of the total cement, which included that in the treated concrete and the fresh cement added to the batch. The general practice, however, would be to dose the activator on the basis of the cement content of the stabilized concrete.Concrete that is stabilized for the purpose of recycling on the next day will be typically over-dosed, to prevent it from setting up in the mixer drum. Prior to batching fresh material, an activator is added to counteract the effect of the stabilizer. In this study, calcium chloride, CaCl2, by weight of cement, added to untreated concrete. This batch was tested at the same times using the same protocols used for the other four original batches of concrete.In order to have a benchmark or control for concrete recycled overnight, one control batch (a fifth batch) was made with 2 % CaClDuring the holding time between initial mixing and 180 min, the original and blended batches were mixed at 20 min intervals for 5 min. The concrete was retempered, if required, to maintain approximately a 75 mm (3 in) slump. This operation was based on real time visual evaluation of slump and adjustments of water content. The water additions were recorded and the water content of concrete samples obtained at different ages was calculated from the measured unit weight.concrete temperature,slump,two 100 mm×200 mm (4 in×8 in) cylinders were molded for a 28 d strength test,one 100 mm×350 mm (4 in×14 in) cylinder was molded with embedded gage studs for drying shrinkage measurements,one 100 mm×100 mm (4 in×4 in) mortar specimen was prepared for measuring the set time, and 6. the 100 mm×200 mm (4 in×8 in) cylinders were weighed to determine the unit weight of fresh concrete.Samples were obtained from the original and blended concrete batches at ages of 8 min, 45 min, and 180 min. Each series of tests required about 23 kg (50 lb) of concrete. The following tests were conducted:not referenced to the time that water touches the cement. The set time, as defined here, is approximately the time it takes for concrete to achieve final set after being placed in its final location. The setting time of the stabilized concrete was also determined and these compared well with the calibration curve shown in The time of set of concrete was determined by a Proctor penetrometer on the mortar fraction of the concrete sample. Concrete was wet-sieved over a hardware mesh with a 6 mm (1/4 in) opening. Sufficient mortar to fill a 100 mm×100 mm (4 in×4 in) cylinder mold was obtained. Time of set was measured by the penetration resistance of the mortar. Set time is defined as the time elapsed between obtaining the sample and the point when the resistance to penetration was 3.5 MPa (500 psi). Note that this definition of set time is Constraints of time and sample quantity prevented the determination of unit weight by ASTM C 138. The 100 mm×200 mm (4 in×8 in) cylinder molds containing fresh concrete were weighed. The volume of the hardened concrete cylinders was determined after they were demolded by weighing them in air and immersed in water. The unit weight was used to calculate the composition of concrete at the various tested ages.The 100 mm×200 mm (4 in×8 in) strength cylinders were cured in a moist room and the compressive strength was determined at 28 d according to ASTM C 39. The compressive strength test result is the average of two cylinder breaks.The 100 mm×350 mm (4 in×14 in) cylinder specimens were cured in saturated lime water solution for 28 d. The specimens were then dried in air at 21 °C (73 °F) and 50 % relative humidity. Length measurements were obtained at 1 d, 28 d, 56 and 91 d, after the drying was initiated. Drying shrinkage data reported is that of one specimen from each concrete sample.The design chosen for this experiment allowed the different treatments to be run over four days, to accommodate time and equipment constraints, as discussed above. A new batch of concrete was mixed each day to run the four treatments specified in the experimental plan for that day. The first step of the data analysis was a comparison of the four original, untreated batches of concrete to see if there was any evidence of significant batch-to-batch differences. Fresh concrete properties of the four original batches are given in In order to make appropriate conclusions on the results of the blended batches of concrete, it was important that the between batch variability of the original batches was low, so that these differences could be ignored in the subsequent analysis. Additionally, the data in the tables indicate that the properties of the original batches of concrete were similar at 45 min and 180 min, at which time the original concrete batches were stabilized.2 had an unexpected effect of the concrete properties. The effect of calcium chloride on properties of the original concrete can be seen in 2 was not expected and may be unique to the brands of cement and calcium chloride used in this study.CaClResults for setting time, strength, and shrinkage of the four original batches were averaged to represent the control responses for blended concrete batches representing the same day recycling of treated concrete (CAT = 45 min). Because of the strong effect of calcium chloride of concrete properties, results from batch OC were used as the control responses for blended batches representing the next day recycling of treated concrete (CAT = 20 h).Data for setting time, strength, and drying shrinkage for the blended batches and the corresponding control batches sampled at 8 min are plotted in 2 effect on concrete properties. The data are paired according to low and high dosage levels for concrete stabilized at the same time, and subsequently blended with fresh materials at the same age. The hollow symbol represents blended concrete containing 5 % treated concrete, and the filled symbol is that for 50 % treated concrete.The data are divided into two sets—the same day recycling case on the left, and the next day on the right—due to the effect of the unusual CaClIn most cases, selecting admixture doses from the “calibration curve,” in In one case (condition 7), the dosage used was too low to hold concrete in a plastic state for the required period. About 45 kg (100 lb) of concrete was stabilized at 180 min for the purpose of holding it for at least 20 h. The admixture was dosed based on our best estimate of the cement content of that concrete. The calibration curve in In If fresh ingredients are batched on top of older plastic concrete (without stabilizing admixtures) and mixed, the setting time of the resulting concrete will be accelerated compared to a similar concrete batch not containing the old concrete. Results from a previous study  indicateWhen stabilizers are used, it appears that two opposing mechanisms control setting time: the accelerating effect of old concrete, and the retarding effect of the admixture. For a particular set of stabilizing conditions, the admixture dosage can be optimized to produce a blended batch with the same setting characteristics as that of a control batch. At this optimum dosage, the accelerating and retarding effects should cancel out. If the admixture dosage is less than that optimum amount, the accelerating effect of old concrete is the dominant mechanism. This is seen for the “low” dosage setting in the blended concrete. Increasing from 5 % to 50 % treated concrete in the blended concrete, decreased the setting time further. At the “high” setting of admixture dosage, apparently, the retardation effect of the admixture was dominant, and the setting time of the blended concrete was longer than that of control. Increasing from 5 % to 50 % treated concrete in the blended concrete resulted in an increase in the net admixture in the batch which further retarded the concrete.If data similar to that in It is important to match the setting time of the control, especially if a blended batch of concrete is one of several batches going out to the same job. Not only is this important so that finishers on the receiving end can expect similar characteristics, but as discussed later, the setting characteristics control water addition rates to maintain concrete slump, which in turn controls the properties of the delivered product.3 (20 lb/yd3). At the later ages of 45 min and 180 min, the strength of concrete was essentially controlled by the amount of water added to maintain a 75 mm (3 in) slump. The average strength of the control concrete was 35.8 MPa (5186 psi), while that of the one control batch containing CaCl2 was about 46.3 MPa (6715 psi).The effects of the stabilizing conditions on compressive strength are illustrated in For concrete recycled on the same day, the strength of the blended concretes were essentially similar to that of the control concrete. The main effects of each factor, that is the differences between averages at the “low” and “high” settings, can also be estimated. No effect of stabilizer dose was evident. Strength results of concrete stabilized at 180 in (SAT) and then blended averaged 3 % lower than control and 4 % lower than when the stabilizer was added at 45 min. When the recycled batches contained 50 % treated concrete, the average strength was 2 % lower than control. These differences are not of practical significance.For the concrete recycled on the next day, only condition 7 resulted in significantly lower strength. In this case, the admixture dosage was insufficient and concrete had stiffened considerably when it was mixed with fresh materials. As indicated earlier this set of conditions was extremely severe and is not recommended in practice.In looking at the main effects for batches containing stabilized concrete blended on the next day, increasing admixture dosage from low to high for the same set of conditions produced slightly higher strengths. Again, this effect was more pronounced for blended concretes containing 50 % treated concrete. The stabilizing admixture disperses cement particles, and this effect is known to produce higher strengths. Concrete that was stabilized at 180 min, on average produced 7 % lower strengths than control and 9 % lower than when the stabilizer was added at 45 min. Strength of blended concrete containing 50 % treated concrete, was 6 % lower than control. While the differences in strength resulting from these conditions may be of practical significance if delivered to the same job, the quality of concrete is still adequate for alternative jobs, if available.Strength of later age samples was essentially controlled by water addition requirements resulting from a modified setting time. Longer setting times of blended batches resulted in lower water contents and higher strengths, as discussed in Sec. 4.3.The shrinkage of 28 d moist cured specimens after 91 d in air is illustrated in A point of reference for differences in drying shrinkage data is that for the four original control batches given in For concrete recycled at 20 h, the overwhelming effect of calcium chloride on shrinkage probably clouded any effect of the admixture and stabilization conditions. No significant difference between the recycled batches and the control were evident. The data does not indicate any conclusive effect of stabilizer addition time, stabilizer dose, or percentage of treated concrete in the recycled batch.At later ages, the shrinkage was controlled by recycling conditions that increased or decreased the setting time with respect to the control batches which in turn determined the water content in the batch at that age, as discussed in Sec. 4.3.The properties of the concrete as delivered to a job site are of primary interest to both the concrete supplier and purchaser. The job site properties are controlled by the haul time of the concrete, which in turn controls the amount of retempering water required to discharge workable concrete. Concrete temperature, which also has a significant effect on water demand, was held constant at 21 °C (70 °F). A batch plant operator familiar with the materials being used has a general idea of the typical water demand of a mixture and will control the amount of added water to furnish concrete at the required slump to the job. A blended concrete batch might need different amounts of retempering water depending on the stabilization conditions.3 (lb/yd3) per min, required to hold concrete at a 75 mm (3 in) slump for 180 min. This quantity is a rough estimate and is assumed to be linear with time. This rate of water addition is plotted against the setting time of the 8 min sample in 3/min (0.4 lb/yd3/min) to hold it at a 75 mm (3 in) slump for 180 min. The control concrete batch containing CaCl2 with a setting time of 2.5 h needed approximately double the rate of water addition.As indicated earlier, the original and blended concretes were periodically agitated and retempered to maintain a 75 mm (3 in) slump. An estimate of the water addition rate was computed by the difference of the calculated water content of the batch at 180 min and that at initial mixing. The rate of water addition is therefore the amount of water, in kg/mWhile the relationship in In general, the data suggest that at similar water contents, the strength and drying shrinkage of blended concrete is essentially similar to control concrete.This study includes only some of the processing factors that may be involved when using extended set-retarding admixtures for the purpose of recycling truck-mixer wash water or returned plastic concrete. Even with this limited scope, it is clear that a significant amount of preliminary testing is necessary to effectively use these admixtures to recycle plastic concrete. However, blended concrete containing stabilized plastic concrete can be used for applications where the setting characteristics are less critical, such as for structural fill or foundation piers. Data representing an upperbound case of recycling stabilized truck-mixer wash water (PCT = 5 %) as batch water in a subsequent concrete batch indicates that the compressive strength and drying shrinkage of the resulting concrete will not be significantly effected.A calibration curve that determines admixture dosage for different holding times should be developed for a particular cement and admixture combination for various concrete ages and temperatures. Set time indicated by a small mortar sample should be used with caution, especially for long holding times. Setting characteristics of blended concrete were controlled by two opposing mechanisms; the accelerating effect of old concrete and the retarding effect of the admixture. The data suggests that the admixture dosage could be optimized, within a relatively small range, to match the setting characteristics of the control batches.Comparing concretes at similar water content, more severe recycling conditions, such as stabilizing at 180 min and recycling 50 % treated concrete in a blended batch, resulted in reduced strengths and increased shrinkage. However, at least for compressive strength, these differences were not of practical consequence.The water demand to maintain slump was related to the modified set time of the blended concrete. Strength and drying shrinkage of blended concrete containing stabilized plastic concrete followed the same relationship with water content as the control concrete.


Antibiotic resistance, a major public health problem, has been linked to antibiotic consumption. In Greece both consumption and resistance rates are among the highest in Europe. A multifaceted campaign targeting both physicians and parents of school children was implemented for the first time in order to educate the public and update doctors, aiming to promote judicious use of antibiotics and hopefully decrease its consumption.The programme consisted of a public education campaign and academic detailing of primary care physicians in the district of Corinth in Peloponnese. The experience and perceptions of parents were recorded in the meetings in the form of course evaluation and assessment, anonymous questionnaires. The use of Rapid Antigen Detection Test (RADT) for streptococcal pharyngitis by primary care physicians was also assessed by use of anonymous questionnaires. Antibiotic consumption was compared before and after the programme between the district of Corinth and the other districts of Peloponnese, as well as at a national level.Antibiotic consumption remained unaltered at 26 Defined daily doses per 1000 Inhabitants per Day (DID) in accordance with the trend in other regions and at a national level. However, the utilization of Amoxycillin and Penicillin was increased by 34.3%, while the use of other antimicrobial classes including macrolides, cephalosporins and fluoroquinolones decreased by 6.4-21.9%. The use of RADT did not lead to a significantly decreased antimicrobial consumption.A multifaceted educational programme targeting both the general public and primary care physicians was associated with rationalization in the choice of antimicrobial. A reduction in the total antimicrobial consumption was not achieved.The online version of this article (doi:10.1186/1471-2458-14-866) contains supplementary material, which is available to authorized users. Antibiotic resistance is an increasing problem of public health. There is an association between antibiotic resistance and antibiotic consumption . In EuroAntibiotic consumption is highest in the community compared to hospitals and is especially associated with upper respiratory tract infections during the winter months . The greA number of interventions has been implemented with variable success . These iBased on the above data a multifaceted regional campaign was performed targeting both the public and primary care physicians in the region of Corinth in Greece in 2009–10.Streptococcus pyogenes group A by the primary care providers (Feb-Apr 2010).The educational material and the programme were organized by the Medical School of the University of Athens in cooperation with the Prefecture of Corinth and the Medical Association of Corinth in Peloponnese. It consisted of a public education campaign and academic detailing of the primary care physicians in the district of Corinth (Jan-Feb 2009) and an assessment of the use of Rapid Antigen Detection Test (RADT) for http://www.ifet.gr/antibiotics_site/FrameSet6.htm. An open discussion in the form of question-and-answer followed between parents and experts. The only mass media intervention in the present campaign was a press-conference organized by the prefecture for the local media at the start of the intervention. Filling the questionnaire by parents was on a voluntary basis and anonymous, in the scope of providing a background for the discussion to follow and the issues raised by the parents.Seventeen two-hourly educational meetings were organized in all major municipalities of the district with parents of children in nursing care and primary school. The title of the meetings was “What Do Greek Parents Need to Know on the Proper Use of Antibiotics”. Before the meeting parents were asked to fill an anonymous questionnaire for their experience and perceptions on the use of antibiotics in common clinical scenarios  and another with the guidelines of the Greek Center for Disease Control and Prevention (KEELPNO) on the treatment of infections .This was carried out in the form of meetings with the physicians who provide primary care for patients with respiratory tract infections including general practitioners, pediatricians, otorhinolaryngologists and chest physicians. The meetings included a lecture entitled “Bugs and antibiotics: the role of the Greek physician in the fight against microbial resistance” followed by four short interactive sessions based on the management of specific cases. Such cases included patients with acute pharyngitis, otitis media and urinary tract infections. The meetings were organized under the auspices of the local Medical Association. The use of bedside RADT for In addition, five meetings were arranged with primary care physicians at the five public primary health care centers. These meetings were based on the same programme with the first meeting. Another meeting was arranged with district dentists under the auspices of the local Dental Association. This meeting was targeted on the use of antibiotics by dentists including endocarditis prophylaxis.In January 2010 primary care physicians in the region including both pediatricians and general practitioners were invited via the local medical association to share their experience with use of RADT test and the management of patients with pharyngo-tonsillitis. Physicians volunteering to participate would fill-in an anonymous form with details on Centor criteria , the useAll questionnaires used in the campaign were anonymous. The questionnaires filled for the RADT use survey were also anonymous and had been subjected to approval by the Ethics Committee of the University General Hospital ATTIKON . The surAntibiotic consumption data was kindly provided by IMS, Greece for the district of Corinth, the other districts of Peloponnese and on a national level. Only antibiotics prescribed for respiratory tract infections were included in the study. These were phenoxymethylpenicillin, amoxicillin and the combination of amoxicillin and clavulanate, 1st and 2nd generation cephalosporins, macrolides and the fluoroquinolones levofloxacin and moxifloxacin. Defined daily doses (DDD) per 1000 inhabitants per day (DID) were calculated based on the ATC/DDD index of the WHO collaborating Centre for Drug Statistics Methodology. The rest of districts in Peloponnese and the national rate served as controls and data were compared before and after the campaign between January 2009 and February 2009 with Fisher’s exact test. In the RADT-related questionnaire, the prescription rate of antibiotics was compared with Fisher’s exact test between those reporting using the RADT vs. those that did not.http://www.statistics.gr for the year 2009).In total 772 parents participated in the meetings . In the academic detailing 111 out of 486 physicians in the prefecture (approximately 20%) and 30 out of 151 dentists (approximately 23%) were present . Demographic data and responses are described in Table p = 0.02)  cases questionnaires. Two physicians – one general practitioner and one pediatrician - reported an empirical approach without RADT treatment group and contributed 54 (20%) cases questionnaires. In the RADT group 177 (85.1%) patients and in the empirical treatment group 34 (64.2%) patients were children (p < 0.001). The utilization of antibiotics in the two groups is presented in Table Antibiotic resistance is an emerging public health issue of major concern worldwide which has been associated with antibiotic consumption , 8. The The present campaign was designed to address antibiotic consumption in upper respiratory tract infections in a primary care setting during the winter months of 2009 and 2010, when the majority of unnecessary antibiotics are usually prescribed. It included initiatives targeting the public and specifically parents of young children, as well as academic detailing of health care providers in the primary care sector. Academic detailing  is effective at inducing change in prescribing behavior in various settings .Antibiotic utilization behavior in the target population was assessed by the use of a questionnaire addressing the main public behaviors associated with antibiotic overuse. In accordance with previous studies in both in Greece  and in oAccording to the results of the study no major change in the total number of antibiotics prescribed was detected. However the observed data indicate that in the targeted district antibiotic utilization was more rational, as reflected by significantly increased use of amoxicillin or penicillin according to the national guidelines and a decrease in the use more broad-spectrum antimicrobials like macrolides, 2nd generation cephalosporins, fluoroquinolones and amoxicillin/clavulanate. However, after the end of the campaign, antibiotic use tended to revert to previous levels, indicating the need for continuous educational initiatives (or at least of longer duration or repetitive).One of the limitations of the analysis of the data is the before-after design with comparisons on the months before and after the intervention. Such analyses do not account for seasonal or other temporal confounding factors. A time-series analysis might be more informative. However, data were available only for limited time points, preventing time-series analysis. A further limitation that may have affected the effectiveness of the intervention is the rate of participation of both healthcare professionals and parents. A bias towards increased participation of physicians and parents already familiar with appropriate antibiotic use cannot be excluded either.It has been shown in various settings that the use of RADT for the detection of streptococcal antigens on pharyngeal swabs improves the diagnostic accuracy of streptococcal pharyngitis compared to the clinical criteria alone, and is comparable to and more practical than the use of pharyngeal swab culture , 15, 16.Even though no reduction was observed in the antibiotic prescription rate, there were significant differences in the type of antibiotics prescribed in the two groups. In the RADT group penicillin and amoxicillin, which are the recommended antibiotics according to the guidelines, were prescribed more frequently compared to all other categories. This was accompanied by a significant decrease in the use of both macrolides and cephalosporins, indicating that the use of RADT encourages treatment with more narrow-spectrum antibiotics. The latter finding is especially important in Greece where consumption of macrolides is especially high with accompanying high rates of resistance.In conclusion, a multifaceted campaign was performed for the first time in Greece, addressing the multiple factors that influence the utilization of antibiotics (providers and public). In general, it revealed the gap that needs to be filled in the proper education of the public in terms of antibiotic use and it provided a starting point for improvement in certain aspects of providers’ practices in terms of antibiotic prescribing. Similar campaigns implemented for longer period of time and at a larger scale may promote the prudent use of antibiotics, with a final aim to reduce antimicrobial resistance in the community.Questionnaire for the evaluation of the parents’ experience, knowledge and behavior concerning antibiotic use.(DOCX 18 KB)Additional file 1:


This audit evaluates the adherence to the prophylactic surgical antibiotic policy at Kingston Hospital. With a greater understanding of the current use of prophylactic surgical antibiotics comes the ability to improve patient care while minimising the development of antibiotic resistance. Surgical site infections are a major source of hospital-acquired infections, causing significant morbidity and mortality. In appropriate cases, surgical antibiotic prophylaxis is essential in preventing such infections; however, this comes with increased risks of antibiotic resistance and antibiotic-associated diarrhoea. Consequently, this institution has extensive guidelines as to the cases in which antibiotic prophylaxis is required and what antibiotics should be administered. This audit examines the adherence to these guidelines. We audited against our local antibiotic prescribing policy, named the Blue Book guidelines.Notes were audited retrospectively for 80 patients undergoing surgery between 19 August and 18 December 2013. These were audited against local antibiotic guidelines (Blue Book guidelines) and analysed using descriptive statistics.Only 57% of operations were compliant with Blue Book antibiotic prophylaxis guidelines. For operations where administration of antibiotics was appropriate, 24% of patients received the incorrect choice, 19% were given the incorrect dose, 28% were given antibiotics for an inappropriate time relative to the procedure, and 38% received antibiotics for the incorrect duration. For operations not requiring prophylaxis, 48% of patients incorrectly received antibiotics.Clostridium difficile colitis.Compliance to local guidelines for prophylactic antibiotics is extremely poor. This not only risks increased morbidity and mortality from surgical site infections but also risks the development of antibiotic resistance and We suggest enhancement of the antibiotic guidelines, and education of those using them. This will improve the provision of antibiotic prophylaxis in the Trust, and ultimately improve patient care.