Introduction: Smear layer (SL) is produced as a result of mechanical instrumentation of the canal(s). Despite the controversies regarding its removal, the evidence-based trend has shifted towards removing and eliminating the SL. Different methods have been used to remove the SL and the aim of this in vitro study was to evaluate the ability of 17% ethylenediaminetetraacetic acid (EDTA), 18% etidronate and Er: YAG on effective removal of the SL. Methods and Materials: Fifty straight single-rooted teeth were divided into three experimental groups (n=15) and one control group of five. The canals were instrumented with HERO 642 rotary files up to 30/0.06. In group 1, canals were irradiated with Er: YAG laser; in groups 2 and 3, canals were irrigated with 17% EDTA and 18% etidronate, respectively. In group 4 (control) distilled water was used for canal irrigation. The amount of remaining SL was quantified according to Hulsmann’s method with scanning electron microscopy (SEM). Data was analyzed by the Kruskal-Wallis and Mann-Whitney U tests (P<0.05). Results: The results showed statistically significant differences in terms of SL removal among the groups (P<0.05). The amount of removed SL by EDTA was significantly greater followed by Er: YAG laser and 18% etidronate. Conclusion: Within the limitations of this study, EDTA was more effective in removing SL compared to Er: YAG and etidronate. The mechanical instrumentation of the root canal produces the amorphous irregular smear layer (SL) containing inorganic debris, organic materials like pulp tissue, odontoblastic processes, necrotic debris, microorganisms and their metabolic byproducts . McComb et al.  it was cDifferent methods, irrigating solutions and chelators have been used to remove the SL . Currenti.e. multiple myeloma and breast/prostate cancer) [Etidronate is a member of the hydroxyethylidene bisphosphonate (HEBP) drug family for prevention of osteoclastic bone resorption in patients suffering from bone diseases such as osteoporosis, Paget`s disease and hypercalcemia associated with malignancies ( cancer) , 14. Eti cancer) .et al. [Moreover, unlike EDTA, etidronate can even be mixed with NaOCl without interfering with the antimicrobial property of this solution , 18. Zehet al.  was the in vitro study was to evaluate the SL removal efficacy of etidronate, EDTA and Er: YAG laser in the apical third of root canals.Although several studies have evaluated the effect of different methods and substances on SL removal, there is no study on the efficacy of EDTA, Er: YAG laser and etidronate. So, the purpose of this Sample collection:Ninety-five freshly extracted human mandibular premolars with straight single roots and closed apices were collected. The teeth were decontaminated by immersion in 5.25% NaOCl for 1 h. After obtaining periapical radiographs, all teeth with external or internal root resorption, calcification, complicated root canal anatomy and previous root canal treatment were excluded.After preparing the access cavity, presence of the canal was confirmed and patency of the canal was established by inserting a #10 K-file  until the file tip emerged from the apical foramen. The working length (WL) was calculated by subtracting 1 mm from this length. Any root with laterally placed apical foramen or an apical constriction diameter wider than a #15 K-file was excluded. The remaining 50 teeth were selected to a standardized WL ranging between 18-20 mm. The sample size was determined after a pilot study.Root canal instrumentation:After coding the teeth, they were instrumented up to #20 K-file to the WL. The canals were then passively instrumented using HERO 642 rotary files  and an electric speed/torque controller device  according to the manufacturer’s instructions in a crown-down manner and with the following sequence: coronal enlargement for all the samples by Gates Glidden drills # 4, 3, 2  and then preparation of the canals with rotary instruments 20/0.04, 25/0.04, 30/0.04, 30/0.06.All the canals were prepared by the same operator. Each rotary instrument was used for preparation of five canals and was applied for 5 sec to the WL. After each rotary file, the canal was rinsed with 1 mL of 1% NaOCl, delivered by 28-guage needles  inserted deeply and passively from coronal to middle third at the end of coronal enlargement. A final rinse with 5 mL of distilled water was used to avoid the development of NaOCl crystals and eliminate the irrigation solution from the canals. During the apical preparation sequence, the needle penetrated within the apical 3 mm. The teeth were randomly divided into 4 groups according to the method used for SL removal.Group1: Fifteen teeth were irradiated with Er: YAG laser  in short pulse (SP) mode through an optical fiber (diameter 0.3 mm), with a wavelength of 2940 nm with an output power of 1 W, pulse energy of 100 mJ and pulse frequency of 10 Hz. The fiber tip was inserted to the WL parallel to the root canal wall and moved with hand circular motion to coronal part of canal during 40 sec . The percentage of water and air during working was 80% and 70%, respectively. After laser irradiation, the canals were irrigated with 5 mL of distilled water.Group 2: Fifteen teeth were irrigated for 2 min with 5 mL of 17% EDTA  followed by 5 mL of 5.25% NaOCl and a final irrigation with 5 mL of distilled water to remove any remnants of irrigants.Group 3: Fifteen teeth were irrigated for 5 min with 5 mL of 5.25% NaOCl followed by 5 mL of 18% etidronate (pH:10.5) for 5 min. Solution of etidronate with 18% concentration (wt/vol) was prepared using purred chemicals  dissolved in deionized water. The solution was stored at 5º C in an airtight dark container. Then it was removed from the refrigerator and stored for 1 min at room temperature, prior to being used.Group 4: Five teeth were washed by 5 mL of distilled water for 2 min as a final irrigation solution.Root sectioning and SEM evaluation:et al. [Two mesial and distal grooves were prepared on apical 6 mm of each root by means of a disc  with no entrance into the canal space. The roots were resected transversely in a mesiodistal direction by a chisel . The same procedure was repeated in a buccolingual direction and the roots were split longitudinally, resulting in 30 samples in each experimental group and 10 control samples. The samples were placed in 2% gluteraldehyde for 24 h and then rinsed 3 times with a sodium cacodylate buffered solution . After incubation in osmium tetroxide for 1 h, the samples were desiccated with ascending concentrations of ethyl alcohol (30-100%), placed in a desiccator for 24 h and mounted on a metallic stub. After coating the samples with 20 μm of gold, the technician who was blind to the samples, prepared the SEM photomicrographs using backscatter mode . The images were analyzed by 3 previously calibrated examiners according to the scoring system by Hulsmann et al.  . A kappa statistical analysis was performed to measure the inter- and intra-examiner variability.P~0.000). The Mann-Whitney U test showed statistically significant differences in the amount of SL between Er: YAG laser and etidronate (P~0.000), EDTA and etidronate (P~0.000) and between EDTA and Er: YAG (P=0.002). EDTA significantly had the best SL removal action followed by Er: YAG laser and etidronate.The kappa values were 0.83 and 0.79, respectively; indicating very good inter- and intra-examiner agreement. Root canal walls absolutely free of SL were not seen in any of the groups . The Kruin vitro study evaluated the various methods to improve SL removal at the apical third of the instrumented straight root canals. In the current study, the samples in all groups were standardized according to the apical diameter of the root canal. Apical preparation was extended to size 30/0.06 to allow adequate apical penetration of irrigations and access for the laser fiberoptic tip (300 µm) to the apical third of the canals. A similar sequence of HERO 642 files were used in all of the samples for canal instrumentation. It is shown that rotary nickel-titanium (NiTi) files produce a significant amount of SL compared to hand instrumentation [This entation , 20. Schentation . Arvanitentation . So the entation , 24; theInjecting the irrigation solution by means of a syringe can control the volume and depth of syringe penetration and results in the flow of the solution to the apical third of the canal . So, allDifferent magnifications of SEM have used to score SL after instrumentation. The benefits of this type of study have been reported by some investigators. In accordance with previous studies, a five score index and a magnification of 2000× were used in our study because it offers a detailed image of the canal walls without limiting the observed field.in vitro, we adjusted the percentage of water and air at 80% and 70%, respectively. The present study demonstrated that Er: YAG laser can remove the SL but not as effective as EDTA. Similar results were obtained in the study by Ramalho et al. [et al. [et al. [Certainly, application of laser has shown great promise in root canal treatment and its main usage is to eliminate the microorganisms and remove the remnants of SL on the instrumented root canal walls. Among the different types of lasers, Er: YAG showed the ability to remove the SL , 11. Its [et al. , 32 also [et al. -35. The  [et al. . So, if Østby was the first investigator who used EDTA to clean the canals [et al. [The most commonly used irrigation solution in endodontics is NaOCl with well established antibacterial properties and dissolving ability of organic components of tissues ; but thie canals . Hashemi [et al.  also conet al. [In this study, the smallest amount of SL removal was observed in 18% etidronate group. This finding confirms that etidronate is a weak chelating agents that had less effect than other commonly used chelators such as EDTA, which is in agreement with previous studies , 37, 38.et al.  showed tCurrent preparation methods of using rotary files and irrigation solutions still fall short in successfully removing the SL from the root canal walls. This was confirmed by the results seen in the control group (group 4) where the distilled water was employed as a final irrigation. Although Er: YAG laser has been proven as an effective disinfecting method for the root canal system, the authors recommend using EDTA solution to achieve the optimum results.in vitro study, EDTA showed significantly greater efficacy in removing smear layer followed by Er: YAG laser and finally etidronate.Within the limitations of this


Antimicrobial stewardship teams have been shown to increase appropriate empirical antibiotic therapy and reduce medical errors and costs in targeted populations, but the effect in non-targeted populations is still unclear. The aim of this study was to determine the prevalence of inappropriate antibiotic use in a large university hospital and identify areas in which antimicrobial stewardship will be the most effective. In a point prevalence survey we assessed the appropriateness of antibiotic therapy using an electronic surveillance system in combination with a standardized method for duration of therapy, dosage, dosage interval, route of administration, and choice of antibiotic drug. Patients using at least one antibiotic drug were included. Among 996 patients admitted in the surveyed wards, 337 patients (33.8 %) used one or more antibiotic drugs. Two hundred and twenty-one patients (22.2 %) used antibiotic medication therapeutically, with a total of 307 antibiotic prescriptions. Antibiotic therapy was deemed inappropriate in 90 (29.3 %) of these prescribed antibiotics, with an unjustified prescription as the most common reason for an inappropriate prescription. Use of fluoroquinolones and amoxicillin/clavulanic acid and a presumed diagnosis of fever of unknown origin, urinary tract infection, and respiratory tract infection were associated with inappropriate antibiotic therapy. Our study provides insight into the (in)appropriateness of antibiotic prescriptions in a tertiary care center in the Netherlands and identifies areas for improvement. The use of an electronic surveillance system for this point prevalence study is easy and may serve as a baseline measurement for the future effect of antibiotic stewardship. Staphylococcus aureus (MRSA) and third-generation cephalosporin-resistant Escherichia coli [Antibiotics are an indispensable part of modern medicine. However, as with all drugs, antibiotics may have adverse effects and medication errors can occur in prescribing. Another untoward effect of antibiotics is the selection of antibiotic-resistant bacteria. In 2007, more than 8,000 excess deaths in Europe were associated with bloodstream infections caused by methicillin-resistant hia coli . This mohia coli . The U.Shia coli , antimichia coli .A clear relationship has been found between the percentage of resistant strains and antimicrobial use . In addiThe aim of this study was to determine the prevalence of inappropriate antibiotic use and to identify the areas in which ASTs can have an important impact hospital-wide.The Erasmus MC, University Medical Center Rotterdam is a 1,320-bed tertiary care center in Rotterdam with all medical specialties available. In 2012, there were 41,773 admissions and 286,155 bed days.The point prevalence survey of antimicrobial use was performed on May 4th and May 16th 2013. Patients were selected with E-Surveillance, an electronic surveillance system, which has been operational in our hospital since 2011 . OriginaAntibiotic drugs were also considered to be prophylactic if they were recorded as such in patient progress notes. Relevant data elements, such as age, sex, ward, and prescribed antibiotic(s) were retrieved from the E-Surveillance database. The appropriateness of antibiotic therapy was determined for each individual patient by both a clinical microbiologist and an infectious disease consultant, using the standardized method developed by Gyssens et al. . Infecti9/l, or erythrocyte sedimentation rate >20 mm/h) A nosocomial infection was defined as infection meeting the CDC criteria and occurring at least 48 h after admission.Prescribing therapeutic antibiotics was considered justified for an infection that was either community-acquired or nosocomial. A community-acquired infection was defined as documented or suspected infection within 48 h after admission with fever (>38 °C) and/or elevated infection parameters  and available microbiological results. The antibiotic prescription was defined as inappropriate due to unjustified prescription, when the use of an antibiotic was not indicated because no infection was present. The antibiotic drug prescription was also considered to be inappropriate when the administered antibiotic drug was not in line with the antibiotic guidelines, in case of allergy to the prescribed antibiotic drug, or when a more effective, less toxic, less expensive, and/or less broad-spectrum alternative agent was available. Additionally, antibiotic drug prescription was considered inappropriate in case of incorrect duration, incorrect dosage, incorrect dosage interval, and/or incorrect route of administration  were using one or more antibiotic drugs. Antibiotic drugs were used prophylactically in 116 patients; these patients were excluded from the analysis. Two hundred and twenty-one patients (22.2 %) used antibiotic medication therapeutically, with a total of 307 antibiotic prescriptions. The median age of patients receiving antibiotic therapy was 62.6 years and 42 % was female. In nearly half of the patients (45.3 %), a clinical microbiologist or infectious diseases specialist was consulted. Twenty patients were admitted on both point prevalence dates. These patients used a total of 64 antibiotic drugs. On both days, 15 of these antibiotics were still prescribed for the same diagnosis. Most patients  were treated with one antibiotic drug, 57 patients (25.8 %) were treated with two, and 13 (5.9 %) were treated with three or more antibiotic drugs. Combinations of beta-lactam antibiotics plus or minus beta-lactamase inhibitors were the most commonly prescribed antibiotic class, followed by fluoroquinolones.In total, 90 (29.3 %) of the 307 prescribed antibiotics were classified as inappropriate antimicrobial drug therapy Table . More spThe rate of inappropriate antibiotic therapy varied from nearly 50 % for broad-spectrum antibiotic drugs to 10 % for narrow-spectrum penicillins Table . AntibioIn our tertiary care hospital, antibiotic drugs are used in 33.8 % of the adult patients in general wards and 22.2 % is used therapeutically. Of the patients prescribed antibiotics therapeutically, 90 (29.3 %) antibiotic prescriptions were inappropriate. The highest percentage of inappropriately prescribed antibiotic drugs was due to unjustified use, i.e., no antibiotic use was deemed indicated. Improper dosing intervals and incorrect duration were also commonly found, as well as prescription of an antibiotic drug when a more effective alternative was available. Urinary tract infection and respiratory tract infection were the infections with the highest inappropriate antimicrobial drug therapy. Our data offer areas of possible intervention by antimicrobial stewardship. In the future, repeated audits of the appropriateness of antimicrobial therapy will give insight into the effectiveness of interventions aimed at improving antibiotic drug use and, thus, the effect of ASTs.Point prevalence surveys are useful tools to assess appropriate antibiotic use . HoweverN = 9,599) received antibiotic drugs [The prevalence of antibiotic use corresponds to the Dutch point prevalence study in 32 hospitals by the Prevention of Nosocomial Infections through Surveillance (PREZIES) network, which showed that 32 % of all admitted patients  , 25. DifIn conclusion, our study provides insight into the appropriateness of antibiotic prescriptions in a tertiary care center in the Netherlands and identifies areas for improvement. We used an electronic surveillance system, thereby making the point prevalence study less time consuming and laborious. A point prevalence study for antibiotic use can be an effective tool to assess the effect of antibiotic stewardship either by an AST or a CDSS.


Clinicians frequently prescribe antibiotics inappropriately for acute respiratory infections (ARIs). Our objective was to test information technology-enabled behavioral interventions to reduce inappropriate antibiotic prescribing for ARIs in a randomized controlled pilot test trial.Primary care clinicians were randomized in a 2 × 2 × 2 factorial experiment with 3 interventions: 1) Accountable Justifications; 2) Suggested Alternatives; and 3) Peer Comparison. Beforehand, participants completed an educational module. Measures included: rates of antibiotic prescribing for: non-antibiotic-appropriate ARI diagnoses, acute sinusitis/pharyngitis, all other diagnoses/symptoms of respiratory infection, and all three ARI categories combined.p < 0.001). There were no significant relationships between any intervention and antibiotic prescribing for non-antibiotic-appropriate ARI diagnoses or sinusitis/pharyngitis. Suggested Alternatives was associated with reduced antibiotic prescribing for other diagnoses or symptoms of respiratory infection  and for all ARI categories combined . Peer Comparison was associated with reduced prescribing for all ARI categories combined .We examined 3,276 visits in the pre-intervention year and 3,099 in the intervention year. The antibiotic prescribing rate fell for non-antibiotic-appropriate ARIs (24.7 % in the pre-intervention year to 5.2 % in the intervention year); sinusitis/pharyngitis (50.3 to 44.7 %); all other diagnoses/symptoms of respiratory infection (40.2 to 25.3 %); and all categories combined  contains supplementary material, which is available to authorized users. Viruses cause the vast majority of acute respiratory infections (ARIs), yet antibiotics remain widely prescribed –3. InappInterventions to reduce antimicrobial prescribing such as physician and patient education, physician audit and feedback, clinical decision support and financial or regulatory incentives have been modestly effective at reducing inappropriate antibiotic prescribing, generally producing approximately 10 % reductions in antibiotic prescribing rates –9. EducaRecognizing the limitations of educational and informational interventions, we developed several interventions that draw on insights from behavioral economics and social psychology . These aThe objective of the study was to pilot test three interventions to improve guideline-concordant antibiotic prescribing as a precursor to inform development of a larger national cluster randomized trial  . The NorThe institutional review boards of Northwestern University and the University of Southern California approved the study. The trial registration identifier is NCT01454960 at ClinicalTrials.gov. The study protocol is available from the corresponding author by request.The NMFF General Internal Medicine clinic is a large adult primary care practice affiliated with an academic medical center and located in Chicago, Illinois. The practice uses the EpicCare electronic health record  for all clinical documentation and for all prescribing. All attending physicians and nurse practitioners were eligible for recruitment . Clinicians provided written informed consent before randomization. Payment for study participation was up to $300 per clinician per month of participation, regardless of intervention assignment . De-identified data from qualifying patient visits that occurred with enrolled clinicians were used in the analyses with an institutional review board waiver of consent.On enrollment, we asked all participating clinicians to complete a 15 to 20 min online survey that included questions about attitudes regarding antibiotic prescribing and an educational module containing information about ARI treatment guidelines –20. The Clinicians randomized to “Accountable Justifications” received EHR alerts in the course of e-prescribing an antibiotic for an ARI diagnosis . The lists of diagnoses we used to suppress study-related clinical decision support were similar to those published in the supplement to the article describing the methods for the multi-site study .This intervention draws from prior studies that show accountability improves decision making accuracy, and that public justification engenders reputational concerns. We expected that the potential to have their non-guideline-concordant choices displayed to others would make clinicians more likely to act in accordance with injunctive norms (guideline recommendations) –26.When clinicians assigned to the Suggested Alternatives intervention entered an ARI diagnosis for a patient visit, a computerized alert presented an order set containing multiple non-antibiotic prescription and non-prescription medication choices as well as educational materials that could be printed and given to the patient . We desiClinicians in the Peer Comparison Intervention group received emailed monthly performance feedback reports. These reports included the clinician’s individual antibiotic prescribing rates for non-antibiotic-appropriate ARIs and as a benchmark, the antibiotic prescribing rate for clinicians who were at the 10th percentile within the clinic . These rates were calculated based on the most recent 20 eligible visits excluding encounters occurring with patients who had certain comorbidities or other diagnosed bacterial infections .If clinicians were among the 10 % of their peers with the “best”—i.e. lowest— prescribing rates the emailed reports told clinicians "You are a top performer.” If clinicians were not among the 10 % best, the emailed report told clinicians “You are not a top performer. You are prescribing too many unnecessary antibiotics.” The proportion of “Top Performers” could be greater than 10 % of clinicians if more than 10 % of clinicians had an inappropriate antibiotic prescribing rate of zero.These peer comparisons were designed to differ from traditional audit and feedback by showing comparisons to top-performing instead of average-performing peers, and its delivery of positive reinforcement to current top-performers. This strategy has been previously shown to help sustain high performance –30.We performed a 2 × 2 × 2 factorial randomized trial, with one arm for each possible combination of the three interventions, corresponding to the eight rows shown in Table We measured physician and nurse practitioner characteristics during the initial online survey. We obtained patient age, sex and race/ethnicity from EHR data.The primary study outcome was the rate of oral antibiotic prescribing during eligible study visits with non-antibiotic-appropriate ARI diagnoses  visits with diagnoses for potentially-antibiotic-appropriate ARI diagnoses  blinded to clinician intervention group judged whether: 1) an antibiotic was prescribed within 24 h without the patient having to reinitiate contact , 2) an antibiotic was not prescribed and it seemed unlikely that an antibiotic at the index visit would have improved the clinical course, or 3) an antibiotic was not prescribed and it may have been clinically useful had an antibiotic been prescribed. An independent data safety and monitoring board reviewed this data during the study.The effects of the study interventions were assessed using separate mixed logistic regression models for each of the primary and secondary outcomes that included each of the three interventions and clinicians’ prior prescribing rate as fixed effects, and individual clinicians as random effects . We tested models with and without two-way and three-way interaction terms between the interventions. Because including these interaction terms had very little impact on the estimated effects of the interventions and complicate the interpretation of the results, we report results of models without interaction terms. Because this was intended as a pilot study, no formal power analysis was conducted. Results are provided with 95 % confidence intervals.In addition to the primary planned analyses, post hoc we constructed models for each of the primary and secondary outcomes that included eligible visits during the year prior to and the year during the intervention period. We tested models that included a term for the intervention year and also a continuous variable for time to assess for evidence of temporal changes in antibiotic prescribing rates.We performed post hoc analyses that included data from the 10 physicians working in the same practice who did not participate in the randomized trial during the year prior to and the year during the intervention period to determine if their antibiotic prescribing rates during these two years differed significantly from the clinicians who participated in the study.Of the 37 internists and 1 nurse practitioner approached, 27 internists and the nurse practitioner enrolled in the trial (74 %). Participating clinicians were 39 male, 61 white, 36 Asian and 4 % African American. Most (75 %) were over 40 years of age. The average number (standard deviation) of distinct patients seen by a participating clinician was 905 (483) in the pre-intervention year and 923 (543) in the intervention year. The flow of participants through the trial is shown in the Additional file The number of clinicians randomized to the different intervention assignments is shown in Table Table In the mixed regression models, antibiotic prescribing was substantially and significantly lower during the intervention year compared to the pre-intervention year across all categories, and the continuous variable for time  was not significant in any of the four models examined except for a slight increase in prescribing rate over time for possibly-antibiotic-appropriate diagnoses , and for all ARI diagnoses combined OR 1.70 (1.31–2.21).In the safety monitoring analyses, we analyzed 18 return visits (0.74 %) with a diagnosis of a bacterial respiratory tract infection that occurred within 30 days of a qualifying study visit at which an antibiotic was not prescribed. Of these, 7 of these received antibiotics within 24 h of the qualifying visit without the patient having to reinitiate contact. In 3 cases, it seemed unlikely that an antibiotic would have improved the course of illness if prescribed at the initial visit. In 8 cases (0.31 %), an antibiotic was not prescribed and may have been clinically useful had it been prescribed at the index visit. None of these 8 patients died or had a prolonged hospitalization. These visits were evenly distributed across the intervention groups  even among clinicians assigned to the control condition . This phSecond, contamination from working in close proximity to peers who received single or multiple interventions related to antibiotic prescribing may have led clinicians to reduce their prescribing more than they otherwise would have. For example, learning that his colleague was a “top performer” relative to a group that includes his own performance may have instilled a sense of competition in a control participant. Such informational contamination between conditions could have been due to information concerning interventions, information about antibiotic prescription rates and/or general conversation about antibiotic over-prescribing. The fact that prescribing rates among non-participating physicians within the same physical practice changed little and remained substantially greater than rates for study participants speaks against this possibility.Third, this study is limited by its sample size. This was a fairly small pilot study meant to test procedures to be employed in a larger multisite study . The numConsistent with prior studies , 35, 36,We also observed a reduction in antibiotic prescribing at potentially-antibiotic-appropriate ARI visits (which consist of acute sinusitis and pharyngitis). These clinical syndromes are frequently caused by viruses, and our safety analysis showed very few patients returning within 30 days following a visit where an antibiotic was not prescribed and it may have been clinically useful had one been given. Therefore, we think it is likely that the reduction in antibiotic use in this diagnostic category was clinically appropriate.An additional potential limitation is worth noting. The baseline antibiotic prescribing rates among clinicians in this group (both among clinicians who participated and those who didn’t) were lower than what has been reported in broader populations of clinicians –3. This The conduct of this pilot study had a large impact on antibiotic prescribing across all study physicians including among control participants (but not among non-participants within the same practice). The results of a larger multisite trial conducted by this same study group help address this study’s aforementioned limitations . The larARI, acute respiratory infection; CI, confidence interval; EHR, electronic health record; NMFF, Northwestern Medical Faculty Foundation; OR, odds ratio


Pregnant couples tend to prefer a maximum of information about the health of their fetus. Therefore, we implemented whole genome microarray instead of conventional karyotyping (CK) for all indications for prenatal diagnosis (PND). The array detects more clinically relevant anomalies, including early onset disorders, not related to the indication and more genetic anomalies of yet unquantifiable risk, so-called susceptibility loci (SL) for mainly neurodevelopmental disorders. This manuscript highlights the psychological challenges in prenatal genetic counselling when using the array and provides counselling suggestions. First, we suggest that pre-test decision counselling should emphasize deliberation about what pregnant couples wish to learn about the future health of their fetus more than information about possible outcomes. Second, pregnant couples need support in dealing with SL. Therefore, in order to consider the SL in a proportionate perspective, the presence of phenotypes associated with SL in the family, the incidence of a particular SL in control populations and in postnatally ascertained patients needs highlighting during post-test genetic counselling. Finally, the decision that couples need to make about the course of their pregnancy is more complicated when the expected phenotype is variable and not quantifiable. Therefore, during post-test psychological counseling, couples should concretize the options of continuing and ending their pregnancy; all underlying feelings and thoughts should be made explicit, as well as the couple’s resources, in order to attain adequate decision-making. As such, pre- and post-test counselling aids pregnant couples in handling the uncertainties that may accompany offering a broader scope of genetic PND using the array. Until recently, conventional karyotyping (CK) was the gold standard to detect chromosomal aberrations in prenatal diagnosis (PND). Whole genome microarray analysis has been introduced in prenatal medicine due to its evident advantages; microarray detects more submicroscopic copy number variants (CNVs) that cause clinically relevant abnormalities ,2,3 and It is remarkable that ample publications voice healthcare professionals’ concerns regarding high resolution invasive prenatal genetic diagnosis ,5,6, butOur lab in Rotterdam has completely replaced CK by a whole genome SNP array for all indications since July, 2012 . Thus, pWhereas PND has always been familiar with the phenomenon of unexpected diagnoses, the number and nature of unexpected diagnoses has expanded with the implementation of the SNP array when compared to CK ,17. We ret al. (2013) [et al. (2009) [The most challenging type of SNP array results are the SL. SL are pathogenic CNVs , with incomplete penetrance or variable expression, that are associated with a yet unquantifiable chance for (mainly) neurodevelopmental disease  ,20. They. (2013)  and Door. (2009) . One recent study suggests that the information of SL may represent a burden to couples and that offering such knowledge should be accompanied by careful pre- and post-test counselling . ConcernOur rationale to decide in favor of the disclosure of SL lies in the necessity of the follow-up of a pregnancy postnatally in order to learn to interpret the SL. If we do not disclose, we cannot justify follow-up. In addition, if we do not disclose and neurodevelopmental problems present in the child, we will have deprived the parents of important information, which might have led to more adequate diagnostics and care. On the other hand, we are well aware of the burden SL might present psychologically. Here, we provide an overview of the possible psychological and counselling challenges, and we share our ideas regarding how to deal with these.Due to the challenges of detecting unexpected diagnoses and SL at relatively low prevalence, there is currently no consensus regarding the introduction of the array for all indications in prenatal medicine. With this paper, we aim to contribute to the development of international consensus. sine qua non; before decision-making, pregnant couples need to clearly oversee the possible consequences of engaging in prenatal genetic diagnosis. Informed decision-making has been defined as the outcome of a process based on sufficient knowledge and deliberation and that is congruent with one’s personal attitudes [Informed consent is a condition ttitudes . Adequatttitudes . Conversttitudes . Informettitudes ,32, and ttitudes . With thttitudes . It is tttitudes ,34. One Often underestimated aspects of counselling are the facilitation of understanding the information, attributing personal meaning to the information and decision-making based upon the information provided ,36. Howei.e., Down syndrome, late onset disorders, SL).In summary, we promote that in addition to the provision of information, during pre-test counselling, more attention is paid to enabling pregnant couples to consider what they wish to learn from prenatal diagnosis. Couples need to be invited to personalize the information by reflecting on what possible outcomes might be generated and what consequences they would attach to various outcomes  might be traumatizing ,38,39. AIt has been consistently reported that understanding and recall of information about (genetic) probabilities and chances are poor ,42. When. Several sessions of posttest genetic counselling may be needed and should be available for the pregnant couple to learn to understand and provide personal meaning to an anomalous test outcome.This psychological task is more difficult with the increase of unquantifiable results, and more couples will be confronted with this when using the SNP array in PNDSince we started to employ the array for all PND indications, we have decided to discuss whether or not to disclose the SL to the patient in a multidisciplinary team. If disclosed, we currently offer these pregnant couples posttest genetic counseling by a clinical geneticist and by a psychologist, if needed. The clinical geneticist informs the pregnant couples about the incidence of this particular SL in control populations and in postnatally ascertained patient populations. The difference between postnatal ascertainment  and the prenatal identification of an SL, because of AMA, for instance, is explained. This approach puts the SL in a proportionate perspective. Furthermore, the specific abnormal phenotypes found after postnatal ascertainment are discussed. It is specified that the chance of an abnormal phenotype after prenatal diagnosis is currently not known in spite of efforts to quantify this , since iThus, we aim to put an SL in a realistic perspective for pregnant couples. The SL implies an increased risk of an abnormal phenotype. We consider SL as actionable findings, leading to extra attention postnatally for development and behavior and easy access to early intervention programs, if necessary.In general, psychological counseling after the disclosure of unfavorable results from PND implies guidance and support in decision-making and the grief process . It may i.e., social, financial, emotional resources, etc.) may incline the couple to prefer to prevent the fetus’ possible suffering. Conversely, couples may accept the risk, because they rely on the possibility of a favorable outcome or on their resources for dealing with unfavorable outcomes. Awareness of such underlying feelings and thoughts may aid pregnant couples in making a decision that is complete and does justice to their capabilities and resources. This may be even more important in the case of SL, because pregnant couples can hardly anticipate the possible consequences. This approach is important to prevent couples from panicking into a decision and to ensure that the decision made is one that has taken into account all of the possible considerations  of the pregnant couple. Couples deciding about ending or continuing their pregnancy often do not have a clear view on what they might expect from either option. When a couple decides to continue their pregnancy, the counseling focuses on adjustment until and after delivery ,46. WhenBecause of the difficulties SL may represent to pregnant couples, we are currently conducting a study in order to evaluate how pregnant couples regard SL, whether they wish to be informed of these and how they respond when confronted with SL. In summary, the array increases tested disorders in genetic PND. This broadening of the scope of PND may be accompanied by a number of psychological challenges for pregnant couples: Deciding what they wish to learn about the (future) health of their fetus, understanding (categories of) the possible outcomes of PND, dealing with unexpected diagnoses and SL, deciding about the course of their pregnancy and adjusting to the decision outcome. Unexpected prenatal test results and SL may complicate deliberation and decision-making regarding the course of pregnancy. During pre-test counselling, we suggest that the counsellor not only provides information regarding possible test outcomes, but also enables deliberation about what pregnant couples wish to learn about the health of their fetus. In addition, it must be specified that array results may not be as certain as pregnant couples might hope for. Increasing the number of genetic disorders in one test in PND will generate more results of variable expressivity and heterogeneity of clinical features, which may induce stress and impede decision-making . We therWith regard to posttest psychological counseling, we recommend that couples are encouraged to concretize as much as possible the options of continuing and ending their pregnancy. All underlying feelings and thoughts should be made explicit, as well as the couple’s resources for adequate decision-making. Pretest genetic and decision counselling and posttest genetic and psychological counselling should be readily available when offering a broader scope of genetic PND using the array . This ma


Antibiotic treatment options for common infections are diminishing due to the proliferation of antimicrobial resistance (AMR). The impact of Antimicrobial Stewardship (AMS) programs seeking to preserve viable antibiotic drugs by governing their use in hospitals has hitherto been limited. Pharmacists have been delegated a critical role in antibiotic governance in AMS teams within hospitals but the experience of pharmacists in influencing antibiotic use has received limited attention. In this study we explore the experiences of pharmacists in antibiotic decision-making in two Australian hospitals.We conducted 19 semi-structured interviews to explore hospital-based pharmacists’ perceptions and experiences of antibiotic use and governance. The analysis was conducted with NVivo10 software, utilising the framework approach.responsibilities of pharmacy in optimising antibiotic use and the interprofessional challenges therein; (2) the importance of antibiotic streamlining and the constraints placed on pharmacists in achieving this; and (3) the potential, but often under-utilised expertise, pharmacists bring to antibiotic optimisation.Three major themes emerged in the pharmacist interviews including (1) the Pharmacists have a critical role in AMS teams but their capacity to enact change is limited by entrenched interprofessional dynamics. Identifying how hospital pharmacy’s antibiotic gatekeeping is embedded in the interprofessional nature of clinical decision-making and limited by organisational environment has important implications for the implementation of hospital policies seeking to streamline antibiotic use. Resource constraints (i.e. time limitation and task prioritisation) in particular limit the capacity of pharmacists to overcome the interprofessional barriers through development of stronger collaborative relationships. The results of this study suggest that to enact change in antibiotic use in hospitals, pharmacists must be supported in their negotiations with doctors, have increased presence on hospital wards, and must be given opportunities to pass on specialist knowledge within multidisciplinary clinical teams.The online version of this article (doi:10.1186/s12913-016-1290-0) contains supplementary material, which is available to authorized users. Antimicrobial resistance (AMR) is a prominent public health concern. While the proliferation of multi-drug resistant pathogens reaches pandemic proportions, the development of new antibiotic drugs has stagnated, presenting the significant potential for a post-antibiotic era in the near future –4. The rantimicrobial stewardship (AMS) ’s deemed inappropriate; asking for advice from people when you’re not sure; taking the time to look things up if you don’t know the answer yourself. And it’s ingrained in us through our practice from the very beginning, this is what you’re taught […], because that's basically what a pharmacist does is they’re a problem solver” .Another participant commented on what hospital pharmacy … as pharmacists we don’t make choices other than to intervene. And our role is predominately advisory” .“… a hands-on approach to them, to keep on advising and recommending and so on. Everyone has access to the antibiotic guidelines, but it’s getting them [doctors] to do it” .“… It’s pretty obvious that they [doctors] aren’t aware of the restrictions to prescribing certain antibiotics and it’s up to pharmacy to make them aware” .All respondents agreed that pharmacy’s main function is to be the clinical team’s experts on medications driven by their professional education, identity and standards. This translates into communication with doctors, giving and seeking advice, keeping records and establishing links across different departments of the hospital. In everyday practice, pharmacists assume a range of different responsibilities when solving problems around antibiotic prescribing:“[pharmacists] spend their day … in the wards rather than living out of a pharmacy department. … the idea is that they’re readily available to all the doctors at any time. … So we do act as a team, so we try and do a little bit of everything. So at any one time one of us could be doing anything for anyone on any ward” .“It’s clear if you have experienced pharmacists as part of the team I could guarantee that would improve antimicrobial prescribing. And that's not just for serious things like septicaemia, that's from everything…trained pharmacists make a huge impact, there's just not enough, and we’re always behind the eight ball” .Pharmacists interpreted their role in antibiotic prescribing within the boundaries of their relationship with doctors—i.e. they acted as doctors’ advisors, provided education and got involved more directly in antibiotic decision-making. Hospital pharmacy’s potential to bring drug expertise to bear is associated with its greater penetration into the provision of care at the bedside. Increasingly, pharmacy assumes duties beyond the dispensing of drugs, requiring stronger involvement in clinical practice and a physical presence on hospital wards. As this senior pharmacist described, ideally:“There's a basic delivery of services which needs to happen and that is the delivery of medication, and everything else is extra. So if you can’t, you have to ensure that the core service is functional … until you can cover your core work … and we’re doing it well, I don’t see how we can then say ’well we’ll take on the responsibility of ensuring that we do antibiotic education, and we’re rolling out guidelines, and we’re making sure we’re picking up suboptimal prescribing and we’re looking at gentamicin lists and we’re doing all the stuff that we can do to capture this problem'” .“… So it’s an enormous amount of work… there's a job where you clock in and you clock out. And I think if you’re going to be a professional maybe there's a perception … it needs to become your vocation as well. So that thing that you do out of hours for enjoyment maybe that needs to be cut down a bit because you’re going to need to put some hours in at home” .The interviews revealed environmental and resource constraints limiting pharmacists’ ability to ensure their clinical presence and contribute proactively to antibiotic decisions. This perception was voiced across all levels of seniority and areas of clinical focus. Pharmacists made sense of these constraints and their implications for everyday work in different ways:It emerged that pharmacy’s ongoing clinical expansion remains somewhat incomplete. The participants expressed an inability to work towards consistent antibiotic practices in accordance with hospital policies during working hours. They acknowledged new responsibilities within the tighter governance of antibiotic use, but described a process of prioritisation as a result of competing demands on their time. This prioritisation revealed that a “basic delivery of services” (i.e. the generic dispensing of drugs) was perceived as core business with responsibilities around promoting the judicious use of antibiotics being described as “extras” by pharmacists on the wards. There are clear role-based limitations on the penetration of pharmacy expertise at the bedside and in the capacity of pharmacy to help streamline antibiotic use.“We cannot supply it … we all carry the list of ID approval requirements. They’re not in the impres[t] rooms [onsite drug supply], so then it does have to go through pharmacy. And then pharmacy can say 'hey we’re not going to do this'. So that's our biggest bargaining chip” .These constraints are made more pertinent by the informal practices pharmacists face when attempting to optimise antibiotic use in line with antibiotic guidelines. Almost all of our participants talked about difficulties to intervene once a prescription was written. Being present at the moment of initiating antibiotic therapy was for most the exception. This, and the lack of a default mechanism to correct suboptimal antibiotic scripts, exposed pharmacy interventions to a range of inconsistencies in practice. We asked pharmacists how they deal with prescriptions that deviate from therapeutic guidelines:“… we seem to be the people that are sort of the gatekeepers … and we seem to always be the ones going 'well no you can’t have that. You have to get ID approval'” .“… we have to be the gatekeeper a bit in the middle of the nurses and the doctors. Yeah we’re very responsible for it, if we end up with none [antibiotics] left to choose from in the future, if we just gave everything out willy nilly, that's the reason I guess why everything’s not on imprest, or everything’s not in the after hours cupboard, because there's meant to be a pharmacist involved in that” .“… We can then go and say ‘okay we’ve now got thirty patients, we’ll look at charts to see how inappropriate prescribing is. Let’s see the areas or the teams or who are prescribing inappropriately.’ We then need to feedback to them to say ‘look there's a problem here. These are actually the guidelines, this is what we need to do. Would you like some assistance in writing a protocol? Do you need education?’” .The imprest stock is an onsite supply of medications accessible by nurses and doctors at any time. All antibiotic drugs that need approval from an Infectious Diseases specialist are stocked only in limited quantity. For a supply of more than 24 h specific dispensing from pharmacy is required for each patient. This gives pharmacy the ability to track the use of restricted drugs. Many of the pharmacists emphasised the importance of this measure of control and regarded themselves as “gatekeepers” watching over the use of antibiotic drugs:“… pharmacy can certainly be a platform because they have the ability to restrict the actual supply. And that's not necessarily the right answer…” “… there isn’t enough time obviously for pharmacy to both review it and supply it. So it’s hard. You’re talking about a system that has to still function. How much do you restrict it? How much can you restrict it?” .Managing and restricting the supply of antibiotic drugs is one of the key antimicrobial stewardship interventions of hospital pharmacy. Pharmacists justified this enactment of interprofessional control emphasising their responsibility to safeguard the viability of antibiotics for the future. The latter interview excerpt above demonstrates how restrictive management of supply is accompanied by attempts to educate and raise awareness about therapeutic guidelines. Participants also discussed the limitations a restrictive approach to streamlining antibiotic use imposes. The same participant as above admitted that:“So it depends on the ward, whether it’s on the impres[t], like in their drug rooms. Then the nurses just have the straight out availability to go and take it. … If a pharmacist doesn’t see it it can just go under the radar. … also, if they don’t have it on their ward they will go to another, the respiratory ward that does have it and get it just to avoid pharmacy” .“… they’ll just go to a different ward, or they’ll change it to something easier that someone's not going to hassle them about, but might not be the best choice. I think that's a big problem is a lot of the time they will do something easier, that doesn’t require ID [Infectious Diseases approval] … they’ll choose a different antibiotic rather than choosing the correct one and just giving them a call” .The participants doubted that gatekeeping practices alone were the best way to optimise antibiotic use. The latter interviewee above viewed it even as potentially harmful to “the functioning of the system”. This argument relates to the hospital as an organisational setting characterised by hierarchically-structured professional relations. Establishing a dispensing authority in the form of the pharmacist as gatekeeper alongside the prescribing authority manifest in the doctor was perceived as a threat to the integrity of that system. The majority of participants voiced concerns about the appropriateness and effectiveness of this part of their work:Restrictive supply management on its own was described by the above, and other participants as ultimately counterproductive and encouraging suboptimal choice of antibiotic drug rather than providing decision aids for problematic cases. These reported informal practices undermined these pharmacists’ capacity to enforce best practice guidelines and put pressure on them to engage directly with doctors in discussions about appropriate antibiotic prescribing. These negotiations take place between distinct professional communities drawing on different types of knowledge resources, cultural idiosyncrasies and sets of responsibilities.“… for us [pharmacy] our main focus all the time is drugs. And I'm aware that for the doctors, they’ve got so many other things that they have to think about. And so sometimes for them they’ve just got to get whatever it is written down and look at everything else” .“… We’re pharmacy based so we’re thinking about drugs all the time … Whereas they [doctors]’ve got a much broader spectrum. … we’re pretty privileged as being pharmacists in hospitals that we get to look at the holistic view of the patient, we are able to look at their labs, we can see their sensitivities, … we’ve had four-and-a-half … years training looking at medications and seeing if they’re appropriate. And then we can relay that to the doctors” .Gatekeeping practices alone are ineffective and may have adverse effects on the choice of antibiotics pushing pharmacy to intervene more directly in the prescribing process. Participants’ accounts of their involvement expanded beyond the choice of drug into common prescribing issues around duration, dosage and mode of delivery. While some pharmacists dismissed doctors as “ignorant” or even “lazy”, others explained suboptimal prescribing practices in terms of different knowledge resources, professional standards, localised norms and differing notions of care, i.e. pharmaceutical care as narrowly focused on individual drug therapy and clinical care as taking a holistic approach to patient assessment and treatment. More precisely they perceived it as a clash between medical judgment and pharmaceutical standards. The following participants discussed how they view pharmacy’s drug expertise fitting into clinical practice:“… if [as a doctor] you've seen something prescribed over ten years and you know it’s going to work you know, it might be 90 % right. … You can go up to a doctor and go ‘why did you choose this’ or whatever, and it’s like’close enough is good enough’, … As long as they’ve got something that's treating the patient and they’re going to get better, then I don’t think they care too much” .“Well close enough is not good enough! … ‘I think the dose is okay, it won’t kill them, it’ll do’, that's terrible, that is absolutely terrible. … we’re in an electronic age, it’s at your fingertips, it’s not that hard to look up. … I would no sooner prescribe something that I wasn't completely sure what the dose was, if I could prescribe, than fly to the moon! You know that's terrible! It’s a patient!” .“… you see it all the time and you go’that's not great but it’s safe, it’s a little bit effective’ and … you've got to pick your battles … we’re still often giving supply because it’s either the patient gets no antibiotics, or a patient gets something that - you know we can sort of look from our end I guess and say ’yeah they’re covering the right bug, it’s probably not the best therapy’” .The pharmacists understood their expertise as unique and highly specialised on drugs as opposed to the broader remit of medical knowledge. They framed this expertise as different from, and more specialised than, the clinical knowledge of the medical profession. Most participants agreed that the value of pharmacy is not to supplant medical judgment but provide practical advice and prompts to complement it, for example, by assisting with precise dosing, raising awareness about AMR and promoting the accessibility of clinical guidelines. Pharmacists built on their professional education and standards to argue for their potential to make meaningful contributions in the clinical field. They situated their argument within an ethical discourse about the “patient good” which served as an overarching framework connecting pharmacy with the medical profession. This subtheme became more evident when they talked about the frustrations they experience when their drug expertise clashed with prescribing decisions made by doctors:There was a high degree of inconsistency within the interviews regarding how the participants resolved the tension between their drug expertise and allowing for medical judgment and thus reducing conflict. Individual pharmacists make choices to what extent they follow-up cases of suboptimal prescribing. While some argued prescribing is either done right or wrong and deviations from best practice were challenged, others engaged in “picking battles” and perceived antibiotic prescribing as a grey zone. The first group of participants drew on collective quality standards within the pharmacy profession to enforce precise dosing, appropriate choice of drug, duration and mode of delivery. They supported their argumentation by pointing out the instant availability of knowledge resources online and off-line, laboratory testing with short turnarounds and pharmacy and ID counsel. Ultimately, they utilised discourses about the “patient good” to justify demands on the medical profession for greater accuracy in antibiotic prescribing. The second group engaged in a process of prioritisation and de-escalation guided by pragmatic principles taking into account resource constraints of time-poor doctors, the multitude of clinical factors doctors have to consider beyond drugs and the apparent—albeit limited—efficacy of the course of antibiotic treatment. They also drew on similarly understood discourses about the “patient good” to defend poor prescribing as long as patient outcomes were positive. Both groups placed short-term patient outcomes rather than long-term concerns about AMR at the centre of their ethical reasoning moderating the moral distress of sub-optimal antibiotic practices . In eithThe proliferation of AMR has resulted in attempts to better govern the use of antibiotics in hospitals. However, recent changes to antibiotic governance with a focus on altering doctors’ behaviour have had limited success. Our previous research revealed the significance of other stakeholders in antibiotic decision-making , and a vFirst, this study indicates that hospital pharmacists perceive themselves as antibiotic gatekeepers seeking to fulfil educational, advisory and supervisory roles within antibiotic governance—they negotiate with other stakeholders caring for patients at the bedside to bring their expertise on drugs to bear and are empowered by their control over the supply of antibiotics. This reaffirms the interprofessional nature of antibiotic decision-making and the need to address potential institutional and informal barriers to enhanced collaboration across professionals and settings in the hospital.Second, we found that some doctors and nurses can undermine pharmacists’ dispensing of restricted antibiotic drugs, thus weakening their ability to prevent deviations from therapeutic guidelines and offer timely advice on drug choice, mode of delivery and duration. This highlights the potential detrimental effects of interprofessional dynamics and resource constraints on hospital pharmacy‘s capacity to perform responsibilities pertaining to antibiotic governance. Increased presence on the hospital wards and better integration into clinical teams would provide more opportunities to pharmacists to get involved in antibiotic decision-making, ideally prior to prescriptions being written.Third, consistent with previous research on the reporting of prescribing errors , pharmacHowever, this study is not without limitations. Given the qualitative approach of the study, the findings within this context cannot be generalized to other settings. The AMS program implemented at the participating hospitals favours, besides educational measures and evaluation, the restrictive management of antimicrobials through its antibiotic approval system in which pharmacy is tasked with gatekeeping duties. Pharmacists working in settings with no antibiotic restrictions, or with AMS programs which rely on other mechanisms may encounter different issues. Our data indicated that there exists a tension between clinical judgment and pharmaceutical expertise which requires significant exploration. Only if the reasoning behind pharmacists’ decisions to prioritise and intervene in the optimisation of antibiotics use and its relation to medical expertise is understood, institutional mechanisms can be designed to promote more consistent engagement of hospital pharmacy in antibiotic governance.Our findings have significant repercussions for antibiotic optimisation and streamlining. The increasing self-perception of pharmacy as antibiotic gatekeeper opens up significant potential for individual pharmacists to influence antibiotic decision-making in everyday practice. However, the resistance of doctors to pharmacy advice and the inconsistencies in pharmacy practice impose severe constraints on the implementation of hospital policies aiming at the optimisation of antibiotic prescribing. These need to be addressed, for example via the design of institutional mechanisms that are sensitive to the differences in professional culture and provide pharmacy with a stronger foothold at the bedside. This includes the reallocation of resources to acknowledge hospital pharmacy’s responsibilities beyond the dispensing of medicines and provide them with opportunities to accumulate specialised clinical experience. Increased presence of pharmacists on hospital wards across high resource and low resource settings would also allow the establishment and maintenance of trusting relationships between pharmacists and doctors that promotes a sense of shared obligation to the “patient good” and a mutual understanding as partners in the effort to curb the proliferation of AMR.


Aberrant activation of the HGF/MET signaling axis has been strongly implicated in the malignant transformation and progression of gastroesophageal cancer (GEC). MET receptor overexpression in tumor samples from GEC patients has been consistently correlated with an aggressive metastatic phenotype and poor prognosis. In preclinical GEC models, abrogation of HGF/MET signaling has been shown to induce tumor regression as well as inhibition of metastatic dissemination. Promising clinical results in patient subsets in which MET is overexpressed have spurned several randomized studies of HGF/MET-directed agents, including two pivotal global Phase III trials. Available data highlight the need for predictive biomarkers in order to select patients most likely to benefit from HGF/MET inhibition. In this review, we discuss the current knowledge of mechanisms of MET activation in GEC, the current status of the clinical evaluation of MET-targeted therapies in GEC, characteristics of ongoing randomized GEC trials and the associated efforts to identify and validate biomarkers. We also discuss the considerations and challenges for HGF/MET inhibitor drug development in the GEC setting. Gastroesophageal adenocarcinoma (GEC) is a common and highly morbid malignancy accounting for ~1 million cases and in excess of 700,000 cancer-related deaths annually . There iBecause of its insidious nature, patients newly diagnosed with GEC often present with advanced incurable disease. For those presenting with potentially resectable cancer that undergo surgery with curative intent in conjunction with perioperative chemotherapy or post-operative chemoradiation, overall survival ranges from 30-35% at five years and recurrence within two years following surgery is commonplace -6. Thus,Patients with metastatic GEC (mGEC) can be subdivided into two populations based on HER2 expression status . HER2-poFor patients with HER2-negative GEC, doublet or triplet combination chemotherapy remains the mainstay of treatment , 9, 10. Effective delivery of conventional cytotoxic therapy is challenging in the setting of GEC by virtue of both substantial disease-related morbidity and the advanced age of patients . These cAs with other cancers, recent progress in the molecular profiling of GEC has lead to the design of several targeted therapies that are currently in clinical development , 14. GasThis Review will focus on the anti-MET agents currently in late stage clinical development in advanced/metastatic GEC. The Review summarizes the available data as well as the current status of ongoing randomized studies of MET-directed agents. Since identification of tumours most likely to respond to MET pathway blockade is a key component of therapeutic development, we also discuss the issues and challenges associated with identification of MET biomarkers to aid patient selection.et al [www.vai.org/met) [MET is a transmembrane receptor tyrosine kinase (RTK) for which hepatocyte growth factor (HGF) is the only known ligand. MET is predominantly expressed on cells of epithelial origin but is also found on non-epithelial tissues such as endothelium, neuronal cells, melanocytes and hematopoietic cells. MET activation induces complex cellular signaling mediated through a variety of transduction pathways driven by a diverse array of adaptors and downstream effectors . MET pret al -22. Undeet al . Aberranorg/met) , 21, 23.MET gene amplification appears rare in treatment-naïve gastric tumors with reported incidences of between 0 – 5% [In vitro, MET-amplified GEC cells are acutely sensitive to HGF/MET pathway blockade [Dysregulated HGF/MET signaling is commonly seen in GEC. Signal activation by HGF in GEC cell lines and tumor models promotes tumorigenesis and metastases. The potentiated capacity for metastatic transformation upon MET activation has been linked with an increased capacity for epithelial-mesenchymal transition (EMT) and inhibiting detachment-mediated apoptosis (anoikis) in GEC models . Perturbn 0 – 5% , 35, 36.n 0 – 5% , 35-39 Iblockade , 41.in vitro resistance to cytotoxic agents known to be active in GEC [In addition to oncogenesis and malignant transformation, aberrant MET signaling has been associated with e in GEC , 43.Collectively, these data provide a compelling rationale to clinically evaluate HGF/MET inhibitors in the setting of GEC.Several drugs targeting the HGF/MET signaling axis, including both antibodies and small molecule inhibitors have been evaluated in the clinic. Antibodies directed against either HGF or MET prevent ligand-receptor interaction and consequently impact downstream MET signaling Figure . Small mBoth rilotumumab  and onartuzumab  are in the latter stages of clinical development for GEC. The primary hypothesis being tested in both studies is whether addition of HGF/MET-targeted therapy to standard platinum-based chemotherapy improves survival in patients with gastroesophageal tumors overexpressing MET.Rilotumumab is a fully human monoclonal IgG2 antibody that binds HGF and prevents its binding to the MET receptor and subsequent signaling . OnartuzThe strongest clinical evidence supporting HGF/MET inhibition in GEC comes from a first-line randomized, placebo-controlled Phase II study of rilotumumab in combination with platinum-based chemotherapy in patients with advanced GEC. In this three arm trial, 121 patients with unresectable locally advanced or metastatic disease were randomized to epirubicin, capecitabine and cisplatin (ECX) plus placebo (n = 39) or ECX plus either 7.5-mg/kg (n=40) or 15-mg/kg rilotumumab (n=42). Patients were primarily drawn from Western countries  with only a minority (17%) from Asia. The combination of rilotumumab (low and high doses combined) plus ECX marginally improved both PFS (HR = 0.64) and OS (HR = 0.73) in the intention-to-treat population. Toxicities more frequently seen with rilotumumab included peripheral edema, thrombocytopenia, neutropenia and deep vein thrombosis (DVT). Of note, peripheral edema is a frequently reported toxicity associated anti-HGF/MET antibodies across multiple tumor types and combination regimens .High i.e. weak (1+) or stronger MET staining in >50% of malignant cells. Patients with METHigh tumors appeared to experience marked clinical benefit from the addition of HGF-targeted therapy to chemotherapy with respect to PFS and OS compared to those treated with chemotherapy alone . On the other hand, worse clinical outcomes were observed in the METLow patient subpopulation receiving anti-HGF therapy compared to those receiving chemotherapy only . Interestingly, a similar trend towards worsened clinical outcomes in MET-negative patients was also observed in study of rilotumumab in patients with castration-resistant prostate cancer [High and METLow tumors receiving chemotherapy alone, the IHC analysis confirmed that high MET expression is a marker of poor prognosis as suggested by prior retrospective analyses. These data provide clinical proof-of-concept supporting HGF/MET axis targeting in GEC and suggest that MET receptor expression measured by IHC could function as a predictive biomarker for patient selection.The relationship between MET expression and clinical outcomes were also evaluated in this trial . Archivae cancer . When coMET gene polysomy, moderate MET expression by IHC and a remarkably high baseline serum HGF level. In addition, intratumoral coexpression of both HGF and MET was observed possibly indicating autocrine signaling [In a Phase I study dose-escalation study of onartuzumab in patients with various locally advanced or metastatic solid tumors, a sustained complete response (>2 years) was noted in a patient with chemorefractory GEC with hepatic metastases , 50. Intignaling . Collectignaling .Several early phase studies have evaluated anti-MET TKIs in patients with GEC, with mixed results. Crizotinib, a tyrosine kinase inhibitor that targets both ALK and MET tyrosine kinases, was originally developed as a MET-specific inhibitor but has been FDA-approved for use in ALK-positive NSCLC. Single-agent clinical responses to crizotinib were noted in two of four patients with relapsed mGEC and increased MET copy number (≥5 copies) . ConversThe evidence linking aberrant HGF/MET signaling to gastric tumorigenesis coupled with encouraging early clinical results has triggered significant clinical development efforts Table . Phase In=450) are randomized 1:1 to ECX  plus double-blind rilotumumab (15 mg/kg IV) or placebo on a three-weekly cycle. Patients can receive up to ten cycles of ECX, with Rilotumumab or placebo continued until disease progression. Randomization is stratified according to disease extent  and ECOG score (0 vs. 1). Eligibility is restricted to patients with MET positive and HER2 negative tumors.RILOMET-1 is a randomized, global, double-blind, placebo-controlled Phase III study of rilotumumab in combination with ECX as first-line treatment for advanced MET-positive GEC (NCT01697072) . PatientThe primary endpoint is OS. Key secondary endpoints include PFS, objective response rate (ORR), OS in MET expression tertiles, safety, and pharmacokinetics. RILOMET-1 is being conducted in approximately 180 sites in Australia, Europe, Africa, North America and South America.Met or EGFR inhibition in Gastroesophageal Adenocarcinoma) study is a randomized Phase II study evaluating the addition of either Rilotumumab or panitumumab  to mFOLFOX6 as first-line treatment for advanced GEC. Patients from 30 French sites (planned n=165) are randomized 1:1 to mFOLFOX6 alone or combined with either panitumumab (6 mg/kg) or rilotumumab (10 mg/kg). Randomization is stratified by extent of disease , histologic subtype  and study center. The primary endpoint is 4-month PFS and key secondary endpoints include ORR, PFS, OS and safety.The MEGA (n=800) are randomized 1:1 to receive either mFOLFOX6  with onartuzumab  or mFOLFOX6 plus placebo. A maximum of 12 cycles of mFOLFOX6 are permitted. From cycle thirteen onwards, onartuzumab or placebo is continued until disease progression. Randomization is stratified by MET expression status, world region (Asia Pacific vs. Rest of World) and history of prior gastrectomy. Only patients with tumors centrally classified as both HER2-negative and MET-positive (by IHC) are eligible. For the purposes of determining eligibility, a MET positive tumor is defined when ≥50% of malignant cells express MET (cytoplasmic and/or membranous staining) at weak, moderate or strong intensity.MetGastric is a randomized placebo-controlled, international Phase III study in patients with previously untreated metastatic GEC (NCT01662869) . PatientThe co-primary endpoint of this study is OS in all patients (ITT) and a subgroup of patients with MET IHC scores of 2+ or 3+. Secondary endpoints include PFS, ORR, safety as well as correlative tissue and biomarker studies.YO28252 is a multicentre, randomized, placebo-controlled Phase II study of onartuzumab in combination with mFOLFOX6 being run in parallel with MetGastric (NCT01590719). Patients (planned n=120) from the US or Asia-Pacific are randomized 1:1 to mFOLFOX6 in combination with either placebo or onartuzumab. While the Phase II study design and patient population is largely similar to MetGastric, there are some important differences. Unlike MetGastric, patients are not required to have MET-positive disease to be eligible for participation as the primary purpose of the study is to evaluate the clinical profile of onartuzumab in both MET-positive and MET-negative tumors. Randomization is according to Lauren histologic subtype  and history of prior gastrectomy. The primary endpoint is PFS in both the ITT population and those defined prior to unblinding as MET-positive by IHC. Key secondary endpoints include OS, ORR, safety and pharmacokinetics.MET gene copy number [One of the key challenges associated with the development of targeted therapeutics is identifying tumors that are sensitive as well as patients likely to derive clinical benefit . Criticay number .www.vai.org/met) [www.vai.org/met). In the absence of clinical validation there is currently no consensus on scoring criteria for MET IHC tests. Studies using IHC to evaluate the prognostic impact of MET expression have primarily utilized retrospective analyses of variably sized and annotated clinical cohorts, resulting in a high degree of variability in the published findings with respect to the prevalence of MET overexpression and/or its prognostic implications. Potential sources of variability include the use of different sample types, inter-reader variation, primary and secondary antibodies, staining protocols, scoring methods as well as differences in tissue processing and storage [MET protein expression using IHC has been extensively studied in GEC with most studies reporting that MET overexpression is associated with poor patient survival (org/met) . Some, b storage , 58. Fur storage -61 and iIn clinical development, both rilotumumab and onartuzumab are being developed in GEC using a strategy of identifying MET-positive patients by means of a centrally conducted IHC assay on FFPE tissue Figure . For botStudy OAM4558g was a randomized Phase II study in which patients with relapsed NSCLC were randomized to receive onartuzumab plus erlotinib  or erlotinib alone . In ordeCollectively these data suggest that MET IHC is a valid biomarker with which to select patients for MET-targeted therapy. Despite the encouraging nature of these data sets, limitations must be considered. Namely both Phase II studies had a small sample sizes and did not prospectively stratify based on MET expression status (definition of MET positivity was determined before unblinding but after random assignment). Several outstanding questions remain with respect to the use of MET IHC. Rigorous clinical validation of the diagnostic cut-off point used to define MET positivity will be critical given the detrimental outcomes seen in the Phase II setting in MET-negative patient subsets , 48, 62.MET gene amplification results in constitutive activation of the MET receptor and an oncogenic addiction to the MET signaling pathway thereby rendering gastric tumor cells acutely sensitive to HGF/MET axis inhibition [MET amplification has been correlated with an increased frequency of distant metastasis, more extensive TNM stage and diminished survival in retrospective clinical series [MET copy number gain has been reported in up to 30% of clinical cases and may be associated with poor prognosis [Aberrations in gene copy number in malignant cells can be driven by either gene amplification or polysomy. Gene amplification refers to a copy number gain for a specific gene (or group of genes) on a given chromosome arm without a change in copy number for genes located in other regions of the chromosome, whereas polysomy gives rise to a copy number gain for a given gene as a result of the presence of extra copies of the entire chromosome . In prechibition , 41. Whihibition , it is hhibition -37, 69. l series , 35. METrognosis , 37. Prerognosis , 51. Thirognosis , in whicrognosis , 31. Ultin situ hybridization , MET amplification and blood-based markers.Defining the patient population most likely to benefit from HGF/MET-targeted therapy is imperative. Multiple biomarker platforms are currently being investigated for this purpose in GEC, the most advanced being IHC. While preliminary data from patient subsets in randomized Phase II trials have shown that high MET protein expression as measured by IHC can differentiate patients who may benefit from MET pathway blockade, the success of MET IHC as a predictive biomarker has not been universal in all tumor types . SusceptShould anti-MET therapy prove to be clinically effective, an understanding of the toxicity implications of MET antagonism will be essential. To date HGF/MET-targeted agents, in particular monoclonal antibodies appear to be relatively well tolerated. Peripheral edema has been associated with treatment with all monoclonal antibodies targeting HGF or MET in combination with various cytotoxic and targeted therapies across multiple tumor types . A recenThe complexity and diversity of HGF/MET signal transduction coupled with the high degree of interplay between MET and other membrane receptors, as well as its role in resistance suggest that combination treatment approaches will be most fruitful.In the context of GEC, combinability of HGF/MET-targeted agents with chemotherapy will be imperative. Both RILOMET-1 and MetGastric are utilizing different platinum-based cytotoxic combinations (ECX vs. mFOLFOX6) and it remains to be seen if the difference in chemotherapy backbone will impact the outcome of the respective trials. Preclinically, aberrant MET signaling has been implicated in resistance to both cisplatin and oxaliplatin , 80. TheA wealth of data indicates a high degree of co-expression and cross-talk with respect to MET and HER family members, which include HER1/EGFR, HER2 and HER3. Amplification or overexpression of HER2 occurs in 10 – 15% of gastroesophageal adenocarcinomas and HER2-targeted therapy (trastuzumab) is approved for use in advanced GEC . While Hin vitro and in vivo and is though to be a key regulator of the angiogenic switch [in vitro and in vivo through activation of common signaling intermediates. Collectively, these data could support combination trials of HGF/MET-directed therapy with anti-angiogenic agents. The results of ongoing trials of MET-targeted drugs with anti-VEGF therapy in other tumor types will shed light on the usefulness of this approach [Anti-angiogenic therapy is likely to become a standard-of-care treatment for relapsed mGEC based on results of a Phase III trial of ramucirumab, a VEGFR2-targeted monoclonal antibody . HGF/METc switch . HGF/METapproach , 84.In vitro mechanisms of resistance to MET-targeted agents include mutation in the MET activation loop, compensatory upregulation of HER kinase signaling as well as amplification of MET and KRAS [Should HGF- or MET-directed therapy prove to be an effective treatment option for patients with GEC, experience with other RTK inhibitors suggests that resistance will invariably develop even in the subset of cancers that initially derive clinical benefit . In vitrand KRAS -87. In tIt is presently unclear to what extent targeting MET or HGF might impact the efficacy of HGF/MET axis inhibition. The results of RILOMET-1 and MetGastric will provide some clinical insight into this issue should the results of the respective trials be divergent. Of interest, a randomized trial of ficlatuzumab  in combination with an EGFR inhibitor in NSCLC failed to show efficacy in patients with high MET expression Mok et al . HoweverAlthough GEC is a global disease, it is not uniform. There are marked differences in patient demographics, treatment practices and treatment outcomes in GEC patients in different countries and regions . It is nTargeting the HGF/Met axis has significant clinical potential in GEC, which will hopefully be realized in the context of well-conducted clinical trials, the development and clinical validation of pathway biomarkers and rational mechanism-based treatment combinations.


By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone;  We demonstrate that this new toxin is similar to GhoT of the type V GhoST TA system, but it lacks an antitoxin and is not regulated by GhoS antitoxin; hence, it is an orphan toxin. We also determine that OrtT functions as a toxin by damaging the cell membrane and reducing intracellular ATP level and that OrtT has a distinct physiological role, which is to reduce cellular growth and metabolism in response to the stringent response induced by depletion of THF.Along with the consistent high specificity of nearly all toxins for their adjacent antitoxins, all but one of the characterized TA systems consists of two-components with the exception of the three-component system ω-ε-ζ of pyogenes . Here, wortT caused severe growth inhibition (TG (Met) to ACG (Thr) to generate pCA24N-ortTACG. The wild-type strain harboring pCA24N-ortTACG with inactivated protein OrtT was able to grow with full induction (OrtT is the same size (57 aa) and shares 63% protein identity with toxin GhoT A. Hence,hibition B,C; hencnduction B. These ortT. As expected, persistence increased 430 fold with ampicillin treatment and 27 fold with ciprofloxacin treatment relative to cells with an empty vector  . To inveortT lyses cells and found that production of OrtT in wild-type cells caused 99.99% of the bacterial population to undergo cellular lysis within two hours after induction  with wild-type cells producing toxin OrtT and compared them to wild-type cells with an empty vector. We found there was a dramatic change in cell morphology due to OrtT production; the two most common phenomena were (i) nucleoid condensation and enlargement of the periplasmic space; and (ii) cellular component leakage leading to almost a hollow cytoplasmic space  were constructed in a way that the toxin and putative antitoxin genes are under the control of the same promoter. Note that such co-expression of toxins and antitoxins from the same promoter has been used for different TA systems such as MqsRA . Nonetheless, we tested for RNA interactions by cloning the upstream and downstream intergenic region between ortT and neighboring genes yncL and ydcY, respectively, to construct pCA24N-ortT-UTR . Therefore, no antitoxin partner for OrtT was identified from these four sets of experiments which confirm that OrtT is an orphan toxin.To verify that an antitoxin for OrtT is not coded elsewhere within the genome, we performed an extensive search for potential antitoxins by constructing a genomic library by partially digesting BW25113 genomic DNA and cloning the DNA fragments downstream of i.e., ∆ortT ∆Kan) had low metabolism like wild-type cells whereas cells lacking GhoT  showed high metabolism under this stressful condition. Note that only strains which lack the antibiotic resistance marker were used so that the only difference between the strains is deletion of the toxin gene.Because both OrtT and GhoT are toxins that corrupt the membrane, we investigated if these two toxins serve a similar role in cell physiology. GhoT decreases cellular metabolism in the presence of carbenicillin and cefoxitin helping cells withstand this kind of membrane stress . Hence, ∆ortT has a negative fitness score for trimethoprim (TMP) [∆ortT ∆Kan mutant had dramatically higher metabolism compared to wild-type strain in response to 1 µg/mL TMP  . TMP is im (TMP) . THF plaim (TMP) . In addiim (TMP) . We foung/mL TMP B, indicape cells B. These ∆ortT ∆Kan mutant showed higher metabolism compared to the wild-type host in response to 60 µg/mL SMZ . SMZ is often used along with TMP for their synergistic effect against bacteria since SMZ inhibits dihydropteroate synthetase, whereas TMP inhibits DHFR  Figure . As expeg/mL SMZ C which cortT ∆Kan cells with or without TMP treatment. As expected, the mutant lacking ortT showed higher growth than that of the wild-type cells in the presence of TMP . Four different conditions were used to induce the stringent response: TMP [ortT expression was induced  [E. coli. OrtT is a small, hydrophobic peptide that shares 63% protein identity with GhoT, and we have demonstrated that: (i) OrtT strongly inhibits growth and causes cell lysis; (ii) removal of the start codon encoding ortT restores growth proving OrtT is active as a protein; (iii) OrtT damages the cell membrane and depletes cellular ATP, (iv) the F38R substitution reduces OrtT toxicity; (v) unlike GhoT, OrtT is most likely an orphan toxin as it is neither encoded by a bicistronic operon nor under in trans regulation by an antitoxin encoded elsewhere in the genome; (vi) OrtT reduces cellular metabolism in the presence of antimicrobials like TMP and SMZ; and (vii) ortT is activated under stringent conditions; so, its role in cell physiology appears to be to reduce growth during nutritional stress.In our previous study, the physiological role of type V toxin GhoT was ascertained; GhoT causes membrane damage by reducing cellular ATP level and reduces metabolism in the presence of various antimicrobials related to cell-wall synthesis (foxitin) . In the i.e., F38R) in OrtT that allowed us previously to create a non-toxic version of GhoT [ortT did not have the same impact on metabolism in presence of carbenicillin as ghoT. In addition, ortT was advantageous for the cells in response to a completely different set of antibiotics, TMP and SMZ, related to THF synthesis whereas the ghoT mutation was unrecognizable under the same conditions. Hence, OrtT serves a different physiological role than GhoT.Based on the structural homology between GhoT and OrtT, we introduced the same mutation .Furthermore, ge sites  which artes GhoT . Unlike tes GhoT . Collectike GhoT ; howeverortT is predicted to be under RpoE (σE) and RpoH (σH) regulation, and our results confirm this prediction. RpoE is the stress response sigma factor for extracytoplasmic stress [ortT expression via activation of RpoH and RpoE. OrtT in turn damages cellular membrane and contributes to reduce the intracellular ATP due to PMF disruption or via direct leakage of intracellular ATP. In this manner, when THF is depleted, the stringent response is activated to shut down growth via accumulation of ppGpp and OrtT  was used to maintain the pCA24N-based plasmids [The bacterial strains and plasmids used in this study are listed in ng pCP20  and the  primers  and by splasmids .ydcY and ghoS were PCR-amplified from E. coli K12 BW25113 chromosomal DNA using primer pair ydcY-SalI-F/ydcY-HindIII-R and ghoS-SalI-F/ghoS-HindIII-R respectively. The forward primers were designed to include the synthetic RBS from pCA24N plasmid [ortT-UTR plasmid was constructed by cloning the intervening DNA flanking ortT from the BW25113 chromosome into pCA24N-ortT. To do so, the 217 bp upstream intergenic region (between yncL and ortT), an inactivated ortT (174 bp), and the 381 bp downstream intergenic region including ydcY  via site-directed mutagenesis using the primer pair ortT-ACG-chrom-F/R  that was used in the growth experiment . PCR products were purified using a Promega PCR product purification kit . The purified PCR product and the vector pCA24N-ortT were digested with SalI and HindIII restriction enzymes  and then ligated using T4 DNA ligase. The ligation mixture was transformed into BW25113 competent cells and the resulted plasmids were verified by sequencing.The putative antitoxin genes,  plasmid  (Table 3periment B had onlBamHI (to cut the vector) and Sau3AI (to cut the genomic DNA) restriction enzymes  that produce compatible overhangs. Since there are two BamHI sites in the ASKA plasmid pCA24N-ortT , the vector was modified by deleting the first BamHI site in the pCA24N-ortT plasmid via site-directed mutagenesis with the BamHI-F/R primer pair (ortT-B (with a single BamHI site) was digested with BamHI and dephosphorylated with rSAP phosphatase  to prevent self-ligation. BW25113 genomic DNA was partially digested with Sau3AI and ~2–4 Kb genome fragments were gel-purified and ligated into the linearized vector using T4 DNA ligase. The ligation mixture was transformed into BW25113 competent cells. The library construction was verified by sequencing three random clones , which had inserts mapping to different places in the genome . The average insert size of the library was 622 bp found by digesting the plasmids obtained from 25 random clones with SapI (cuts near the origin of the plasmids). The library was then screened by growing the clones with 1 mM IPTG to induce ortT expression. Surviving clones (~0.25% of the library) that grew in presence of IPTG were analyzed by digesting their plasmids with AflII which cuts within the ortT coding region; none of the plasmids were cut by AflII whereas the plasmids could be digested with SapI. Moreover, the linearized plasmids were shorter compared to the plasmid that contains ortT (pCA24N-ortT-B) which indicates the toxin gene was missing in the survivor plasmids. Approximately, 21,314 clones were screened in this experiment; hence, the genome coverage was 2.9 times .The genome library was constructed by using mer pair . The resortT  or 0.1 mM IPTG (for growth assays with F38R mutants) and turbidity at 600 nm was recorded every hour for monitoring cell growth. For performing streak toxicity tests, overnight cultures were streaked on LB plates supplemented with chloramphenicol and with or without IPTG (same as liquid growth test) and incubated overnight.2 to 107 via 10-fold serial dilution steps in 0.85% NaCl solution and 10 µL of diluted cell suspension was plated on LB agar plate with chloramphenicol for enumerating colony forming unit (CFU) per mL [To determine whether OrtT overexpression causes cell lysis, overnight cultures were refreshed in fresh LB medium with chloramphenicol to an initial turbidity at 600 nm of 0.05, cells were grown to turbidity of 0.5, then induced with 2 mM IPTG (time zero). Cell sample was collected every 30 min by harvesting 1 mL culture for assessing cell viability. Harvested cell pellets were washed and diluted by 10) per mL .ortT and BW25113/pCA24N-(-gfp) were refreshed to a turbidity at 600 nm of 0.05, grown in LB-chloramphenicol to a turbidity at 600 nm of 0.2, and then 1 mM of IPTG was added to induce OrtT production for 4 h. Cells were harvested and fixed with primary fixative  at room temperature for 15 min, then 4 °C for 12 h. Cell pellets were then washed three times with buffer  for 5 min. Secondary fixation was carried by fixing the cell pellets with 1% osmium tetroxide in buffer for 1 h in dark, followed by three washes with buffer and one wash with sterile MilliQ water. Cells were then subjected to en bloc staining by treating with 2% uranyl acetate in the dark for 1 h. Next, dehydration step was performed by applying a graded ethanol series , and 100% acetone . The dehydrated cells were then embedded into epoxy resins after three epoxy exchange for at least 12 h at 60 °C. The hardened cell samples were then sectioned into thin specimens (70 nm thick) using an ultramicrotome . Positive staining was performed by staining the specimens with uranyl acetate for 6 min and lead citrate for another 6 min. The stained specimens were then examined on a FEI Tecnai G2 Spirit BioTwin TEM  at an accelerating voltage of 120 kV.TEM was performed for overexpressing OrtT as described previously . For preThe ATP assay was conducted as described previously  using thThe persister assay was conducted as described . Brieflyi.e., carbenicillin, TMP, and SMZ), or solvent only (water or DMSO). The metabolic activity (NADH production) was monitored by taking hourly measurement of the absorbance at 590 nm which indicates the intracellular reducing state based on the generation of formazane (purple) from the tetrazolium dye. For performing the growth assay under these conditions, the BioLog Redox Dye was replaced with water and growth was monitored by measuring the absorbance at 600 nm.Metabolic activities in response to antimicrobials were assayed using reagents from Biolog, Inc.  following BioLog’s procedure . Brieflyi.e., 75 µg/mL TMP, 100 µg/mL MUP, 1 mg/mL SHX or 1 mM IPTG for relA induction) was added when the cells reached the exponential phase (turbidity at 600 nm of 0.2~0.4), and the cells were induced for 2 h before isolating RNA. For TMP, MUP, and SHX treatment, BW25113 was used. For RelA production, BW25113 carrying pCA24N-relA  using 100 ng of total RNA as the template. Primers (−∆∆CT formula [gyrA as the internal control for normalizing all data.Total RNA was isolated as described previously  using Qi24N-relA  or empty Primers  were ann formula  with gyrConditions for tricine-SDS-PAGE and the Western blot analysis are described in the Supplementary Methods.ortT. Further research is required to determine how OrtT is inactivated after stress; however, our results create a new simpler paradigm for reducing cellular metabolism during stress using an orphan toxin.Although previous studies have predicted independent occurrences of toxins ,68,69, d