Immunoglobulin G4-related disease (IgG4-RD) and systemic sclerosis (SSc) are rare autoimmune diseases characterized by the presence of CD4+ cytotoxic T cells in the blood as well as inflammation and fibrosis in various organs, but they have no established etiologies. Similar to other autoimmune diseases, the gut microbiome might encode disease-triggering or disease-sustaining factors.The gut microbiomes from IgG4-RD and SSc patients as well as healthy individuals with no recent antibiotic treatment were studied by metagenomic sequencing of stool DNA. De novo assembly-based taxonomic and functional characterization, followed by association and accessory gene set enrichment analysis, were applied to describe microbiome changes associated with both diseases.Clostridium and typically oral Streptococcus species were significantly overabundant, while Alistipes, Bacteroides, and butyrate-producing species were depleted in the two diseases compared to healthy controls. Accessory gene content analysis in these species revealed an enrichment of Th17-activating Eggerthella lenta strains in IgG4-RD and SSc and a preferential colonization of a homocysteine-producing strain of Clostridium bolteae in SSc. Overabundance of the classical mevalonate pathway, hydroxyproline dehydratase, and fibronectin-binding protein in disease microbiomes reflects potential functional differences in host immune recognition and extracellular matrix utilization associated with fibrosis. Strikingly, the majority of species that were differentially abundant in IgG4-RD and SSc compared to controls showed the same directionality in both diseases. Compared with multiple sclerosis and rheumatoid arthritis, the gut microbiomes of IgG4-RD and SSc showed similar signatures; in contrast, the most differentially abundant taxa were not the facultative anaerobes consistently identified in inflammatory bowel diseases, suggesting the microbial signatures of IgG4-RD and SSc do not result from mucosal inflammation and decreased anaerobism.Microbiomes of IgG4-RD and SSc patients distinctly separated from those of healthy controls: numerous opportunistic pathogenic These results provide an initial characterization of gut microbiome ecology in fibrosis-prone IgG4-RD and SSc and reveal microbial functions that offer insights into the pathophysiology of these rare diseases.The online version contains supplementary material available at 10.1186/s13073-021-00853-7. Ruminococcus gnavus, Prevotella copri) that may act as sources of disease-triggering or disease-sustaining molecules and antigens.The gut microbiome exists in an essential symbiosis with its human host by serving as a source of nutrients and small molecules, informing the development and activity of the immune system and providing colonization resistance against pathogens. Strains of gut bacteria stimulate the expansion of various immune cell populations  and provMicrobial antigens are recognized by the immune system via presentation on major histocompatibility complex (MHC) class II molecules to CD4+ T cells. Recently, an unusual subset of cytotoxic CD4+ T cells has been described in patients with two rare, fibrosis-prone autoimmune disorders: immunoglobulin G4-related disease (IgG4-RD) and systemic sclerosis (SSc) –5. IgG4-The immunological characteristics of IgG4-RD and SSc overlap, where CD4+ T cells—including the unusual subpopulation of IFN-γ, IL-1β, and TGF-β secreting cytotoxic CD4+ T cells (CTLs)—play a key role in disease pathogenesis . AccordiEggerthella lenta enrichment in RA) but not IBD. We also identified overabundance of microbiome pathways related to nutrition (ethanolamine utilization) and fibrosis (hydroxyproline utilization and fibronectin binding). Finally, strain-level analysis showed preferential colonization of autoimmune patients by Clostridium bolteae encoding a strain-specific cystine uptake and metabolism locus as well as by a proinflammatory strain of E. lenta.Given the pivotal role of the immune system in the pathogenesis of IgG4-RD and SSc, it is important to identify triggers of inflammatory responses and the potential contribution from the human gut microbiome. To our knowledge, there have been no reports on the microbiome in IgG4-RD, and only a few 16S studies in SSc described microbiome changes . Here weN = 58) and systemic sclerosis  were recruited at Massachusetts General Hospital (MGH) and the University of Michigan, respectively. The IgG4-RD cohort included patients in remission (N = 45) and with active disease (N = 13) assessed using the IgG4-RD responder index [N = 39), diffuse cutaneous SSc , sine scleroderma , and overlap scleroderma . Available patient information included age, sex, and current medication. As controls, healthy, non-medicated individuals (N = 165) were recruited during screening visits at MGH. For the purpose of this study, we only collected stool samples from the study participants. Human patient research in the IgG4-RD cohort was reviewed and approved by the Partners Human Research Committee (2008P002154). Human patient research in the SSc cohort, who meet the 2013 classification criteria, was approved by the Institutional Review Board of the University of Michigan Medical School (HUM00101836). Human patient research in the healthy control cohort was reviewed and approved by the Partners Human Research Committee (2015P000275). The study was approved by the Office of Research Subject Protection at the Broad Institute of MIT and Harvard. All experiments adhered to the regulations of these review boards. Study procedures were performed in compliance with all relevant ethical and federal regulations. This research conformed to the principles of the Helsinki Declaration. All study participants gave their written informed consent for sample collection and to participate in the study.Patients with immunoglobulin G4-related disease .Stool samples were collected by study participants at home using self-collection kits in 100% ethanol and stored at room temperature for less than 48 h prior to dissection and long-term storage at − 80 °C, as previously described . To extrThe quality control for the metagenomic data was conducted using Trim Galore! to detect and remove sequencing adapters (minimum overlap of 5 bp) and kneadData v0.7.2 to remove human DNA contamination and trim low-quality sequences , retaining reads that were at least 50 bp long. Metagenomic reads were assembled individually for each sample into contigs using MEGAHIT , followeR package vegan). The significance of differences in alpha diversity between disease and control cohorts was determined using linear fixed effects modeling with covariates . Alpha diversity differences in IgG4-RD patients due to the disease activity and stratified by SSc subgroups were also evaluated using linear fixed effects modeling with age, sex, and treatment information as covariates.Alpha diversities were calculated using Shannon and beta diversity was calculated using Bray-Curtis dissimilarity based on relative abundances at species and MSP levels  in the adonis function’s model formula, was performed prior to PERMANOVA analysis. Each covariate was individually analyzed for its association to the microbial dataset and then ordered based on the effect size  from the most significant to the least. The pairwise distances between the species were determined using the Bray-Curtis dissimilarity measure and 1000 permutations were performed per analysis. Other parameters of the analysis were kept as default.The permutational multivariate analysis of variance (PERMANOVA) analysis was performed on the species-level data to identify correlation between the fixed effect covariates  and the composition of the gut microbiome as a whole. The PERMANOVA implementation in the E. lenta’s cgr locus [cgr locus gene homologs as a signal for abundance of the cgr locus. It is often difficult to disentangle the absence of a gene or operon in a bacterial genome from a missing observation due to an insufficient depth of sequencing. Yet the absence of a gene can readily be assessed when the counts of the core genes are proportional to the counts of the accessory genes, as identified by MSPminer [cgr genes can be reliably detected only in samples where the coverage of the E. lenta genome is sufficient. We used the proportionality between the cgr locus counts and the counts of the top 30 top representative core genes reported by MSPminer for E. lenta to derive the minimal number of reads mapping to E. lenta core genes that would allow the observation of at least 1 read mapping to the cgr locus. In that way, the metagenomic samples with less than 16 reads mapping to E. lenta core genes were discarded from the analysis. Odds ratios and significance of enrichment of the cgr locus in disease compared to controls were determined using a one-sided Fisher’s test. We note here that in the read mapping procedure, after the BWA step, we filter alignments to only retain uniquely mapped reads at 95% or greater level of nucleotide identity along the read length. This alleviates the risk of recruiting reads originating from identical or highly homologous regions in other genes.We used USEARCH to identify, in the gene catalog, homologs of the 7 genes specific to gr locus , and detMSPminer . AccordiP values were adjusted for multiple hypothesis testing using Benjamini-Hochberg correction.For the microbiome-wide search for enriched accessory modules, we used a similar approach. A threshold of at least 1 mapped read was used to analyze the presence and absence of accessory modules reported by MSPminer and associated with specific MSPs; a minimal number of reads mapping to the top 30 representative core genes reported by MSPminer for a given MSP was similarly derived based on the proportionality rule to determine samples with enough signal for that MSP to be included in the analysis [lm function from stats-package in R, was performed to identify differentially abundant features  between cohorts, SSc subgroups, and individuals with active and inactive IgG4-RD status. Prior to linear modeling, features present in less than 20% of the samples were filtered out. In analyses involving multiple cohorts, the less prevalent species (< 20% samples) that are specifically absent in either the control or disease cohorts  were included in downstream analyses. Furthermore, the zeros were replaced by half of the smallest non-zero measurement on a per-feature basis and log10 transformation was applied on the relative abundances for normality. In analyses studying differences between the cohorts , linear modeling included fixed effect covariates: age, sex, cohort information and treatment information. Variable “treatment information” represented 6 different treatment categories: no treatment, rituximab (RTX), prednisone, other medication, RTX with prednisone and prednisone with other medication. Moreover, in analyses within a particular cohort, such as between individuals with the active and inactive IgG4-RD status and the SSc subgroups, covariates related to the IgG4-RD status and SSc subgroups were added to the respective models, while the cohort information was excluded. Comparison between the SSc subgroups and the control cohort was performed by expanding the cohort information with the four SSc subgroup classifications (i.e. classifying the samples belonging to the SSc category in the cohort-covariate into their respective subgroups). Nominal P values from the lm output were adjusted for multiple testing using Benjamini-Hochberg correction and associations at FDR < 0.05 (unless stated differently) were considered as significant.Linear fixed effects modeling, as implemented in the N = 58) and SSc (N = 90), respectively recruited at Massachusetts General Hospital and University of Michigan (Table N = 39), diffuse cutaneous SSc (N = 39), sine scleroderma (N = 7), and overlap scleroderma (N = 5). Individuals in both disease cohorts had a similar age distribution (mean age ~ 60 years old) and approximately half were on treatment with immunosuppressive agents, such as prednisone and rituximab . A few individuals received a combination of drugs, such as RTX and prednisone or prednisone and another form of treatment   and de Comparison of the Shannon diversity index revealed that IgG4-RD had a decreased alpha diversity compared to healthy controls Fig. b. Using −3) with a concordant increase of Firmicutes in SSc (FDR = 0.06) and Actinobacteria in IgG4-RD (FDR = 0.15) relative to controls . Among the Firmicutes, numerous opportunistic pathogenic Clostridium species were overabundant . These Clostridia are well-adapted to invade host tissue by encoding pathogenicity factors, such as antibiotic resistance genes and flagellins, and have been identified as extraintestinal infectious agents [Veillonella and five Streptococcus species. Colonization of the lower parts of the gastrointestinal (GI) tract by oral microbes is a recognized phenomenon in systemic diseases such as RA [Faecalibacterium species from group IV Clostridium  that are known for their beneficial butyrate production were depleted in the two diseases. Lastly, an additional 67 MSPs that were differentially abundant in only one disease showed a consistent trend of overabundance or depletion in the other disease . E. lenta is also overabundant in the gut microbiome of RA and multiple sclerosis patients [E. lenta might be playing an important role in multiple autoimmune diseases. Apart from the species with known taxonomy, we observed 36 differentially abundant MSPs with no species-level information , diffuse cutaneous SSc (dcSSc), sine scleroderma (ssSSc), and overlap scleroderma (osSSc) to healthy controls. We observed 16 MSPs that were commonly differentially abundant between at least two SSc subgroups; most notably, overabundance of three K. pneumoniae show variable potential in activating type 1 T helper (Th) cells, highlighting the importance of accessory genes on modulating immune function [E. lenta, which was one of the top differentially abundant species in our comparison  operon [cgr locus has been attributed immunomodulatory function: cgr locus positive (cgr+) E. lenta drive activation of Th17 cells and production of pro-inflammatory cytokines, while cgr− strains do not show this phenotype [cgr+ E. lenta strains in SSc and IgG4-RD patients, we identified five genes in the assembled gene catalog that comprise the cgr locus and evaluated their abundances in each sample. Using that signal as the marker for presence or absence of the cgr locus, in samples with sufficient coverage of E. lenta . These observations implicate E. lenta as a potential microbiome factor with strain-dependent enzymatic activity that might lead to the breakdown of immune homeostasis and expansion of its population in colonized subjects.A species-level focus in human microbiome studies can lack the specificity necessary for attributing functional roles, as different strains of the same species can harbor vastly different accessory genomes. These genomic differences can be particularly relevant in the context of immune system activation. For instance, different strains of function . With thson Fig. c. E. len) operon . The cgrhenotype . To detesee the “” sectionpulation . E. lentClostridium scindens, contained multiple proteins involved in nutrient transport across membranes . In SSc-enriched acc 058 from C. bolteae, five of its six genes co-assembled next to each other on the same contig in our samples; two additional genes were predicted in the contig using Glimmer [C. bolteae reference genomes and represent a variation present only in some strains of this species , which is consistent with the ability of C. bolteae to bloom with cysteine as a sole carbon source [Our assembly-based microbiome characterization through MSPs grouped genes into clusters based on their presence and co-abundance pattern such that each species is represented by its core genes and often multiple clusters of different accessory genes. In order to perform a microbiome-wide screen for disease-related accessory modules, we evaluated whether any specific accessory module was differentially distributed in IgG4-RD and SSc compared to healthy controls. We detected 53 accessory modules (acc) that were enriched or depleted in SSc  that is encoded by specific microbial families [In addition to strain-specific functional potential, multiple species can contribute to similar functional capabilities. To investigate this in an untargeted manner, we summarized the relative abundance of microbial pathways using HUMAnN2 . Consistfamilies . This ovfamilies .Fig. 4FR. gnavus, Blautia producta, or C. clostridioforme  to be overabundant in SSc and IgG4-RD compared to healthy controls  in the assembled gene catalog and detected 17 homologs with at least 62% amino acid identity [Finally, we specifically focused on relative abundance changes in functions related to extracellular matrix (ECM) binding and fibrosis. Using eggNOG-Mapper , we detefb1 K1373 to be ovidentity . The comfb1 K1373 to be ovHere we characterized a strikingly similar gut microbiome architecture between two fibrotic autoimmune diseases: IgG4-RD and SSc. Consistent sampling, data generation, and processing allowed us to avoid technical biases and identify biologically relevant features common between the disorders. The gut microbiomes in both diseases were significantly different from healthy controls and showed a depletion of typical, health-associated commensals and expansion of potentially pathogenic and pro-inflammatory species.Bacteroides and Faecalibacterium species as well as an overabundance of Bifidobacterium dentium and several Lactobacillus species. We additionally identified different Clostridium and Streptococcus species to be overabundant in SSc compared to healthy controls, a signature that indicates either fewer GI symptoms in our cohort or a cohort-specific signal [Our analysis greatly expanded the number of samples analyzed and added species-level and functional depth to a previous 16S rRNA-based report on the SSc microbiome . Consistc signal , 37. In c signal . We detec signal  and glucc signal . Given acgr+ E. lenta strains, a clade that increasingly is being attributed pro-inflammatory functions [C. bolteae encoding a homocysteine metabolism locus. SSc is associated with vasculopathy that may be promoted by elevated levels of homocysteine in circulation [sfb1) and an enzyme that potentially allows for energy extraction from ECM (HypD homologs). These enrichments might enhance the availability of ECM to specific microbial species for attachment and additional nutrient sources [By employing a de novo assembly approach to study IgG4-RD and SSc patient microbiomes, we pinpointed changes to the as-yet uncultured constituents of the human gut and performed strain analysis focused on the variation in accessory gene content. This revealed an enrichment in IgG4-RD and SSc of unctions . We furtculation , which h sources , 59.cgr+ E. lenta [cgr+ E. lenta, the levels of circulating Th17 cells are elevated in patients with IgG4-RD and SSc [Several functional signatures link the microbiomes of IgG4-RD and SSc patients to inflammation. The classical mevalonate pathway that leads to IPP synthesis was increased in both diseases. IPP and other metabolites from this pathway are recognized by ɣδ T cells as a part of surveillance against microbial infections ; differeE. lenta . Th17 ceE. lenta . Consist and SSc , 67. Whi and SSc –70.Such disease-relevant taxonomical and functional characteristics encourage further study into the identification of specific microbiome-derived molecules that can drive autoimmune pathologies , such asE. lenta and taxa typical of the oral cavity that are similar to autoimmune diseases including the neuroimmune disorder multiple sclerosis and RA, a disease with multiple joint pathologies [S. parasanguinis, B. producta, Lactobacillus gasseri, and R. gnavus) as well as common signals with 11 out of 42 species depleted in IBD [Escherichia coli to be overabundant, albeit weakly, in SSs but not IgG4-RD. This pathobiont has been associated with mucosal inflammation and decreased gut anaerobism in IBD [The shared microbiome signature in IgG4-RD and SSc raises a question about the existence of a more universal microbiome architecture in immune-mediated disorders. We observed expansions of hologies , 44, 73.d in IBD . We obsem in IBD . Gut fibm in IBD , 56 but E. lenta that encodes a cgr locus, indicating a potential microbiome-driven skewing of the immune cell population in the context of these rare autoimmune diseases. We also found that microbiome changes in IgG4-RD and SSc partially recount observations from other autoimmune diseases and that IgG4-RD- and SSc-specific effects likely further shape the landscape of the associated gut microbiomes.Our characterization of the gut microbiome in fibrosis-prone IgG4-RD and SSc revealed taxonomic and functional gut microbiome features that are common in these two diseases, including reduction of health-associated commensals and expansion of potentially pathogenic and pro-inflammatory species. IgG4-RD and SSc patients showed expansion of a Th17-inducing strain of Additional file 1. Supplementary Tables.Additional file 2. Supplementary Figures.


At Children’s Hospital and Medical Center in Omaha, Nebraska, the intraoperative antibiotic redosing guidelines and the time frame considered compliant for redosing were unclear. This lack of clarity plus an ill-defined process for ensuring intraoperative antibiotic redosing resulted in a compliance rate of 11%. The organization’s surgical site infection (SSI) rate was 3.19%, above the national benchmark of 1.87%. The primary project goal was to increase intraoperative antibiotic redosing compliance. The secondary project goal was to decrease SSIs.With recommendations from the Infectious Disease Society of America, we developed new organizational redosing guidelines, as well as a new antibiotic-specific reminder alert in the electronic medical record. Implementation of the new guidelines and processes occurred after providing education to the anesthesiologists, surgeons, and circulating nurses. Monthly evaluation of data allowed for quick recognition of oversights followed by the initiation of process updates.Data showed that the initial compliance rate for the intraoperative redosing of antibiotics was 11%. Following interventions, compliance has reached and sustained an average of 99%. Survey results show that provider knowledge of the guidelines and process has improved. Though not directly related, the National Surgical Quality Improvement Program observed that the SSI rate decreased from 3.19% in 2014 to 2.3% in 2018.This project demonstrates that comprehensive education along with antibiotic-specific electronic medical record alerts significantly increased the compliance of intraoperative antibiotic redosing at Children’s Hospital & Medical Center. Continuous education and monthly updates sustained results for over 40 months. Approximately 40% to 60% of SSIs are preventable.7 Correctly administered perioperative prophylactic antibiotics decrease the rate of SSIs.2 Clinical practice guidelines for antimicrobial prophylaxis published in the American Journal of Health-System Pharmacy recommend intraoperative antibiotic dosing intervals during surgical procedures based on the pharmacokinetics of each drug.8 In the cardiac surgical patient population, correct intraoperative antibiotic redosing has decreased SSI rates.3Surgical site infections (SSIs) are infections of the incision or organ space that occur after surgery.At the Children’s Hospital & Medical Center in Omaha, Nebraska, the SSI rate was higher than the national benchmark based on data from the National Surgical Quality Improvement Program (NSQIP) database. NSQIP uses a systematic sampling process to ensure a representative sample of surgical cases. It excludes cardiac, ophthalmology, interventional radiology, dental procedures, and endoscopies. The hospital contributes to the NSQIP database entering 13% of cases annually. In 2014, Children’s SSI rate for all cases was 3.19%, whereas the benchmark was 1.88%. The NSQIP database identified 42 SSIs. Therefore, based on the hospital’s total surgical volume, approximately 315 SSIs occurred in 2014.9 SSIs are the fourth largest contributor to harm reported across the SPS network.9 SPS designed a bundle to reduce harm from SSIs to hospitalized children.9 Bundle elements include preoperative baths, no razor use, appropriate preoperative antibiotic timing, appropriate skin antisepsis, and appropriate antibiotic redosing  project was to increase intraoperative antibiotic redosing compliance from 11.2% to 100% for qualifying cases. As referenced, the time frame for this project aligned with our hospital’s focus on improving patient safety and decreasing hospital-acquired conditions. Therefore, a secondary project goal was to decrease SSIs.10We identified a need to establish consistent intraoperative antibiotic redosing guidelines and define the exact time frame, in minutes, considered compliant for intraoperative antibiotic redosing. Additionally, a well-defined process for ordering intraoperative antibiotics was necessary. QI projects are exempt from institutional review board application and approval. This project utilized the Plan Do Check Act (PDCA) methodology Table , thoroug12 This work outlined procedures and drug-specific timeframes for intraoperative antibiotic redosing. To be deemed compliant with the redosing, antibiotics needed to be administered 30 minutes before or after the electronic medical record (EMR) redosing due time, determined by the antibiotic administration start time. If a procedure was longer than 2 half-lives, the drug was to be readministered. Renal function did not play a specific role in the creation of the guidelines; however, the pharmacy looks at the creatinine clearance if available on each patient before fulfilling a requested redose.Our project team consisted of 2 anesthesiologists, a surgeon, an operating room (OR) nurse, a pharmacist, and an information technology (IT) specialist. In May 2015, we gathered baseline data. Concurrently, monthly meetings were held with the SSI workgroup to review current processes. The SSI workgroup consisted of a surgeon from each subspecialty, a pharmacist from the Antimicrobial Stewardship Program, members from the Performance Improvement Department, an IT specialist, OR nursing managers, and 2 anesthesiologists. At these meetings, research into existing intraoperative antibiotic redosing guidelines and policies took place. Most committee members, including the anesthesiologists, surgeons, and OR nurses, were unaware of formal antibiotic redosing guidelines. However, we discovered that hospital guidelines did exist via a paper ordering sheet. In June 2015, members of the project team worked with members of the hospital’s Antimicrobial Stewardship Program to establish perioperative antimicrobial guidelines utilizing information from the Infectious Disease Society of America and information compiled from hospitals that are part of the Children’s Hospital Association Network Table .8,11,12 With new guidelines established, the project team collected baseline data from all qualifying intraoperative cases. This analysis excluded antibiotics given outside of the OR. Initial data analysis was calculated using the antibiotic-specific redosing time frames from the preexisting guidelines. These guidelines required antibiotic redoses to be given within a 5-minute window on either side of the due time. If a case required more than 1 redose, all required redoses needed to be administered correctly, or the case was considered noncompliant. As stated, most physicians were unaware of these guidelines. In June 2015, we assessed surgeon and anesthesia provider knowledge using a survey. Of the 48 who received the survey, 20 responded for a response rate of 41.7%. Only 41% of responders felt that they had a clear understanding of the redosing guidelines. The lack of clarity is evident by the average 2014 compliance rate of 11.2%  and in non-OR locations . To account for this, we updated the EMR antibiotic reminder to alert 30 minutes before the time due, allowing staff more time to order and physically receive the antibiotics. Project leaders discussed these obstacles at group meetings and encouraged anesthesia providers to order the antibiotics early in these circumstances. The pharmacy representative on the team was also made aware of this problem to provide education to the pharmacy staff. Providers received updates when new PDCA cycles or updates in the process occurred.Following the project initiation, monthly evaluation of EMR data occurred. The Performance Improvement Department conducted an initial data review with potential cases of noncompliance sent to the anesthesiologists on the project team. The review of these noncompliant cases involved peer-to-peer conversations and revisions in the process followed by further education if required Table . AnestheThe framework for the composition of this article utilized the Standards for QUality Improvement Reporting Excellence (SQUIRE) guidelines.Following project implementation, compliance with intraoperative antibiotic redosing increased from the 2014 average of 11.2% to 89% within the first month. Analysis of August data started on the implementation date of August 10. At the end of the second month, compliance rates had increased to 100% (Fig. In April 2018, anesthesia providers were resurveyed using the same set of questions. Seventy-five percent of anesthesia providers who responded had a clear understanding of the frequency at which commonly used antibiotics should be redosed intraoperatively according to the hospital guidelines, a 33% improvement. Based on new survey results, anesthesia provider knowledge regarding the correct redosing timeframe considered complaint was also 75%, an improvement of 50% from project initiation.The antibiotic redosing team and hospital’s SSI workgroup worked well together. The SSI workgroup’s goal was to institute the bundle modeled off of the SPS bundle to decrease the rate of SSIs Table . In JanuThis QI initiative defined evidence-based intraoperative antibiotic redosing guidelines and the timeframe considered compliant for antibiotic redosing. It utilized an antibiotic-specific EMR alert to remind anesthesia providers when an antibiotic was due. Throughout the project, monthly evaluation of data allowed for quick recognition of oversights or problems followed by the initiation of process updates. Although not all PDCA cycles or revisions resulted in noticeable change, they did ensure that high compliance rates were sustained.7 One university-associated hospital incorporated EMR reminders as part of a multifaceted approach to improving intraoperative antibiotic redosing. Their compliance rates improved by 15% to approximately 80%.2 Another study looking only at the use of EMR alerts to improve the administration of the second antibiotic dose found an absolute improvement of 17.1%, with approximately 70% compliance rate.7 This study showed sustained compliance rates 7 months later. Our compliance rates increased the first month significantly (Fig. The use of EMR alerts as part of single or multifaceted improvement initiatives has been reported in other studies.Additionally, providers involved in noncompliance would get an individual email as a reminder to maintain vigilance. By evaluating all possible cases of noncompliance for accuracy and any notes made by the anesthesia providers, we were able to implement changes to ensure sustainability. Combining extensive education with the EMR antibiotic-specific reminders highlights the strengths of this project.Although timely notification in the EMR is likely the most important factor in achieving compliance, several other factors likely contributed. The new guidelines were widely disseminated and accessible, whereas the previous guidelines were not. Additionally, the new guidelines were updated to reflect the definition of compliance used throughout the rest of the institution, which states that redoses should be administered within a 30-minute window on either side of the due time. Analysis of baseline data using the preexisting guidelines required a stricter 5-minute redosing window to be compliant.One limitation of the project is that antibiotic ordering still requires diligence on the part of the OR team and, ultimately, the anesthesia providers. Upon initial development of the reordering process, the project team wanted the EMR to alert the OR pharmacy when an antibiotic redose was near due; however, limitations in the EMR made this impossible, and we were unable to identify an alternative solution for pharmacy involvement in the reordering process. Per hospital policy, physicians order all antibiotics from the pharmacy to ensure administration of the correct dose, which can take additional time. And, as with any QI project, communication is imperative. Some providers remained unaware of the changes implemented with this project. The education initially excluded residents and new providers, as we identified with monthly audits. An additional limitation is that OR nurses do not always write the antibiotic redosing time on the whiteboard despite this being part of the time-out process. We have not initiated any PDCA cycles to improve this, but it is something we could consider in the future. Finally, we did not have a control group without intervention because the project included all cases meeting requirements for redosing by nature of the QI initiative.Due to the shortened redosing interval of some antibiotics, there is a concern for acute kidney injury, so in September 2015, the team worked on updating the guidelines to address renal dosing. To our knowledge, there have not been any cases of acute kidney injury reported.Improved compliance with intraoperative antibiotic redosing was one component of the bundle initiative instituted by the hospital’s SSI workgroup. We cannot delineate the impact of the correct antibiotic redosing on the overall decrease in the SSIs. Although the antibiotic redosing portion of the SSI bundle has remained consistent, data on the compliance rate of all portions of the bundle are not available due to inconsistent record keeping.3 and this QI project demonstrates that comprehensive education along with antibiotic-specific EMR alerts significantly increased the compliance of intraoperative antibiotic redosing. Through effort by many individuals, the changes have now become standard practice. Utilizing continual education and monthly updates, we have sustained results for over 40 months.Although we cannot draw a clear correlation between the SSI rate and our improved intraoperative compliance redosing, correct intraoperative antibiotic redosing has been shown to decrease SSIs,The authors have no financial interest to declare in relation to the content of this article.The authors would like to acknowledge the Children’s Hospital & Medical Center Performance Improvement Department, IT Department, Pharmacy, and Antimicrobial Stewardship Program for their contributions and assistance throughout the project.


Overall antibiotic consumption decreased from 16.01 to 13.31 DID (−16.85%). Consumption of amoxicillin/clavulanic acid and quinolones decreased from 6.04 to 4.72 DID (−21.88%) and 1.64 to 1.23 DID (−25.06%), respectively. The percentage of patients treated with antibiotics decreased from 26.99 to 22.41%. The intervention resulted in cost savings of €72,673. Use of antibiotics to treat pharyngotonsillitis, UTI, and acute otitis media, sinusitis, and bronchitis decreased significantly. Our antimicrobial stewardship program led to a decrease in antibiotic consumption and significantly improved the use of antibiotics for the most prevalent PHC infections.The aim of the study was to evaluate the impact of a multifaceted antimicrobial stewardship intervention on antibiotic consumption in a primary health care (PHC) area in Spain. Quasi-experimental study conducted in a PHC area with nine PHC centers, a 400-bed acute care teaching hospital, and 18 nursing homes serving a population of 260,561. The intervention was based on the 2016 CDC Core Elements of Outpatient Antibiotic Stewardship publication and targeted 130 PHC physicians, 41 PHC pediatricians, 19 emergency physicians, and 18 nursing home physicians. The components were commitment, actions for improving antibiotic prescribing, tracking and feedback, and education and experience. The primary outcome was overall antibiotic consumption. Secondary outcomes were consumption of antibiotics to treat pharyngotonsillitis, acute otitis media, acute sinusitis, acute bronchitis, and urinary tract infection (UTI), percentage of patients treated with specific antibiotics, and dispensing costs. Consumption was measured in defined daily doses per 1,000 inhabitants per day (DID) and compared pre- and postintervention (2016  Antimicrobial resistance is growing and is now recognized as a major global health threat . It is rSpain is one of the highest consumers of antibiotics in the developed world. Total antibiotic consumption in 2016 was 32.9 defined daily doses per 1,000 inhabitants per (DID) . compareEscherichia coli resistance to third-generation cephalosporins increased from 0.6 to 15.0% in Spain and from 0.6 to 6.4% in the Netherlands  publication on outpatient antibiotic stewardship describes four core elements for assessing and improving antibiotic prescribing in outpatient settings: 1) commitment, 2) action for policy and practice, 3) tracking and reporting, 4) and education and expertise . In 2014We performed a quasi-experimental study to evaluate the program. Data were collected in three phases: a baseline phase (January–December 2016), an intervention phase (January–December 2017), and a sustainability phase (January–December 2018).The setting for the intervention was a PHC area in the province of Barcelona that serves a population of 260,657 inhabitants and has nine PHC centers, a 400-bed acute care teaching hospital managed by the same health organization, together with 18 nursing homes. The Spanish national health care system offers universal coverage to a population of 46.4 million inhabitants (2016) . The couThe ASP targeted 130 PHC physicians, 41 PHC pediatricians, 19 emergency physicians, and 18 nursing home physicians. It also included an educational campaign targeting patients. All physicians and patients were included, no sampling was made.All components of the ASP were implemented simultaneously by the PHC antimicrobial stewardship team set up in 2016 to work in coordination with the hospital-based team formed 3 years earlier. The PHC team is composed of 10 PHC physicians, four PHC pediatricians, three clinical pharmacists, an infectious disease specialist, and a microbiologist.The components of the intervention, all evidence-based and detailed below, were selected to align with the CDC’s four core elements of outpatient antibiotic stewardship .Commitment to the ASP. Thirty minute face-to-face sessions were organized to present the ASP to all relevant stakeholders in the PHC area . Twenty-five sessions were held to ensure 100% coverage and 150 poster reminders were distributed among the physicians’ offices and the emergency areas of the participating PHC centers. In case that a new physician was hired during the intervention or sustainability phase the AS Team presented the project and provided the educational material through a face-to-face meeting. A member of the antimicrobial stewardship team visited each of the nursing homes to explain the intervention to the physicians working there and provide them with educational material on good antibiotic prescribing practices. All care providers were asked to commit to the ASP but were not required to sign any documents to this effect. No financial incentives were offered.Actions for improving antibiotic prescribing. All PHC centers were supplied with a rapid test for detecting Streptococcus pyogenes and an accompanying protocol based on the Centor clinical scoring scale (modified McIsaac score) to ensure correct use of the test  and 85% participated in at least one session. All physicians received the workshop material by email. Over 600 project-related emails were sent. Finally, purpose-designed educational leaflets and posters (n = 150) on safe antibiotic use targeting patients were placed in waiting rooms and common areas.Antibiotic consumption was measured in DID day calculated with drug packages in all cases. The primary outcome was overall antibiotic consumption. Secondary outcomes were i) consumption of amoxicillin (ATC: J01CA04), amoxicillin/clavulanic acid (ATC: J01CR02), macrolides , and quinolones  (the main antibiotics prescribed in PHC); ii) consumption of narrow-spectrum antibiotics ; Antibiotics with less than 0.00 DID was considered negligible. iii) percentage of patients treated with antibiotics for common PHC conditions ; iv) direct costs associated with antibiotic consumption; v) unique patients prescribed at least one course of antibiotics; and vi) antibiotic consumption in nursing homes measured as defined daily doses per 100 bed days (DDBs). Indicators were measured at baseline and during the intervention and sustainability phases.Data were extracted from the patients’ electronic medical records throughout the study period. Patient contacts were traced by checking records for ICD-9 codes for relevant infections and ATC codes for antibiotic prescriptions. In order to calculate the economic indicator, the reference prices for drugs stablished by the Ministry of Health, Consumer Affairs and Social Welfare of Spain were applied .vs. sustainability phase/2018). Proportions and SDs were used for the descriptive statistics. Proportions of categorical variables in two or more independent groups were compared using the Chi-square test. P-values ≤ 0.05 were considered statistically significant. When required, statistical trend analyses were carried out to evaluate the significance of temporal variations. All endpoint definitions and statistical methods were predefined in a statistical analysis plan before evaluation of the data. Analyses were performed using Stata 13 .A descriptive analysis was performed for each outcome indicator using data collected before and after the intervention  age was 40.85 (22.81) years; 20.98% of the patients were pediatric (0–14 years old) and 16.07% were aged 65 years or older. The study population included 1,964 nursing home residents with a mean age of 79.99 (8.22) years. A mean of 181,863 unique-patient visits per year was registered over the study period.Overall antibiotic consumption decreased by 16.85% (16.01 to 13.31 DID) between 2016 (baseline) and 2018 (sustainability period). It decreased by 16.94% in the adult population (17.34 to 14.41 DID) and by 19.22% in the pediatric population (10.05 to 8.12 DID). The percentage of patients treated with antibiotics also decreased, from 26.99% in 2016 to 22.41% in 2018 . Overall antibiotic consumption decreased in both adults and children but the largest decrease (26.00%) was observed in nursing home residents (7.74 DDB in 2016 to 5.72 DDB in 2018).On analyzing consumption of specific antibiotics, significant reductions were observed for amoxicillin , amoxicillin/clavulanic acid , quinolones , and macrolides . Consumption of narrow-spectrum antibiotics increased from 31.32 to 32.35% . The overall results of the ASP are summarized in The percentage of adults diagnosed with pharyngotonsillitis and treated with antibiotics decreased significantly from 64.81% in 2016 to 54.38% in 2018 . The corresponding reduction for children was 12.68% . In both adult and pediatric patients, there was a significant increase in the use of amoxicillin to treat pharyngotonsillitis and a decrease in the use of amoxicillin/clavulanic acid and macrolides. Further information over qualitative data for the treatment of pharyngotonsillitis in the three periods of study is shown in The proportion of patients with acute otitis media treated with antibiotics also decreased: from 58.26 to 49.53%  in the case of adults and from 72.37 to 68.09%  in the case of children. As with pharyngotonsillitis, there was a significant increase in the use of amoxicillin and a decrease in that of amoxicillin/clavulanic acid and macrolides in the adult and pediatric populations. Further information over qualitative data for the treatment of acute otitis media in the three periods of study is shown in Nonsignificant changes were observed for the percentage of patients with urinary tract infections treated with antibiotics. Adults increased from 70.94 to 71.40% , and children decreased from 51.15 to 49.28% . In adults, the use of fosfomycin and cefuroxime increased from 39.58 to 45.36% and 26.76 to 29.93% while that of amoxicillin/clavulanic acid and quinolones decreased from 6.15 to 4.24% and 27.52 to 20.47%, respectively. Further information over qualitative data for the treatment of UTI in the three periods of study is shown in Finally, there was a significant decrease in the use of antibiotics to treat acute bronchitis in both adults  and children . In the case of acute sinusitis, antibiotic use fell significantly in adults  but increased, albeit insignificantly, in children . Further information over the percentage of patients treated with antibiotic with diagnosis of acute sinusitis and acute bronchitis is shown in Total spending on antibiotics fell from €905,700.76 during the baseline phase to €793,765.89 during the sustainability phase , reductions were observed in the total spending of Amoxicillin/Clavulanic  and Quinolones  in correlation with the decrease in DID. Economic data for the baseline, intervention, and sustainability phases are shown in The findings of this quasi-experimental study show that a multifaceted ASP reduced overall antibiotic consumption and costs in a PHC area. Our study supports previous reports that behavioral and educational interventions lead by pharmacists can improve antibiotic prescribing practices . In 2017The primary aim of our intervention was to reduce antibiotic consumption and we achieved an absolute reduction of 16.85% between 2016 and 2018. The corresponding reduction in antibiotic prescriptions in the Spanish national health care system was 4.99% , which iUse of narrow-spectrum antibiotics in our PHC area increased by 1.04%, which is just slightly higher than the increase of 0.44% reported for the Spanish public health care system for the same period . Use of Most antimicrobial stewardship interventions to date have targeted single conditions . In a syversus 60.4% of those in the Netherlands , the following licenses/restrictions apply Organic Law 15/1999, de 13 de December, Protection of personal Data.BOENum. 298, 14th December de 1999, pages 43088 a 43099. Requests to access these datasets should be directed to Servei Català de Salut , atenciociutadana@catsalut.cat.All authors contributed in the design of the antimicrobial stewardship interventions and in the development of local guidelines on antibiotic. PM-L, RM, and RT implemented the interventions in all primary health care centers. EC and LB acted as expert consultants on antibiotic use.The project received a grant within the “Mútua Terrassa Primary Care Research fellowship 2017 (BE0106)”. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. This study was conducted as a quality program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses.Coatings containing gentamicin at a concentration of 1.25% weight/volume (wt/vol), similar to that found in commercially available antibiotic-loaded bone cement, were prepared and tested in the laboratory for: kinetics of antibiotic release; activity against planktonic and biofilm bacterial cultures; biocompatibility with cultured mammalian cells; and physical bonding to the material . The sol-gel coatings and controls were then tested in vivo in a small animal healing model , and applied to the surface of commercially pure HA-coated titanium rods.The coating released gentamicin at > 10 × minimum inhibitory concentration (MIC) for sensitive staphylococcal strains within one hour thereby potentially giving effective prophylaxis for arthroplasty surgery, and showed > 99% elution of the antibiotic within the coating after 48 hours. There was total eradication of both planktonic bacteria and established bacterial biofilms of a panel of clinically relevant staphylococci. Mesenchymal stem cells adhered to the coated surfaces and differentiated towards osteoblasts, depositing calcium and expressing the bone marker protein, osteopontin. In the in vivo small animal bone healing model, the antibiotic sol-gel coated titanium (Ti)/HA rod led to osseointegration equivalent to that of the conventional HA-coated surface., bone healing in an animal model is not impaired. This, thus, represents a biomaterial modification that may have the potential to protect at-risk patients from implant-related deep infection.In this study we report a new sol-gel technology that can release gentamicin from a bioceramic-coated cementless arthroplasty material. In vitro, local gentamicin levels are in excess of what can be achieved by antibiotic-loaded bone cement. In vivoBone Joint J 2021;103-B(3):522–529.Cite this article:  While prophylactic antibiotics are used to reduce the risk of PJI during primary surgery, once an infection becomes established on the prosthesis, antibiotics alone will not cure a PJI. This is because a biofilm has now become established and the only solution is radical surgery with appropriate antibiotic therapy. If, however, microorganisms entering the wound can be neutralized before a biofilm becomes established, the incidence of infection could be further reduced in both primary and revision surgery.The incidence of periprosthetic joint infection (PJI) is between 1% and 3% after primary arthroplasty and up to 15% after revision procedures.2 local antibiotic delivery is not therefore available for cementless arthroplasty. In order to provide a local antibiotic delivery system for cementless components, we developed a controlled-release antibiotic hybrid organic-inorganic sol-gel coating system for application onto a cementless implant material that is already coated with growth-promoting hydroxyapatite (HA).The local delivery of antibiotic provides higher concentrations of antibiotic at the site of infection than can be achieved by systemic therapy. While this delivery can be provided using antibiotic-loaded bone cement, it can obviously only be used with cemented components. While there is now an established trend for the use of cementless components at both primary and revision surgery,5 It required a multilayer application and had limited opportunities for the tuning of its properties, since it only contained inorganic silica precursors. This study, using a rat Staphylococcus aureus osteomyelitis model, demonstrated antimicrobial activity in vivo when vancomycin was in the sol-gel.5Sol-gel systems are a group of materials which are prepared as a liquid sol that undergoes curing to produce a cross-linked matrix, which can be used as a coating. A range of these systems have been explored for biomedical applications, although there has been only one previous system tested as a drug-delivery coating for orthopaedic prostheses. This previous antibiotic-loaded sol-gel coating contained vancomycin and was tested on a titanium (Ti) alloy.7 We have added the growth factor, bone morphogenetic protein-2 (BMP-2),9 to the coating to investigate any potential enhancement of bone-healing properties.We have developed a hybrid sol-gel process  to prepare thin silica-based films with controlled pore size, hydrophobicity, and toughness for delivery of an antibiotic from the surface of the implant. The broad-spectrum antibiotic, gentamicin, was used as the antimicrobial agent as it is the antibiotic which is most frequently used in bone cement today. It is active against both Gram-negative and Gram-positive bacteria, which are responsible for the majority of PJIs. The coating was developed to provide the release of gentamicin during the time taken to undertake a routine primary hip or knee arthroplasty, typically between one and two hours. Furthermore, it was predicted that a rapid antibiotic-releasing coating of a few micrometres in thickness would avoid long-term low levels of antibiotic elution. Such low levels have been observed with bone cement, and have been implicated in the development of resistant microorganisms during prolonged infections.-1 to 2 µg ml-1, respectively. Where necessary, bone cement containing 1.25% (w/v) of gentamicin was used as a control material. Elution kinetics of gentamicin and BMP-2 were monitored analytically in vitro from coated glass samples. Antimicrobial activity of eluted gentamicin was confirmed against key pathogens in minimum inhibitory concentration (MIC) assays, as well as assays of biofilm formation and eradication. In vitro cytocompatibility testing and phenotypic assessment were performed using human mesenchymal stem cells (MSCs). In vivo biocompatibility analysis of the coatings on HA-coated Ti was performed in a rat healing model system. Full details of the methods can be found in the Supplementary Material, which also includes a completed ARRIVE checklist to show that we adhered to the ARRIVE guidelines.Sol-gel coatings were prepared and cured at room temperature on 20 mm × 20 mm glass coverslips (for in vitro characterization) or HA-coated Ti cylinders . Where stated, gentamicin and BMP-2 were incorporated into the coatings at 1.25% weight/volume (w/v) and 0.5 µg mlStatistical analyses were achieved using SPSS statistics software v23  and GraphPad Prism 6 software . For pull-out test analysis, the three coating types were compared using the Kruskal–Wallis test for non-parametric data. Statistical significance was set at p < 0.05.-2 (SD 0.296) after 48 hours, representing > 99% of gentamicin contained within the coating. The mean amount of gentamicin eluted from bone cement reached a maximum of 0.775 µg mm-2 (SD 0.159) after 48 hours, representing only 4.98% of the original gentamicin contained within the cement , respectively) monitored over a 78-hour period. Elution was monitored into ammonium acetate buffer and into Dulbecco's Modified Eagle Medium (DMEM) cell culture medium . The mean gentamicin concentrations eluted from the sol-gel into the ammonium acetate buffer reached a maximum of 1.557 µg mme cement . Similare cement . The coa-1) were coated onto HA/Ti squares and eluted into DMEM cell culture media, and the eluted BMP-2 concentration was measured by enzyme-linked immunosorbent assay (ELISA). After one week, concentrations ranged from 180 pg ml-1 to 256 pg ml-1, corresponding to only 0.2% to 0.7% of the BMP-2 eluting from the coating (data not shown).Sol-gel preparations containing gentamicin (1.25% w/v) and BMP-2  were assessed by a tensile pull-out test . All but two of nine samples that were tested snapped before pulling out of the bone cement (150 N to 220 N load). It should be noted that the cement into which the coated samples were embedded in these tests was antibiotic-free and used solely as a means to apply force to the outside of the coatings; there was no intention that the coatings should be embedded in cement during surgery. The samples that pulled out (~150 N) were not coated with sol-gel and failed at the interface between HA and Ti wire. There was no significant difference between the pull-out strengths of the samples .The in vitro adhesive properties of the sol-gel on HA-coated Ti, HA-Ti wire coated with sol-gel + gentamicin (1.25% w/v), and HA-Ti wire coated with sol-gel + gentamicin (1.25% w/v) + BMP2 -irradiated prior to in vivo implantation. Samples were examined by scanning electron microscopy (SEM) before and after γ-irradiation to verify that the coating was still intact and that no delamination had occurred at the rod ends due to cutting. No delamination could be seen at the rod ends , and cryRat MSCs seeded onto glass coverslips coated with sol-gel adhered and spread onto the surface of all sol-gel surfaces, regardless of the agents which were incorporated. Clear filamentous actin staining was seen, demonstrating cell spreading on the surface of sol-gel coated surfaces . AlizariWhen the implants were tested in the rat healing model, no clinical signs of infection were observed on any implant and all were immobile, suggesting that osseointegration had occurred. Micro-CT (µCT) analysis demonstrated that implants coated with sol-gel had comparable new bone volume to that of HA-coated Ti controls . Vital bThese results show that sol-gel coating has suitable properties to provide controlled local gentamicin prophylaxis for cementless orthopaedic prostheses, with bone healing properties that are comparable to an HA-coated surface currently in use. We found that the coating releases gentamicin in a controlled manner within a desirable time frame, with > 99% of the total antibiotic eluted within 48 hours. This time frame is compatible with effective prophylaxis during the peri- and postoperative period, to kill microorganisms resulting from surgical contamination and exclude the possibility of long-term elution.When added, BMP-2 was retained within the coating and did not substantially elute. However, in vitro studies showed that BMP-2 was not required within the sol-gel coating to promote differentiation of MSCs, or required for effective in vivo bone healing.11 This is a resorbable hydrogel, which can incorporate different antimicrobials and is applied by syringe during the operation. In vitro analysis of this coating has demonstrated elution of 99% after 96 hours of various antibiotics including vancomycin, gentamicin, and N-acetylcysteine. There was a reduction but not total eradication of S. aureus and S. epidermidis established biofilms in vitro over a 48-hour period.12 Clinical trials have shown that this system can reduce the rate of early surgical site infections and has not shown any adverse side effects.11 The physical nature of the hydrogel is somewhat different from the sol-gel coating that has been developed in this study. The more physically robust and thinner sol-gel may be less likely to be removed from the surface during insertion into bone. The DAC system does, however, enable tailoring of local antibiotic delivery to individual patients who undergo a two-stage revision procedure, and in a recent case-controlled study the results with respect to the control of infection were better in the coated group.13 This is something we plan to evaluate in due course, along with a potential use of the coating in single-stage revision surgery for PJI. The high concentration of eluted local antibiotic may well be very efficacious in this surgical scenario, in which high concentrations of antibiotic are essential for the success of single-stage surgery for PJI.Currently there are few options for local antibiotic delivery for cementless arthroplasty. A Defensive Antibacterial Coating (DAC) has been developed and trialled for use in hip and knee arthroplasties.14 A similar silver-based coating, Modular Universal Tumour And Revision System  uses galvanic deposition to provide a coating of elemental silver.15 This technology reduced infection in a rabbit model. Clinical studies have also shown a reduction in infection rates compared with non-coated implants. In practice, these coatings have mainly been applied to tumour megaprostheses for use in major skeletal reconstruction and not for routine orthopaedic implants. A recent animal test model of a silver-containing HA coating found that it significantly reduced the burden of methicillin-resistant Staphylococcus aureus (MRSA) infection, and that this effect was enhanced by introducing a vancomycin solution at the surgical site.16 A question still remains about the cytotoxicity of silver-based products, particularly long term.17A silver-based coating, Agluna, has been developed by Accentus Medical , and uses Ti nanotubes containing silver ions to provide an antimicrobial coating for metal implants. Retrospective analysis has shown that rates of infection were reduced using this technology, particularly in two-stage revision procedures where antibiotic-loaded cement was also used.S. aureus over 24 hours. In vivo elution studies showed similar results, although trace levels of gentamicin were detected after six weeks.18 However, this application is currently limited to the surgical treatment of tibial fractures and the technology has not been expanded for use with arthroplasties.19 A gentamicin-loaded ultra-high molecular weight polyethylene material enables local delivery of antibiotic, although its use is limited to spacers used in two-stage revision surgery.20Synthes GmbH  produce the Expert tibial nail PROtect, a tibial nail with a gentamicin coating that has shown promising results in preventing surgical site infections. This system uses a biodegradable polylactic acid coating for the release of gentamicin to provide antibiotic prophylaxis. Elution of 75% of gentamicin from the coating over 42 days was demonstrated in vitro, with a significant reduction in adhesion of 5 The single-layer hybrid sol-gel technology described in this paper targets a wider range of Gram-positive and Gram-negative organisms by using gentamicin. It is easy to apply and has the potential to adjust its hydrophobicity for elution of a wide range of therapeutic molecules, including antibiotics. Moreover, its antimicrobial and biocompatibility properties were demonstrated when it was applied to HA-coated implant material of the type widely used for promoting bone growth on uncemented prostheses. In vivo bone attachment was shown to be comparable to the HA-coated Ti surface of a type currently used in orthopaedic prostheses. In future, we propose to investigate the suitability of the coating for delivering a wide range of antibacterial and antifungal drugs, including combinations of antimicrobials, and application of the coating onto a range of materials used in orthopaedic surgery. We also propose to investigate a range of antibiotic loading levels and perform further in vivo testing, in particular to establish whether the coating can be modified quickly to kill biofilms as well as planktonic bacteria. In this way, it may be possible to design coatings which can protect against a wider range of organisms, facilitating a stratified or patient-specific approach.The sol-gels which have previously been shown to have potential as controlled release antimicrobial coatings used vancomycin, which provides no protection against infection caused by Gram-negative organisms.Take home message- There is a need for new systems to provide local antimicrobial prophylaxis for uncemented orthopaedic prostheses.- Here we report a single-layer gentamicin-containing hybrid organic-inorganic coating capable of controlled release of active antibiotic.- The coating is fully biocompatible with bone healing in a small animal in vivo system, suggesting promise for development as a coating for orthopaedic implants.


The technological process of concrete production consists of several parts, including concrete mix design, concrete mix production, transportation of fresh concrete mix to a construction site, placement in concrete framework, and curing. Proper execution of these steps provides good quality concrete. Some factors can disturb the technological process, mainly temperature and excessive precipitation. Changing daily temperature and rainfall during fabrication, transportation, and placement can shape not only the properties of the concrete mix but also the compressive strength of hardened concrete. In this paper, we tried to answer the question of how temperature and precipitation affect concrete production. The scope of this study was to determine the change of compressive strength of the hardened concrete in a specific period for selected concrete mix recipes, taking into account changing daily temperature and precipitation magnitude. The investigated concrete mixes concrete compressive strength beyond that of the concrete grade, termed “concrete superstrength”. This concrete post limiting behaviour of concrete is also discussed. One of the most widespread and accessible construction materials used in civil engineering is concrete. The significant uses of concrete are dams, residential and commercial buildings, roads and driveways, marine constructions, culverts and sewers, foundations, concrete bridges, fences, and many more. Many standards specify basic requirements for the design of structural concrete, such as the Eurocodes in Europe  was faster, and there were some differences for different replacement materials. The hydration of belite was slowed down at higher temperatures by the pulverised fuel ash admixture, but augmented by volcanic ash. The last study [One of the first studies of concrete produced in different environmental conditions is the work of W.H. Price from 1951 . W.H. PrJ. Komonen et al.  studied In our research, we aimed to investigate the impacts of changing temperature and precipitation during the production and transport stages on concrete compressive strength. The parameters we followed were the mean daily compressive strength of concrete, the composition and features of the concrete mix, the average daily air temperature, and the daily rainfall. The problem of excessive compressive strength beyond that of the concrete grade is also discussed. The concrete that we tested was both produced and transported to a construction site located in Gdańsk, Poland.ck,cube) for C30/37 concrete grade is 37 MPa (N/mm2) and for C50/60 concrete grade is 60 MPa (N/mm2). The characteristic compressive strength is specified at 28 days on concrete cubical samples with dimensions equal to 150 mm . There is also the minimum characteristic cylindrical strength , which for C30/37 concrete grade is 30 MPa (N/mm2), and for C50/60 concrete grade is 50 MPa (N/mm2). We adopted an S3 fresh concrete consistency class (with a slump from 100 to 150 mm) and a maximum size of aggregate 16 mm according to EN 206 [3 to 2397 kg/m3  directly before concrete placement (on average about 15 min after delivery) from January 2016 to October 2016; see  12390-2  standard 12390-2 .Concrete superstrength is excessive compressive strength beyond that designed for the concrete grade. In the EN 206 standard  and otheULCS=1.25ce range .The excessive compressive strength beyond that of the concrete grade may have negative outcomes for some types of structural elements. Based on this concept, it is possible to evaluate the phenomenon of the concrete superstrength that occurred in the investigated concrete mixes.We took meteorological data from the Gdansk Rebiechowo station (number code: 254180090) operated by the Polish Institute of Meteorology and Water Management—National Research Institute  in PolanIn Fi is a maximum load at failure, Aci is a cross-sectional area of the specimen, and n is the number of samples (n = 3 or 4). The daily mean compressive strength of concrete was calculated for each investigated concrete mix type used on that day on the building site; see The uniaxial compressive experimental laboratory tests were performed using a standard computer-operated testing machine (Advantest 9 C300KN) on standard cubical concrete samples with dimensions of 15 × 15 × 15 cm. In accordance with EN 12390-3 , samplesIn In The mean strength of non-overlapping consecutive results Each test result and concrete with production control certification:mean of three or four results for a single-family member Conformity of compressive strength was assessed on specimens with full concrete compressive strength (after 28 days) following EN 206-1 standard  for concThe second criterion satisfied each test results for all tested specimens. In For the CM_A concrete mix, about 69% in criterion 1 and 51% in criterion 3 test results can be assigned to C30/37 concrete grade. Nearly 30% in criterion 1 and 49% in criterion 3 of the daily mean compressive strength values of concrete can be assigned to higher concrete grade, and only 1.4% in criterion 1 did not achieve the designed compressive strength. In the case of CM_Aw concrete mix, only 2.5% in criterion 1 of test results can be directly assigned to C30/37 concrete grade. The vast majority in criterion 1 (97.5%) and all in criterion 3 of the daily mean compressive strength values were classified as higher concrete grades. There were similarities in the case of CM_B concrete mix samples, where we also observed that a significant number (86% in criterion 1 and 92% in criterion 3) of daily mean compressive strength readings exceeding the range of concrete grade expected.In the results of our study, we observed the influence of average daily temperature on mean compressive strength a. For thMost of our results from concrete mix type CM_A  and The concrete mixes that we examined also showed excessive compressive strength beyond the designed concrete grade. That can raise a fundamental problem. Material that has a compressive strength higher than the designed one also has different stiffness, which is not beneficial from a structural point of view. Excessive stiffness of some parts of structural elements may cause a different redistribution of internal forces, so that elements with higher stiffness may exhibit greater than assumed stress and deformation. Uncontrolled cracks may appear at the joints of structural elements, which are supposed to be uniform in terms of strength and made of concrete with specific material properties. It would be valuable to explore this topic more by merging analytical finite element method modelling of buildings or civil engineering structures and varying material features within individual elements of the construction, supported by according laboratory tests. Comparison of the static-strength analysis, backed by experimental data and actual design state would allow us to estimate the impact of the high variability of concrete mechanical properties within the structure. We plan to undertake this topic in our future research.The conformity criterion for the specification of concrete grade only determines requirements for minimal compressive strength. In the European Union, the current norm for ready mixed concrete is ΕΝ 206 , which iThe results we obtained encourage us to continue the research and might be a base for numerous new investigations. In our future research, we plan to expand the database of measured parameters through new laboratory tests, especially those related to cement hydration and heat of hydration changes. We also plan to supplement our study with numerical analyses and field measurements of a built concrete structure to estimate an influence of material variability expressed by excessive compressive strength beyond the designed concrete grade. We hope that our findings spark a vital interest in the community of scientists and civil engineers to take into consideration the influences of environmental conditions during the manufacture of concrete on its mechanical behaviour.


Co-delivery of POSS and anthracyclines led to higher anti-cancer activity towards HeLa  and MCF-7 (human breast adenocarcinoma) cell lines. The obtained supramolecular hybrid complexes were characterised by nuclear magnetic resonance (NMR) spectroscopy (nuclear Overhauser effect spectroscopy [NOESY] and homonuclear correlation spectroscopy [COSY]), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). The two-dimensional (2D) NOESY spectra of the complexes showed the cross-correlation peaks for hydroxyl groups of POSS (~4.3–4.8 ppm) with OH groups of DOX and DAU. FTIR showed that hydroxyl group of POSS can interact with amine and hydroxyl groups of DOX and DAU. The viability of HeLa and MCF-7 was analysed with the MTT assay to evaluate the cytotoxicity of free DOX and DAU and the relevant complexes with POSS at different molar ratios. At a low DOX concentration (2.5 µM), for molar ratios 1:1, 1:4, and 1:8 (POSS:DOX), the complexes showed two and three times higher cytotoxicity towards HeLa and MCF-7 cells, respectively, than DOX itself after both 24- and 48-h incubation. The 1 µM concentration for a 1:4 POSS:DOX molecular ratio and the 2.5 µM concentration for all complexes were more toxic towards MCF-7 cells than free DOX after 48-h incubation. In the case of POSS:DAU complexes, there was higher toxicity than that of free drug after 48-h incubation. It can be concluded that the formation of non-covalent complexes increases toxicity of anthracycline drugs towards Hela and MCF-7 cells. The novel complexes are inexpensive to prepare and more effective than free drugs at low systemic toxicity. A novel strategy, recently developed by us, to use polyhedral oligomeric silsesquioxanes (POSS) as an anti-cancer drug carrier is presented. Anthracycline:POSS complexes were prepared by simple co-addition of doxorubicin (DOX) or daunorubicin (DAU) with hydrophilic POSS(OH) These findings pointed to increased transport effectiveness of DOX to the cells by means of octa(3-aminopropyl)silsesquioxane as the complexing nanocarrier [32 (The anthracycline antibiotics doxorubicin (DOX) and daunorubicin (DAU) are anti-cancer drugs that are highly effective in the clinical treatment of a wide variety of tumours. However, administration of anthracyclines can be limited by the occurrence of undesirable side-effects such as cardiotoxicity and inherent drug resistance. Drug delivery via nanocarriers offers an important approach that can improve anti-tumour activity while at the same time decreasing toxicity of anti-cancer drugs towards normal cells . Drugs, ocarrier . Similarocarrier . Thus, srier [32 . The POS32, was carried out according to the procedure previously described [1H, 13C, 29Si, 1H-13C Heteronuclear Single Quantum Coherence (HSQC) and 1H-1H Correlation Spectroscopy (COSY) NMR, Fourier Transform Infrared Spectroscopy (FTIR) and elemental analysis (see ESI†). Hydrochlorides of DOX and DAU were obtained from Sigma-Aldrich . Dimethyl-d6-sulfoxide , deuterium oxide  and phosphate-buffered saline  were used as supplied. Complexes of DOX and DAU with POSS(OH)32 were prepared by dissolving the anthracyclines in PBS (pH 7.4)/DMSO-d6 or D2O, followed by addition of POSS solution in relevant solvents. The molar ratio of DOX/DAU: POSS(OH)32 was 8:1. All the preparation steps were carried out in oven-dried glassware and under an argon atmosphere. All cell culture reagents were purchased from Gibco . Flasks and multi-well plates were obtained from Nunc . MTT  and DOX were supplied by Sigma-Aldrich, Poznan, Poland. Microvascular endothelial (HMEC-1), MCF-7 and HeLa cell lines were obtained from American Type Culture Collection .The synthesis of hydroxyl functionalised silsesquioxanes, referred to in the literature as POSS(OH)escribed . These s1H NMR spectra were recorded using a 500 MHz Bruker Avance III  instrument. Chemical shifts are reported in ppm downfield from TMS using DMSO-d6 and D2O as solvents (6. The complexes were prepared at room temperature with a 5 × 10−3 g·mL−1 DOX/DAU concentration in DMSO-d6. Size of fid (TD): 4096-512; spectral width (SW): 16.0214 ppm (8012.820 Hz); acquisition time (AQ): 0.2555904–0.0319488 s; number of scans: 16; TM1: 0.2-0.1, TM2: 0.7-0.9, delays D1:1 s 1H-1H COSY analyses were carried out in D2O. POSS:DOX and POSS:DAU complexes were prepared at room temperature with 2.22 × 10−2 g·mL−1 DOX and 2.20 × 10−2 g·mL−1 DAU, respectively, in D2O. Size of fid (TD): 4096-256; spectral width (SW): 10.9942 ppm (5498.534 Hz); acquisition time (AQ): 0.3724629–0.0465579 s; number of scans: 6; TM1: 0-0.1; TM2: 0-0.9; delays D1:1 s.Two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) solvents . The 2D −1 resolution  equipped with a deuterated triglycine sulfate detector and attenuated total reflectance (ATR) for 64 scans at a 2 cmsolution . Anthrac−1) were added to all cell culture media. The cells were grown in T-75 culture flasks at 310 K in an atmosphere containing 5% CO2. The cells were sub-cultured every 2–3 days. Cell viability was determined by the trypan blue exclusion assay with the use of a Countess Automated Cell Counter . Cells were seeded either in 96-well plates at 1.5 × 104 cells/well in 100 µL of an appropriate medium or in 12-well plates at 2.0 × 105 cells/well. After seeding, the plates were incubated for 24 h in a humidified atmosphere containing 5.0% CO2 at 310 K in order to allow cells to attach to the plates.HMEC-1 cells were grown in an MCDB131 medium supplemented with L-glutamine, hydrocortisone and epidermal growth factor. MCF-7 cells were grown in Minimum Essential Medium (MEM), while HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM). Ten per cent foetal bovine serum (FBS) and streptomycin (100 mg·mLHydrodynamic diameters of particles were measured in deionised water at 310 K by dynamic light scattering (DLS) using a Zeta Sizer Nano ZS . Hydrodynamic diameter measurements were performed for pure POSS (50 µM) and for complexes formed by POSS with DOX or DAU at different molar ratios  immediately after mixing the components and after 24-h incubation. For each sample, measurements were performed three times in three subsequent replications.−4 g·mL−1. Samples were prepared by placing a drop of an PBS solution of the anthracycline’s complexes on the carbon coated copper grid, and then the solvent was evaporated at room temperature.Transmission electron microscopy (TEM) was performed using a Talos F200X electron microscope  operating at 200 kV. Anthracycline complexes were prepared by mixing DOX/DAU with POSS in 8:1 ratio  to a final DOX/DAU concentration in PBS of 1 × 10−1 MTT solution in PBS was added to each well and cells were further incubated under normal culture conditions for 4 h. After incubation, the MTT solution was removed and the obtained formazan precipitate was dissolved in DMSO (100 µL/well). The conversion of the tetrazolium salt (MTT) to a coloured formazan by mitochondrial and cytosolic dehydrogenases was a marker of cell viability. Before the absorbance measurement, plates were shaken for 1 min, and the absorbance at 570 nm was measured with a PowerWave HT Microplate Spectrophotometer .The cytotoxicity of free drugs and their complexes with POSS at different molar ratios  was determined by the MTT assay  using 1 4 cells/well). Cells were incubated with the compounds for a specific period of time (5 min to 48 h) in a humidified atmosphere containing 5.0% CO2 at 310 K. After the appropriate incubation period, cells were washed with PBS, suspended in 500 µL of medium and immediately analysed with a Becton Dickinson LSR II flow cytometer  using a blue laser at 488 nm and phycoerythrin (PE) band pass filter  at 575/26 nm.In vitro uptake studies were carried out using fluorescent DOX or DAU and their complexes with POSS at different molar ratios . DOX/DAU (1 µM) and their complexes with POSS were added to 12-well plates containing cells (2.0 × 10p < 0.05 was considered significant.Data are expressed as mean standard deviation (SD). Analysis of variance (ANOVA) with post hoc test (Tukey) was used for multiple comparisons. Statistics were calculated using Statistica software v13.1 , and 4’ (~5.4 ppm) and OH14 (~4.9 ppm). There were analogous interactions in the 2D NOESY spectrum for the DAU:POSS complex. The protons of POSS hydroxyl groups interact with the OH4’ and OMe substituents in DAU. The nuclear Overhauser effect (NOE) is an especially useful tool to determine both the structure and dynamics of molecules. The observed NOE signals (using 2D NOESY NMR) for anthracycline:POSS complexes allowed us to determine which hydroxyl groups of POSS and protons of the anthracycline (DOX or DAU)  are enga1H-1H COSY analysis was run for DOX:POSS, DAU:POSS and POSS alone (2 (~1.57 ppm) and H3 (~2.65 ppm) towards higher δ values .The SS alone C,D to co−1 (νO-H); 3321 cm−1 ; 2974, 2954, 2932 and 2896 cm−1 , 1724 cm−1 ; 1616 , 1580 cm−1 ; 1525 cm−1 ; 1413 cm−1 ; 1283 cm−1 ; 1201 cm−1 ; 1234 cm−1  and 1205 cm−1 ; and 1191 cm−1  [−1 , 1723 cm−1 , 1234 cm−1 , 1283 cm−1  and 1201 cm−1  decreased. The FTIR spectrum of the DAU:POSS complex  and 1191 cm−1  decreased in the DAU:POSS complex.We used FTIR to study the structural characteristics of the DOX/DAU:POSS complexes in PBS (pH 7.4) after 24 h at 310 K . The FTI complex  in PBS d32 and anthracyclines, we employed DLS to measure the hydrodynamic diameter. The diameter of complexes was measured immediately after the addition of POSS to DOX or DAU and after 24 h. In the case of DOX, the initial diameter of the complexes was similar to that of POSS and did not change as the POSS:DOX molar ratio increased. However, after 24-h incubation at 310 K, the diameters increased. The highest values occurred for 1:16 molar ratio (~800 nm) (To further confirm the formation of complexes between POSS(OH)~800 nm) A. On the~800 nm) B. After We observed the interaction between DOX/DAU and POSS in PBS using TEM. As shown in Anthracyclines contain flat aromatic moieties that intercalate between DNA bases. DOX binds to DNA and topoisomerase II isoenzymes to form the ternary TopII–DOX–DNA complex, which leads to double-stranded DNA breakage . Even afDOX was more toxic after 48 h, but the use of POSS intensified the cytotoxic effect for all cell lines. The obtained results are consistent with the mechanism of drug action and the time of doubling applied cell lines. After intercalation of DOX in DNA in the cell’s nucleus, the cell must reach one of the checkpoints in the cell cycle, when the repair system directs the cell to programmed death: apoptosis . In HeLa32 with DAU at the molar ratios 1:4 and 1:12 and a 1.5 µM DAU concentration showed higher toxicity towards HeLa cells then free drug after 48 h. In the case of the MCF-7 cell line, the improved cytotoxic effect occurred after 24- and 48-h incubation for the 1:8 and 1:16 molar ratios in the 2.5–10 µM concentration range. Similar to DOX, there was a surprising lack of sensitivity towards POSS:DAU complexes by the non-cancer HMEC-1 cell line. The MTT test using DAU was repeated with the same conditions and analogous complexes. The results obtained from hydrodynamic diameter measurements are in good agreement with the results of cytotoxicity assessed with MTT. DOX formed complexes after 24-h incubation and, therefore, their greatest effectiveness occurred after 48 h. In comparison, DAU formed less stable complexes, the effect of which occurred after 24-h incubation. Using the autofluorescence of anthracyclines, we measured the fluorescence of the POSS:DOX and POSS:DAU complexes as a function of time with a flow cytometer in order to confirm the postulated mechanism of action. Moreover, as predicted, in the first few hours, there was an increase in the amount of DOX in both the cell lines, with the fastest penetrating complex having a 1:4 POSS:DOX molar ratio , because the signals from the OHAr functions are significantly broadened. We demonstrated that POSS can form non-covalent supramolecular complexes with anthracyclines. We showed the formation of such complexes by NOESY NMR analysis, which revealed cross-correlation peaks for POSS hydroxyl groups and OHAr groups with POSS. The decrease in the absorption intensity indicates the interaction of DOX/DAU with POSS via hydrogen bonding between the OH groups in the anthraquinone ring with the POSS OH moieties [1H-1H COSY NMR revealed chemical shift changes of the resonances originating from POSS protons (H2 and H3) towards higher δ values, supporting formation of POSS:DOX/DAU complexes in D2O.However, we obtained such information from FTIR analysis because it clearly indicates that there are interactions of anthracycline OHmoieties . Moreove32. On the contrary, the diameters of the POSS:DOX complex increased after 24-h or 3-h incubation; these data demonstrated that POSS:DOX complexes are much more stable. This phenomenon has a direct impact on the rate at which the complexes penetrate cancer cells. The more stable POSS:DOX complexes penetrated cells evenly over the first 18 h of incubation. In the case of POSS:DAU complexes, the highest cellular uptake occurred within the first hour of incubation and, interestingly, depended on the cell line. The transport rate was up to fourfold higher for HeLa than for MCF-7 cells. The cytotoxicity results confirmed that POSS facilitated the DOX/DAU transport; therefore, faster penetrating complexes are more toxic than free drugs. The observed differences between cell lines may also indicate a different POSS-mediated transport mechanism and action depending on the type of tumour. HeLa cells have very low p53 functional activity [The decrease in the diameter of the POSS:DAU complexes after 24-h (for DLS) or 3-h (for TEM) incubation may indicate that DAU forms much weaker complexes with POSS(OH)activity  due to cactivity  and may activity . Therefo32 to be another useful silsesquioxane [Our studies demonstrated that even small structural differences between anthracyclines have a significant impact on their ability to form complexes with POSS-type nanocarriers. Their distribution, cytotoxic activity, and mode of action depend on the cell line. Based on our observations of the effects of POSS on co-delivery with anthracyclines, we can conclude that POSS-type nanocarriers reduce the need for high concentrations of anti-cancer drugs. Therefore, the use of POSS to facilitate anthracycline drug delivery drug seems to be a promising approach in potential future anti-cancer therapy. The novel complexes are inexpensive to prepare, more effective than free drugs at low systemic toxicity. We found POSS(OH)quioxane  that inc