Outcomes from an inpatient beta-lactam allergy guideline across a large US health system.

OBJECTIVE: To assess the safety of, and subsequent allergy documentation associated with, an antimicrobial stewardship intervention consisting of test-dose challenge procedures prompted by an electronic guideline for hospitalized patients with reported ß-lactam allergies. DESIGN: Retrospective cohort study. SETTING: Large healthcare system consisting of 2 academic and 3 community acute-care hospitals between April 2016 and December 2017. METHODS: We evaluated ß-lactam antibiotic test-dose outcomes, including adverse drug reactions (ADRs), hypersensitivity reactions (HSRs), and electronic health record (EHR) allergy record updates. HSR predictors were examined using a multivariable logistic regression model. Modification of the EHR allergy record after test doses considered relevant allergy entries added, deleted, and/or specified. RESULTS: We identified 1,046 test-doses: 809 (77%) to cephalosporins, 148 (14%) to penicillins, and 89 (9%) to carbapenems. Overall, 78 patients (7.5%; 95% confidence interval [CI], 5.9%-9.2%) had signs or symptoms of an ADR, and 40 (3.8%; 95% CI, 2.8%-5.2%) had confirmed HSRs. Most HSRs occurred at the second (ie, full-dose) step (68%) and required no treatment beyond drug discontinuation (58%); 3 HSR patients were treated with intramuscular epinephrine. Reported cephalosporin allergy history was associated with an increased odds of HSR (odds ratio [OR], 2.96; 95% CI, 1.34-6.58). Allergies were updated for 474 patients (45%), with records specified (82%), deleted (16%), and added (8%). CONCLUSION: This antimicrobial stewardship intervention using ß-lactam test-dose procedures was safe. Overall, 3.8% of patients with ß-lactam allergy histories had an HSR; cephalosporin allergy histories conferred a 3-fold increased risk. Encouraging EHR documentation might improve this safe, effective, and practical acute-care antibiotic stewardship tool.

Evidence that regulation of intramembrane proteolysis is mediated by substrate gating during sporulation in Bacillus subtilis.

During the morphological process of sporulation in Bacillus subtilis two adjacent daughter cells (called the mother cell and forespore) follow different programs of gene expression that are linked to each other by signal transduction pathways. At a late stage in development, a signaling pathway emanating from the forespore triggers the proteolytic activation of the mother cell transcription factor σK. Cleavage of pro-σK to its mature and active form is catalyzed by the intramembrane cleaving metalloprotease SpoIVFB (B), a Site-2 Protease (S2P) family member. B is held inactive by two mother-cell membrane proteins SpoIVFA (A) and BofA. Activation of pro-σK processing requires a site-1 signaling protease SpoIVB (IVB) that is secreted from the forespore into the space between the two cells. IVB cleaves the extracellular domain of A but how this cleavage activates intramembrane proteolysis has remained unclear. Structural studies of the Methanocaldococcus jannaschii S2P homolog identified closed (substrate-occluded) and open (substrate-accessible) conformations of the protease, but the biological relevance of these conformations has not been established. Here, using co-immunoprecipitation and fluorescence microscopy, we show that stable association between the membrane-embedded protease and its substrate requires IVB signaling. We further show that the cytoplasmic cystathionine-ß-synthase (CBS) domain of the B protease is not critical for this interaction or for pro-σK processing, suggesting the IVB-dependent interaction site is in the membrane protease domain. Finally, we provide evidence that the B protease domain adopts both open and closed conformations in vivo. Collectively, our data support a substrate-gating model in which IVB-dependent cleavage of A on one side of the membrane triggers a conformational change in the membrane-embedded protease from a closed to an open state allowing pro-σK access to the caged interior of the protease.

Addressing Inpatient Beta-Lactam Allergies: A Multihospital Implementation.

Addressing inaccurate penicillin allergies is encouraged as part of antibiotic stewardship in the inpatient setting. However, implementing interventions targeted at the 10% to 15% of inpatients reporting a previous penicillin allergy can pose substantial logistic challenges. We implemented a computerized guideline for patients with reported beta-lactam allergy at 5 hospitals within a single health care system in the Boston area. In this article, we describe our implementation roadmap, including both successes achieved and challenges faced. We explain key implementation steps, including assembling a team, stakeholder engagement, developing or selecting an approach, spreading the change, establishing measures, and measuring impact. The objective was to detail the lessons learned while empowering others to be part of this important, multidisciplinary work to improve the care of patients with reported beta-lactam allergies.

Evaluation of a Pharmacist-Directed Vancomycin Dosing and Monitoring Pilot Program at a Tertiary Academic Medical Center.

BACKGROUND: Consensus guidelines recommend vancomycin doses of 15 to 20 mg/kg every 8 to 12 hours in patients with normal renal function. OBJECTIVE: To evaluate the effect of a pharmacist-directed vancomycin dosing and monitoring pilot program on the percentage of patients receiving targeted weight-based dosing recommendations. METHODS: This was a pre-/postevaluation study, approved by the institutional review board at our institution, comparing retrospectively reviewed vancomycin dosing practices hospital-wide between September 1 and September 30, 2010 to patients prospectively managed by a pharmacist-directed vancomycin pilot program between February 1 and April 26, 2011. All adult inpatients receiving intravenous vancomycin were included, unless patients had a creatinine clearance less than or equal to 60 mL/min or indication for therapy was surgical prophylaxis or febrile neutropenia. The primary outcome was the percentage of patients who received optimal vancomycin dosing defined as ≥30 mg/kg/d within 24 hours of initiation of therapy. Secondary outcomes included number of pharmacist interventions, length of therapy and incidence of nephrotoxicity while receiving vancomycin. RESULTS: A total of 319 patients were analyzed, 161 preimplementation and 158 postimplementation. The percentage of patients who received optimal vancomycin dosing was significantly higher postimplementation of the pilot program, 96.8 versus 40.4% (P < 0.001). Pharmacist-directed interventions postimplementation, resulted in 50% more patients being dosed optimally (P < 0.001). Patients in the pilot program also had a shorter length of therapy (10.0 vs 8.4 days, P < 0.003) and a lower incidence of nephrotoxicity (8.7% vs 3.2%, P = 0.006). CONCLUSIONS: This pharmacist-directed vancomycin pilot program significantly increased the percentage of patients optimally dosed according to consensus guidelines within 24 hours of initiation of therapy.

Implementation of a dexmedetomidine stewardship program at a tertiary academic medical center.

BACKGROUND: Brigham and Women's Hospital implemented a dexmedetomidine stewardship program in October 2010 beginning with an institution-specific prescribing guideline. To ensure continued adherence to the prescribing guideline, a pharmacist-driven quality assurance program was implemented in November 2011. OBJECTIVE: The primary objective of this study is to describe the role and impact of a dexmedetomidine stewardship program on dexmedetomidine use at a tertiary academic medical center. METHODS: This is a prospective descriptive analysis of a dexmedetomidine stewardship program. Dexmedetomidine stewardship data were collected prospectively from January 2012 through June 2012, in all intensive care units (ICUs) at a single academic medical center. Adult patients (>18 years old) receiving dexmedetomidine therapy continuously for sedation and in the ICU were included in the analysis. RESULTS: A total of 99 patients were identified during the study time frame, during which 71 (71.7%) were identified as compliant with the institutional guideline. The total number of patients receiving dexmedetomidine for greater than 24 hours was 13 (13.1%), of whom 10 (76.9%) received targeted interventions; 15 (15.2%) targeted interventions were made on all patients receiving dexmedetomidine during the study time frame. The total use of dexmedetomidine during the study period was 1310 vials, compared with 5404 vials during the equivalent time frame in 2010-a 76% reduction. CONCLUSIONS: A dexmedetomidine stewardship program, including an institution-specific prescribing guideline and a pharmacist-driven quality assurance program may ensure guideline compliance and decreased use of dexmedetomidine at an academic medical center.

Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis.

Sporulating Bacillus subtilis cells assemble a transenvelope secretion complex that connects the mother cell and developing spore. The forespore protein SpoIIQ and the mother-cell protein SpoIIIAH interact across the double membrane septum and are thought to assemble into a channel that serves as the basement layer of this specialized secretion system. SpoIIQ is absolutely required to recruit SpoIIIAH to the sporulation septum on the mother-cell side, however the mechanism by which SpoIIQ is localized has been unclear. Here, we show that SpoIIQ localization requires its partner protein SpoIIIAH and degradation of the septal peptidoglycan (PG) by the two cell wall hydrolases SpoIID and SpoIIP. Our data suggest that PG degradation enables a second mother-cell-produced protein to interact with SpoIIQ. Cells in which both mother-cell anchoring mechanisms have been disabled have a synergistic sporulation defect suggesting that both localization factors function in the secretion complex. Finally, we show that septal PG degradation is critical for the assembly of an active complex. Altogether, these results suggest that the specialized secretion system that links the mother cell and forespore has a complexity approaching those found in Gram-negative bacteria and reveal that the sporulating cell must overcome similar challenges in assembling a transenvelope complex.

FisB mediates membrane fission during sporulation in Bacillus subtilis.

How bacteria catalyze membrane fission during growth and differentiation is an outstanding question in prokaryotic cell biology. Here, we describe a protein (FisB, for fission protein B) that mediates membrane fission during the morphological process of spore formation in Bacillus subtilis. Sporulating cells divide asymmetrically, generating a large mother cell and smaller forespore. After division, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Membrane fission releases the forespore into the mother cell cytoplasm. Cells lacking FisB are severely and specifically impaired in the fission reaction. Moreover, GFP-FisB forms dynamic foci that become immobilized at the site of fission. Purified FisB catalyzes lipid mixing in vitro and is only required in one of the fusing membranes, suggesting that FisB-lipid interactions drive membrane remodeling. Consistent with this idea, the extracytoplasmic domain of FisB binds with remarkable specificity to cardiolipin, a lipid enriched in the engulfing membranes and regions of negative curvature. We propose that membrane topology at the final stage of engulfment and FisB-cardiolipin interactions ensure that the mother cell membranes are severed at the right time and place. The unique properties of FisB set it apart from the known fission machineries in eukaryotes, suggesting that it represents a new class of fission proteins.

Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis.

How bacteria respond to chromosome replication stress has been traditionally studied using temperature-sensitive mutants and chemical inhibitors. These methods inevitably arrest all replication and lead to induction of transcriptional responses and inhibition of cell division. Here, we used repressor proteins bound to operator arrays to generate a single stalled replication fork. These replication roadblocks impeded replisome progression on one arm, leaving replication of the other arm and re-initiation unaffected. Remarkably, despite robust generation of RecA-GFP filaments and a strong block to cell division during the roadblock, patterns of gene expression were not significantly altered. Consistent with these findings, division inhibition was not mediated by the SOS-induced regulator YneA nor by RecA-independent repression of ftsL. In support of the idea that nucleoid occlusion prevents inappropriate cell division during fork arrest, immature FtsZ-rings formed adjacent to the DNA mass but rarely on top of it. Furthermore, mild alterations in chromosome compaction resulted in cell division that guillotined the DNA. Strikingly, the nucleoid occlusion protein Noc had no discernable role in division inhibition. Our data indicate that Noc-independent nucleoid occlusion prevents inappropriate cell division during replication fork arrest. They further suggest that Bacillus subtilis normally manages replication stress rather than inducing a stress response.

A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis.

How proteins catalyze morphogenesis is an outstanding question in developmental biology. In bacteria, morphogenesis is intimately linked to remodeling the cell wall exoskeleton. Here, we investigate the mechanisms by which the mother cell engulfs the prospective spore during sporulation in Bacillus subtilis. A membrane-anchored protein complex containing two cell wall hydrolases plays a central role in this morphological process. We demonstrate that one of the proteins (SpoIIP) has both amidase and endopeptidase activities, such that it removes the stem peptides from the cell wall and cleaves the cross-links between them. We further show that the other protein (SpoIID) is the founding member of a new family of lytic transglycosylases that degrades the glycan strands of the peptidoglycan into disaccharide units. Importantly, we show that SpoIID binds the cell wall, but will only cleave the glycan strands after the stem peptides have been removed. Finally, we demonstrate that SpoIID also functions as an enhancer of SpoIIP activity. Thus, this membrane-anchored enzyme complex is endowed with complementary, sequential, and stimulatory activities. These activities provide a mechanism for processive cell wall degradation, supporting a model in which circumferentially distributed degradation machines function as motors pulling the mother cell membranes around the forespore.

SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis.

How cells maintain their ploidy is relevant to cellular development and disease. Here, we investigate the mechanism by which the bacterium Bacillus subtilis enforces diploidy as it differentiates into a dormant spore. We demonstrate that a sporulation-induced protein SirA (originally annotated YneE) blocks new rounds of replication by targeting the highly conserved replication initiation factor DnaA. We show that SirA interacts with DnaA and displaces it from the replication origin. As a result, expression of SirA during growth rapidly blocks replication and causes cell death in a DnaA-dependent manner. Finally, cells lacking SirA over-replicate during sporulation. These results support a model in which induction of SirA enforces diploidy by inhibiting replication initiation as B. subtilis cells develop into spores.

Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation.

Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation.

SpoIIIE strips proteins off the DNA during chromosome translocation.

The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.

SpoIIQ anchors membrane proteins on both sides of the sporulation septum in Bacillus subtilis.

During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation.

The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis.

The FtsK/SpoIIIE family of ATP-dependent DNA transporters mediates proper chromosome segregation in dividing bacteria. In sporulating Bacillus subtilis cells, SpoIIIE translocates much of the circular chromosome from the mother cell into the forespore, but the molecular mechanism remains unclear. Using a new assay to monitor DNA transport, we demonstrate that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. The fusion of the septal membranes during cytokinesis precedes DNA translocation and does not require SpoIIIE, as suggested by analysis of lipid dynamics, serial thin-section electron microscopy, and cell separation by protoplasting. These data support a model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum.

Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum.

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.