Abstract
Background
Within a sufficiently large bacterial population, some members will naturally adopt an alternate, metabolically-active state that favors small molecule synthesis over cell ...division. In Staphylococcus aureus this process can be sharply accelerated by multiple factors present during infection including nutrient limitation, host cationic peptide exposure and polymorphonuclear neutrophil internalization. These isogenic “tolerant” subpopulations have variable responses during antibiotic exposure and can remain viable in the presence of typically bactericidal concentrations. Survivors of antibiotic exposure can restart cell division upon cessation of antibiotics and cause relapse or recurrent infection. In this study we determine the ability of typical and atypical antistaphylococcal therapies to reduce the viability of tolerant Staphylococcus aureus bacteria.
Methods
S. aureus strain ATCC29213 as well as four clinical isolates (two MSSA, two MRSA) were selected for analysis. Overnight cultures were diluted in pre-warmed broth (MHB50) to 1×106 cfu/mL. Tolerance was induced by exposure to mupirocin (low 0.032 µg/mL or high 3.2 µg/mL) for 30 min. Tolerant cultures were exposed to vancomycin (35 µg/mL), cefazolin (25 µg/mL), daptomycin (7 µg/mL), telavancin (10 µg/mL), dalbavancin (6 µg/mL) or oritavancin (14 µg/mL) and viability was assessed by dilution plating at pre-defined time points (0, 2, 6, 24, 48 h). The minimum duration for 3-log viability reduction from baseline (MDK99.9) and culture viability at 48h were calculated independently for each of three biological replicates.
Results
The rate of bacterial killing (MDK99.9) was reduced for all study antibiotics by the addition of mupirocin in a dose-dependent manner. In contrast to all other regimens, including lipoglycopeptide comparators, oritavancin was the only antimicrobial agent that maintained a similar extent of bacterial killing against tolerant staphylococci.
Conclusion
Antimicrobial tolerant staphylococci exhibit a decreased rate of killing by antistaphylococcal agents. However, oritavancin remained effective at maintaining a similar extent of killing. Further studies to investigate the role of otritavancin against recurrent or relapse staphylococcal infection is warranted.
Disclosures
All Authors: No reported disclosures
Abstract
Background
We report a patient case of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia which over 30 days developed resistance to all three primary antistaphylococcal ...antibiotics. Index blood cultures displayed susceptibility to vancomycin (VAN, MIC=1), ceftaroline (CPT, MIC=0.5) and daptomycin (DAP, MIC=0.5). The patient was maintained on VAN/CPT with negative blood cultures by hospital day 7. The regimen was later modified to DAP/piperacillin-tazobactam. One month after initial presentation, during the same encounter, blood cultures were again positive for MRSA, now displaying intermediate resistance to VAN (MIC=2) and CPT (MIC=2), and resistance to DAP (MIC=4).
Methods
Isolates were collected from the initial bacteremia episode (B325) and the recurrence (B326). In-vitro one-compartment pharmacokinetic/pharmacodynamic modeling was performed on each isolate to determine which antibiotics or combinations would effectively eradicate cultures. Regimens examined included DAP (10mg/kg), DAP/cefazolin (CFZ), VAN/CFZ and penicillin/clavulanate. Draft whole-genome sequences are pending for each clinical isolate using hybrid assembly (Unicycler v0.4.2) of MinION and Illumina (150bp PE) reads.
Results
DAP/CFZ combination reduced viability of both B325 and B326 below limit of detection by 12 hours and maintained efficacy for 72 h. DAP, initially effective in reducing B326 cell concentrations below limit of detection, allowed regrowth by 36 h. All other modeled therapies were less effective. Interestingly, DAP took significantly longer to kill B325 relative to S. aureus collected contemporaneously from other patients. For DAP-containing regimens, the time to 3-log viability reduction (MDK99.9) was five-fold longer in B325 compared to similar clinical isolates with the same DAP MIC. Comparative genomics of sequential isolates is pending, although single nucleotide polymorphisms in vraT and mprF were identified in B326 compared to B325.
Conclusion
Delayed kill kinetics of B325 when exposed to DAP may indicate antibiotic tolerance, which could partially account for B325’s rapid transition to a multi-drug resistant organism. DAP/CFZ therapy appears to be the most active combination against both antibiotic-tolerant and multidrug resistant S. aureus strains.
Disclosures
All Authors: No reported disclosures
Abstract
Background
Genetic manipulation of Staphylococcus aureus (both methicillin sensitive S. aureus, MSSA, and methicillin resistant S. aureus, MRSA) poses a technical challenge due to poor ...transformation efficiency, limited endogenous DNA repair activity, lineage-specific methylation patterns and intrinsic resistance to common selectable markers.
Methods
To address transformation efficiency we have optimized electrocompetent cell preparation and electroporation protocols for staphylococci. Further, we have improved a CRISPR counterselection platform that delivers a heterologous ssDNA recombinase and an inducible Cas9 endonuclease. When used for recombineering and counterselection, this strategy allows minimization of the number of elements necessary to transform in a single electroporation event. The Cas9 delivery platform has been modified to include a range of selectable markers including resistance to apramycin, erythromycin, kanamycin, nourseothricin, spectinomycin or trimethoprim.
Results
Overall electroporation efficiency increased by multiple orders of magnitude (> 100×) using the optimized cell preparation protocol. The CRISPR delivery platform can be stably maintained in a repressed state for multiple generations and induced with anhydrotetracycline. We have introduced targeted mutations in multiple loci using this system with an average turnaround time of 12 days.
Conclusion
This improved dual-plasmid CRISPR platform is robust and allows the investigator to rapidly and specifically alter the genomes of staphylococci. These tools will facilitate the study of how specific genetic polymorphisms contribute to various phenotypes in S. aureus, including the virulence of MRSA.
Disclosures
All Authors: No reported disclosures
Abstract
Background
During the early COVID-19 pandemic a large number of investigational agents were utilized due to lack of therapeutic options. We evaluate the utility of commonly-used ...investigational agents combined with hydroxychloroquine (HCQ).
Methods
This multicenter observational cohort study included patients admitted with COVID-19 between March - May 2020 in Detroit, Michigan who received at least 2 doses of HCQ. Our primary outcome was the change in Sequential Organ Failure Assessment (SOFA) score from presentation to day 5 of HCQ therapy with a secondary outcome of in-hospital mortality. Data collected included demographics, Charlson Comorbidity index (CCI), daily SOFA score, laboratory data and COVID-directed therapies. Multiple linear regressions were performed to control for potential confounders between different therapies and change in SOFA score.
Results
Three hundred thirty-five patients receiving HCQ were included. Patients were 62 ± 14.8 years of age, male (54%) and African-American (82%) with a mean CCI of 1.7 ± 1.9. In our cohort, 32% were admitted to the intensive care unit and 35% expired. Therapies received by more than 20% of patients in addition to HCQ included azithromycin (80%), zinc (76%) and vitamin D (29%). In our unadjusted analysis, a significant improvement in SOFA score was observed with zinc (0.76) while no significant change was observed with azithromycin (-0.46) or vitamin D (0.05). However, there was no significant change in SOFA score after adjusting for confounders for azithromycin, zinc and vitamin D. No difference in mortality was observed between the groups.
Conclusion
Overall, no benefit in end-organ damage or mortality was observed with the addition of azithromycin, zinc or vitamin D to HCQ. Further studies are needed to confirm this observation.
Disclosures
All Authors: No reported disclosures
Abstract
Background
Previous studies demonstrate that adding oxacillin during daptomycin (DAP) exposure can prevent DAP resistance development in community-acquired (ST8/USA300) MRSA, presumably by ...preventing mprF mutation. Hospital-acquired strains, such as MRSA sequence types 5 and 239, typically have higher β-lactam (BL) minimum inhibitory concentrations (MICs) than their community-acquired counterparts and are often less toxigenic, more multidrug-resistant and more refractory to primary antistaphylococcal therapies. It is unknown whether DAP resistance prevention occurs in hospital-acquired MRSA lineages or if augmenting DAP therapy with BL antibiotics other than oxacillin would prevent DAP resistance development.
Methods
MRSA ST5/USA100 (D592) and ST239 (JKD6004) differ in the degree to which BL enhances DAP activity. D592 and JKD6004 were passaged in escalating concentrations of DAP in a stepwise fashion in vitro as described previously. Following 28 days of serial passage all replicates were passaged twice on mannitol-salt agar and tested for DAP MIC by Etest. Parallel passages were performed in media supplemented with BL antibiotics. Between-group differences in DAP MIC suppression effectiveness among individual BLs compared with nafcillin was evaluated using Kruskal–Wallis rank-sum testing with Holm-adjusted post-hoc Dunn testing.
Results
Passage of D592 or JKD6004 in DAP resulted in highly DAP-resistant isolates (median ≥256 mg/L, IQR 96,256). In contrast, when passages were performed in the presence of DAP+BL, DAP resistance development was suppressed. No between-group differences in DAP MIC suppression effectiveness were observed among individual BLs compared with nafcillin. Highly DAP-resistant isolates demonstrated variable collateral susceptibility to BL monotherapy but were frequently susceptible to combination antibiotic exposure.
Conclusion
Addition of β-lactams to DAP can prevent DAP resistance development in vitro in ST5/USA100 and ST239 MRSA, consistent with findings in ST8/USA300 lineages. Furthermore, this ability appears to be a class effect of β-lactam antibiotics and is independent of the extent of DAP-BL synergy. This provides evidence to support the use of BL combination therapy without regard to staphylococcal lineage or specific BL used.
Disclosures
All authors: No reported disclosures.
Abstract
Background
Loss of daptomycin susceptibility in Staphylococcus aureus is often associated with sequestered foci of infection, driven by selection pressure from both administered antibiotics ...and host defense peptides. Susceptibility testing of the organism cultured from blood is assumed to parallel that of the infectious foci, such as heart valves. We studied a case of tricuspid valve endocarditis where one leaflet yielded exclusively daptomycin-nonsusceptible S. aureus and another leaflet yielded purely daptomycin-susceptible S. aureus. We examined the responses of the two populations to different anti-staphylococcal therapies to identify regimens effective against both isolates.
Methods
Both isolates were whole-genome-sequenced using Illumina technologies. The presence of heterogeneous daptomycin-resistant subpopulations was assessed by dilution plating and population analysis profiling. One compartment pharmacokinetic/pharmacodynamic modeling was used to simulate different potential antistaphylococcal pharmacotherapies against each isolate. Hemolysin activity was evaluated as a surrogate for accessory gene regulator function.
Results
The daptomycin-susceptible isolate did not demonstrate heteroresistance while the daptomycin-resistant population was uniformly daptomycin nonsusceptible. The daptomycin non-susceptible isolate demonstrated regrowth by 72 hours of simulated treatment with vancomycin (2 g Q12H) or daptomycin (10 mg/kg daily). Adding cefazolin (2 g Q8H) to vancomycin or daptomycin prevented regrowth at 72 hours. The daptomycin-resistant isolate was deficient in hemolysin production suggesting agr dysfunction. Comparative sequencing identified daptomycin-resistant isolate mutations in mprF, purR and agrA.
Conclusion
This case underscores the complex dynamics of the emergence of S. aureus resistance to daptomycin in vivo. Our pharmacokinetic modeling supports combination therapy in the treatment of endovascular MRSA infection. Reduced hemolytic activity supports the hypothesis that agr modulation is associated with persistent infection and/or treatment failure. Ongoing studies will identify features of distinct bacterial populations that promote ecological succession during infection at a sequestered anatomical site.
Disclosures
All authors: No reported disclosures.
Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased ...approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient‐matched SCD and non‐stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican‐positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub‐clusters. Intra sub‐cluster gene analysis detected 13 co‐regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.
Prevailing agricultural systems dominated by annual crop monocultures, and the landscapes that contain them, lack resilience and multifunctionality. They are vulnerable to extreme weather events, ...contribute to degradation of soil, water, and air quality, reduce biodiversity, and negatively impact human health, social engagement, and equity. To achieve greater resilience, stability, and multiple ecosystem services therein, and to improve socioeconomic outcomes, we propose a practical framework to gain multifunctionality at multiple scales. This framework includes forages within agroecosystems that have the essential structural features of diversity, perenniality, and circularity. These three structural features are associated with increased resilience, stability, and provision of several ecosystem services, which in turn improve human health and socioeconomic outcomes. This framework improves understanding of, and access to, tools and materials for promoting the adoption of diverse circular agroecosystems with perennial forages. Application of this framework can result in land transformations that solve sustainability challenges in agriculture if policy, economic, and social barriers can be overcome by a transdisciplinary process of equitable knowledge production.
Prevailing agroecosystems have multiple environmental and socioeconomic problems. Diverse, perennial, circular forage systems (DPCFS) foster resilience to climate change while providing multiple ecosystem services and socioeconomic benefits. A landscape transition from prevailing to DPCFS requires overcoming socioeconomic and policy barriers to adoption while creating enabling conditions through a transdisciplinary process of research and outreach.
BACKGROUND:Carbon dioxide embolus has been reported as a rare but clinically important risk associated with transanal total mesorectal excision surgery. To date, there exists limited data describing ...the incidence, risk factors, and management of carbon dioxide embolus in transanal total mesorectal excision.
OBJECTIVE:This study aimed to obtain data from the transanal total mesorectal excision registries to identify trends and potential risk factors for carbon dioxide embolus specific to this surgical technique.
DESIGN:Contributors to both the LOREC and OSTRiCh transanal total mesorectal excision registries were invited to report their incidence of carbon dioxide embolus. Case report forms were collected detailing the patient-specific and technical factors of each event.
SETTINGS:The study was conducted at the collaborating centers from the international transanal total mesorectal excision registries.
MAIN OUTCOME MEASURES:Characteristics and outcomes of patients with carbon dioxide embolus associated with transanal mesorectal excision were measured.
RESULTS:Twenty-five cases were reported. The incidence of carbon dioxide embolus during transanal total mesorectal excision is estimated to be ≈0.4% (25/6375 cases). A fall in end tidal carbon dioxide was noted as the initial feature in 22 cases, with 13 (52%) developing signs of hemodynamic compromise. All of the events occurred in the transanal component of dissection, with mean (range) insufflation pressures of 15 mm Hg (12–20 mm Hg). Patients were predominantly (68%) in a Trendelenburg position, between 30° and 45°. Venous bleeding was reported in 20 cases at the time of carbon dioxide embolus, with periprostatic veins documented as the most common site (40%). After carbon dioxide embolus, 84% of cases were completed after hemodynamic stabilization. Two patients required cardiopulmonary resuscitation because of cardiovascular collapse. There were no deaths.
LIMITATIONS:This is a retrospective study surveying reported outcomes by surgeons and anesthetists.
CONCLUSIONS:Surgeons undertaking transanal total mesorectal excision must be aware of the possibility of carbon dioxide embolus and its potential risk factors, including venous bleeding (wrong plane surgery), high insufflation pressures, and patient positioning. Prompt recognition and management can limit the clinical impact of such events. See Video Abstract at http://links.lww.com/DCR/A961.
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