Systematic genome-wide discovery of host factors governing bacteriophage infectivity.

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host-gene impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

Spatial and temporal control of lysis by the lambda holin.

The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.

ViWrap: A modular pipeline to identify, bin, classify, and predict viral-host relationships for viruses from metagenomes.

Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of the difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate the screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis, including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub (https://github.com/AnantharamanLab/ViWrap). A detailed description of the software, its usage, and interpretation of results can be found on the website.

Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota.

Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters.

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous or homologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of protein-encoding ortholog groups for individual genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of statistics for each inferred ortholog group. These programs are showcased through application to: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-genetic insights of two common BGCs in a fungal species, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

ViWrap: A modular pipeline to identify, bin, classify, and predict viral-host relationships for viruses from metagenomes.

Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub (https://github.com/AnantharamanLab/ViWrap). A detailed description of the software, its usage, and interpretation of results can be found on the website.

Butyrate Differentiates Permissiveness to Clostridioides difficile Infection and Influences Growth of Diverse C. difficile Isolates.

A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota-accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome is short-chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known to decrease the fitness of C. difficile in vitro, and high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI than in those from healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCE Clostridioides difficile is a leading cause of infectious diarrhea in humans, and it imposes a tremendous burden on the health care system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI, but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

Operating Room Fire During Total Knee Arthroplasty Tibial Impaction: A Case Report and Review of the Literature.

A fire in the operating room is a rare but potentially deadly occurrence. We present an operating room fire during an elective total knee arthroplasty with an unclear ignition source. Flames were visualized originating from the excess bone cement while impacting the tibial component. The electrocautery device was not in use during impaction and was in a plastic sheath at the head of the bed. To our knowledge, this is the first reported case of an operating room fire involving bone cement not caused by an electrocautery device.

Phage-encoded cationic antimicrobial peptide required for lysis.

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Complete Genome Sequence of Klebsiella pneumoniae Myophage Muenster.

Klebsiella pneumoniae is associated with antibiotic-resistant nosocomial infections. Here, we present the annotated genome sequence of the Klebsiella jumbo phage Muenster. The Muenster genome sequence (346,937 bp) encodes 6 tRNAs and 561 putative protein-coding genes, including 9 tail fibers, suggesting a genetic mechanism to broaden the host range.

Intra-articular Corticosteroid Injections for Symptomatic Knee Osteoarthritis: What the Orthopaedic Provider Needs to Know.

Intra-articular corticosteroid injections have been used for decades in the management of symptomatic osteoarthritis of the knee and remain a common practice. The pain relief from a steroid injection is thought to work by reducing inflammation within the arthritic knee. Substantial variability remains among providers with regard to the technique used to perform the procedure, including the site of the injection, the medications injected, and the level of sterility. The success of steroid injections in relieving arthritic knee pain most often occurs in the short term. However, the efficacy of intra-articular corticosteroid injections varies within the published literature. The latest American Academy of Orthopaedic Surgeons clinical practice guideline does not support conclusive recommendations about the use of intra-articular corticosteroid injections for symptomatic knee osteoarthritis. Providers should be aware of the adverse effects and potential complications of these injections when using them in clinical practice.

Special Considerations for Mass Violence Events in Senior Living Facilities: A Case Report on the Pinelake Health and Rehab Center Shooting.

The 2009 Pinelake Health and Rehab Center shooting in Carthage, North Carolina, presents a unique case study for examining the specific considerations for mass violence events in senior living facilities. A variety of factors, including reduced sensory perception, reduced mobility, and cognitive decline, may increase the vulnerability of the populations of senior living facilities during mass violence events. Management of response aspects such as evacuation, relocation, and reunification also require special consideration in the context of mass violence at senior living facilities. Better awareness of these vulnerabilities and response considerations can assist facility administrators and emergency managers when preparing for potential mass violence events at senior living facilities. (Disaster Med Public Health Preparedness. 2017;11:150-152).

Opening a New Level II Trauma Center Near an Established Level I Trauma Center: Is This Good for Trauma Care?

OBJECTIVES: To describe how the initiation and later removal of a provisional level II trauma center (PL2TC) status at a community hospital affected the volume and severity of injured patients seen at an established academic level 1 trauma center (AL1TC). METHODS: Census data including counts of injury ICD-9 codes and patients seen in the emergency department (ED) and trauma center at an AL1TC were collected monthly from January 2010 to October 2014. An interrupted time series analysis was used to model the monthly census data with 2 time interruptions to describe the change in patient volume at the interruptions. The interruptions were (1) the initiation of the PL2TC status at a nearby community hospital and (2) the subsequent removal of the PL2TC status. RESULTS: The number of diagnoses, encounters, and patients seen at the AL1TC ED decreased while the PL2TC was operating. After the removal of the PL2TC status, there was a 19.4% increase in the ED patient volume per month at the AL1TC. The number of orthopaedic trauma patients seen through the ED at the AL1TC dropped 11.1% per month when the PL2TC began functioning as a trauma center. However, the volume of orthopaedic patients at the AL1TC did not recuperate after the PL2TC lost level 2 status. CONCLUSIONS: A significant decrease in patient volume was seen at the AL1TC with the initiation of the PL2TC in close proximity. Orthopaedic patient volume did not recuperate after the removal of the PL2TC status.

Blood transfusion associated with shoulder arthroplasty.

BACKGROUND: Studies have reported high rates of transfusion in shoulder arthroplasty. This study was conducted to evaluate the rate of transfusion at our institution, to confirm reported risk factors for transfusion, and to look for changes over time.We hypothesized that transfusion rates associated with shoulder arthroplasty at our institution are lower than those recently reported and that the incidence of transfusion is higher in individuals with low preoperative hemoglobin, with revision arthroplasty, and in older individuals. MATERIALS AND METHODS: A retrospective review of 366 shoulder arthroplasties (323 patients) was performed. This included total shoulder arthroplasties, hemiarthroplasties, revision arthroplasties, and reverse total shoulder arthroplasties. Logistic regression analysis evaluated the association of clinical variables with transfusion. Early (1996-2005) and late (2006-2009) groups were compared to evaluate changes in demographics and transfusion rates over time. RESULTS: The overall transfusion rate was 7.4% (27 of 339). Predictors of transfusion were higher intraoperative blood loss, low preoperative hemoglobin level, and humeral cement fixation. Procedure type was not predictive of transfusion. There was no difference in transfusion rates between the early and late groups, but the late group had an increased use of general anesthesia combined with a regional block, increased intraoperative blood loss, and increased use of sequential compression devices for venous thromboembolism prophylaxis. CONCLUSIONS: Lower preoperative hemoglobin, higher intraoperative blood loss, and humeral cement fixation were predictors of transfusion, but not female sex, increasing age, type of procedure, or comorbidities.

Involuntary memory chaining versus event cueing: Which is a better indicator of autobiographical memory organisation?

Involuntary memory chains are spontaneous recollections of the past that occur in a sequence. Much like semantic memory priming, this memory phenomenon has provided some insights into the nature of associations in autobiographical memory. The event-cueing procedure (a laboratory-based memory sequencing task) has also provided some insights into the nature of autobiographical memory organisation. However, while both of these memory-sequencing phenomena have exhibited the same types of memory associations (conceptual associations and general-event or temporal associations), both have also produced discrepant results with respect to the relative proportions of such associations. This study investigated the possibility that the results from event cueing are artefacts of various memory production responses. Using a number of different approaches we demonstrated that these memory production responses cause overestimates of general-event association. We conclude that for this reason, the data from involuntary memory chains provide a better picture of the organisation of autobiographical memory.