ABD-3, the confluence of powerful antibacterial modalities: ABDs delivering and expressing lss, the gene encoding lysostaphin.

In response to the antimicrobial resistance crisis, we have developed a powerful and versatile therapeutic platform, the Antibacterial Drone (ABD) system. The ABD consists of a highly mobile staphylococcal pathogenicity island re-purposed to deliver genes encoding antibacterial proteins. The chromosomally located island is induced by a co-resident helper phage, packaged in phage-like particles, and released in very high numbers upon phage-induced lysis. ABD particles specifically adsorb to bacteria causing an infection and deliver their DNA to these bacteria, where the bactericidal cargo genes are expressed, kill the bacteria, and cure the infection. Here, we report a major advance of the system, incorporation of the gene encoding a secreted, bactericidal, species-specific lytic enzyme, lysostsphin. This ABD not only kills the bacterium that has been attacked by the ABD, but also any surrounding bacteria that are sensitive to the lytic enzyme which is released by secretion and by lysis of the doomed cell. So while the killing field is thus expanded, there are no civilian casualties (bacteria that are insensitive to the ABD and its cargo protein(s) are not inadvertently killed). Without amplifying the number of ABD particles (which are not re-packaged), the expression and release of the cargo gene's product dramatically extend the effective reach of the ABD. A cargo gene that encodes a secreted bactericidal protein also enables the treatment of a mixed bacterial infection in which one of the infecting organisms is insensitive to the ABD delivery system but is sensitive to the ABD's secreted cargo protein.

Cardiac surgery elicits pericardial inflammatory responses that are distinct compared with postcardiopulmonary bypass systemic inflammation.

Objectives: Cardiac surgery using cardiopulmonary bypass contributes to a robust systemic inflammatory process. Local intrapericardial postsurgical inflammation is believed to trigger important clinical implications, such as postoperative atrial fibrillation and postsurgical intrathoracic adhesions. Immune mediators in the pericardial space may underlie such complications. Methods: In this prospective pilot clinical study, 12 patients undergoing isolated coronary artery bypass graft surgery were enrolled. Native pericardial fluid and venous blood samples (baseline) were collected immediately after pericardiotomy. Postoperative pericardial fluid and venous blood samples were collected 48-hours after cardiopulmonary bypass and compared with baseline. Flow cytometry determined proportions of specific immune cells, whereas multiplex analysis probed for inflammatory mediators. Results: Neutrophils are the predominant cells in both the pericardial space and peripheral blood postoperatively. There are significantly more CD163lo macrophages in blood compared with pericardial effluent after surgery. Although there are significantly more CD163hi macrophages in native pericardial fluid compared with baseline blood, after surgery there are significantly fewer of these cells present in the pericardial space compared with blood. Postoperatively, concentration of interleukin receptor antagonist 6, and interleukin 8 were significantly higher in the pericardial space compared with blood. After surgery, compared with blood, the pericardial space has a significantly higher concentration of matrix metalloproteinase 3, matrix metalloproteinase 8, and matrix metalloproteinase 9. The same trend was observed with transformational growth factor ß. Conclusions: Cardiac surgery elicits an inflammatory response in the pericardial space, which differs from systemic inflammatory responses. Future work should determine whether or not this distinct local inflammatory response contributes to postsurgical complications and could be modified to influence clinical outcomes.

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Brain death post cardiac surgery: A modified apnea test to confirm death by neurologic criteria for a patient on extracorporeal membrane oxygenation.

Death by neurologic criteria (DNC) requires coma, absent brainstem reflexes, and the inability to breathe independently during apnea testing (AT). For patients on extracorporeal membrane oxygenation (ECMO), this clinical determination is more challenging. Herein, we report the case of a patient with DNC on ECMO post cardiac surgery, highlighting various considerations to the process and modifications required for AT.

Comprehensive characterization of the postoperative pericardial inflammatory response: Potential implications for clinical outcomes.

Objective: There is a paucity of data on the inflammatory response that takes place in the pericardial space after cardiac surgery. This study provides a comprehensive assessment of the local postoperative inflammatory response. Methods: Forty-three patients underwent cardiotomy, where native pericardial fluid was aspirated and compared with postoperative pericardial effluent collected at 4, 24, and 48 hours' postcardiopulmonary bypass. Flow cytometry was used to define the levels and proportions of specific immune cells. Samples were also probed for concentrations of inflammatory cytokines, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Results: Preoperatively, the pericardial space mainly contains macrophages and T cells. However, the postsurgical pericardial space was populated predominately by neutrophils, which constituted almost 80% of immune cells present, and peaked at 24 hours. When surgical approaches were compared, minimally invasive surgery was associated with fewer neutrophils in the pericardial space at 4 hours' postsurgery. Analysis of the intrapericardial concentrations of inflammatory mediators showed interleukin-6, MMP-9, and TIMP-1 to be highest postsurgery. Over time, MMP-9 concentrations decreased significantly, whereas TIMP-1 levels increased, resulting in a significant reduction of the ratio of MMP:TIMP after surgery, suggesting that active inflammatory processes may influence extracellular matrix remodeling. Conclusions: These results show that cardiac surgery elicits profound alterations in the immune cell profile in the pericardial space. Defining the cellular and molecular mediators that drive pericardial-specific postoperative inflammatory processes may allow for targeted therapies to reduce immune-mediated complications.

Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.

The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Antibacterial particles and predatory bacteria as alternatives to antibacterial chemicals in the era of antibiotic resistance.

This review is focused on the subset of antibacterial agents whose action involves one-on-one targeting of infecting bacteria. These agents target individual bacteria and their efficacy is based on particle numbers in contrast to chemical agents such as antibiotics, whose efficacy is based on minimal inhibitory concentrations. Four extant members of this class are predatory bacteria, functional (plaque-forming) phages, and engineered particulate systems, phagemids (plasmids that contain a phage packaging signal) and antibacterial drones (ABDs) that package chromosomal island DNA carrying antibacterial genes. We differentiate the natural predators, phages and predatory bacteria, from the engineered delivery vehicles, phagemids and ABDs, because the latter are much more versatile and can largely bypass the historical warfare that informs the predator-prey interactions.

A regulatory cascade controls Staphylococcus aureus pathogenicity island activation.

Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.

Delirium and depression in cardiac surgery: A comprehensive review of risk factors, pathophysiology, and management.

BACKGROUND: Mental health and wellbeing continue to gain more attention as they are inextricably associated with clinical outcomes, particularly quality of life. Many medical ailments and major surgeries affect patients' mental health, including depression and delirium. AIMS: The objective of this manuscript was to comprehensively review and critically examine the literature pertaining to cardiac surgery, depression, and delirium. METHODS: This is a narrative review article. We performed our search analysis by using the following key words: "Cardiac Surgery", "Depression", "Delirium", "Clinical outcomes", and "Mental Health". Search analysis was done on MedLine PubMed, accessing indexed peer-reviewed publications. RESULTS: Cardiac Surgery is a life-altering intervention indicated to improve morbidity and mortality in patients with cardiovascular diseases. Psychiatric conditions before and after cardiac surgery worsen patient prognosis and increase mortality rate. Specifically, preoperative depression increases postoperative depression and is associated with impaired functional status, slow physical recovery, and an increased readmission rate. DISCUSSION: Although the exact pathophysiology between depression and cardiovascular disease (CVD) is unknown, several pathways have been implicated. Unmanaged depression can also lead to other psychological conditions such as delirium. Like depression, the exact association between delirium and CVD is not well understood, but believed to be multifactorial. CONCLUSION: Herein, we provide a comprehensive review of the links between depression, delirium, and cardiovascular surgery. We critically examine the current data that pertains to the pathophysiology of these debilitating mental health issues in the context of cardiac surgery. Finally, we summarize the various treatment options available for managing depression and delirium in the cardiac surgery patient population.

Starch or Saline After Cardiac Surgery: A Double-Blinded Randomized Controlled Trial.

BACKGROUND: Despite decades of investigation, the balance of clinical risks and benefits of fluid supplementation with starch remain unresolved. Patient-centered outcomes have not been well explored in a "real-world" trial in cardiac surgery. OBJECTIVE: We sought to compare a starch-based fluid strategy with a saline-based fluid strategy in the cardiac surgery patient. DESIGN: A pragmatic blinded randomized controlled trial comparing starch-based with saline-based fluid strategy. SETTING: A large tertiary academic center in London Ontario between September 2009 and February 2011. PARTICIPANTS: Patients undergoing planned, isolated coronary revascularization. MEASUREMENTS: Serum creatinine and patient weight were measured daily postoperatively. METHODS: Patients were randomized to receive 6% hydroxyethyl starch (Voluven) or saline for perioperative fluid requirements. Fluid administration was not protocolized. Co-primary outcomes were incidence of acute kidney injury (AKI) and maximum postoperative weight gain. Secondary outcomes included bleeding, transfusion, inotropic and ventilator support, and fluid utilization. RESULTS: The study was prematurely terminated due to resource limitations. A total of 69 patients (19% female, mean age = 65) were randomized. Using RIFLE criteria for AKI, "risk" occurred in 12 patients in each group (risk ratio [RR] = 1.0; 95% confidence interval [CI] = 0.5-1.9; P = 1.00), whereas "injury" occurred in 7 of 35 (20%) and 3 of 34 (9%) of patients in the starch and saline groups, respectively (RR = 2.3; 95% CI = 0.6-8.1; P = .31). Maximum weight gain, bleeding and blood product usage, and overall fluid requirement were similar between groups. LIMITATIONS: The study had to be prematurely terminated due to resource limitations which led to a small sample size which was not sufficiently powered to detect a difference in the primary outcomes. CONCLUSIONS: This pragmatic double-blinded randomized controlled trial revealed a number of interesting hypothesis-generating trends and confirmed the feasibility of undertaking a logistically complex trial in a pragmatic fashion.

CONTEXTE: L'équilibre entre les avantages et les risques cliniques d'une supplémentation en fluides à base d'amidon n'est toujours pas établi malgré des décennies d'études. Les résultats des patients subissant une chirurgie cardiaque n'ont pas été explorés suffisamment dans le cadre d'un essai concret. OBJECTIF: Comparer deux stratégies de supplémentation liquidienne, une solution à base d'amidon et une solution saline, chez des patients subissant une chirurgie cardiaque. TYPE D'ÉTUDE: Un essai pragmatique, contrôlé, à répartition aléatoire et mené en double insu comparant deux stratégies de supplémentation liquidienne une solution à base d'amidon et une solution saline. CADRE: Un grand centre universitaire de soins tertiaires de London (Ontario) entre septembre 2009 et février 2011. SUJETS: Des patients subissant une revascularisation coronarienne planifiée et isolée. MESURES: La créatinine sérique et le poids du patient ont été mesurés quotidiennement à la suite de l'intervention. MÉTHODOLOGIE: Les patients ont été répartis aléatoirement pour recevoir du Voluven (solution d'amidon hydroxyéthylé à 6 %) ou une solution saline pour les fluidiques périopératoires. L'administration ne s'est pas faite selon un protocole établi. L'incidence d'insuffisance rénale aiguë (IRA) et un gain pondéral maximal après l'intervention constituaient les deux principaux résultats mesurés. Les résultats secondaires incluaient une hémorragie, l'utilisation de transfusion sanguine, d'inotrope, d'assistance respiratoire et l'administration de fluides. RÉSULTATS: L'étude a été interrompue prématurément par manque de ressources. Les 69 patients (19 % de femmes) répartis aléatoirement avaient en moyenne 65 ans. La classification RIFLE avait permis de détecter un « risque ¼ d'IRA chez 12 patients dans chacun des groupes (RC: 1,0; IC 95 %: 0,5-1,9; p=1,00) et une « insuffisance ¼ chez 7 patients sur 35 (20 %) du groupe « amidon ¼ et 3 patients sur 34 (9 %) du groupe « saline ¼ (RC: 2,3; IC 95 %: 0,6-8,1; p=0,31). Le gain pondéral maximal, le nombre d'hémorragies, l'utilisation de produits sanguins et les besoins liquidiens étaient similaires dans les deux groupes. LIMITES: L'étude a été interrompue prématurément en raison d'un manque de ressources. Par conséquent, le faible échantillon de patients s'avère insuffisamment puissant pour détecter des différences significatives entre les deux groupes. CONCLUSIONS: Cette étude a mis en lumière quelques tendances permettant d'émettre des hypothèses intéressantes. L'étude a également confirmé la possibilité d'entreprendre un essai logistique complexe de manière pragmatique.

Discovery of quorum quenchers targeting the membrane-embedded sensor domain of the Staphylococcus aureus receptor histidine kinase, AgrC.

We combined mRNA display technology with lipid-nanodisc based selections and identified high-affinity ligands targeting the integral membrane sensor domain of the histidine kinase AgrC as potent inhibitors of Staphylococcus aureus quorum sensing-modulated virulence. Our study highlights the potential of this integrated approach for identifying functional modulators of integral membrane proteins.

Pathogenicity Islands and Their Role in Staphylococcal Biology.

Pathogenicity islands are members of a vast collection of genomic islands that encode important virulence, antibiotic resistance and other accessory functions and have a critical role in bacterial gene transfer. Staphylococcus aureus is host to a large family of such islands, known as SaPIs, which encode super antigen and other virulence determinants, are mobilized by helper phages and transferred at extremely high frequencies. They benefit their host cells by interfering with phage predation and enhancing horizontal gene transfer. This chapter describes their life cycle, the bases of their phage interference mechanisms, their transfer system and their conversion to antibacterial agents for treatment ofstaphylococcal infections.

SUPERIOR SVG: no touch saphenous harvesting to improve patency following coronary bypass grafting (a multi-Centre randomized control trial, NCT01047449).

BACKGROUND: Single centre studies support No Touch (NT) saphenous vein graft (SVG) harvesting technique. The primary objective of the SUPERIOR SVG study was to determine whether NT versus conventional (CON) SVG harvesting was associated with improved SVG patency 1 year after coronary artery bypass grafting surgery (CABG). METHODS: Adults undergoing isolated CABG with at least 1 SVG were eligible. CT angiography was performed 1-year post CABG. Leg adverse events were assessed with a questionnaire. A systematic review was performed for published NT graft patency studies and results aggregated including the SUPERIOR study results. RESULTS: Two hundred and-fifty patients were randomized across 12-centres (NT 127 versus CON 123 patients). The primary outcome (study SVG occlusion or cardiovascular (CV) death) was not significantly different in NT versus CON (NT: 7/127 (5.5%), CON 13/123 (10.6%), p = 0.15). Similarly, the proportion of study SVGs with significant stenosis or total occlusion was not significantly different between groups (NT: 8/102 (7.8%), CON: 16/107 (15.0%), p = 0.11). Vein harvest site infection was more common in the NT patients 1 month postoperatively (23.3% vs 9.5%, p < 0.01). Including this study's results, in a meta-analysis, NT was associated with a significant reduction in SVG occlusion, Odds Ratio 0.49, 95% Confidence Interval 0.29-0.82, p = 0.007 in 3 randomized and 1 observational study at 1 year postoperatively. CONCLUSIONS: The NT technique was not associated with improved patency of SVGs at 1-year following CABG while early vein harvest infection was increased. The aggregated data is supportive of an important reduction of SVG occlusion at 1 year with NT harvesting. TRIAL REGISTRATION: NCT01047449 .

Optimizing Surgical Skills in Cardiac Surgery Residents with Cardiac Transplant in the High-Fidelity Porcine Model.

OBJECTIVES: Simulation is a pivotal tool within cardiac surgery to facilitate learner growth and skill acquisition. There are many methods of simulation and it is possible to develop and implement new curricula incorporating these modalities. The objective of this paper is to describe the feasibility of a high-fidelity cardiac transplant simulation curriculum for surgical residents. METHODS: Our simulation setting was the Animal Resource Center at the University of Calgary. It was set up with 4 separate operating rooms, 2 for donor heart retrievals and 2 for heart implantations. This was done to allow 2 learners to participate with each animal, replicating the true intraoperative environment. Our teaching sessions were facilitated by 2 surgeons experienced in cardiac transplantation. In addition, we had support staff including multiple perfusionists, nurses, and anesthesia technologists. RESULTS: The curriculum was evaluated from many perspectives in real time throughout the simulation as well as afterward in posttest qualitative interviews with all participants. The residents readily identified the acquisition of and increased proficiency in specifically targeted surgical skills. In addition, the residents were able to practice communication, collaboration, and management. Furthermore, the simulation session and our debriefings contributed significantly to fostering a team approach. CONCLUSIONS: The pig is an excellent preclinical model for acquiring and developing the skills necessary for human cardiac transplantation. The residents partaking in the curriculum were satisfied with the learning they received and saw value in the swine transplant curriculum. The overall curriculum was cost-effective, due to the low overall operating costs associated with it.

Identification of a Molecular Latch that Regulates Staphylococcal Virulence.

Virulence induction in the Staphylococcus aureus is under the control of a quorum sensing (QS) circuit encoded by the accessory gene regulator (agr) locus. Allelic variation within agr produces four QS specificity groups, each producing a unique secreted autoinducer peptide (AIP) and receptor histidine kinase (RHK), AgrC. Cognate AIP-AgrC interactions activate virulence through a two-component signaling cascade, whereas non-cognate pairs are generally inhibitory. Here we pinpoint a key hydrogen-bonding interaction within AgrC that acts as a switch to convert helical motions propagating from the receptor sensor domain into changes in inter-domain association within the kinase module. AgrC mutants lacking this interaction are constitutively active in vitro and in vivo, the latter leading to a pronounced attenuation of S. aureus biofilm formation. Thus, our work sheds light on the regulation of this biomedically important RHK.

Conversion of staphylococcal pathogenicity islands to CRISPR-carrying antibacterial agents that cure infections in mice.

Staphylococcus aureus and other staphylococci continue to cause life-threatening infections in both hospital and community settings. They have become increasingly resistant to antibiotics, especially ß-lactams and aminoglycosides, and their infections are now, in many cases, untreatable. Here we present a non-antibiotic, non-phage method of treating staphylococcal infections by engineering of the highly mobile staphylococcal pathogenicity islands (SaPIs). We replaced the SaPIs' toxin genes with antibacterial cargos to generate antibacterial drones (ABDs) that target the infecting bacteria in the animal host, express their cargo, kill or disarm the bacteria and thus abrogate the infection. Here we have constructed ABDs with either a CRISPR-Cas9 bactericidal or a CRISPR-dCas9 virulence-blocking module. We show that both ABDs block the development of a murine subcutaneous S. aureus abscess and that the bactericidal module rescues mice given a lethal dose of S. aureus intraperitoneally.

Staphylococcal pathogenicity islands-movers and shakers in the genomic firmament.

The staphylococcal pathogenicity islands (SaPIs) are highly mobile 15kb genomic islands that carry superantigen genes and other virulence factors and are mobilized by helper phages. Helper phages counteract the SaPI repressor to induce the SaPI replication cycle, resulting in encapsidation in phage like particles, enabling high frequency transfer. The SaPIs split from a protophage lineage in the distant past, have evolved a variety of novel and salient features, and have become an invaluable component of the staphylococcal genome. This review focuses on recent studies describing three different mechanisms of SaPI interference with helper phage reproduction and other studies demonstrating that helper phage mutations to resistance against this interference impact phage evolution. Also described are recent results showing that SaPIs contribute in a major way to lateral transfer of host genes as well as enabling their own transfer. SaPI-like elements, readily identifiable in the bacterial genome, are widespread throughout the Gram-positive cocci, though functionality has thus far been demonstrated for only a single one of these.