A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. While phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates, and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat Vancomycin Resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.

Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform.

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of ∼24 µM.

Pearls and pitfalls of imaging metastatic disease from pancreatic adenocarcinoma: a systematic review.

Pancreatic adenocarcinoma is a systemic disease due to the presence of metastatic disease at the time of diagnosis and local recurrence as well as distant metastatic disease after treatment in a majority of patients. Recognition of these metastatic sites may help in accurate staging and assessment of therapeutic response. The authors discuss and illustrate imaging findings of metastatic disease from pancreatic adenocarcinoma in different organ systems with emphasis on entities that can mimic metastatic pancreatic cancer.

Hand assisted laparoscopic radical nephrectomy for renal cell carcinoma with inferior vena caval thrombus.

PURPOSE: To our knowledge we present the initial clinical report of hand assisted laparoscopic radical nephrectomy for renal cell carcinoma with tumor thrombus extending into the inferior vena cava. MATERIALS AND METHODS: A 76-year-old man was referred to our medical center with a 12.5 x 10 cm. stage T3b right renal tumor extending into the inferior vena cava. The caval thrombus was limited and completely below the level of the hepatic veins. After preoperative renal embolization via the hand assisted transperitoneal approach the right kidney was completely dissected with the renal hilum. Proximal and distal control of the inferior vena cava was obtained with vessel loops and a single lumbar vein was divided between clips. An endoscopic Satinsky vascular clamp was placed on the inferior vena cava just beyond its juncture with the right renal vein, thereby, encompassing the caval thrombus. The inferior vena cava was opened above the Satinsky clamp and a cuff of the inferior vena cava was removed contiguous with the renal vein. The inferior vena cava was repaired with continuous 4-zero vascular polypropylene suture and the Satinsky clamp was then removed. A literature search failed to reveal any similar reports of laparoscopic radical nephrectomy for stage T3b renal cell cancer. RESULTS: Surgery was completed without complication with an estimated 500 cc blood loss. Pathological testing confirmed stage T3b grade 3 renal adenocarcinoma with negative inferior vena caval and soft tissue margins. CONCLUSIONS: The introduction of vascular laparoscopic instrumentation and the hand assisted approach enabled us to extend the indications for laparoscopic radical nephrectomy to patients with minimal inferior venal caval involvement.

Management of the postpubertal patient with cryptorchidism: an updated analysis.

PURPOSE: Management of the postpubertal cryptorchid testis depends on patient age at presentation. Based on the belief that the risk of death from surgery first exceeds the risk of death from testis cancer at age 32 years patients younger than 32 years are advised to undergo orchiectomy, while those older than 32 years are advised to remain under close observation. However, the data on which this recommendation is based are now a quarter-century old. During this interval significant improvements have been made in perioperative care and germ cell tumor therapy. We revisited the topic using contemporary data to determine whether and how recommendations on management of the postpubertal cryptorchid testis should be changed. MATERIALS AND METHODS: Contemporary data on germ cell mortality in the United States were obtained from the National Center for Health Statistics. From these data the lifetime risk of death from germ cell cancer in the general population was calculated for each 5-year interval between ages 15 and 60 years. Since the lifetime risk of germ cell tumor is believed to be higher in patients with cryptorchidism than in the general population, the lifetime risk of eventual death from germ cell tumor in the cryptorchid population was calculated by multiplying each 5-year lifetime risk by 9.7, which is the generally accepted relative risk of germ cell tumor in a cryptorchid testis. Contemporary literature on perioperative mortality was reviewed and we estimated the current mortality of orchiectomy based on American Society of Anesthesiologists (ASA) class. Mortality rates were plotted to determine the age when operative mortality exceeds the risk of mortality from germ cell malignancy. RESULTS: While perioperative mortality and germ cell neoplasia mortality decreased in the last 25 years, the relative decrease in perioperative mortality was significantly greater. Thus, in ASA class I or II cases mortality from orchiectomy began to exceed mortality from germ cell cancer at age 50 years. CONCLUSIONS: Improvements in therapy for germ cell neoplasia and perioperative care in the last 25 years have dramatically decreased the mortality of each cause. However, the decrease in perioperative mortality has been greater. In contrast to a generation ago, accidental death during routine elective surgery is now extremely rare in healthy patients. Thus, we advocate orchiectomy in all healthy males (ASA I and II) who present with postpubertal cryptorchidism until age 50 years.