I attended the 6th Annual Meeting of the Boston Area Resistance Network Meeting this past Friday. A fabulous meeting. Kudos to the organizers. I was particularly riveted by a presentation from Erin Duffy, CSO, of Melinta Pharmaceuticals: "On the design and optimization of pyrrolocytosines." It was a beautiful example of structure-guided design. Melinta was originally called Rib-X Pharmaceuticals. Rib-X was founded on the idea that new drugs could be developed based on the structure of the 50S ribosome (therefore Rib-X) solved by Tom Steitz and Peter Moore. A series of compounds were synthesized to explore a binding site in the 50S ribosome that were not known to be engaged by existing natural products. Therefore, ribosome modifying enzymes conferring resistance through modification of this binding site would presumably not have previously evolved. A point was made of exploring the empty space within the ribosome. I don't remember all of the details, but it went along the lines of by compound 500 had great Gram-positive activity; by compound 1000 had great Gram-negative and Gram-positive activity inclusive of Acinetobacter and Pseudomonas, Interestingly, the molecular characteristics associated with Gram-negative penetrance did not follow eNTRy rules. By compound 2000, toxicity issues had been worked out inclusive of mammalian testing. So seemingly a really promising first in class agent. The final slide for this impressive development effort was a picture of the research group and then a comment that they were all let go the previously week. The company redirected all of its efforts to sales for approved agents and pulled out of discovery efforts. This follows on Achaogen eliminating their discovery program after approval of plazomicin, which follows on Novartis exiting the antibiotic development space. Disconcerting juxtaposition of success and abandonment.
New antibiotics that may offer additional and much needed treatment options will only be used in hospital systems if the clinical microbiology laboratory can provide timely antimicrobial susceptibility testing data. Historically there has been a time lag in the availability of susceptibility testing methods either at reference laboratories (long delay to results), incorporation in automated commercial systems (4 years), or simpler methods like disk diffusion and gradient strips that can be performed manually on an as needed basis. I was excited to learn that delafloxacin and meropenem/vaborbactam disk diffusion and gradient strip methods finally became available. Fantastic. However, before we can introduce those methods in the clinical laboratory, we need to validate performance of these methods per CLIA '88 regulations and good laboratory practice. This requires either comparing the new methods to a reference standard (broth microdilution -- need antibiotic powder, and a lot of set up time) or a set of strains that has been previously characterized by a reference dilution method and has a good representation of susceptible and resistant isolates. Those are serious roadblocks. By chance, I happened to give a talk at the Northeast Branch of the American Society of Microbiology and a someone from Melinta Pharmaceuticals happened to be there, and that someone referred me to our local Key Account Manager who offerred a solution "on request". Specifically, Melinta or other pharmaceutical companies are not allowed to approach me and tell me about a solution, but if I inquire independently and ask for a previously characterized set of bacterial isolates, they are allowed to tell me that in fact they have a series of previously characterized isolates for both drugs available for validation. These isolates are provided free of charge from Laboratory Specialist, Inc, Sent by FEDEX with first class documentation, data for broth microdilution performed circa ten times on each isolates with modal MICs and MIC distribution - wow, that is awesome. Methods validated beautifully. It just seems odd to me that such a valuable resource, providing clinical laboratories the ability to robustly evaluate AST methods for newly marketed drugs, needs to remain on a need to know basis. Clinical labs, please take note of this available resource. The same proved to be true for plazomicin. Achaogen uses the same Laboratory Specialists, Inc. to provide 30 characterized isolates on request; however, the FEDEX chargers are not absorbed. The FDA-CDC Biobank also now has a set of isolates characterized for plazomicin susceptibility and I see now also delafloxacin., I suspect the exact same set of organisms. The existing rule restricting such company-clinical laboratory communication about these important resources should be relaxed! We need facile access to validation strains sets to bring new testing into our laboratories in a timely fashion, to facilitate the availability of new antibiotics when they are most needed, and to help support profitability of antibiotic development by pharmaceutical companies.
JoVE manuscript accepted on use of Inkjet Printing to perform antimicrobial susceptibility testing for single and drug combinations and follow up time-kill study methodology
Postdoctoral fellow, Thea Brennan-Krohn, recently had a manuscript accepted in the Journal of Visualized Experimentation, aka JoVE. The title of the manuscript and link to the abstract are "Antimicrobial Synergy Testing by the Inkjet Printer-Assisted Automated Checkerboard Array and the Manual Time-Kill Method." We have been fielding a lot of questions over the past two years about implementation of inkjet printing antimicrobial susceptibility testing technology and thought it would be useful to share a video of the technique as well as classic time-kill analysis to analyze antimicrobial synergy. We are excited to learn that the CDC has decided to implement the technology in the near future in their Antimicrobial Resistance Laboratory Network (ARLN), initially to test, the combination of ceftazidime-avibactam and aztreonam for activity against multidrug-resistant Gram-negatives.
Postdoctoral fellow KP Smith discusses how to use "time to positivity" information in interpreting the meaning of blood cultures. His blog article is titled: "Predictive Value of Blood Culture Time to Positivity."
We have been taking advantage of Addgene for years. About to deposit about twenty plasmids form Yoon-Suk Kang's bioreporter tool kit. Assembling plasmid maps in SnapGene. Thank you, Addgene!
Apramycin scaffold exploration using novel glycochemistry and SAR studies to enhance activity against Acinetobacter baumannii and other multidrug-resistant Gram-negative pathogens.
KP Smith Speaking tomorrow at Harvard Antimicrobial Resistance Laboratory Network as well as Broad Institute collaborator, Alejandro Pironti
Congratulations to postdoctoral fellow, Yoon-Suk Kang, on acceptance of his manuscript in Infection and Immunity. The manuscript is titled: "Brucella neotomae recapitulates attributes of zoonotic human disease in a murine infection model." One of my favorite figures are images of tdTomato-labeled Brucella detected by confocal microscopy replicating in the middle of a liver granulomas visualized by counterstaining with H&E. In contrast to wild type organisms, virB4 mutant bacteria are found as single cells in sinusoids presumably within Kuffpfer cells. Wild type bacteria are found at high levels in mouse thymus at all time points examined; virB4 mutant bacteria are undetectable in thymus. B. neotomae induces a Th-1 immune response. More information in the manuscript.
Excel and Powerpoint have been banned for figure preparation. Converted my lab over to GraphPad Prism. A very well designed graphing and statistics program. Love several features including the ability to apply standard dimension formats for charts, and wand tool to covert charts to exact format used previously. Application of statistics to data is facile, also a wand tool to apply the same statistics to multiple tables. Grateful to Assay and Drug Development Technology for their requirement (recommendation?) to use this or similar programs in figure preparation. Powerpoint never worked well for figure preparation and conversion to vector-graphics format, for use with illustrator was a nightmare. Now we just start out using a freeware vector graphics program or Adobe Illustrator directly. Greatly speeds up manuscript preparation.
P.S. I have no financial relationship with GraphPad Prism, but it is a good product so therefore my endorsement!
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