Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

• Premise of the study: Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• Methods: The fi rst 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• Key results: Morphometric comparison between Arizona-and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identifi ed a previously undefi ned cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• Conclusions: Our B73-specifi c model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis , barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize.

Erratum for Schooley et al., "Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection." Antimicrob Agents Chemother 62:e02221-18.

Public health and decontamination decisions following an event that causes indoor contamination with a biological agent require knowledge of the environmental persistence of the agent. The goals of this study were to develop methods for experimentally depositing bacteria onto indoor surfaces via aerosol, evaluate methods for sampling and enumerating the agent on surfaces, and use these methods to determine bacterial surface decay. A specialized aerosol deposition chamber was constructed, and methods were established for reproducible and uniform aerosol deposition of bacteria onto four coupon types. The deposition chamber facilitated the control of relative humidity (RH; 10 to 70%) following particle deposition to mimic the conditions of indoor environments, as RH is not controlled by standard heating, ventilation, and air conditioning (HVAC) systems. Extraction and culture-based enumeration methods to quantify the viable bacteria on coupons were shown to be highly sensitive and reproducible. To demonstrate the usefulness of the system for decay studies, Yersinia pestis persistence as a function of surface type at 21°C and 40% RH was determined to be >40%/min for all surfaces. Based upon these results, at typical indoor temperature and RH, a 6-log reduction in titer would expected to be achieved within 1 h as the result of environmental decay on surfaces without active decontamination. The developed approach will facilitate future persistence and decontamination studies with a broad range of biological agents and surfaces, providing agent decay data to inform both assessments of risk to personnel entering a contaminated site and decontamination decisions following biological contamination of an indoor environment. IMPORTANCEPublic health and decontamination decisions following contamination of an indoor environment with a biological agent require knowledge of the environmental persistence of the agent. Previous studies on Y. pestis persistence have utilized large liquid droplet deposition to provide persistence data. As a result, methods were developed to deposit aerosols containing bacteria onto indoor surfaces, reproducibly enumerate bacteria harvested from coupons, and determine surface decay utilizing Y. pestis. The results of this study provide foundational methods required to evaluate surface decay of bacteria and potentially other biological agents, such as viruses, in aerosol particles as a function of surface type and environment. Integrating the data from both aerosol and liquid deposition surface decay studies will provide medical and public health personnel with a more complete understanding of agent persistence on surfaces in contaminated areas for assessment of health risks and to inform decontamination decisions.

No abstract

Scanning Electron Microscopy (SEM) was used to ascertain the binding of bacteriophage (phage) to clinically relevant A. baumannii isolates. The use of phage for successful treatment of pathogenic bacterial infections in conjunction with antibiotics has been documented [1]. The current study examines bacteriophage samples from the US Navy phage library that were previously screened for the ability to inhibit the growth of A. baumannii. The phage (AB-Navy1, AB-Navy4, AB-Navy71, and ABNavy97 from the Myoviridae Family, and AbTP31 from the Podoviridae Family) were examined for their ability to bind to the surface of A. baumannii. These phage were used in conjunction with the three clinical isolates of A. baumannii TP1, TP2, and TP3 [1, 2]. The clinical isolates were taken at different time points during the infection [1].The objective of this study was to determine the binding of specific phage to different clinical isolates of A. baumannii. Figure 1 (A-C) demonstrates that the size, shape and surface texture of uninfected TP1, TP2 and TP3 were similar in appearance. While the concentration of each phage was similar upon infection, there is a large diversity in the number of phage seen bound to the surface of the bacteria. The binding of AB-Navy1 (D-F) and AB-Navy71 (M-O) is high for TP1, but becomes reduced for TP2 and selective for TP3. The binding levels for AB-Navy4 (G-I), AbTP31 (J-L) and AB-Navy97 (P-R) remain relatively consistent for TP1, TP2 and TP3. Although the binding remains consistent, previous data demonstrate that TP3 is resistant to lysis by AB-Navy4 and ABNavy97 [1]. The examination of the TP1 column shows that AB-Navy1, AB-Navy4, AB-Navy71, and AB-Navy97 (D, G, M, and P respectively) bound to TP1 with high efficiency. While the binding efficiency of AbTP31 was significantly lower, this specific phage was previously shown to cause the highest level of growth inhibition of TP1, TP2, and TP3 [2]. References:[1] Schooley RT et al, Antimicrobial Agents and Chemotherapy 61(10) (2017), p. e00954.[2] All bacteria and phage were supplied by the Navy

The use of standards and the development of validated methods of sample processing and analysis for microscopy increases the repeatability, reliability, consistency, and overall quality of output. ISO 17025 accreditation ensures that all personnel are competent to perform assays, all assays are validated to perform properly in the specific space accredited, and that the laboratory as a whole is proficient in the performance of the assay. Additionally, ISO 17025 accreditation ensures that critical supplies are from approved vendors and equipment is maintained properly. This includes the use of traceable ASTM standards, quality controls and a quality management system [1]. ISO 9001 is a set of standards and guidelines for good quality management practices, while ISO 17025 goes beyond the ISO 9001 standard to include additional mechanisms for quality control, document control, quality analysis, and trending of data. The National Bioforensic Analysis Center's bioforensic electron microscopy laboratory is ISO 17025 accredited to analyze samples using Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive X-ray Spectroscopy (EDS). When used properly, the inclusion of standards and approved methods provide additional layers of quality to increase the validity of the data in law enforcement court proceedings.

This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process.