Robert K. Pope

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

ABSTRACT 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 patients 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.

Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

Characterization of novel Staphylococcus aureus lytic phage and defining their combinatorial virulence using the OmniLog® system

ABSTRACT Skin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70–91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires β-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections.

Inactivation and ultrastructure analysis of Bacillus spp. and Clostridium perfringens spores.

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

The Use of Scanning Electron Microscopy for the Analysis of Bacteriophage Binding to Acinetobacter baumanii

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].

Inactivation of Bacterial Endospores in an Artificial Tissue for Electron Microscopy Analysis

Removal of tissue samples that contain, or potentially contain, pathogenic organisms from containment laboratories for electron microscopy analysis poses unique challenges. While the extraction of nucleic acids or proteins for molecular biology or immunology analysis is fairly straightforward, these methods of extraction destroy ultrastructure, and are not suitable for samples intended for microscopy. Fixation of tissue samples containing pathogenic organisms traditionally requires several days to inactivate the organisms. Furthermore, sterility testing for pathogenic organisms can take from 2-21 days, depending on the organism. This lengthy fixation with sterility testing is not feasible for the rapid turnaround required for forensic samples. Previously, data demonstrated that treatment of bacterial endospores with 4% paraformaldehyde/1% glutaraldehyde for 240 minutes inactivates most bacterial endospores. [1]

Non Spore-Forming Bacteria: Sterility and Ultrastructure Study

Live organisms from containment laboratories must be inactivated with a valid protocol, or subsequently sterility tested prior to removal from containment and worked with in a lower level of containment. For bioforensic analysis, many specimens that are imaged using electron microscopy (EM) come from BSL2, BSL-3 and BSL-4 containment laboratories. Current literature documents many procedures for fixation of pathogenic organisms, but very few actually document sterility after fixation. For safety and bioforensic analysis, it has been necessary to identify the minimum times required to inactivate vegetative bacteria while preserving ultrastructure for EM. Recent information from this lab demonstrates that most bacterial endospores are inactivated in less than 240 minutes in Universal fixative (4% paraformaldehyde/1% glutaraldehyde (Brantner, et al. in press). Because spores are the most resistant form of bacteria, the data was useful in developing timelines for the treatment of vegetative cells. It has been considered necessary to treat samples containing pathogenic bacteria for periods up to 21 days for inactivation. In addition to potentially causing damage to the ultrastructure of samples, this prolonged fixation period increases the time required to process bioforensic samples. There are many protocols in the literature for inactivating bacterial samples, but they are either of long duration, or utilize harsh chemicals that destroy ultrastructure for subsequent analysis. This study demonstrates that all vegetative bacterial species, resuspended in fixative and tested, were successfully inactivated in 5 minutes. To standardize the laboratory protocols, the same samples were tested for 5, 15, 30, 60, 120, and 240 minutes.