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

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including 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 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 isolate from the patient.

BLASTPLOT: a PERL module to plot next generation sequencing NCBI-BLAST results.

Background: The development of Next Generation Sequencing (NGS) during the last decade has created an unprecedented amount of sequencing data, as well as the ability to rapidly sequence specimens of interest. Read-based BLAST analysis of NGS data is a common procedure especially in the case of metagenomic samples. However, coverage is usually not enough to allow for de novo assembly.

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood.

Background: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks.

Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption.

Background: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix.

Methods: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection.

Results: Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB.

Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis.

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.

Genetic variation and linkage disequilibrium in Bacillus anthracis.

We performed whole-genome amplification followed by hybridization of custom-designed resequencing arrays to resequence 303 kb of genomic sequence from a worldwide panel of 39 Bacillus anthracis strains. We used an efficient algorithm contained within a custom software program, UniqueMER, to identify and mask repetitive sequences on the resequencing array to reduce false-positive identification of genetic variation, which can arise from cross-hybridization. We discovered a total of 240 single nucleotide variants (SNVs) and showed that B.

The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax.

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses.

Arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing.

Background: Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown.

High-redundancy draft sequencing of 15 clinical and environmental Burkholderia strains.

The Gram-negative Burkholderia genus includes several species of intracellular bacterial pathogens that pose substantial risk to humans. In this study, we have generated draft genome sequences of 15 strains of B. oklahomensis, B.

Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.

Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e.

In vitro analysis of acetalated dextran microparticles as a potent delivery platform for vaccine adjuvants.

Toll-like receptor (TLR) agonists induce potent innate immune responses and can be used in the development of novel vaccine adjuvants. However, access to TLRs can be challenging as exemplified by TLR 7, which is located intracellularly in endosomal compartments. To increase recognition and subsequent stimulatory effects of TLR 7, imiquimod was encapsulated in acetalated dextran (Ac-DEX) microparticles.

Genomic characterization of the Yersinia genus.

Background: New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.

Molecular characterization of a variant of Bacillus anthracis-specific phage AP50 with improved bacteriolytic activity.

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.

Recovery efficiencies of anthrax spores and ricin from nonporous or nonabsorbent and porous or absorbent surfaces by a variety of sampling methods*.

The 2001 anthrax letter cases brought into focus the need to establish the most effective environmental sampling procedures. Results are presented from two studies aimed at establishing the best procedures for everyday surfaces likely to be contaminated after the release of environmentally stable bioaggressive agents, as exemplified by anthrax spores and ricin. With anthrax spores, contact plates, with mean retrieval rates of 28-54%, performed better than other methods by a wide margin for flat nonporous, nonabsorbent surfaces.

Genotyping of Bacillus cereus strains by microarray-based resequencing.

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases.

Analysis of peptide mimotopes of Burkholderia pseudomallei exopolysaccharide.

Previously two capsule-specific monoclonal antibodies (4VA5 and 3VIE5) were identified as protective against Burkholderia pseudomallei in passive transfer experiments. Panning these antibodies against evolutionary phage libraries identified reactive peptides capable of inhibiting its parent monoclonal from binding to B. pseudomallei.

Immune response to two different dosing schedules of the anthrax vaccine precipitated (AVP) vaccine.

A pilot study compared the immune response of regular (0, 3, 6, 32 weeks) and extended (0, 10, 13, 32 weeks) schedules of the UK anthrax vaccine (anthrax vaccine precipitated, AVP). Concentrations of antibodies to protective antigen (PA) were higher (p<0.05) among recipients of the extended (n=7) versus regular schedule (n=6) at week 32, and 2 weeks after the second and third vaccinations.

The US capitol bioterrorism anthrax exposures: clinical epidemiological and immunological characteristics.

Background: Bioterrorism-related anthrax exposures occurred at the US Capitol in 2001. Exposed individuals received antibiotics and anthrax vaccine adsorbed immunization.

Methods: A prospective longitudinal study of 124 subjects--stratified on the basis of spore exposure, nasopharyngeal culture results, and immunization status from inside and outside an epidemiologically defined exposure zone--was performed to describe clinical outcome and immune responses after Bacillus anthracis exposure.

The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages.

Background: Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages.

Microarray-based resequencing of multiple Bacillus anthracis isolates.

We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.

A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores.

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA).

Specificity of an immunochromatographic test for anthrax.

Objective: To evaluate the specificity of an immunochromatographic test (ICT) for anthrax in cattle.

Design: A comparison of an ICT with blood smear and culture in uninfected cattle.

Procedure: Two hundred and forty blood samples were collected from dead cattle at two knackeries within Victoria and tested on-site with an ICT for the detection of protective antigen (PA) of Bacillus anthracis.

Genetic immunization against anthrax.

The objective of this study was to determine whether a DNA prime-protein boost immunization against the Bacillus anthracis protective antigen (PA) and lethal factor (LF) antigens could induce a protective immune response against significant aerosol challenge in the rabbit model. Rabbits were vaccinated with different regimens of DNA vaccines (Table 1) and aerosol challenged with B. anthracis spores, Ames strain, with an average dose of 50 LD(50s) with a range from 18 to 169 LD(50s.