Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Virome profiling of fig wasps (Ceratosolen spp.) reveals virus diversity spanning four realms.

We investigated the virome of agaonid fig wasps (Ceratosolen spp.) inside syconia ("fruits") of various Ficus trees fed upon by frugivores such as pteropodid bats in Sub-Saharan Africa. This virome includes representatives of viral families spanning four realms and includes near-complete genome sequences of three novel viruses and fragments of five additional potentially novel viruses evolutionarily associated with insects, fungi, plants, and vertebrates. Our study provides evidence that frugivorous animals are exposed to a plethora of viruses by coincidental consumption of fig wasps, which are obligate pollinators of figs worldwide.

Genomic Characterization of a Relative of Mumps Virus in Lesser Dawn Bats of Southeast Asia.

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats (Eonycteris spelaea). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

Comparative genomics of Acinetobacter baumannii and therapeutic bacteriophages from a patient undergoing phage therapy.

In 2016, a 68-year-old patient with a disseminated multidrug-resistant Acinetobacter baumannii infection was successfully treated using lytic bacteriophages. Here we report the genomes of the nine phages used for treatment and three strains of A. baumannii isolated prior to and during treatment. The phages used in the initial treatment are related, T4-like myophages. Analysis of 19 A. baumannii isolates collected before and during phage treatment shows that resistance to the T4-like phages appeared two days following the start of treatment. We generate complete genomic sequences for three A. baumannii strains (TP1, TP2 and TP3) collected before and during treatment, supporting a clonal relationship. Furthermore, we use strain TP1 to select for increased resistance to five of the phages in vitro, and identify mutations that are also found in phage-insensitive isolates TP2 and TP3 (which evolved in vivo during phage treatment). These results support that in vitro investigations can produce results that are relevant to the in vivo environment.

Corrigendum: Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

[This corrects the article DOI: 10.3389/fmicb.2018.02664.].

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats (Eonycteris spelaea) in Singapore.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection.

Bacterial infections pose a challenge to human health and burden the health care system, especially with the spread of antibiotic-resistant populations. To provide effective treatment and improved prognosis, effective diagnostic methods are of great importance. Here we present phage-mediated molecular detection (PMMD) as a novel molecular method for the detection and assessment of bacterial antibiotic resistance. This technique consists of a brief incubation, of approximately ten minutes, of the biological sample with a natural bacteriophage (phage) targeting the bacteria of interest. This is followed by total RNA extraction and RT-PCR. We applied this approach to Staphylococcus aureus (SA), a major causative agent of human bacterial infections. PMMD demonstrated a high sensitivity, rapid implementation, and specificity dependent on the phage host range. Moreover, due to the dependence of the signal on the physiological state of the bacteria, PMMD can discriminate methicillin-sensitive from methicillin-resistant SA (MSSA vs. MRSA). Finally, we extended this method to the detection and antibiotic sensitivity determination of other bacteria by proving PMMD efficacy for Bacillusanthracis.

Microbial Dysbiosis During Simian Immunodeficiency Virus Infection is Partially Reverted with Combination Anti-retroviral Therapy.

Human immunodeficiency virus (HIV) infection is characterized by a massive loss of CD4 T cells in the gastrointestinal tract (GIT) that is accompanied by changes in the gut microbiome and microbial translocation that contribute to inflammation and chronic immune activation. Though highly active antiretroviral therapy (HAART) has led to better long-term outcomes in HIV infected patients, it has not been as effective at reverting pathogenesis in the GIT. Using the simian immunodeficiency virus (SIV) infection model, we show that combination antiretroviral therapy (c-ART) partially reverted microbial dysbiosis observed during SIV infection. Though the relative abundance of bacteria, their richness or diversity did not significantly differ between infected and treated animals, microbial dysbiosis was evident via multiple beta diversity metrics: Jaccard similarity coefficient, Bray-Curtis similarity coefficient, and Yue & Clayton theta similarity coefficient. Principal coordinates analysis (PCoA) clustered SIV-infected untreated animals away from healthy and treated animals that were clustered closely, indicating that c-ART partially reversed the gut dysbiosis associated with SIV infection. Metastats analysis identified specific operational taxonomic units (OTUs) falling within the Streptococcus, Prevotella, Acinetobacter, Treponema, and Lactobacillus genera that were differentially represented across the three groups. Our results suggest that complete viral suppression with c-ART could potentially revert microbial dysbiosis observed during SIV and HIV infections.

The temporal RNA virome patterns of a lesser dawn bat (Eonycteris spelaea) colony revealed by deep sequencing.

The virosphere is largely unexplored and the majority of viruses are yet to be represented in public sequence databases. Bats are rich reservoirs of viruses, including several zoonoses. In this study, high throughput sequencing (HTS) of viral RNA extracted from swabs of four body sites per bat per timepoint is used to characterize the virome through a longitudinal study of a captive colony of fruit nectar bats, species Eonycteris spelaea in Singapore. Through unbiased shotgun and target enrichment sequencing, we identify both known and previously unknown viruses of zoonotic relevance and define the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. To our knowledge, this is the first study that combines probe-based viral enrichment with HTS to create a viral profile from multiple swab sites on individual bats and their cohort. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Given the known risk for bat-human zoonoses, a more complete understanding of the viral dynamics in South-eastern Asian bats has significant implications for disease prevention and control. The findings of this study will be of interest to U.S. Department of Defense personnel stationed in the Asia-Pacific region and regional public health laboratories engaged in emerging infectious disease surveillance efforts.

Early clinical experience of bacteriophage therapy in 3 lung transplant recipients.

Bacteriophage therapy (BT) uses bacteriophages to treat pathogenic bacteria and is an emerging strategy against multidrug-resistant (MDR) infections. Experience in solid organ transplant is limited. We describe BT in 3 lung transplant recipients (LTR) with life-threatening MDR infections caused by Pseudomonas aeruginosa (n = 2) and Burkholderia dolosa (n = 1). For each patient, lytic bacteriophages were selected against their bacterial isolates. BT was administered for variable durations under emergency Investigational New Drug applications and with patient informed consent. Safety was assessed using clinical/laboratory parameters and observed clinical improvements described, as appropriate. All patients received concurrent antibiotics. Two ventilator-dependent LTR with large airway complications and refractory MDR P. aeruginosa pneumonia received BT. Both responded clinically and were discharged from the hospital off ventilator support. A third patient had recurrent B. dolosa infection following transplant. Following BT initiation, consolidative opacities improved and ventilator weaning was begun. However, infection relapsed on BT and the patient died. No BT-related adverse events were identified in the 3 cases. BT was well tolerated and associated with clinical improvement in LTRs with MDR bacterial infection not responsive to antibiotics alone. BT may be a viable adjunct to antibiotics for patients with MDR infections.

multiPhATE: bioinformatics pipeline for functional annotation of phage isolates.

SUMMARY: To address the need for improved phage annotation tools that scale, we created an automated throughput annotation pipeline: multiple-genome Phage Annotation Toolkit and Evaluator (multiPhATE). multiPhATE is a throughput pipeline driver that invokes an annotation pipeline (PhATE) across a user-specified set of phage genomes. This tool incorporates a de novo phage gene calling algorithm and assigns putative functions to gene calls using protein-, virus- and phage-centric databases. multiPhATE's modular construction allows the user to implement all or any portion of the analyses by acquiring local instances of the desired databases and specifying the desired analyses in a configuration file. We demonstrate multiPhATE by annotating two newly sequenced Yersinia pestis phage genomes. Within multiPhATE, the PhATE processing pipeline can be readily implemented across multiple processors, making it adaptable for throughput sequencing projects. Software documentation assists the user in configuring the system. AVAILABILITY AND IMPLEMENTATION: multiPhATE was implemented in Python 3.7, and runs as a command-line code under Linux or Unix. multiPhATE is freely available under an open-source BSD3 license from https://github.com/carolzhou/multiPhATE. Instructions for acquiring the databases and third-party codes used by multiPhATE are included in the distribution README file. Users may report bugs by submitting to the github issues page associated with the multiPhATE distribution. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples.

BACKGROUND: Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing. RESULTS: This study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage. CONCLUSIONS: Hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations.

Genomic epidemiology of MRSA infection and colonization isolates among military trainees with skin and soft tissue infection.

PURPOSE: Individuals with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection (SSTI) can be simultaneously colonized with MRSA on multiple body sites. Using whole genome sequencing (WGS), the intrahost relatedness of MRSA colonization and infection isolates was investigated. METHODS: In the context of a prospective case-control study of SSTI, we analyzed colonization and infection isolates from US Army Infantry trainees with purulent infection due to MRSA. At the time of clinical presentation for SSTI, culture swabs were obtained from the infection site, as well as from the patient's nasal, oral, inguinal, and perianal regions. S. aureus culture and susceptibility was performed by standard methods. DNA from MRSA isolates was extracted and libraries were produced. Sequences were generated on an Illumina MiSeq, sequence reads were assembled, and single nucleotide variant (SNV) data were analyzed. RESULTS: Of 74 trainees with MRSA SSTI, 19 (25.7%) were colonized with MRSA. Ten (52.6%) were colonized on more than one body site. Colonization frequency by anatomic site was as follows: inguinal region (33%), nasal region (30%), perianal region (22%), and oral region (14%). A total of 36 MRSA colonization isolates were characterized. The intrahost median number of SNVs between infection and colonization isolates was 17. Among trainees with recurrent MRSA SSTI, limited intrahost diversity suggests that persistent colonization is a major contributor to recurrence risk. CONCLUSIONS: Among military trainees with MRSA SSTI, genomic characterization of infection and colonization isolates revealed a high degree of strain relatedness. Single acquisition events may account for MRSA colonization and infection in this population.

High-Quality Draft Genome Sequence of Pseudomonas aeruginosa 268 Isolated from a Patient with a Left Ventricular Assist Device.

Pseudomonas aeruginosa is known to cause persistent bloodstream infections associated with left ventricular assist devices (LVAD). Here, we present the high-quality draft genome assembly for a clinical isolate, P. aeruginosa 268. The genome sequence is available in GenBank under accession number CP032761.

Conjugative Transfer of a Novel Staphylococcal Plasmid Encoding the Biocide Resistance Gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate.

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

Phage Therapy for a Multidrug-Resistant Acinetobacter baumannii Craniectomy Site Infection.

In the era of antibiotic resistance, alternative treatment options for multidrug-resistant bacterial infections are being explored. We present a case of multidrug-resistant Acinetobacter baumannii infection treated with bacteriophages. Clinical trials are needed to further investigate bacteriophage therapy as an option to treat multidrug-resistant bacterial infections.

Characterizing Phage Genomes for Therapeutic Applications.

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.

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 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.