Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. IMPORTANCE: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Assembly and Annotation of Viral Metagenomes from Short-Read Sequencing Data.

Viral metagenomics enables the detection, characterization, and quantification of viral sequences present in shotgun-sequenced datasets of purified virus-like particles and whole metagenomes. Next generation sequencing (Illumina) derived short single or paired-end read runs are a principal platform for metagenomics, and assembly of short reads allows for the identification of distinguishing viral signatures and complex genomic features for taxonomy and functional annotation. Here we describe the identification and characterization of viral genome sequences, bacteriophages, and eukaryotic viruses, from a cohort of human stool samples, using multiple methods. Following the purification of virus-like particles, sequencing, quality refinement, and genome assembly, we begin the protocol with raw short reads deposited in an open-source nucleotide archive. We highlight the use of VIBRANT, an automated computational tool for the characterization of microbial viruses and their viral community function. Finally, we also describe an alternative assembly-free option of mapping reads to established databases of reference genomes and previously characterized metagenome-assembled viral genomes.

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium.

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis.

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages select for genetic alterations in medically relevant bacteria is important as phages become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages can be widely used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole-genome sequencing of E. faecalis following continuous exposure to these two phages revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome that was selected by both phages tested, demonstrating that infection by two genetically distinct phages selects for diverse variants. This knowledge serves as the basis for the continued study of E. faecalis genome evolution during phage infection and can be used to inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis.

Disruption of c-di-GMP Signaling Networks Unlocks Cryptic Expression of Secondary Metabolites during Biofilm Growth in Burkholderia pseudomallei.

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soilborne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at various temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypobiofilm- and hyperbiofilm-forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) 2, 11, 14 (syrbactin), and 15 (malleipeptin) in parental and ΔII2523 backgrounds also reveal the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under different environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions. IMPORTANCE Burkholderia pseudomallei is a saprophytic bacterium residing in the environment that switches to a pathogenic lifestyle during infection of a wide range of hosts. The environmental cues that serve as the stimulus to trigger this change are largely unknown. However, it is well established that the cellular level of c-di-GMP, a secondary signal messenger, controls the switch from growth as planktonic cells to growth as a biofilm. Disrupting the signaling mediated by c-di-GMP allows for a better understanding of the regulation and the contribution of the surface associated and secreted molecules that contribute to the various lifestyles of this organism. The genome of B. pseudomallei also encodes cryptic biosynthetic gene clusters predicted to encode small molecules that potentially contribute to growth as a biofilm, adaptation, and interactions with other organisms. A better understanding of the regulation of these molecules is crucial to understanding how this versatile pathogen alters its lifestyle.

The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic bacterium endemic throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei. We have previously shown that B. pseudomallei responds to nitrate and nitrite by inhibiting biofilm formation and altering cyclic di-GMP signaling. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei, we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of a conserved nitrate-sensing two-component system. We observed transcriptional downregulation in key components of the biofilm matrix in response to nitrate and nitrite. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, and an uncharacterized cryptic NRPS biosynthetic cluster. RNA expression patterns were reversed in ∆narX and ∆narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, ∆narX and ∆narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing contributes to survival in the host.

Individuals at risk for rheumatoid arthritis harbor differential intestinal bacteriophage communities with distinct metabolic potential.

Rheumatoid arthritis (RA) is an autoimmune disease characterized in seropositive individuals by the presence of anti-cyclic citrullinated protein (CCP) antibodies. RA is linked to the intestinal microbiota, yet the association of microbes with CCP serology and their contribution to RA is unclear. We describe intestinal phage communities of individuals at risk for developing RA, with or without anti-CCP antibodies, whose first-degree relatives have been diagnosed with RA. We show that at-risk individuals harbor intestinal phage compositions that diverge based on CCP serology, are dominated by Streptococcaceae, Bacteroidaceae, and Lachnospiraceae phages, and may originate from disparate ecosystems. These phages encode unique repertoires of auxiliary metabolic genes, which associate with anti-CCP status, suggesting that these phages directly influence the metabolic and immunomodulatory capability of the microbiota. This work sets the stage for the use of phages as preclinical biomarkers and provides insight into a possible microbial-based causation of RA disease development.

Fitness Trade-Offs Resulting from Bacteriophage Resistance Potentiate Synergistic Antibacterial Strategies.

Bacteria that cause life-threatening infections in humans are becoming increasingly difficult to treat. In some instances, this is due to intrinsic and acquired antibiotic resistance, indicating that new therapeutic approaches are needed to combat bacterial pathogens. There is renewed interest in utilizing viruses of bacteria known as bacteriophages (phages) as potential antibacterial therapeutics. However, critics suggest that similar to antibiotics, the development of phage-resistant bacteria will halt clinical phage therapy. Although the emergence of phage-resistant bacteria is likely inevitable, there is a growing body of literature showing that phage selective pressure promotes mutations in bacteria that allow them to subvert phage infection, but with a cost to their fitness. Such fitness trade-offs include reduced virulence, resensitization to antibiotics, and colonization defects. Resistance to phage nucleic acid entry, primarily via cell surface modifications, compromises bacterial fitness during antibiotic and host immune system pressure. In this minireview, we explore the mechanisms behind phage resistance in bacterial pathogens and the physiological consequences of acquiring phage resistance phenotypes. With this knowledge, it may be possible to use phages to alter bacterial populations, making them more tractable to current therapeutic strategies.

Complete Genome Sequence of Pandoraea pnomenusa TF-18, a Multidrug-Resistant Organism Isolated from the Rhizosphere of Rice (Oryza sativa L. subsp. japonica).

Pandoraea pnomenusa strain TF-18 was isolated from the roots of rice seedlings on selective medium containing four classes of antibiotics for isolation of Burkholderia pseudomallei Using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing technology, we report here a complete genome of 5,499,432 bases, a GC content of 64.8%, and 4,849 coding sequences.

Model of Chronic Equine Endometritis Involving a Pseudomonas aeruginosa Biofilm.

Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux-engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux-labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria (P < 0.05). A greater incidence (P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences (P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased (P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly (P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP.

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b.

Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of B. pseudomallei and improve diagnostics and therapeutic treatment strategies.

Clay Components in Soil Dictate Environmental Stability and Bioavailability of Cervid Prions in Mice.

Chronic wasting disease (CWD) affects cervids and is the only known prion disease to affect free-ranging wildlife populations. CWD spread continues unabated, and exact mechanisms of its seemingly facile spread among deer and elk across landscapes in North America remain elusive. Here we confirm that naturally contaminated soil contains infectious CWD prions that can be transmitted to susceptible model organisms. We show that smectite clay content of soil potentiates prion binding capacity of different soil types from CWD endemic and non-endemic areas, likely contributing to environmental stability of bound prions. The smectite clay montmorillonite (Mte) increased prion retention and bioavailability in vivo. Trafficking experiments in live animals fed bound and unbound prions showed that mice retained significantly more Mte-bound than unbound prions. Mte promoted rapid uptake of prions from the stomach to the intestines via enterocytes and M cells, and then to macrophages and eventually CD21+ B cells in Peyer's patches and spleens. These results confirm clay components in soil as an important vector in CWD transmission at both environmental and organismal levels.

The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD.

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.