Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

IMPORTANCE: Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

First Report of Bacterial Leaf Scald of Plum Caused by Xylella fastidiosa in Texas.

Xylella fastidiosa is the etiological agent of Plum Leaf Scald (Greco et al. 2021). The disease was first reported in Argentina (Fernandez-Valiela et al. 1954) and then Brazil and Paraguay (French et al. 1978). In the USA, Plum Leaf Scald has been reported in the Southeastern United States (Wells et al. 1981a) and California (Hernandez-Martinez et al. 2009). In August 2021, during the Stone Fruit Survey of FY2020, plum trees (Mexican variety, Prunus mexicana) with symptoms of leaf scald, were observed in a Central Texas orchard with approximately 7% of trees exhibiting symptoms. Leaf margins were asymmetrically scorched, with necrotic areas that transitioned into chlorotic and healthy green tissues. To detect the presence of the pathogen, leaf sample petioles were tested using a double-antibody sandwich (DAS) ELISA® with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) according to manufacturer's guidelines. X. fastidiosa was detected in 20 of the 35 symptomatic samples. To confirm ELISA results, total DNA was extracted from the plant samples using the Plant DNeasy® kit (Qiagen Co. Hilden, Germany) following the manufacturer's protocol. All 20 ELISA-positive samples tested positive in a X. fastidiosa-specific real time PCR assay, using the primers XF1F and XF1R and probe XF1p (Schaad et al. 2002). Moreover, the ELISA-negative samples were also negative for PCR assay. Symptomatic samples were used to isolate the pathogen. Samples were debarked, surface-sterilized and xylem fluid collected. The fluid was gently imprinted on buffered charcoal yeast extract (BCYE) (Wells et al. 1981b) or periwinkle wilt modified (PWM) agar plates (Summer et al. 2010). After 10 days of incubation, individual colonies were observed. The colonies were slightly convex, white, opalescent, mucoid, circular with entire margins and with smooth surfaces on both media plates. Isolated colonies were triple-streak single colony purified and archived. Genomic DNA was extracted from four purified isolates using the DNeasy Blood and Tissue Qiagen® Kit, to conduct conventional PCR using HL5/HL6 (Francis et al. 2006), which identified the isolates as X. fastidiosa. Using the 16S rRNA primer pair U3/U4 (James 2010), amplicons were sequenced and compared against the NCBI database using the BLASTn algorithm. Comparative sequence analysis of amplicons from the four isolates were identical and indicated that the isolates were 100% identical to X. fastidiosa subsp. multiplex RIV5 (CP064326.1) from cherry plum, and IVIA5901 (CP047134.1) from almond. The sequences of all four isolates were deposited into NCBI GenBank, with the accession numbers OM617940 (467), OM617941 (470), OM617942 (471) and OM617943 (468). To our knowledge, this is the first report of X. fastidiosa associated with plum leaf scald in Texas, extending the geographical range of this important bacterial disease, in the Southern United States. This study highlights the importance of routine scouting of agricultural settings with a view to assessing and detecting early threats from either pests or disease and implementing relevant management strategies.

Complete Genome Sequence of Stenotrophomonas Phage Mendera.

Stenotrophomonas maltophilia is an emerging opportunistic human pathogen. In this report, we describe the isolation and genomic annotation of the S. maltophilia-infecting bacteriophage Mendera. A myophage of 159,961 base pairs, Mendera is T4-like and related most closely to Stenotrophomonas phage IME-SM1.

Complete Genome Sequence of Stenotrophomonas maltophilia Myophage Moby.

Stenotrophomonas maltophilia is a prevalent nosocomial pathogen with multidrug resistance. Here, we describe the complete genome of S. maltophilia myophage Moby, which shares characteristics with Enterobacteria phage T4 and is closely related to Stenotrophomonas phage IME-SM1. Moby has a 159,365-bp genome with 271 predicted protein-coding genes and 24 predicted tRNAs.

Complete Genome Sequence of Xanthomonas Siphophage Samson.

The Xanthomonas genus includes many Gram-negative plant-associated bacteria. Here, we report a virulent Xanthomonas siphophage called Samson. A siphophage isolated from sewage, Samson contains a 43,314-bp genome with 58 predicted genes. Samson has high nucleotide identity with Pseudomonas phage PaMx42.

Complete Genome Sequence of Stenotrophomonas Phage Pokken.

Stenotrophomonas maltophilia is a Gram-negative bacterium associated with multidrug-resistant nosocomial infections, a problem for immunocompromised patients and those with cystic fibrosis. Here, we present the new S. maltophilia-infecting podophage Pokken. Its 76,239-bp genome, with 92 protein-coding genes and 5 tRNA genes predicted, is similar to that of phage N4.

Complete Genome Sequence of Xanthomonas Phage Pagan.

The T7-like podophage Pagan infects Xanthomonas sp. strain ATCC PTA-13101, which was isolated from rice. The 44-kbp Pagan genome contains direct terminal repeats and contains 59 genes, 27 of which have a predicted function. Pagan is most closely related to Xanthomonas phage phi Xc10 and Xylella phage Prado.

Complete Genome Sequence of Stenotrophomonas maltophilia Podophage Ponderosa.

Stenotrophomonas maltophilia is a Gram-negative bacterium that is emerging as a multidrug-resistant global opportunistic pathogen. Here, we describe the genome of the T7-like S. maltophilia podophage Ponderosa, with 54 predicted protein-coding genes and a 493-bp terminal repeat.

Complete Genome Sequence of Agrobacterium tumefaciens Myophage Milano.

Agrobacterium tumefaciens C58 is a tumor-causing pathogen targeting plants and is ubiquitously found in soil. Here, the complete genome sequence of Milano, a myophage infecting A. tumefaciens C58, is presented. Milano encodes 127 proteins, of which 45 can be assigned a predicted function, and it is most similar to the flagellotropic Agrobacterium phage 7-7-1.

A Broad-Host-Range Tailocin from Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of 20 closely related Gram-negative bacterial species that are significant pathogens for persons with cystic fibrosis (CF). Some Bcc strains are highly transmissible and resistant to multiple antibiotics, making infection difficult to treat. A tailocin (phage tail-like bacteriocin), designated BceTMilo, with a broad host range against members of the Bcc, was identified in B. cenocepacia strain BC0425. Sixty-eight percent of Bcc representing 10 species and 90% of non-Bcc Burkholderia strains tested were sensitive to BceTMilo. BceTMilo also showed killing activity against Pseudomonas aeruginosa PAO1 and derivatives. Liquid chromatography-mass spectrometry analysis of the major BceTMilo proteins was used to identify a 23-kb tailocin locus in a draft BC0425 genome. The BceTMilo locus was syntenic and highly similar to a 24.6-kb region on chromosome 1 of B. cenocepacia J2315 (BCAL0081 to BCAL0107). A close relationship and synteny were observed between BceTMilo and Burkholderia phage KL3 and, by extension, with paradigm temperate myophage P2. Deletion mutants in the gene cluster encoding enzymes for biosynthesis of lipopolysaccharide (LPS) in the indicator strain B. cenocepacia K56-2 conferred resistance to BceTMilo. Analysis of the defined mutants in LPS biosynthetic genes indicated that an α-d-glucose residue in the core oligosaccharide is the receptor for BceTMilo.IMPORTANCE BceTMilo, presented in this study, is a broad-host-range tailocin active against Burkholderia spp. As such, BceTMilo and related or modified tailocins have potential as bactericidal therapeutic agents against plant- and human-pathogenic Burkholderia.

Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa.

Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants.

Biocompatibility assessment of spark plasma-sintered alumina-titanium cermets.

Alumina-titanium materials (cermets) of enhanced mechanical properties have been lately developed. In this work, physical properties such as electrical conductivity and the crystalline phases in the bulk material are evaluated. As these new cermets manufactured by spark plasma sintering may have potential application for hard tissue replacements, their biocompatibility needs to be evaluated. Thus, this research aims to study the cytocompatibility of a novel alumina-titanium (25 vol. % Ti) cermet compared to its pure counterpart, the spark plasma sintered alumina. The influence of the particular surface properties (chemical composition, roughness and wettability) on the pre-osteoblastic cell response is also analyzed. The material electrical resistance revealed that this cermet may be machined to any shape by electroerosion. The investigated specimens had a slightly undulated topography, with a roughness pattern that had similar morphology in all orientations (isotropic roughness) and a sub-micrometric average roughness. Differences in skewness that implied valley-like structures in the cermet and predominance of peaks in alumina were found. The cermet presented a higher surface hydrophilicity than alumina. Any cytotoxicity risk associated with the new materials or with the innovative manufacturing methodology was rejected. Proliferation and early-differentiation stages of osteoblasts were statistically improved on the composite. Thus, our results suggest that this new multifunctional cermet could improve current alumina-based biomedical devices for applications such as hip joint replacements.

Peptide-functionalized zirconia and new zirconia/titanium biocermets for dental applications.

OBJECTIVE: Titanium materials have been functionalized with biomolecules as a modern strategy to incorporate bioactive motifs that will expand and improve their biomedical applications. Here, we have biofunctionalized biomaterials based on zirconia of much interest for dentistry: the widely used bioceramic 3Y-TZP and a newly developed 3Y-TZP/Ti biocermet. METHODS: The biosurfaces were activated, silanized, and functionalized with coatings made of oligopeptides. Surface activation by plasma or alkaline-etching was optimized. The surfaces were coated by tethering a purposely-designed RGD-containing peptide. We selected this oligopeptide as a model peptide to validate the effectiveness of the biofunctionalization process. Successful treatments after each step of the process were assessed by surface physical and chemical characterization with water contact angles and XPS, respectively. Coatings' stability was evaluated after 2h sonication in water. Pre-osteoblasts adhesion on the functionalized surfaces was also studied. RESULTS: 10-min air-plasma treatment effectively activated all types of materials with no detrimental effects on the material structure and hardness. Nitrogen XPS-peak confirmed that RGD-peptides were chemically-attached on the silanized samples. This was further confirmed by visualizing the functionalized surfaces with flourescence-labelled RGD-peptides before and after ultrasonication. Furthermore, RGD-functionalized surfaces significantly enhanced osteoblast adhesion on all types of substrates, which demonstrated their successful bioactivation. CONCLUSIONS: We successfully developed stable functional biocoatings on zirconia and biocermets made of oligopeptides. Surface bioactivation of zirconia-containing components for dental implant applications will enable their improved clinical performance by incorporating signalling oligopeptides to accelerate osseointegration, improving permucosal sealing, and/or incorporating antimicrobial properties to prevent peri-implant infections.

Control of Pierce's Disease by Phage.

Pierce's Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella.

Osteoblastic cell response to spark plasma-sintered zirconia/titanium cermets.

Ceramic/metal composites, cermets, arise from the idea to combine the dissimilar properties in the pure materials. This work aims to study the biocompatibility of new micro-nanostructured 3 Y-TZP/Ti materials with 25, 50 and 75 vol.% Ti, which have been successfully obtained by spark slasma sintering technology, as well as to correlate their surface properties (roughness, wettability and chemical composition) with the osteoblastic cell response. All samples had isotropic and slightly waved microstructure, with sub-micrometric average roughness. Composites with 75 vol.% Ti had the highest surface hydrophilicity. Surface chemical composition of the cermets correlated well with the relative amounts used for their fabrication. A cell viability rate over 80% dismissed any cytotoxicity risk due to manufacturing. Cell adhesion and early differentiation were significantly enhanced on materials containing the nanostructured 3 Y-TZP phase. Proliferation and differentiation of SaOS-2 were significantly improved in their late-stage on the composite with 75 vol.% Ti that, from the osseointegration standpoint, is presented as an excellent biomaterial for bone replacement. Thus, spark plasma sintering is consolidated as a suitable technology for manufacturing nanostructured biomaterials with enhanced bioactivity.

Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

Genomes and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia.

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.

Genomic and biological analysis of phage Xfas53 and related prophages of Xylella fastidiosa.

We report the plaque propagation and genomic analysis of Xfas53, a temperate phage of Xylella fastidiosa. Xfas53 was isolated from supernatants of X. fastidiosa strain 53 and forms plaques on the sequenced strain Temecula. Xfas53 forms short-tailed virions, morphologically similar to podophage P22. The 36.7-kb genome is predicted to encode 45 proteins. The Xfas53 terminase and structural genes are related at a protein and gene order level to P22. The left arm of the Xfas53 genome has over 90% nucleotide identity to multiple prophage elements of the sequenced X. fastidiosa strains. This arm encodes proteins involved in DNA metabolism, integration, and lysogenic control. In contrast to Xfas53, each of these prophages encodes head and DNA packaging proteins related to the siphophage lambda and tail morphogenesis proteins related to those of myophage P2. Therefore, it appears that Xfas53 was formed by recombination between a widespread family of X. fastidiosa P2-related prophage elements and a podophage distantly related to phage P22. The lysis cassette of Xfas53 is predicted to encode a pinholin, a signal anchor and release (SAR) endolysin, and Rz and Rz1 equivalents. The holin gene encodes a pinholin and appears to be subject to an unprecedented degree of negative regulation at both the level of expression, with rho-independent transcriptional termination and RNA structure-dependent translational repression, and the level of holin function, with two upstream translational starts predicted to encode antiholin products. A notable feature of Xfas53 and related prophages is the presence of 220- to 390-nucleotide degenerate tandem direct repeats encoding putative DNA binding proteins. Additionally, each phage encodes at least two BroN domain-containing proteins possibly involved in lysogenic control. Xfas53 exhibits unusually slow adsorption kinetics, possibly an adaptation to the confined niche of its slow-growing host.

Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection.

The therapeutic potential of bacteriophages (phages) in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed. Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. Bacterial density, macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (P < .05). No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with intranasal phages, intraperitoneal ultraviolet-inactivated phages, or intraperitoneal lambda phage control mice. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNF-alpha levels compared with mock-infected/mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages.

Fabrication and evaluation of a ratchet type dielectrophoretic device for particle analysis.

Dielectrophoresis is an electrokinetic phenomenon that utilizes an asymmetric electric field to separate analytes based on differences in their polarizabilities relative to that of the suspending medium. One dielectrophoretic device architecture that offers interesting possibilities for particle transport without the use of external flow is the ratchet geometry. This paper describes the fabrication and evaluation of a novel dielectrophoretic ratchet device using a series of fine particles as test probes. The asymmetrical electric field required to selectively transport target analytes was produced using electroformed electrodes which offer the possibility of reducing convective heating and which can be used to construct a device in which all particles located within the fluidic channel are exposed to the applied field. Initial tests of this device were conducted using magnetite and polystyrene fine particles to demonstrate selective particle collection and a separation based on differences in the electrical properties of the analytes employed.