Prophylactic phage biocontrol prevents Burkholderia gladioli infection in a quantitative ex planta model of bacterial virulence.

Agricultural crop yield losses and food destruction due to infections by phytopathogenic bacteria such as Burkholderia gladioli, which causes devastating diseases in onion, mushroom, corn, and rice crops, pose major threats to worldwide food security and cause enormous damage to the global economy. Biocontrol using bacteriophages has emerged as a promising strategy against a number of phytopathogenic species but has never been attempted against B. gladioli due to a lack of quantitative infection models and a scarcity of phages targeting this specific pathogen. In this study, we present a novel, procedurally straightforward, and highly generalizable fully quantitative ex planta maceration model and an accompanying quantitative metric, the ex planta maceration index (xPMI). In utilizing this model to test the ex planta virulence of a panel of 12 strains of B. gladioli in Allium cepa and Agaricus bisporus, we uncover substantial temperature-, host-, and strain-dependent diversity in the virulence of this fascinating pathogenic species. Crucially, we demonstrate that Burkholderia phages KS12 and AH2, respectively, prevent and reduce infection-associated onion tissue destruction, measured through significant (P < 0.0001) reductions in xPMI, by phytopathogenic strains of B. gladioli, thereby demonstrating the potential of agricultural phage biocontrol targeting this problematic microorganism.IMPORTANCEAgricultural crop destruction is increasing due to infections caused by bacteria such as Burkholderia gladioli, which causes plant tissue diseases in onion, mushroom, corn, and rice crops. These bacteria pose a major threat to worldwide food production, which, in turn, damages the global economy. One potential solution being investigated to prevent bacterial infections of plants is "biocontrol" using bacteriophages (or phages), which are bacterial viruses that readily infect and destroy bacterial cells. In this article, we demonstrate that Burkholderia phages KS12 and AH2 prevent or reduce infection-associated plant tissue destruction caused by strains of B. gladioli, thereby demonstrating the inherent potential of agricultural phage biocontrol.

Bacteriophage steering of Burkholderia cenocepacia toward reduced virulence and increased antibiotic sensitivity.

Antibiotic resistance in bacteria is a growing global concern and has spurred increasing efforts to find alternative therapeutics, such as the use of bacterial viruses, or bacteriophages. One promising approach is to use phages that not only kill pathogenic bacteria but also select phage-resistant survivors that are newly sensitized to traditional antibiotics, in a process called "phage steering." Members of the bacterial genus Burkholderia, which includes various human pathogens, are highly resistant to most antimicrobial agents, including serum immune components, antimicrobial peptides, and polymixin-class antibiotics. However, the application of phages in combination with certain antibiotics can produce synergistic effects that more effectively kill pathogenic bacteria. Herein, we demonstrate that Burkholderia cenocepacia serum resistance is due to intact lipopolysaccharide (LPS) and membranes, and phage-induced resistance altering LPS structure can enhance bacterial sensitivity not only to immune components in serum but also to membrane-associated antibiotics such as colistin. IMPORTANCE Bacteria frequently encounter selection pressure from both antibiotics and lytic phages, but little is known about the interactions between antibiotics and phages. This study provides new insights into the evolutionary trade-offs between phage resistance and antibiotic sensitivity. The creation of phage resistance through changes in membrane structure or lipopolysaccharide composition can simultaneously be a major cause of antibiotic sensitivity. Our results provide evidence of synergistic therapeutic efficacy in phage-antibiotic interactions and have implications for the future clinical use of phage steering in phage therapy applications.

Isolation and Characterization of the Novel Bacteriophage AXL3 against Stenotrophomonas maltophilia.

The rapid increase in the number of worldwide human infections caused by the extremely antibiotic resistant bacterial pathogen Stenotrophomonas maltophilia is cause for concern. An alternative treatment solution in the post-antibiotic era is phage therapy, the use of bacteriophages to selectively kill bacterial pathogens. In this study, the novel bacteriophage AXL3 (vB_SmaS-AXL_3) was isolated from soil and characterized. Host range analysis using a panel of 29 clinical S. maltophilia isolates shows successful infection of five isolates and electron microscopy indicates that AXL3 is a member of the Siphoviridae family. Complete genome sequencing and analysis reveals a 47.5 kb genome predicted to encode 65 proteins. Functionality testing suggests AXL3 is a virulent phage and results show that AXL3 uses the type IV pilus, a virulence factor on the cell surface, as its receptor across its host range. This research identifies a novel virulent phage and characterization suggests that AXL3 is a promising phage therapy candidate, with future research examining modification through genetic engineering to broaden its host range.

Novel Stenotrophomonas maltophilia temperate phage DLP4 is capable of lysogenic conversion.

BACKGROUND: Temperate bacteriophages are capable of lysogenic conversion of new bacterial hosts. This phenomenon is often ascribed to "moron" elements that are acquired horizontally and transcribed independently from the rest of the phage genes. Whereas some bacterial species exhibit relatively little prophage-dependent phenotypic changes, other bacterial species such as Stenotrophomonas maltophilia appear to commonly adopt prophage genetic contributions. RESULTS: The novel S. maltophilia bacteriophage DLP4 was isolated from soil using the highly antibiotic-resistant S. maltophilia strain D1585. Genome sequence analysis and functionality testing showed that DLP4 is a temperate phage capable of lysogenizing D1585. Two moron genes of interest, folA (BIT20_024) and ybiA (BIT20_065), were identified and investigated for their putative activities using complementation testing and phenotypic and transcriptomic changes between wild-type D1585 and the D1585::DLP4 lysogen. The gp24 / folA gene encodes dihydrofolate reductase (DHFR: FolA), an enzyme responsible for resistance to the antibiotic trimethoprim. I-TASSER analysis of DLP4 FolA predicted structural similarity to Bacillus anthracis DHFR and minimum inhibitory concentration experiments demonstrated that lysogenic conversion of D1585 by DLP4 provided the host cell with an increase in trimethoprim resistance. The gp65 / ybiA gene encodes N-glycosidase YbiA, which in E. coli BW25113 is required for its swarming motility phenotype. Expressing DLP4 ybiA in strain ybiA770(del)::kan restored its swarming motility activity to wildtype levels. Reverse transcription-PCR confirmed the expression of both of these genes during DLP4 lysogeny. CONCLUSIONS: S. maltophilia temperate phage DLP4 contributes to the antibiotic resistance exhibited by its lysogenized host strain. Genomic analyses can greatly assist in the identification of phage moron genes potentially involved in lysogenic conversion. Further research is required to fully understand the specific contributions temperate phage moron genes provide with respect to the antibiotic resistance and virulence of S. maltophilia host cells.

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity.

BACKGROUND: A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is "phage therapy", the clinical application of bacteriophages to selectively kill bacteria. RESULTS: Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics. CONCLUSIONS: The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence.

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O-linked protein glycosylation system in B. cenocepacia K56-2. The PglLBc O-oligosaccharyltransferase (O-OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N-glycosylation system to a Neisseria meningitides-derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56-2, PglLBc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc-HexNAc-Hex, which is unrelated to the O-antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post-translational modification in Bcc with implications for pathogenesis.

Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.

Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.

BacMap: an up-to-date electronic atlas of annotated bacterial genomes.

Originally released in 2005, BacMap is an electronic, interactive atlas of fully sequenced bacterial genomes. It contains fully labeled, zoomable and searchable chromosome maps for essentially all sequenced prokaryotic (archaebacterial and eubacterial) species. Each map can be zoomed to the level of individual genes and each gene is hyperlinked to a richly annotated gene card. The latest release of BacMap (http://bacmap.wishartlab.com/) now contains data for more than 1700 bacterial species (~10× more than the 2005 release), corresponding to more than 2800 chromosome and plasmid maps. All bacterial genome maps are now supplemented with separate prophage genome maps as well as separate tRNA and rRNA maps. Each bacterial chromosome entry in BacMap also contains graphs and tables on a variety of gene and protein statistics. Likewise, every bacterial species entry contains a bacterial 'biography' card, with taxonomic details, phenotypic details, textual descriptions and images (when available). Improved data browsing and searching tools have also been added to allow more facile filtering, sorting and display of the chromosome maps and their contents.

Genomic characterization of JG068, a novel virulent podovirus active against Burkholderia cenocepacia.

BACKGROUND: As is true for many other antibiotic-resistant Gram-negative pathogens, members of the Burkholderia cepacia complex (BCC) are currently being assessed for their susceptibility to phage therapy as an antimicrobial treatment. The objective of this study was to perform genomic and limited functional characterization of the novel BCC phage JG068 (vB_BceP_JG068). RESULTS: JG068 is a podovirus that forms large, clear plaques on Burkholderia cenocepacia K56-2. Host range analysis indicates that this phage can infect environmental, clinical, and epidemic isolates of Burkholderia multivorans, B. cenocepacia, Burkholderia stabilis, and Burkholderia dolosa, likely through interaction with the host lipopolysaccharide as a receptor. The JG068 chromosome is 41,604 base pairs (bp) in length and is flanked by 216 bp short direct terminal repeats. Gene expression originates from both host and phage promoters and is in the forward direction for all 49 open reading frames. The genome sequence shows similarity to Ralstonia phage ϕRSB1, Caulobacter phage Cd1, and uncharacterized genetic loci of blood disease bacterium R229 and Burkholderia pseudomallei 1710b. CoreGenesUniqueGenes analysis indicates that JG068 belongs to the Autographivirinae subfamily and ϕKMV-like phages genus. Modules within the genome encode proteins involved in DNA-binding, morphogenesis, and lysis, but none associated with pathogenicity or lysogeny. Similar to the signal-arrest-release (SAR) endolysin of ϕKMV, inducible expression of the JG068 SAR endolysin causes lysis of Escherichia coli that is dependent on the presence of an N-terminal signal sequence. In an in vivo assay using the Galleria mellonella infection model, treatment of B. cenocepacia K56-2-infected larvae with JG068 results in a significant increase in larval survival. CONCLUSIONS: As JG068 has a broad host range, does not encode virulence factors, is obligately lytic, and has activity against an epidemic B. cenocepacia strain in vivo, this phage is a highly promising candidate for BCC phage therapy development.

PHAST: a fast phage search tool.

PHAge Search Tool (PHAST) is a web server designed to rapidly and accurately identify, annotate and graphically display prophage sequences within bacterial genomes or plasmids. It accepts either raw DNA sequence data or partially annotated GenBank formatted data and rapidly performs a number of database comparisons as well as phage 'cornerstone' feature identification steps to locate, annotate and display prophage sequences and prophage features. Relative to other prophage identification tools, PHAST is up to 40 times faster and up to 15% more sensitive. It is also able to process and annotate both raw DNA sequence data and Genbank files, provide richly annotated tables on prophage features and prophage 'quality' and distinguish between intact and incomplete prophage. PHAST also generates downloadable, high quality, interactive graphics that display all identified prophage components in both circular and linear genomic views. PHAST is available at (http://phast.wishartlab.com).

Synergistic Interactions among Burkholderia cepacia Complex-Targeting Phages Reveal a Novel Therapeutic Role for Lysogenization-Capable Phages.

Antimicrobial resistance is a danger to global public health and threatens many aspects of modern medicine. Bacterial species such as those of the Burkholderia cepacia complex (Bcc) cause life-threatening respiratory infections and are highly resistant to antibiotics. One promising alternative being explored to combat Bcc infections is phage therapy (PT): the use of phages to treat bacterial infections. Unfortunately, the utility of PT against many pathogenic species is limited by its prevailing paradigm: that only obligately lytic phages should be used therapeutically. It is thought that 'lysogenic' phages do not lyse all bacteria and can transfer antimicrobial resistance or virulence factors to their hosts. We argue that the tendency of a lysogenization-capable (LC) phage to form stable lysogens is not predicated exclusively on its ability to do so, and that the therapeutic suitability of a phage must be evaluated on a case-by-case basis. Concordantly, we developed several novel metrics-Efficiency of Phage Activity, Growth Reduction Coefficient, and Stable Lysogenization Frequency-and used them to evaluate eight Bcc-specific phages. Although these parameters vary considerably among Bcc phages, a strong inverse correlation (R2 = 0.67; P < 0.0001) exists between lysogen formation and antibacterial activity, indicating that certain LC phages with low frequency of stable lysogenization may be therapeutically efficacious. Moreover, we show that many LC Bcc phages interact synergistically with other phages in the first reported instance of mathematically defined polyphage synergy, and that these interactions result in the eradication of in vitro bacterial growth. Together, these findings reveal a novel therapeutic role for LC phages and challenge the current paradigm of PT. IMPORTANCE The spread of antimicrobial resistance is an imminent threat to public health around the world. Particularly concerning are species of the Burkholderia cepacia complex (Bcc), which cause life-threatening respiratory infections and are notoriously resistant to antibiotics. Phage therapy is a promising alternative being explored to combat Bcc infections and antimicrobial resistance in general, but its utility against many pathogenic species, including the Bcc, is restricted by the currently prevailing paradigm of exclusively using rare obligately lytic phages due to the perception that 'lysogenic' phages are therapeutically unsuitable. Our findings show that many lysogenization-capable phages exhibit powerful in vitro antibacterial activity both alone and through mathematically defined synergistic interactions with other phages, demonstrating a novel therapeutic role for LC phages and therefore challenging the currently prevailing paradigm of PT.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Characterization of Bacteriophage cd2, a Siphophage Infecting Carnobacterium divergens and a Representative Species of a New Genus of Phage.

Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. IMPORTANCE Currently, very little is known about phages that infect carnobacteria, an important genus of lactic acid bacteria with both beneficial and detrimental effects in the food and aquaculture industries. This report provides a detailed characterization of phage cd2, a novel siphophage that targets Carnobacterium divergens, and sets the groundwork for understanding the biology of these phages and their potential use in the detection and biocontrol of C. divergens isolates.

Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages.

BACKGROUND: Genomic analysis of bacteriophages infecting the Burkholderia cepacia complex (BCC) is an important preliminary step in the development of a phage therapy protocol for these opportunistic pathogens. The objective of this study was to characterize KL1 (vB_BceS_KL1) and AH2 (vB_BceS_AH2), two novel Burkholderia cenocepacia-specific siphoviruses isolated from environmental samples. RESULTS: KL1 and AH2 exhibit several unique phenotypic similarities: they infect the same B. cenocepacia strains, they require prolonged incubation at 30°C for the formation of plaques at low titres, and they do not form plaques at similar titres following incubation at 37°C. However, despite these similarities, we have determined using whole-genome pyrosequencing that these phages show minimal relatedness to one another. The KL1 genome is 42,832 base pairs (bp) in length and is most closely related to Pseudomonas phage 73 (PA73). In contrast, the AH2 genome is 58,065 bp in length and is most closely related to Burkholderia phage BcepNazgul. Using both BLASTP and HHpred analysis, we have identified and analyzed the putative virion morphogenesis, lysis, DNA binding, and MazG proteins of these two phages. Notably, MazG homologs identified in cyanophages have been predicted to facilitate infection of stationary phase cells and may contribute to the unique plaque phenotype of KL1 and AH2. CONCLUSIONS: The nearly indistinguishable phenotypes but distinct genomes of KL1 and AH2 provide further evidence of both vast diversity and convergent evolution in the BCC-specific phage population.

Characterization of DC1, a broad-host-range Bcep22-like podovirus.

Bcep22-like phages are a recently described group of podoviruses that infect strains of Burkholderia cenocepacia. We have isolated and characterized a novel member of this group named DC1. This podovirus shows many genomic similarities to BcepIL02 and Bcep22, but it infects strains belonging to multiple Burkholderia cepacia complex (BCC) species.

Cangene gold medal award lecture - Genomic analysis and modification of Burkholderia cepacia complex bacteriophages.

The Burkholderia cepacia complex (Bcc) is a group of 17 Gram-negative predominantly environmental bacterial species that cause potentially fatal opportunistic infections in cystic fibrosis (CF) patients. Although its prevalence in these individuals is lower than that of Staphylococcus aureus and Pseudomonas aeruginosa , the Bcc remains a serious problem in the CF community because of the pathogenicity, transmissibility, and inherent antibiotic resistance of these organisms. An alternative treatment for Bcc infections that is currently being developed is phage therapy, the clinical use of viruses that infect bacteria. To assess the suitability of individual phage isolates for therapeutic use, the complete genome sequences of a panel of Bcc-specific phages were determined and analyzed. These sequences encode a broad range of proteins with a gradient of relatedness to phage and bacterial gene products from Burkholderia and other genera. The majority of these phages were found not to encode virulence factors, and despite their predominantly temperate nature, a proof-of-principle experiment has shown that they may be modified to a lytic form. Both the genomic characterization and subsequent engineering of Bcc-specific phages are fundamental to the development of an effective phage therapy strategy for these bacteria.

Antimicrobial activity in the egg wax of the tick Amblyomma hebraeum (Acari: Ixodidae) is associated with free fatty acids C16:1 and C18:2.

Untreated eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) exhibited antimicrobial activity (AMA) against Gram-negative but not Gram-positive bacteria; eggs denuded of wax by solvent extraction showed no AMA. The unfractionated egg wax extract, however, showed AMA against Gram-positive but not Gram-negative bacteria, as also shown by Arrieta et al. (Exp Appl Acarol 39: 297-313, 2006). In this study we partitioned the egg wax into various fractions, using a variety of techniques, analyzed their compositions, and tested them for AMA. The crude aqueous extract exhibited AMA. However, although more than 30 metabolites were identified in this extract by nuclear magnetic resonance analysis, none of them seemed likely to be responsible for the observed AMA. In the crude organic extract, cholesterol esters were the most abundant lipids, but were devoid of AMA. Fatty acids (FAs), with chain lengths between C13 and C26 were the next most abundant lipids. After lipid fractionation and gas chromatography/mass spectroscopy, free FAs, especially C16:1 and C18:2, accounted for most of the AMA in the organic extract. The material responsible for AMA in the crude aqueous extract remains unidentified. No AMA was detected in the intracellular contents of untreated eggs.

Characterization of Stenotrophomonas maltophilia phage AXL1 as a member of the genus Pamexvirus encoding resistance to trimethoprim-sulfamethoxazole.

Stenotrophomonas maltophilia is a ubiquitous environmental bacterium capable of causing disease in humans. Antibiotics are largely ineffective against this pathogen due to numerous chromosomally encoded antibiotic resistance mechanisms. An alternative treatment option is phage therapy, the use of bacteriophages to selectively kill target bacteria that are causing infection. To this aim, we isolated the Siphoviridae bacteriophage AXL1 (vB_SmaS-AXL_1) from soil and herein describe its characterization. Host range analysis on a panel of 30 clinical S. maltophilia strains reveals a moderate tropism that includes cross-species infection of Xanthomonas, with AXL1 using the type IV pilus as its host surface receptor for infection. Complete genome sequencing and analysis revealed a 63,962 bp genome encoding 83 putative proteins. Comparative genomics place AXL1 in the genus Pamexvirus, along with seven other phages that infect one of Stenotrophomonas, Pseudomonas or Xanthomonas species. Functional genomic analyses identified an AXL1-encoded dihydrofolate reductase enzyme that provides additional resistance to the antibiotic combination trimethoprim-sulfamethoxazole, the current recommended treatment option for S. maltophilia infections. This research characterizes the sixth type IV pilus-binding phage of S. maltophilia and is an example of phage-encoded antibiotic resistance.

The Exploration of Complement-Resistance Mechanisms of Pathogenic Gram-Negative Bacteria to Support the Development of Novel Therapeutics.

Resistance to antibiotics in Bacteria is one of the biggest threats to human health. After decades of attempting to isolate or design antibiotics with novel mechanisms of action against bacterial pathogens, few approaches have been successful. Antibacterial drug discovery is now moving towards targeting bacterial virulence factors, especially immune evasion factors. Gram-negative bacteria present some of the most significant challenges in terms of antibiotic resistance. However, they are also able to be eliminated by the component of the innate immune system known as the complement system. In response, Gram-negative bacteria have evolved a variety of mechanisms by which they are able to evade complement and cause infection. Complement resistance mechanisms present some of the best novel therapeutic targets for defending against highly antibiotic-resistant pathogenic bacterial infections.