Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

In an era of proliferating multidrug resistant bacterial infections that are exhausting the capacity of existing chemical antibiotics and in which the development of new antibiotics is significantly rarer, Western medicine must seek additional therapeutic options that can be employed to treat these infections. Among the potential antibacterial solutions are bacteriophage therapeutics, which possess very different properties from broad spectrum antibiotics that are currently the standard of care, and which can be used in combination with them and often provide synergies. In this review we summarize the state of the development of bacteriophage therapeutics and discuss potential paths to the implementation of phage therapies in contemporary medicine, focused on fixed phage cocktail therapeutics since these are likely to be the first bacteriophage products licensed for broad use in Western countries.

Background. Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. Methods. Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. Results. Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. Conclusions. We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Background: Infections by pan-drug resistant Acinetobacter baumannii plague military and civilian healthcare systems. Previous A. baumannii pan-genomic studies used modest sample sizes of low diversity and comparisons to a single reference genome, limiting our understanding of gene order and content. A consensus representation of multiple genomes will provide a better framework for comparison. A large-scale comparative study will identify genomic determinants associated with their diversity and adaptation as a successful pathogen. Results: We determine draft-level genomic sequence of 50 diverse military isolates and conduct the largest bacterial pan-genome analysis of 249 genomes. The pan-genome of A. baumannii is open when the input genomes are normalized for diversity with 1867 core proteins and a paralog-collapsed pan-genome size of 11,694 proteins. We developed a novel graph-based algorithm and use it to assemble the first consensus pan-chromosome, identifying both the order and orientation of core genes and flexible genomic regions. Comparative genome analyses demonstrate the existence of novel resistance islands and isolates with increased numbers of resistance island insertions over time, from single insertions in the 1950s to triple insertions in 2011. Gene clusters responsible for carbon utilization, siderophore production, and pilus assembly demonstrate frequent gain or loss among isolates. Conclusions: The highly variable and dynamic nature of the A. baumannii genome may be the result of its success in rapidly adapting to both abiotic and biotic environments through the gain and loss of gene clusters controlling fitness. Importantly, some archaic adaptation mechanisms appear to have reemerged among recent isolates.

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis , we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis . Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis , we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. ThewboAgene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain inBrucella. Deletion ofwboAin smooth, virulentB. melitensis16M results in a rough mutant designated WRR51. UnlikeB. abortus, both smooth and rough strains ofB. melitensisare resistant to complement-mediated killing. To determine the role of surface OPS in the interactions ofB. melitensiswith monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. SmoothB. melitensissurvived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on liveB. melitensisbenefits the bacterium by preventing the death of macrophages,Brucella's preferred target for intracellular replication.