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.

Extensive population-level genetic variability at the Salmonella rfb locus, which encodes enzymes responsible for synthesis of the O-antigen polysaccharide, is thought to have arisen through frequency-dependent selection (FDS) by means of exposure of this pathogen to host immune systems. The FDS hypothesis works well for pathogens such as Haemophilus influenzae and Neisseria meningitis, which alter the composition of their O-antigens during the course of bloodborne infections. In contrast, Salmonella remains resident in epithelial cells or macrophages during infection and does not have phase variability in its O-antigen. More importantly, Salmonella shows host-serovar specificity, whereby strains bearing certain O-antigens cause disease primarily in specific hosts; this behavior is inconsistent with FDS providing selection for the origin or maintenance of extensive polymorphism at the rfb locus. Alternatively, selective pressure may originate from the host intestinal environment itself, wherein diversifying selection mediated by protozoan predation allows for the continued existence of Salmonella able to avoid consumption by host-specific protozoa. This selective pressure would result in high population-level diversity at the Salmonella rfb locus without phase variation. We show here that intestinal protozoa recognize antigenically diverse Salmonella with different efficiencies and demonstrate that differences solely in the O-antigen are sufficient to allow for prey discrimination. Combined with observations of the differential distributions of both serotypes of bacterial species and their protozoan predators among environments, our data provides a framework for the evolution of high genetic diversity at the rfb locus and host-specific pathogenicity in Salmonella.T he enteric pathogen Salmonella enterica presents at least 70 different O-antigens [the outermost structure of the Gramnegative lipopolysaccharide (LPS)] to mammalian immune systems (1); this polysaccharide decorates the outer surface of the cell. Historically, extensive genetic diversity at the rfb locus, which encodes enzymes directing O-antigen synthesis (2-6), has been attributed to frequency-dependent selection (FDS) (7,8) imposed by the host immune system (5, 9, 10). Novel rfb loci would have an advantage because their cognate O-antigens would be unrecognized by immune systems (Fig. 1A); strains carrying rare loci would have higher fitness and would avoid rapid stochastic loss, rising to higher frequency. Yet selective advantages decrease with abundance; as a result, strains with common rfb loci cannot dominate the population or elicit a selective sweep (7) because their fitnesses become lower as they become more abundant. In concert, FDS prevents the loss of rare alleles or the dominance of common alleles, thus maintaining diversity (8).According to the FDS model, expression of different LPS molecules through gene regulation allows invading bacteria to escape host immunity, survive, and proceed throughout its life cycle; this hypothesis explains...

Here, we report a complex case that involved a pediatric patient who experienced recalcitrant multidrug-resistant Pseudomonas aeruginosa infection complicated by bacteremia/sepsis; our antibacterial options were limited because of resistance, allergies, and suboptimal source control. A cocktail of 2 bacteriophages targeting the infectious organism introduced on 2 separate occasions sterilized the bacteremia.

The AmtB protein transports uncharged NH 3 into the cell, but it also interacts with the nitrogen regulatory protein P II , which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P II homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to NH + 4 addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2 , have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB 1 are defective in their response to both NH + 4 addition and darkness, while mutants lacking AmtB 2 show little effect on the regulation of nitrogenase activity. These responses to darkness and NH + 4 appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB 1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB 1 -P II regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P II is proposed. Other results demonstrate a degree of specificity in interaction of AmtB 1 with the different P II homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P II proteins regulate processes involved in nitrogen acquisition and utilization.

The nitrogen regulatory protein P II and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P II homolog, GlnJ, has higher affinity for an AmtB 1 -containing membrane than the other two P II homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB 1 complex in the presence of ATP and 2-KG, supporting a role for P II as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K d of 2-KG for GlnJ bound to AmtB 1 is 340 M, which is higher than that required for uridylylation of GlnJ in vitro, about 5 M. This supports a model where multiple 2-KG and ATP molecules must bind a P II trimer to stimulate release of P II from AmtB 1 , in contrast to the lower 2-KG requirement for productive uridylylation of P II by GlnD.The ammonium channel/rhesus (Amt/Rh) family of proteins is a widely distributed group of trimeric integral membrane proteins found in all domains of life that can function as gas channels of ammonia and perhaps carbon dioxide (13,20,29,39,43). A subset of this family, the AmtB proteins, is found in bacteria, archaea, some lower eukaryotes and plants. Homologs of amtB are often found in close proximity to genes encoding P II homologs (46). P II regulatory proteins are also found in most prokaryotes and some plant chloroplasts (3). P II is a small, soluble, trimeric protein that regulates the functions of several other proteins involved in nitrogen metabolism (3,4,35,36). AmtB appears to have two roles in the cell. The first function, to act as a channel for uncharged ammonia, has been explored physiologically, structurally, and computationally (28,33,44). The second function of AmtB is to interact with P II and has only recently been described (6,9,21,22,53).AmtB proteins have been shown to interact with homologs of P II in several bacteria and in the archaeon Methanococcus jannaschii (8,10,17,18,44,45,47,50,53). The association of P II with AmtB can physically block the ammonia gas channels of AmtB under conditions of nitrogen sufficiency in the cell. In addition, the AmtB-P II complex is able to recruit at least one other protein to the membrane, dinitrogenase reductase-activating glycohydrolase (DRAG), in organisms capable of nitrogen fixation (22,48). This membrane sequestration requires both an Amt protein and a P II protein and results in the inability of DRAG to activate dinitrogenase reductase in vivo. Finally, AmtB is able to remove equimolar amounts of P II from the cytoplasm, preventing P II from interacting with at least some other proteins. Although membrane sequestration of P II has b...