Phage-Antibiotic Combinations Against Klebsiella pneumoniae
Ontology highlight
ABSTRACT: hvKP ATCC43816 and its lytic phage H5 were employed as a phage-antibiotic combination model. Based on the comprehensive characterization of phages, including cryo-electron microscopy, we evaluated the synergic effect of H5 on bacterial killing in vitro when combined with multiple antibiotics, and analyzed the advantages of phage-antibiotic combinations from an evolutionary perspective and proposes a novel PAS mechanism by using ceftazidime as an example.
Project description:Rapidly growing antibiotic resistance among gastrointestinal pathogens, and the ability of antibiotics to induce the virulence of these pathogens makes it increasingly difficult to rely on antibiotics to treat gastrointestinal infections. The probiotic E. coli strain Nissle 1917 (EcN) is the active component of the pharmaceutical preparation Mutaflor® and has been successfully used in the treatment of gastrointestinal disorders. Gut bacteriophages are dominant players in maintaining the microbial homeostasis in the gut, however, their interaction with incoming probiotic bacteria remains to be at conception. The presence of bacteriophages in the gut makes it inevitable for any probiotic bacteria to be phage resistant, in order to survive and successfully colonize the gut. This study addresses the phage resistance of EcN, specifically against lytic T4 phage infection. From various experiments we could show that i) EcN is resistant towards T4 phage infection, ii) EcN’s K5 polysaccharide capsule plays a crucial role in T4 phage resistance and iii) EcN’s lipopolysaccharide (LPS) inactivates T4 phages and notably, treatment with the antibiotic polymyxin B which neutralizes the LPS destroyed the phage inactivation ability of isolated LPS from EcN. Our results further indicate that N-acetylglucosamine at the distal end of O6 antigen in EcN’s LPS could be the interacting partner with T4 phages. From our findings, we have reported for the first time, the role of EcN’s K5 capsule and LPS in its defense against T4 phages. In addition, by inactivating the T4 phages, EcN also protects E. coli K-12 strains from phage infection in tri-culture experiments. The combination of the identified properties is not found in other tested commensal E. coli strains. Furthermore, our research highlights phage resistance as an additional safety feature of EcN, a clinically successful probiotic E. coli strain.
Project description:Antibiotic use can lead to expansion of multi-drug resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank that enables the rapid design of antimicrobial bacteriophage cocktails to treat multi-drug resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identified host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank and experimental evolution strategies, we formulated combinations of phages that minimize the occurrence of phage resistance in vitro. Optimized bacteriophage cocktails selectively suppressed the burden of multi-drug resistant K. pneumoniae in the mouse gut microbiome and drove bacterial populations to lose key virulence factors that act as phage receptors. Further, phage-mediated diversification of bacterial populations in the gut enabled co-evolution of phage variants with higher virulence and a broader host range. Altogether, the Klebsiella PhageBank represents a roadmap for both phage researchers and clinicians to enable phage therapy against a critical multidrug-resistant human pathogen.
Project description:By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to contend with suboptimal conditions that would otherwise reduce their fitness. In addition, certain types of dormancy like sporulation, can serve as a refuge from parasitic infections. Phages are unable to attach to spores, but their genomes can be entrapped in the resting structures and are able to resume infection upon host germination. Thus, dormancy has the potential to affect both the reproductive and survival components of phage fitness. Here, we characterized the distribution and diversity of sigma factors in nearly 3,500 phage genomes. Homologs of bacterial sigma factors that are responsible for directing transcription during sporulation were preferentially recovered in phages that infect spore-forming hosts. While non-essential for lytic infection, when expressed in Bacillus subtilis, we demonstrate that phage-encoded sigma factors activated sporulation gene networks and reduced spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate the expression of complex traits, like the transitions involved in bacterial dormancy.
Project description:Multiple immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defense. Here we studied an anti-phage defense system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fiber, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defense system release a mixture of partially assembled, tailless phage particles, and fully assembled phages in which the central tail fiber is obstructed by the covalently attached ubiquitin-like protein. These phages exhibit severely impaired infectivity, explaining how the defense system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.
Project description:Bacterial populations face the constant threat of viral predation exerted by bacteriophages (or phages). In response, bacteria have evolved a wide range of defense mechanisms against phage challenges. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection. We observed a broad phage inhibition by aminoglycosides. We demonstrate that aminoglycosides do not prevent the injection of phage DNA into bacterial cells but instead block an early step of the viral life cycle. In this context, we used RNA sequencing of S. venezuelae cells infected with phage Alderaan to comparatively investigate the influence of apramycin on phage DNA tanscription at two different time points after inital infection.
Project description:During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.
Project description:To extend the applicability of our approach, we tested a more extensively multiplexed pool of phages which contained ~50 clones with each phage equally distributed across the pool. Most of the factors tested are associated with cancer progression.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.