Complete genome sequence of Pseudomonas aeruginosa phage Knedl.

Bacteriophage Knedl is the first reported Pseudomonas aeruginosa phage that targets the Psl exopolysaccharide as receptor. Here, we report the genome of Knedl, demonstrating that it belongs to the genus Iggyvirus of the Queuovirinae subfamily. Future studies on the infection mechanism of Knedl could inform phage-based approaches to eradicate biofilms.

Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication.

Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates ("hibernation"). However, a newly isolated Pseudomonas aeruginosa phage, named Paride, can directly replicate and induce the lysis of deep-dormant hosts. While non-growing bacteria are notoriously tolerant to antibiotic drugs, the combination with Paride enables the carbapenem meropenem to eradicate deep-dormant cultures in vitro and to reduce a resilient bacterial infection of a tissue cage implant in mice. Our work might inspire new treatments for persistent bacterial infections and, more broadly, highlights two viral strategies to infect dormant bacteria (hibernation and direct replication) that will guide future studies on phage-host interactions.

Messages from the dead protect bacteria from viral attack.

Bacterial populations are ubiquitously threatened by viral predation. In this issue, Tzipilevich and colleagues show that bacteria killed by viruses release a danger signal that warns neighboring cells to ramp up their defenses. This "message from the dead" thereby induces phenotypic tolerance to infections and slows down viral spread in the population.

Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection.

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage-host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage-host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages' host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

Antibiotic Tolerance and Persistence Studied Throughout Bacterial Growth Phases.

Antibiotic tolerance and persistence allow bacteria to survive lethal doses of antibiotic drugs in the absence of genetic resistance. Despite the urgent need to address these phenomena as a cause of clinical antibiotic treatment failure, studies on antibiotic tolerance and persistence are notorious for contradictory and inconsistent findings. Many of these problems are likely caused by differences in the methodology used to study antibiotic tolerance and persistence in the laboratory. Standardized experimental procedures would therefore greatly promote research in this field by facilitating the integrated analysis of results obtained by different research groups. Here, we present a robust and adaptable methodology to study antibiotic tolerance/persistence in broth cultures of Escherichia coli and Pseudomonas aeruginosa . The hallmark of this methodology is that the formation and disappearance of antibiotic-tolerant cells is recorded throughout all bacterial growth phases from lag after inoculation over exponential growth into early and then late stationary phase. In addition, all relevant experimental conditions are rigorously controlled to obtain highly reproducible results. We anticipate that this methodology will promote research on antibiotic tolerance and persistence by enabling a deeper view at the growth-dependent dynamics of this phenomenon and by contributing to the standardization or at least comparability of experimental procedures used in the field.

Defining Proteomic Signatures to Predict Multidrug Persistence in Pseudomonas aeruginosa.

Bacterial persisters are difficult to eradicate because of their ability to survive prolonged exposure to a range of different antibiotics. Because they often represent small subpopulations of otherwise drug-sensitive bacterial populations, studying their physiological state and antibiotic stress response remains challenging. Sorting and enrichment procedures of persister fractions introduce experimental biases limiting the significance of follow-up molecular analyses. In contrast, proteome analysis of entire bacterial populations is highly sensitive and reproducible and can be employed to explore the persistence potential of a given strain or isolate. Here, we summarize methodology to generate proteomic signatures of persistent Pseudomonas aeruginosa isolates with variable fractions of persisters. This includes proteome sample preparation, mass spectrometry analysis, and an adaptable machine learning regression pipeline. We show that this generic method can determine a common proteomic signature of persistence among different P. aeruginosa hyper-persister mutants. We propose that this approach can be used as diagnostic tool to gauge antimicrobial persistence of clinical isolates.

Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections.

The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed the inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro, and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multidrug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.IMPORTANCE Over the past decades, pan-resistant strains of major bacterial pathogens have emerged and have rendered clinically available antibiotics ineffective, putting at risk many of the major achievements of modern medicine, including surgery, cancer therapy, and organ transplantation. A thorough understanding of processes leading to the development of antibiotic resistance in human patients is thus urgently needed. We show that drug tolerance, the ability of bacteria to survive prolonged exposure to bactericidal antibiotics, rapidly evolves in the opportunistic human pathogen Pseudomonas aeruginosa upon recurrent exposures to antibiotics. Our studies show that tolerance protects P. aeruginosa against different classes of antibiotics and that it generally precedes and promotes resistance development. The rapid evolution of tolerance during treatment regimens may thus act as a strong driving force to accelerate antibiotic resistance development. To successfully counter resistance, diagnostic measures and novel treatment strategies will need to incorporate the important role of antibiotic tolerance.