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Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.Antibiotic persistence contributes to the survival of bacteria during antibiotic treatment. In this Consensus Statement, scientists working on the response of bacteria to antibiotics define antibiotic persistence and provide practical guidance on how to study bacterial persisters.

Abstract Multi-species biofilm communities are environments in which complex but ill understood exchanges between bacteria occur. Although monospecies cultures are still widely used in the laboratory, new approaches have been undertaken to study interspecies interactions within mixed communities. This review describes our current understanding of competitive relationships involving nonbiocidal biosurfactants, enzymes, and metabolites produced by bacteria and other microorganisms. These molecules target all steps of biofilm formation, ranging from inhibition of initial adhesion to matrix degradation, jamming of cell–cell communications, and induction of biofilm dispersion. This review presents available data on nonbiocidal molecules and provides a new perspective on competitive interactions within biofilms that could lead to antibiofilm strategies of potential biomedical interest. This review describes how nonbiocidal competitive interactions can profoundly impact on microbial behavior in biofilm environments and discusses the potential biological roles and use of bio'active molecules targeting adhesion and biofilm formation without affecting growth and overall bacterial fitness.

Bacterial interactions with neighboring microorganisms via production of small metabolites enable bacteria to respond and adapt to environmental changes. The study of intercellular interactions primarily focused on soluble metabolites, but bacteria also produce and release into their headspace a wide variety of volatile secondary metabolites, the ecological roles of which have generally been overlooked. However, bacterial volatile compounds are known to contribute to interkingdom interactions (plant, fungi and nematodes), and recent studies also identified their at-a-distance influence on bacterial behavior. The present review describes the biological roles of bacterial volatile compounds in inter- and intraspecies bacterial interactions, a new and yet unexplored research area, with potential clinical and industrial applications. The present review describes how airborne volatile compounds produced by bacteria can influence bacterial physiology and behavior, which constitutes an unexplored aspect of bacterial interactions.

Targeted killing of pathogenic bacteria without harming beneficial members of host microbiota holds promise as a strategy to cure disease and limit both antimicrobial-related dysbiosis and development of antimicrobial resistance. We engineer toxins that are split by inteins and deliver them by conjugation into a mixed population of bacteria. Our toxin–intein antimicrobial is only activated in bacteria that harbor specific transcription factors. We apply our antimicrobial to specifically target and kill antibiotic-resistant Vibrio cholerae present in mixed populations. We find that 100% of antibiotic-resistant V. cholerae receiving the plasmid are killed. Escape mutants were extremely rare (10−6–10−8). We show that conjugation and specific killing of targeted bacteria occurs in the microbiota of zebrafish and crustacean larvae, which are natural hosts for Vibrio spp. Toxins split with inteins could form the basis of precision antimicrobials to target pathogens that are antibiotic resistant.Selective killing of bacteria in a mixed population is enabled by engineering an antimicrobial that is toxic only in pathogens harboring specific genetic elements.

Use of totally implantable venous-access ports (TIVAPs) is standard practice for patients with diseases such as solid-tumour cancers, haematological malignancies, and chronic digestive diseases. Use of TIVAPs allows long-term administration of venotoxic compounds, improves patients' quality of life, and reduces the risk of infection. Microbial contamination, formation of pathogenic biofilms, and infection, however, are associated with morbidity, mortality, and increased health-care costs. Local and systemic complications or infections related to specific pathogens might lead to device removal. Alternatively, conservative treatment with combined systemic antibiotics and antibiotic lock therapy might be useful. We discuss in-vitro and in-vivo basic and clinical research findings on the epidemiology, diagnosis, and prevention of TIVAP-related infections, the current challenges to management, promising strategies, and some treatments in development that are likely to improve outcomes of TIVAP-related infections, with a particular focus on antibiotic lock therapy.

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E . coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure–function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation. Many Gram-negative bacteria secrete exopolysaccharides via functionally homologous synthase-dependent systems. Here the authors use electron microscopy to reveal that biofilm-promoting cellulose in E . coli is secreted by a conserved multi-component secretion system with a megadalton-sized asymmetric architecture.

Bacterial antibiotic resistance is a global health concern of increasing importance and intensive study. Although biofilms are a common source of infections in clinical settings, little is known about the development of antibiotic resistance within biofilms. Here, we use experimental evolution to compare selection of resistance mutations in planktonic and biofilm Escherichia coli populations exposed to clinically relevant cycles of lethal treatment with the aminoglycoside amikacin. Consistently, mutations in sbmA , encoding an inner membrane peptide transporter, and fusA , encoding the essential elongation factor G, are rapidly selected in biofilms, but not in planktonic cells. This is due to a combination of enhanced mutation rate, increased adhesion capacity and protective biofilm-associated tolerance. These results show that the biofilm environment favors rapid evolution of resistance and provide new insights into the dynamic evolution of antibiotic resistance in biofilms. Mutations in sbmA and fusA are rapidly selected in biofilm but not planktonic E. coli when exposed to intermittent amikacin antibiotic treatment, which suggests that the biofilm environment favors rapid evolution of resistance.

Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria–substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation. We find that adhesion forces of the rod-shaped bacteria Escherichia coli and Pseudomonas aeruginosa are polar. This asymmetry induces mechanical tension, and drives daughter cell rearrangements, which eventually determine the shape of the microcolonies. Informed by experimental data, we develop a quantitative model of microcolony morphogenesis that enables the prediction of bacterial adhesion strength from simple time-lapse measurements. Our results demonstrate how patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope. It is unclear how cell adhesion and elongation coordinate during formation of bacterial microcolonies. Here, Duvernoy et al. monitor microcolony formation in rod-shaped bacteria, and show that patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope.

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.