Hydrodynamic flow and concentration gradients in the gut enhance neutral bacterial diversity

The gut microbiota features important genetic diversity, and the specific spatial features of the gut may shape evolution within this environment. We investigate the fixation probability of neutral bacterial mutants within a minimal model of the gut that includes hydrodynamic flow and resulting gradients of food and bacterial concentrations. We find that this fixation probability is substantially increased, compared with an equivalent well-mixed system, in the regime where the profiles of food and bacterial concentration are strongly spatially dependent. Fixation probability then becomes independent of total population size. We show that our results can be rationalized by introducing an active population, which consists of those bacteria that are actively consuming food and dividing. The active population size yields an effective population size for neutral mutant fixation probability in the gut.

Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host’s ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Protozoan predation drives adaptive divergence in Pseudomonas fluorescens SBW25; ecology meets experimental evolution.

Protozoan predators can affect the structure of bacterial communities, but investigations of how predation might influence bacterial evolution and antagonistic behaviours are scarce. Here, we performed a 20-day predator-prey evolution experiment on solid media to investigate the effect of continuous protozoan predation on bacterial traits using Pseudomonas fluorescens SBW25 as prey and Naegleria gruberi as an amoeboid predator. We observed the divergence of colony morphotypes coincident with an increase in bacterial grazing resistance and relative prey fitness in selected bacterial isolates. When subjected to these resistant prey, N. gruberi show reduced activity (increased encystment) and limited replication. An investigation of the mutations responsible for predation resistance reveals mutations in wspF and amrZ genes, affecting biofilm formation and motility. The bacterial mutants in the wspF gene successfully colonise the air-liquid interface and produce robust cellulose biofilms that prevent predation. The mutation in the amrZ mutant withstands predation but this variant produces low levels of cellulose and limited swarming motility. Our findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in a short period.

Genomic and functional characterization of Pseudomonas aeruginosa-targeting bacteriophages isolated from hospital wastewater

Pseudomonas aeruginosa infections can be difficult to treat and new therapeutic approaches are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 genetically and phenotypically diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and characterized their genetic, phenotypic, and prophage diversity. We then used these isolates to screen and isolate 14 new P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. For four different phages, we evolved bacterial mutants that were resistant to phage infection. We then used genome sequencing and functional analysis of the resistant mutants to study their mechanisms of phage resistance as well as changes in virulence factor production and antibiotic resistance, which differed from corresponding parent bacterial isolates. Finally, we tested two phages for their ability to kill P. aeruginosa grown in biofilms in vitro, and observed that both phages reduced viable bacteria in biofilms by least one order of magnitude. One of these phages also showed activity against P. aeruginosa biofilms grown on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

Evolution of Bacterial Cross-Resistance to Lytic Phages and Albicidin Antibiotic

Due to concerns over the global increase of antibiotic-resistant bacteria, alternative antibacterial strategies, such as phage therapy, are increasingly being considered. However, evolution of bacterial resistance to new therapeutics is almost a certainty; indeed, it is possible that resistance to alternative treatments might result in an evolved trade-up such as enhanced antibiotic resistance. Here, we hypothesize that selection for Escherichia coli bacteria to resist phage T6, phage U115, or albicidin, a DNA gyrase inhibitor, should often result in a pleiotropic trade-up in the form of cross-resistance, because all three antibacterial agents interact with the Tsx porin. Selection imposed by any one of the antibacterials resulted in cross-resistance to all three of them, in each of the 29 spontaneous bacterial mutants examined in this study. Furthermore, cross-resistance did not cause measurable fitness (growth) deficiencies for any of the bacterial mutants, when competed against wild-type E. coli in both low-resource and high-resource environments. A combination of whole-genome and targeted sequencing confirmed that mutants differed from wild-type E. coli via change(s) in the tsx gene. Our results indicate that evolution of cross-resistance occurs frequently in E. coli subjected to independent selection by phage T6, phage U115 or albicidin. This study cautions that deployment of new antibacterial therapies such as phage therapy, should be preceded by a thorough investigation of evolutionary consequences of the treatment, to avoid the potential for evolved trade-ups.

A phage mechanism for selective nicking of dUMP-containing DNA

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

Hydrodynamic flow and concentration gradients in the gut enhance neutral bacterial diversity

The gut microbiota features important genetic diversity, and the specific spatial features of the gut may shape evolution within this environment. We investigate the fixation probability of neutral bacterial mutants within a minimal model of the gut that includes hydrodynamic flow and resulting gradients of food and bacterial concentrations. We find that this fixation probability is substantially increased compared to an equivalent well-mixed system, in the regime where the profiles of food and bacterial concentration are strongly spatially-dependent. Fixation probability then becomes independent of total population size. We show that our results can be rationalized by introducing an active population, which consists of those bacteria that are actively consuming food and dividing. The active population size yields an effective population size for neutral mutant fixation probability in the gut.

Absence of osmoregulated periplasmic glucan confers antimicrobial resistance and increases virulence in Escherichia coli

Clarifying the molecular mechanisms by which bacteria acquire virulence traits is important toward understanding the bacterial virulence system. In the present study, we utilized a bacterial evolution method in a silkworm-infection model and revealed that deletion of the opgGH operon encoding synthases for osmoregulated periplasmic glucan (OPG) increased the virulence of non-pathogenic laboratory strain of Escherichia coli against silkworms. The opgGH knockout mutant exhibited resistance to the host antimicrobial peptides and antibiotics. Compared with the parent strain, the opgGH knockout mutant produced greater amounts of colanic acid, which is involved in E. coli resistance to antibiotics. RNA sequence analysis revealed that the opgGH knockout altered the expression of various genes, including the evgS/evgA two-component system that functions in antibiotic resistance. In both a colanic acid-negative background and evgS-null background, the opgGH knockout increased E. coli resistance to antibiotics and increased the silkworm killing activity of E. coli. In the null background of the envZ/ompR two-component system, which genetically interacts with opgGH, the opgGH knockout increased the antibiotic resistance and the virulence in silkworms. These findings suggest that the absence of OPG confers antimicrobial resistance and virulence of E. coli in a colanic acid-, evgS/evgA-, and envZ/ompR- independent manner. IMPORTANCE The gene mutation types that increase bacterial virulence of Escherichia coli remain unclear, in part due to the limited number of methods available for isolating bacterial mutants with increased virulence. We utilized a bacterial evolution method in the silkworm infection model, in which silkworms were infected with mutagenized bacteria and highly virulent bacterial mutants were isolated from dead silkworms. We revealed that knockout of OPG synthases increases E. coli virulence against silkworms. The OPG-knockout mutants were resistant to host antimicrobial peptides as well as antibiotics. Our findings not only suggest a novel mechanism for virulence acquisition in E. coli, but also support the usefulness of utilizing the bacterial experimental evolution method in the silkworm infection model.

Effect of diet modification in intestinal Escherichia coli population and antimicrobial resistance

The fluctuations in the number of some intestinal bacterial lineages may be associated with increased antimicrobial resistance and disease. Adaptation to a given environment may select bacterial mutants that have reduced ability to adapt to new environments and changes in diet have been associated with alterations in microbiome taxon composition. We wanted to see the effect of diet change in linage composition and antimicrobial resistance profiles of numerically dominant E. coli. We subjected 50 chickens from an industrial operation (under corn-based diet supplemented with antimicrobials) to 2 antimicrobial-free diets; one based on corn and the other based on alfalfa. Fecal samples were obtained from all animals at arrival and after five weeks under different diets. Five E. coli colonies (from each sample) were subjected to genetic typing and antimicrobial susceptibility testing. We observed high diversity and high turnover rate of numerically dominant E. coli strains from animals from both diet groups. We did not find differences in antimicrobial resistance profiles in isolates from different diet groups. Our results suggest that there is high diversity and high turnover rate of E. coli strains in the intestines regardless of the diet. Chicken intestines seemed to contain many E. coliThe fluctuations in the number of some intestinal bacterial lineages may be associated with increased antimicrobial resistance and disease. Adaptation to a given environment may select bacterial mutants that have reduced ability to adapt to new environments and changes in diet have been associated with alterations in microbiome taxon composition. We wanted to see the effect of diet change in linage composition and antimicrobial resistance profiles of numerically dominant E. coli. We subjected 50 chickens from an industrial operation (under corn-based diet supplemented with antimicrobials) to 2 antimicrobial-free diets; one based on corn and the other based on alfalfa. Fecal samples were obtained from all animals at arrival and after five weeks under different diets. Five E. coli colonies (from each sample) were subjected to genetic typing and antimicrobial susceptibility testing. We observed high diversity and high turnover rate of numerically dominant E. coli strains from animals from both diet groups. We did not find differences in antimicrobial resistance profiles in isolates from different diet groups. Our results suggest that there is high diversity and high turnover rate of E. coli strains in the intestines regardless of the diet. Chicken intestines seemed to contain many E. coli lineages able to thrive in different substrates. The absence of differences in antimicrobial resistance among bacteria, from animals in different diets, may indicate that the carriage of antimicrobial resistance genes does not affect the bacterial ability to adapt to different substrates. lineages able to thrive in different substrates. The absence of differences in antimicrobial resistance among bacteria, from animals in different diets, may indicate that the carriage of antimicrobial resistance genes does not affect the bacterial ability to adapt to different substrates.

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.