Identifying more targeted antimicrobials active against selected bacterial phytopathogens.

AIMS: Phytopathogens are a global threat to the world's food supply. The use of broad-spectrum bactericides and antibiotics to limit or eliminate bacterial infections is becoming less effective as levels of resistance increase, while concurrently becoming less desirable from an ecological perspective due to their collateral damage to beneficial members of plant and soil microbiomes. Bacteria produce numerous antimicrobials in addition to antibiotics, such as bacteriocins with their relatively narrow activity spectra, and inhibitory metabolic by-products, such as organic acids. There is an interest in developing these naturally occurring antimicrobials for use as alternatives or supplements to antibiotics. METHODS AND RESULTS: In this study, we investigate the inhibitory potential of 217 plant-associated bacterial isolates from 44 species including plant pathogens, plant growth promoting rhizobacteria and plant commensals. Over half of the isolates were found to produce antimicrobial substances, of which 68% were active against phytopathogens. Even more intriguing, 98% of phytopathogenic strains were sensitive to the compounds produced specifically by plant growth promoting rhizobacteria. CONCLUSION: These data argue that plant-associated bacteria produce a broad range of antimicrobial substances, and that the substances produced preferentially target phytopathogenic bacteria. SIGNIFICANCE AND IMPACT OF STUDY: There is a need for novel antimicrobials for use in agriculture. The methods presented here reveal the potential for simple phenotypic screening methods to provide a broad range of potential drug candidates.

Anion Exchange Doping: Tuning Equilibrium to Increase Doping Efficiency in Semiconducting Polymers.

High electron affinity (EA) molecules p-type dope low ionization energy (IE) polymers, resulting in an equilibrium doping level based on the energetic driving force (IE-EA), reorganization energy, and dopant concentration. Anion exchange doping (AED) is a process whereby the dopant anion is exchanged with a stable ion from an electrolyte. We show that the AED level can be predicted using an isotherm equilibrium model. The exchange of the dopant anion (FeCl3-) for a bis(trifluoromethanesulfonamide) (TFSI-) anion in the polymers poly(3-hexylthiophene-2,5-diyl) (P3HT) and poly[3-(2,2-bithien-5-yl)-2,5-bis(2-hexyldecyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione-6,5-diyl] (PDPP-2T) highlights two cases in which the process is nonspontaneous and spontaneous, respectively. For P3HT, FeCl3 provides a high doping level but an unstable counterion, so exchange results in an air stable counterion with a marginal increase in doping. For PDPP-2T, FeCl3 is a weak dopant, but the exchange of FeCl3- for TFSI- is spontaneous, so the doping level increases by >10× with AED.

Evaluation of the potential of colicins to prevent extraluminal contamination of urinary catheters by Escherichia coli.

The feasibility of using colicins to create an antimicrobial lubricant to prevent extraluminal catheter contamination during urinary catheter insertion was assessed. Levels of resistance of uropathogenic Escherichia coli to antibiotics and colicins were compared. The results showed that antibiotics and colicins possess similar frequencies of resistance to a single drug, whereas colicins exhibit significantly lower levels of multidrug resistance (22%) than antibiotics (42%). Colicins and antibiotics showed complementary inhibitory activity, with each targeting different subsets of pathogenic isolates. The collateral impact of these two antimicrobials on genera that are members of the fecal/vaginal/urinary microbiome was assessed, with colicins showing significantly less collateral damage than antibiotics. Using a novel colicin, SR4, minimum inhibitory concentrations (MICs) for a panel of 30 uropathogenic isolates were determined and showed that SR4 achieved the same antimicrobial efficacy as gentamicin using 20-30% less drug. An SR4-impregnated catheter lubricant was created and its ability to prevent extraluminal urinary catheter contamination in vitro was demonstrated. These data indicate that a colicin-impregnated lubricant may provide a viable prophylactic option for preventing catheter-associated urinary tract infections.

Effect of a Medical Student-Led Curriculum on Teen Health Knowledge and Intentions: An Evaluation of the MiHealth Pilot Program.

INTRODUCTION: Adolescents are often thought of as a healthy population, however, they routinely engage in high-risk behaviors that can lead to health problems. Medical students designed MiHealth, a program in which medical students teach health lessons in the high school classroom to help address these behaviors. METHODS: A series of six lessons were created and implemented in the classroom for this pilot study focused on sexual health, intimate partner violence, mental health, smoking and marijuana, nutrition, and physical fitness. High school students in grades nine through twelve at a public high school in southeast Michigan receiving the MiHealth curriculum (N=52) or the standard health education curriculum (N=61) were surveyed on health knowledge, attitudes, and intentions before and after the program. RESULTS: Six weeks after program completion, high school students who received the MiHealth curriculum scored significantly higher on health knowledge ( P=0.007), and expressed significantly healthier attitudes and intentions toward risk behavior compared to controls (P=0.025). Among individual themes, MiHealth resulted in significant knowledge gains in sexual health ( P=0.001) and mental health (P<0.025), and significantly healthier attitudes regarding sexual health (P=0.047), nutrition (P=0.040), and smoking and marijuana (P=0.012). CONCLUSIONS: MiHealth demonstrated promising improvements in health knowledge retention and attitude changes in adolescents 6 weeks after program completion. An interactive curriculum targeting key adolescent health topics given by near-peer medical student educators may provide benefits beyond traditional high school health curricula.

Pheromonicins: an ecologically sound family of bacteriocin-based antibiotics for use in the age of the microbiome.

The time is ripe to usher in a new paradigm in infection control and to move beyond our sole reliance on broad-spectrum antibiotics whose use results in extensive collateral damage to our microbiome and, in so doing, exerts significant selective pressures for resistance to emerge. We propose to supplement the existing pharmacy of conventional antibiotics, with a new drug family, the pheromonicins. These bacteriocin-based antimicrobials are stable, nontoxic proteins that possess potent antibacterial activities, and which can be easily and rapidly retargeted against any bacteria desired. Here we discuss colicin Ia, a pore forming bacteriocin, as the base of a novel drug development platform, the pheromonicins. Recent work suggests this versatile drug development platform can be used to generate pheromonicins active against enveloped viruses, fungi and human cancer cells. Pheromonicins provide a less toxic, more ecologically sound alternative to conventional antibiotics, and their use will help limit our sole reliance on broad-spectrum drugs.

Rapid identification of bacterial biofilms and biofilm wound models using a multichannel nanosensor.

Identification of infectious bacteria responsible for biofilm-associated infections is challenging due to the complex and heterogeneous biofilm matrix. To address this issue and minimize the impact of heterogeneity on biofilm identification, we developed a gold nanoparticle (AuNP)-based multichannel sensor to detect and identify biofilms based on their physicochemical properties. Our results showed that the sensor can discriminate six bacterial biofilms including two composed of uropathogenic bacteria. The capability of the sensor was further demonstrated through discrimination of biofilms in a mixed bacteria/mammalian cell in vitro wound model.

Functional gold nanoparticles as potent antimicrobial agents against multi-drug-resistant bacteria.

We present the use of functionalized gold nanoparticles (AuNPs) to combat multi-drug-resistant pathogenic bacteria. Tuning of the functional groups on the nanoparticle surface provided gold nanoparticles that were effective against both Gram-negative and Gram-positive uropathogens, including multi-drug-resistant pathogens. These AuNPs exhibited low toxicity to mammalian cells, and bacterial resistance was not observed after 20 generations. A strong structure-activity relationship was observed as a function of AuNP functionality, providing guidance to activity prediction and rational design of effective antimicrobial nanoparticles.

The evolutionary histories of clinical and environmental SHV ß-lactamases are intertwined.

The rise of antibiotic-resistant pathogens focuses our attention on the source of antibiotic resistance genes, on the existence of these genes in environments exposed to little or no antibiotics, and on the relationship between resistance genes found in the clinic and those encountered in non-clinical settings. Here, we address the evolutionary history of a class of resistance genes, the SHV ß-lactamases. We focus on bla SHV genes isolated both from clinical and non-clinical sources and show that clinically important resistance determinants arise repeatedly from within a diverse pool of bla SHV genes present in the environment. While our results argue against the notion of a single common origin for all clinically derived bla SHV genes, we detect a characteristic selective signature shaping this protein in clinical environments. This clinical signature reveals the joint action of purifying and positive selection on specific residues, including those known to confer extended-spectrum activity. Surprisingly, antibiotic resistance genes isolated from non-clinical-and presumably antibiotic-free-settings also experience the joint action of purifying and positive selection. The picture that emerges undercuts the notion of a separate reservoir of antibiotic resistance genes confined only to clinical settings. Instead, we argue for the presence of a single extensive and variable pool of antibiotic resistance genes present in the environment.

Rethinking the composition of a rational antibiotic arsenal for the 21st century.

The importance of the human microbiome in health may be the single most valuable development in our conception of the microbial world since Pasteur's germ theory of the 1860s. Its implications for our understanding of health and pathogenesis are profound. Coupled with the revolution in diagnostics that we are now witnessing - a revolution that changes medicine from a science of symptoms to a science of causes - we cannot continue to develop antibiotics as we have for the past 80 years. Instead, we need to usher in a new conception of the role of antibiotics in treatment: away from single molecules that target broad phylogenetic spectra and towards targeted molecules that cripple the pathogen while leaving the rest of the microbiome largely intact.

Resistance is futile: the bacteriocin model for addressing the antibiotic resistance challenge.

Pathogenic bacteria resistant to many or all antibiotics already exist. With the decline in microbiological research at pharmaceutical companies, the high rate at which resistance has evolved and spread has demanded a novel approach to addressing this critical human health issue. In the present paper, we propose a new paradigm in antibiotic discovery and development, one that applies ecological and evolutionary theory to design antimicrobial drugs that are more difficult and/or more costly to resist. In essence, we propose to simply adopt the strategies invented and applied by bacteria for hundreds of millions of years. Our research focuses on bacteriocins, powerful biological weapons, and their use as alternative therapeutics in human health.

Competitive interactions in Escherichia coli populations: the role of bacteriocins.

Explaining the coexistence of competing species is a major challenge in community ecology. In bacterial systems, competition is often driven by the production of bacteriocins, which are narrow-spectrum proteinaceous toxins that serve to kill closely related species, providing the producer better access to limited resources. Bacteriocin producers have been shown to competitively exclude sensitive, nonproducing strains. However, the dynamics between bacteriocin producers, each lethal to its competitor, are largely unknown. In this study, we used in vitro, in vivo and in silico models to study competitive interactions between bacteriocin producers. Two Escherichia coli strains were generated, each carrying a DNA-degrading bacteriocin (colicins E2 and E7). Using reporter-gene assays, we showed that each DNase bacteriocin is not only lethal to its opponent but, at lower doses, can also induce the expression of its opponent's toxin. In a well-mixed habitat, the E2 producer outcompeted its adversary; however, in structured environments (on plates or in mice colons), the two producers coexisted in a spatially 'frozen' pattern. Coexistence occurred when the producers were initiated with a clumped spatial distribution. This suggests that a 'clump' of each producer can block invasion of the other producer. Agent-based simulation of bacteriocin-mediated competition further showed that mutual exclusion in a structured environment is a relatively robust result. These models imply that colicin-mediated colicin induction enables producers to successfully compete and defend their niche against invaders. This suggests that localized interactions between producers of DNA-degrading toxins can lead to stable coexistence of heterogeneously distributed strains within the bacterial community and to the maintenance of diversity.

Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli.

DNA supercoiling is the master function that interconnects chromosome structure and global gene transcription. This function has recently been shown to be under strong selection in Escherichia coli. During the evolution of 12 initially identical populations propagated in a defined environment for 20,000 generations, parallel increases in DNA supercoiling were observed in ten populations. The genetic changes associated with the increased supercoiling were examined in one population, and beneficial mutations in the genes topA (encoding topoisomerase I) and fis (encoding a histone-like protein) were identified. To elucidate the molecular basis and impact of these changes, we quantified the level of genetic, phenotypic, and molecular parallelism linked to DNA supercoiling in all 12 evolving populations. First, sequence determination of DNA topology-related loci revealed strong genetic parallelism, with mutations concentrated in three genes (topA, fis, and dusB), although the populations had different alleles at each locus. Statistical analyses of these polymorphisms implied the action of positive selection and, moreover, suggested that fis and dusB, which belong to the same operon, have related functions. Indeed, we demonstrated that DusB regulates the expression of fis by both experimental and phylogenetic analyses. Second, molecular analyses of five mutations in fis and dusB affecting the transcription, translation, and protein activity of Fis also revealed strong parallelism in the resulting phenotypic effects. Third, artificially increasing DNA supercoiling in one of the two populations that lacked DNA topology changes led to a significant fitness increase. The high levels of molecular and genetic parallelism, targeting a small subset of the many genes involved in DNA supercoiling, indicate that changes in DNA superhelicity have been important in the evolution of these populations. Surprisingly, however, most of the evolved alleles we tested had either no detectable or slightly deleterious effects on fitness, despite these signatures of positive selection.

Role of bacteriocins in mediating interactions of bacterial isolates taken from cystic fibrosis patients.

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97 % of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections.

Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract.

BACKGROUND: The ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins. In an attempt to evaluate the importance of bacteriocin production for Escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken. RESULTS: Six colicin-producing, yet otherwise isogenic, E. coli strains were administered and established in the large intestine of streptomycin-treated mice. The strains' persistence, population density, and doubling time were monitored over a period of 112 days. Early in the experiment only minor differences in population density between the various colicin-producing and the non-producing control strains were detected. However, over time, the density of the control strains plummeted, while that of the colicin-producing strains remained significantly higher (F(7,66) = 2.317; P < 0.0008). CONCLUSION: The data presented here support prior claims that bacteriocin production may play a significant role in the colonization of E. coli in the gastrointestinal tract. Further, this study suggests that the ability to produce bacteriocins may prove to be a critical factor in determining the success of establishing probiotic E. coli in the gastrointestinal tract of humans and animals.

Population genomics and the bacterial species concept.

In recent years, the importance of horizontal gene transfer (HGT) in bacterial evolution has been elevated to such a degree that many bacteriologists now question the very existence of bacterial species. If gene transfer is as rampant as comparative genomic studies have suggested, how could bacterial species survive such genomic fluidity? And yet, most bacteriologists recognize, and name, as species, clusters of bacterial isolates that share complex phenotypic properties. The Core Genome Hypothesis (CGH) has been proposed to explain this apparent paradox of fluid bacterial genomes associated with stable phenotypic clusters. It posits that there is a core of genes responsible for maintaining the species-specific phenotypic clusters observed throughout bacterial diversity and argues that, even in the face of substantial genomic fluidity, bacterial species can be rationally identified and named.

The evolution of reduced microbial killing.

Bacteria engage in a never-ending arms race in which they compete for limited resources and niche space. The outcome of this intense interaction is the evolution of a powerful arsenal of biological weapons. Perhaps the most studied of these are colicins, plasmid-based toxins produced by and active against Escherichia coli. The present study was designed to explore the molecular responses of a colicin-producing strain during serial transfer evolution. What evolutionary changes occur when colicins are produced with no target present? Can killing ability be maintained in the absence of a target? To address these, and other, questions, colicinogenic strains and a noncolicinogenic ancestor were evolved for 253 generations. Samples were taken throughout the experiment and tested for killing ability. By the 38th transfer, a decreased killing ability and an increase in fitness were observed in the colicin-producing strains. Surprisingly, DNA sequence determination of the colicin plasmids revealed no changes in plasmid sequences. However, a set of chromosomally encoded loci experienced changes in gene expression that were positively associated with the reduction in killing. The most significant expression changes were observed in DNA repair genes (which were downregulated in the evolved strains), Mg ion uptake genes (which were upregulated), and late prophage genes (which were upregulated). These results indicate a fine-tuned response to the evolutionary pressures of colicin production, with far more genes involved than had been anticipated.

The role of SOS boxes in enteric bacteriocin regulation.

Bacteriocins are a large and functionally diverse family of toxins found in all major lineages of Bacteria. Colicins, those bacteriocins produced by Escherichia coli, serve as a model system for investigations of bacteriocin structure-function relationships, genetic organization, and their ecological role and evolutionary history. Colicin expression is often dependent on host regulatory pathways (such as the SOS system), is usually confined to times of stress, and results in death of the producing cells. This study investigates the role of the SOS system in mediating this unique form of toxin expression. A comparison of all the sequenced enteric bacteriocin promoters reveals that over 75 % are regulated by dual, overlapping SOS boxes, which serve to bind two LexA repressor proteins. Furthermore, a highly conserved poly-A motif is present in both of the binding sites examined, indicating enhanced affinity of the LexA protein for the binding site. The use of gene expression analysis and deletion mutations further demonstrates that these unique LexA cooperative binding regions result in a fine tuning of bacteriocin production, limiting it to times of stress. These results suggest that the evolution of dual SOS boxes elegantly accomplishes the task of increasing the amount of toxin produced by a cell while decreasing the rate of uninduced production, effectively reducing the cost of colicin production. This hypothesis may explain why such a promoter motif is present at such high frequencies in natural populations of bacteriocin-producing enteric bacteria.

Tests of parallel molecular evolution in a long-term experiment with Escherichia coli.

The repeatability of evolutionary change is difficult to quantify because only a single outcome can usually be observed for any precise set of circumstances. In this study, however, we have quantified the frequency of parallel and divergent genetic changes in 12 initially identical populations of Escherichia coli that evolved in identical environments for 20,000 cell generations. Unlike previous analyses in which candidate genes were identified based on parallel phenotypic changes, here we sequenced four loci (pykF, nadR, pbpA-rodA, and hokB/sokB) in which mutations of unknown effect had been discovered in one population, and then we compared the substitution pattern in these "blind" candidate genes with the pattern found in 36 randomly chosen genes. Two candidate genes, pykF and nadR, had substitutions in all 11 other populations, and the other 2 in several populations. There were very few cases, however, in which the exact same mutations were substituted, in contrast to the findings from conceptually related work performed with evolving virus populations. No random genes had any substitutions except in four populations that evolved defects in DNA repair. Tests of four different statistical aspects of the pattern of molecular evolution all indicate that adaptation by natural selection drove the parallel changes in these candidate genes.

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates / Divergencia genómica de algunas cepas de Escherichia coli: pruebas de la transferencia horizontal y variación en las velocidades de mutación

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago (AU)

Se secuenciaron 36 regiones del genoma de Escherichia coli B elegidas al azar y que están presentes en la mayoría o en todos los genomas de E. coli secuenciados. Se examinaron las relaciones filogenéticas entre cepas de E. coli y se buscaron pruebas de transferencia génica horizontal y de variación en la tasa de mutación. El árbol filogenético conjunto de genes indica que E. coli B y K-12 son las cepas con un parentesco más estrecho, mientras que E. coli O157:H7 se encuentra más alejada y aún más lo están Shigella flexneri 2a y E. coli CFT073, siendo esta última la más distante de todas. En el grupo B, K-12 y O157:H7, varias regiones indican que hay topologías alternativas. La transferencia génica horizontal es una explicación plausible de estas incongruencias filogenéticas, pero también hemos hallado pruebas de una evolución más rápida en sitios sinónimos en el linaje O157:H7. Así pues, una interpretación más profunda de estos resultados queda confundida por una asociación entre unos genes que muestran una evolución más rápida y otros que son transferidos horizontalmente. Usando genes que apoyan los grupos B y K-1, y empleando una estima de la tasa de mutación obtenida a partir de un experimento de evolución a largo plazo con E. coli B y suponiendo 200 generaciones por año, se estimó que las cepas B y K-12 divergieron hace varios cientos de miles de años, mientras que O157:H7 se separó de su ancestro común hace entre 1,5 y 2 millones de años (AU)

Phage associated bacteriocins reveal a novel mechanism for bacteriocin diversification in Klebsiella.

Ninety-six isolates of Klebsiella pneumoniae and K. oxytoca were recovered from wild mammals in Australia. 14.6% of these bacteria produce killing phenotypes that suggest the production of bacteriocin toxins. Cloning and sequencing of the gene clusters encoding two of these killing phenotypes revealed two instances of a bacteriocin associated with a bacteriophage gene, the first such genetic organization described. The newly identified klebicin C gene cluster was discovered in both K. pneumoniae and K. oxytoca. The newly identified klebicin D gene cluster was detected in K. oxytoca. Protein sequence comparisons and phylogenetic inference suggest that klebicin C is most closely related to the rRNase group of colicins (such as colicin E4), while klebicin D is most closely related to the tRNase group of colicins (such as colicin D). The klebicin C and D gene clusters have similar genetic and regulatory organizations. In both cases, an operon structure is inferred consisting of a phage-associated open reading frame and klebicin activity and associated immunity genes. This novel bacteriophage/bacteriocin organization may provide a novel mechanism for the generation of bacteriocin diversity in Klebsiella.