Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient. - Related Documents




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900601.0000Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient. Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.201930663644
892110.9998Multivariate approach to comparing whole-cell proteomes of Bacillus cereus indicates a biofilm-specific proteome. Biofilm bacteria are widely held to exhibit a unique phenotype, typified by their increased resistance to antimicrobial agents. Numerous studies have been devoted to the identification of biofilm-specific genes, but surprisingly few have been reported to date. We compared the whole cell proteomes of 24 h old Bacillus cereus biofilms and the associated suspended population to exponential, transient and stationary phase planktonic cultures using the unbiased approach of principal component analysis, comparing the quantity variations of the 823 detected spots. The analyses support the hypothesis that biofilms of Gram positive bacteria have a unique pattern of gene expression. The data provides proteomic evidence for a new biofilm and surface influenced planktonic population which is distinct to both planktonic and biofilm cells.200616889414
426120.9997Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage. Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations.201829119434
892030.9997A systems biology approach to drug targets in Pseudomonas aeruginosa biofilm. Antibiotic resistance is an increasing problem in the health care system and we are in a constant race with evolving bacteria. Biofilm-associated growth is thought to play a key role in bacterial adaptability and antibiotic resistance. We employed a systems biology approach to identify candidate drug targets for biofilm-associated bacteria by imitating specific microenvironments found in microbial communities associated with biofilm formation. A previously reconstructed metabolic model of Pseudomonas aeruginosa (PA) was used to study the effect of gene deletion on bacterial growth in planktonic and biofilm-like environmental conditions. A set of 26 genes essential in both conditions was identified. Moreover, these genes have no homology with any human gene. While none of these genes were essential in only one of the conditions, we found condition-dependent genes, which could be used to slow growth specifically in biofilm-associated PA. Furthermore, we performed a double gene deletion study and obtained 17 combinations consisting of 21 different genes, which were conditionally essential. While most of the difference in double essential gene sets could be explained by different medium composition found in biofilm-like and planktonic conditions, we observed a clear effect of changes in oxygen availability on the growth performance. Eight gene pairs were found to be synthetic lethal in oxygen-limited conditions. These gene sets may serve as novel metabolic drug targets to combat particularly biofilm-associated PA. Taken together, this study demonstrates that metabolic modeling of human pathogens can be used to identify oxygen-sensitive drug targets and thus, that this systems biology approach represents a powerful tool to identify novel candidate antibiotic targets.201222523548
891340.9997The gut environment regulates bacterial gene expression which modulates susceptibility to bacteriophage infection. Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.202235421351
899050.9997Enhanced virulence of Salmonella enterica serovar typhimurium after passage through mice. The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro, and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica.201121098099
382460.9997Screening for novel antibiotic resistance genes. Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. The bacterial strains are screened for antibiotic resistance. Cultures of the strain are grown, and DNA is extracted. A partial digest of the extraction is cloned into Escherichia coli, and the transformants are plated on selective media. Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen.201020830570
631170.9997Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces. BACKGROUND: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. RESULTS: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. CONCLUSIONS: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.201728950904
940580.9997Functional Metagenomic Screening for Antimicrobial Resistance in the Oral Microbiome. A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.202134410638
891590.9997Genetic regulation of host responses to Salmonella infection in mice. Salmonella spp are Gram-negative bacteria capable of infecting a wide range of host species, including humans, domesticated and wild mammals, reptiles, birds and insects. The outcome of an encounter between Salmonella and its host is dependent upon multiple factors including the host genetic background. To facilitate the study of the genetic factors involved in resistance to this pathogen, mouse models of Salmonella infection have been developed and studied for years, allowing identification of several genes and pathways that may influence the disease outcome. In this review, we will cover some of the genes involved in mouse resistance to Salmonella that were identified through the study of congenic mouse strains, cloning of spontaneous mouse mutations, use of site-directed mutagenesis or quantitative trait loci analysis. In parallel, the relevant information pertaining to genes involved in resistance to Salmonella in humans will be discussed.200212424619
3810100.9997The Effect of the Presence and Absence of DNA Repair Genes on the Rate and Pattern of Mutation in Bacteria. Bacteria lose and gain repair genes as they evolve. Here, we investigate the consequences of gain and loss of 11 DNA repair genes across a broad range of bacteria. Using synonymous polymorphisms from bacteria and a set of 50 phylogenetically independent contrasts, we find no evidence that the presence or absence of these 11 genes affects either the overall level of diversity or the pattern of mutation. Using phylogenetic generalized linear squares yields a similar conclusion. It seems likely that the lack of an effect is due to variation in the genetic background and the environment which obscures any effects that the presence or absence of individual genes might have.202439376054
8919110.9997Gene expression in Pseudomonas aeruginosa biofilms. Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.200111677611
6310120.9997Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes. BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.200616984631
8384130.9997In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors. Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.201425408687
9433140.9997The relative contributions of physical structure and cell density to the antibiotic susceptibility of bacteria in biofilms. For many bacterial infections, noninherited mechanisms of resistance are responsible for extending the term of treatment and in some cases precluding its success. Among the most important of these noninherited mechanisms of resistance is the ability of bacteria to form biofilms. There is compelling evidence that bacteria within biofilms are more refractory to antibiotics than are planktonic cells. Not so clear, however, is the extent to which this resistance can be attributed to the structure of biofilms rather than the physiology and density of bacteria within them. To explore the contribution of the structure of biofilms to resistance in a quantitative way, we developed an assay that compares the antibiotic sensitivity of bacteria in biofilms to cells mechanically released from these structures. Our method, which we apply to Escherichia coli and Staphylococcus aureus each with antibiotics of five classes, controls for the density and physiological state of the treated bacteria. For most of the antibiotics tested, the bacteria in biofilms were no more resistant than the corresponding populations of planktonic cells of similar density. Our results, however, suggest that killing by gentamicin, streptomycin, and colistin is profoundly inhibited by the structure of biofilms; these drugs are substantially more effective in killing bacteria released from biofilms than cells within these structures.201222450987
9007150.9997Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces. AIMS: To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces. METHODS AND RESULTS: Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined. CONCLUSIONS: Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.200919239551
8923160.9997The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli. Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. IMPORTANCE: With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.201627879333
9670170.9997An Approach to In Silico Dissection of Bacterial Intelligence Through Selective Genomic Tools. All the genetic potential and the intelligence a bacteria can showcase in a given environment are embedded in its genome. In this study, we have presented systematic guidelines to understand a bacterial genome with the relevant set of in silico tools using a novel bacteria as an example. This study presents a multi-dimensional approach from genome annotation to tracing genes and their network of metabolism operating in an organism. It also shows how the sequence can be used to mine the enzymes and construction of its 3-dimensional structure so that its functional behavior can be predicted and compared. The discriminating algorithm allows analysis of the promoter region and provides the insight in the regulation of genes in spite of the similarity in its sequences. The ecological niche specific bacterial behavior and adapted altered physiology can be understood through the presence of secondary metabolite, antibiotic resistance genes, and viral genes; and it helps in the valorization of genetic information for developing new biological application/processes. This study provides an in silico work plan and necessary steps for genome analysis of novel bacteria without any rigorous wet lab experiments.201830013271
9406180.9997Proteomics as the final step in the functional metagenomics study of antimicrobial resistance. The majority of clinically applied antimicrobial agents are derived from natural products generated by soil microorganisms and therefore resistance is likely to be ubiquitous in such environments. This is supported by the fact that numerous clinically important resistance mechanisms are encoded within the genomes of such bacteria. Advances in genomic sequencing have enabled the in silico identification of putative resistance genes present in these microorganisms. However, it is not sufficient to rely on the identification of putative resistance genes, we must also determine if the resultant proteins confer a resistant phenotype. This will require an analysis pipeline that extends from the extraction of environmental DNA, to the identification and analysis of potential resistance genes and their resultant proteins and phenotypes. This review focuses on the application of functional metagenomics and proteomics to study antimicrobial resistance in diverse environments.201525784907
8388190.9997Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines. All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 degrees C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.201020624965