Showing papers on "Bacteria published in 2008"

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria

Abstract: Bacteria have existed on Earth for three billion years or so and have become adept at protecting themselves against toxic chemicals. Antibiotics have been in clinical use for a little more than 6 decades. That antibiotic resistance is now a major clinical problem all over the world attests to the success and speed of bacterial adaptation. Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Acquisition of genes needed to elaborate the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids, transposable elements and integron systems, that move genes from one DNA system to another and from one bacterial cell to another, not necessarily one related to the gene donor. Bacterial plasmids serve as the scaffold on which are assembled arrays of antibiotic resistance genes, by transposition (transposable elements and ISCR mediated transposition) and site-specific recombination mechanisms (integron gene cassettes).The evidence suggests that antibiotic resistance genes in human bacterial pathogens originate from a multitude of bacterial sources, indicating that the genomes of all bacteria can be considered as a single global gene pool into which most, if not all, bacteria can dip for genes necessary for survival. In terms of antibiotic resistance, plasmids serve a central role, as the vehicles for resistance gene capture and their subsequent dissemination. These various aspects of bacterial resistance to antibiotics will be explored in this presentation.

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

Abstract: Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.

Bacteria Subsisting on Antibiotics

Abstract: Antibiotics are a crucial line of defense against bacterial infections. Nevertheless, several antibiotics are natural products of microorganisms that have as yet poorly appreciated ecological roles in the wider environment. We isolated hundreds of soil bacteria with the capacity to grow on antibiotics as a sole carbon source. Of 18 antibiotics tested, representing eight major classes of natural and synthetic origin, 13 to 17 supported the growth of clonal bacteria from each of 11 diverse soils. Bacteria subsisting on antibiotics are surprisingly phylogenetically diverse, and many are closely related to human pathogens. Furthermore, each antibiotic-consuming isolate was resistant to multiple antibiotics at clinically relevant concentrations. This phenomenon suggests that this unappreciated reservoir of antibiotic-resistance determinants can contribute to the increasing levels of multiple antibiotic resistance in pathogenic bacteria.

Targeted photothermal lysis of the pathogenic bacteria, Pseudomonas aeruginosa, with gold nanorods.

Abstract: Increases in the prevalence of antibiotic resistant bacteria require new approaches for the treatment of infectious bacterial pathogens. It is now clear that a nanotechnology-driven approach using nanoparticles to selectively target and destroy pathogenic bacteria can be successfully implemented. We have explored this approach by using gold nanorods that have been covalently linked to primary antibodies to selectively destroy the pathogenic Gram-negative bacterium, Pseudomonas aeruginosa. We find that, following nanorod attachment to the bacterial cell surface, exposure to near-infrared radiation results in a significant reduction in bacterial cell viability.

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.

Abstract: Successful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms--organized communities of surface-attached cells--but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs

Abstract: Aims: To investigate whether the relative abundance of the Bacteroidetes and Firmicutes divisions in pigs is different between obese and lean animals. Methods and Results: Group-specific primers were designed to target the 16S rRNA genes of Bacteroidetes and Firmicutes present in the gut. After the validation of their specificity, these primers were used in the real-time PCR quantification of all Bacteria, Firmicutes division, Bacteroidetes division and Bacteroides spp. in the faecal samples of obese and lean pigs from Banna mini-pig inbred line. The obese pigs had a ∼61% fewer percentage (based on all Bacteria) of Bacteroidetes division (P = 0·033) and a ∼56% fewer proportion of Bacteroides spp. (P = 0·047) than the lean pigs. The proportions of both Bacteroidetes and Bacteroides had a negative correlation (P < 0·01) with the body weight. Conclusion: The results suggested that the fat storage might affect the proportion of Bacteroidetes division in the gut. Significance and Impact of the Study: The real-time PCR assays developed for Firmicutes and Bacteroidetes will be useful for investigating the composition of gut microbiota.

Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea.

Abstract: Recently, a microbial consortium was shown to couple the anaerobic oxidation of methane to denitrification, predominantly in the form of nitrite reduction to dinitrogen gas. This consortium was dominated by bacteria of an as yet uncharacterized division and archaea of the order Methanosarcinales. The present manuscript reports on the upscaling of the enrichment culture, and addresses the role of the archaea in methane oxidation. The key gene of methanotrophic and methanogenic archaea, mcrA, was sequenced. The associated cofactor F(430) was shown to have a mass of 905 Da, the same as for methanogens and different from the heavier form (951 Da) found in methanotrophic archaea. After prolonged enrichment (> 1 year), no inhibition of anaerobic methane oxidation was observed in the presence of 20 mM bromoethane sulfonate, a specific inhibitor of MCR. Optimization of the cultivation conditions led to higher rates of methane oxidation and to the decline of the archaeal population, as shown by fluorescence in situ hybridization and quantitative MALDI-TOF analysis of F(430). Mass balancing showed that methane oxidation was still coupled to nitrite reduction in the total absence of oxygen. Together, our results show that bacteria can couple the anaerobic oxidation of methane to denitrification without the involvement of Archaea.

Metabolic aspects of aerobic obligate methanotrophy.

Abstract: Publisher Summary This chapter discusses the metabolic aspects of aerobic obligate methanotrophy. Aerobic methanotrophs are a unique group of gram-negative bacteria that use methane as carbon and energy source. Methanotrophs have been studied intensively over the past 40 years since these bacteria possess significant metabolic potential for practical use in the biotransformation of a variety of organic substrates, bioremediation of pollutants the production of single-cell protein (SCP), and value-added products. They also play a vital role in the global methane cycle, mitigating the emissions and green-house effects of methane on the Earth's climate. Methanotrophs build all of their cell constituents from C1 compounds by employing special biosynthetic pathways for phosphotrioses, which are different from those of heterotrophic bacteria.

Redox-Active Antibiotics Control Gene Expression and Community Behavior in Divergent Bacteria

Abstract: It is thought that bacteria excrete redox-active pigments as antibiotics to inhibit competitors. In Pseudomonas aeruginosa, the endogenous antibiotic pyocyanin activates SoxR, a transcription factor conserved in Proteo- and Actinobacteria. In Escherichia coli, SoxR regulates the superoxide stress response. Bioinformatic analysis coupled with gene expression studies in P. aeruginosa and Streptomyces coelicolor revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses, despite the fact that many of these organisms still produce redox-active small molecules, which indicates that redox-active pigments play a role independent of oxidative stress. These compounds had profound effects on the structural organization of colony biofilms in both P. aeruginosa and S. coelicolor, which shows that "secondary metabolites" play important conserved roles in gene expression and development.

Comparison of Carbon Nutrition for Pathogenic and Commensal Escherichia coli Strains in the Mouse Intestine

Abstract: The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.

Antimicrobial defense and persistent infection in insects.

Abstract: During 400 million years of existence, insects have rarely succumbed to the evolution of microbial resistance against their potent antimicrobial immune defenses. We found that microbial clearance after infection is extremely fast and that induced antimicrobial activity starts to increase only when most of the bacteria (99.5%) have been removed. Our experiments showed that those bacteria that survived exposure to the insect's constitutive immune response were subsequently more resistant to it. These results imply that induced antimicrobial compounds function primarily to protect the insect against the bacteria that persist within their body, rather than to clear microbial infections. These findings suggest that understanding of the management of antimicrobial peptides in natural systems might inform medical treatment strategies that avoid the risk of drug resistance.

Vancomycin-modified nanoparticles for efficient targeting and preconcentration of Gram-positive and Gram-negative bacteria.

Abstract: A series of vancomycin-modified nanoparticles were developed and employed in magnetic confinement assays to isolate a variety of Gram-positive and Gram-negative bacteria from aqueous solution. We determined that the orientation/architecture of vancomycin on the surface of the nanoparticles and the overall surface coverage is critical in mediating fast and effective interactions between the nanoparticle and the pathogen cell wall surface and only one orientation/architecture in a series of modified nanoparticles leads to the efficient and reproducible capture of several important pathogenic bacteria. Interestingly, as the nanoparticles increase in diameter (from approximately 50 to 2800 nm), it is necessary to incorporate a long linker between the nanoparticle surface and the vancomycin moiety in order for the surface bound probe to efficiently confine Gram-positive bacteria. Finally, we also determined that the time required for efficient labeling and subsequent magnetic confinement significantly decreases as the size of the nanoparticle and the vancomycin surface coverage on the nanoparticle increases. As disease detection technologies transition to "lab-on-a-chip" based platforms it is necessary to develop strategies to effectively and inexpensively preconcentrate cells from large volume to volumes more amenable to these types of microfluidic devices. These small molecule-modified superparamagnetic nanoparticles can provide a means by which this can be accomplished.

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli

Abstract: We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

Abstract: The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol−1, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol−1, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.

Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi.

Abstract: Summary. This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236].

Protein oxidation: key to bacterial desiccation resistance?

Abstract: For extremely ionizing radiation-resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the US Department of Energy's Hanford Site in Washington State. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of the genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms, which limit protein oxidation during dehydration.

Nucleotide Biosynthesis Is Critical for Growth of Bacteria in Human Blood

Abstract: Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another Gram-negative pathogen, Salmonella enterica, and in the Gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections.

Plant Cell Wall Breakdown by Anaerobic Microorganisms from the Mammalian Digestive Tract

Abstract: Degradation of lignocellulosic plant material in the mammalian digestive tract is accomplished by communities of anaerobic microorganisms that exist in symbiotic association with the host. Catalytic domains and substrate-binding modules concerned with plant polysaccharide degradation are found in a variety of anaerobic bacteria, fungi, and protozoa from the mammalian gut. The organization of plant cell wall-degrading enzymes, however, varies widely. The cellulolytic gram-positive bacterium Ruminococcus flavefaciens produces an elaborate cellulosomal enzyme complex that is anchored to the bacterial cell wall; assembly of the complex involves at least five different dockerin:cohesin specificities, and the R. flavefaciens genome encodes at least 180 dockerin-containing proteins that encompass a wide array of catalytic and binding activities. On the other hand, in the cellulolytic protozoan, Polyplastron multivesiculatum, individual plant cell wall-degrading enzymes appear to be secreted into food vacuoles, while the gram-negative bacterium Prevotella bryantii appears to possess a sequestration-type system for the utilization of soluble xylans. The system that is employed for polysaccharide utilization must play a major role in defining the ecological niche that each organism occupies within a complex gut community. 16S rRNA analyses are also revealing uncultured bacterial species closely adherent to fibrous substrates in the rumen and in the large intestine of animals and humans. The true complexity, both at a single organism and community level, of the microbial enzyme systems that allow animals to digest plant material is beginning to become apparent.

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

Abstract: Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when

Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria.

Abstract: Bacterial aerobic ammonium oxidation and anaerobic ammonium oxidation (anammox) are important processes in the global nitrogen cycle. Key enzymes in both processes are the octahaem cytochrome c (OCC) proteins, hydroxylamine oxidoreductase (HAO) of aerobic ammonium-oxidizing bacteria (AOB), which catalyses the oxidation of hydroxylamine to nitrite, and hydrazine oxidoreductase (HZO) of anammox bacteria, which converts hydrazine to N(2). While the genomes of AOB encode up to three nearly identical copies of hao operons, genome analysis of Candidatus'Kuenenia stuttgartiensis' showed eight highly divergent octahaem protein coding regions as possible candidates for the HZO. Based on their phylogenetic relationship and biochemical characteristics, the sequences of these eight gene products grouped in three clusters. Degenerate primers were designed on the basis of available gene sequences with the aim to detect hao and hzo genes in various ecosystems. The hao primer pairs amplified gene fragments from 738 to 1172 bp and the hzo primer pairs amplified gene fragments from 289 to 876 bp in length, when tested on genomic DNA isolated from a variety of AOB and anammox bacteria. A selection of these primer pairs was also used successfully to amplify and analyse the hao and hzo genes in community DNA isolated from different ecosystems harbouring both AOB and anammox bacteria. We propose that OCC protein-encoding genes are suitable targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing bacteria.