[Show abstract][Hide abstract] ABSTRACT: Vancomycin-resistant Enterococcus faecium has emerged as a multidrug-resistant pathogen in hospital settings. Here, we present the draft genome sequence of a high-level
vancomycin-resistant strain, E. faecium ATCC 51559, which is employed as a standard laboratory vanA genotype-positive control strain for clinical and laboratory studies.
[Show abstract][Hide abstract] ABSTRACT: The Burkholderia cepacia complex (BCC) includes opportunistic pathogenic bacteria that have occasionally been recovered from various pharmaceutical products, including antiseptics and disinfectants. Plausible reasons for the contamination include intrinsic sources, such as inadequate process controls, especially for water or equipment used during product manufacture, or extrinsic sources, such as improper handling and dilution or distribution in contaminated containers. Because the survival of BCC in antiseptics is a concern to the public health and pharmaceutical industry, we determined minimum inhibitory concentrations (MICs) of 36 BCC strains against the antiseptics, following exposure to chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions (1-500 µg/ml for each chemical). Susceptibility to CHX and BZK varied across the BCC strains and was recorded as mean 90.3 and 111.1 µg/ml, respectively, at initial inoculation, which was significantly higher than the 46.4 and 61.1 µg/ml levels measured for BCC incubated in water for 40 days. After determining antiseptic MICs of individual BCC strains, BCC recovery was measured on Tryptic Soy Agar (TSA), Reasoner's Second Agar (R2A) and diluted preparations of these media under their sub-MICs. The survival of BCC was monitored for 14 days (336 h) in sub-MICs diluted to less than their antiseptic susceptible concentration value. Diluted TSA and R2A media exhibited greater efficiency of recovery for most BCC strains from the CHX and BZK solutions than full strength TSA or R2A. For BCC survival in antiseptic solutions, the cell number of BCC decreased rapidly within the first 20 min in both antiseptics, but after this, recovery remained constant in CHX and increased in BZK over the 14 day incubation period. The results indicate that BCC in water can remain viable with low susceptibility to antiseptics for 14 days, which suggests the necessity for improved detection methods and control measures to monitor BCC contamination in pharmaceutical products.
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes.
Journal of Industrial Microbiology and Biotechnology 02/2015; 42(5). DOI:10.1007/s10295-015-1599-4 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The bacterial genus Mycobacterium is of great interest in the medical and biotechnological fields. Despite a flood of genome sequencing and functional genomics data, significant gaps in knowledge between genome and phenome seriously hinder efforts toward the treatment of mycobacterial diseases and practical biotechnological applications. In this study, we propose the use of systematic, comparative functional pan-genomic analysis to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon (PAH) metabolism in the genus Mycobacterium.
Phylogenetic, phenotypic, and genomic information for 27 completely genome-sequenced mycobacteria was systematically integrated to reconstruct a mycobacterial phenotype network (MPN) with a pan-genomic concept at a network level. In the MPN, mycobacterial phenotypes show typical scale-free relationships. PAH degradation is an isolated phenotype with the lowest connection degree, consistent with phylogenetic and environmental isolation of PAH degraders. A series of functional pan-genomic analyses provide conserved and unique types of genomic evidence for strong epistatic and pleiotropic impacts on evolutionary trajectories of the PAH-degrading phenotype. Under strong natural selection, the detailed gene gain/loss patterns from horizontal gene transfer (HGT)/deletion events hypothesize a plausible evolutionary path, an epistasis-based birth and pleiotropy-dependent death, for PAH metabolism in the genus Mycobacterium. This study generated a practical mycobacterial compendium of phenotypic and genomic changes, focusing on the PAH-degrading phenotype, with a pan-genomic perspective of the evolutionary events and the environmental challenges.
Our findings suggest that when selection acts on PAH metabolism, only a small fraction of possible trajectories is likely to be observed, owing mainly to a combination of the ambiguous phenotypic effects of PAHs and the corresponding pleiotropy- and epistasis-dependent evolutionary adaptation. Evolutionary constraints on the selection of trajectories, like those seen in PAH-degrading phenotypes, are likely to apply to the evolution of other phenotypes in the genus Mycobacterium.
Keywords: Mycobacterium ; PAH metabolism; Pan-genome; Functional genomics; Functional pan-genome; Phenotype network; Evolution; Epistasis; Pleiotropy
[Show abstract][Hide abstract] ABSTRACT: Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and their pleiotropic and epistatic behavior(s) responsible for low-molecular weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparison using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild-type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity toward three-ring PAHs. Structure analysis and PAH substrates docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taken together, the Pdo system is a RHO system with pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also a crucial for metabolic quality and quantity of the PAH-MN.
Journal of Bacteriology 07/2014; 196(19). DOI:10.1128/JB.01945-14 · 2.81 Impact Factor
Encyclopedia of Food Microbiology, 2nd Edition, 2nd edited by Carl A Batt, 06/2014: chapter Metabolic Pathways | Production of secondary metabolites of bacteria: pages 561-569; Elsevier., ISBN: ISBN-13: 978-0123847300
[Show abstract][Hide abstract] ABSTRACT: Ceftiofur is a highly effective veterinary cephalosporin, yet it is rapidly degraded by bacteria in the gut. The goal of this work was to directly determine the mechanism of ceftiofur degradation by the bovine intestinal isolate Bacillus cereus P41. B. cereus P41 was isolated from the feces of a cow that had not been treated with cephalosporins, and was found to rapidly degrade ceftiofur in culture. Analysis of spent culture media by HPLC/UV and HPLC/MS revealed one major metabolite of ceftiofur, with a negative ion m/z of 127. Comparison of ceftiofur, ceftriaxone, and cefpodoxime degradation suggested that the major stable ceftiofur metabolite was the thiofuroic acid group eliminated from the C-3 position of the drug after hydrolysis by β-lactamase. Genomic DNA from B. cereus P41 was cloned into Escherichia coli, and the transformants were screened for growth in the presence of ceftiofur. DNA sequencing of the plasmid pHSG299-BC-3 insert revealed the presence of a gene encoding a metallo-β-lactamase. Incubation of ceftiofur with either the E. coli transformant or a commercial B. cereus metallo-β-lactamase showed degradation of the drug and formation of the same major metabolite produced by B. cereus P41. These data demonstrate that a metallo-β-lactamase plays a major role in the degradation of ceftiofur by the bovine intestinal bacterium B. cereus P41.
[Show abstract][Hide abstract] ABSTRACT: Abstract Silver nanoparticles (AgNP) are widely used for their antibacterial properties. Incorporation of AgNP into food-related products and health supplements represents a potential route for oral exposure to AgNP; however, the effects of such exposure on the gastrointestinal system are mostly unknown. This study evaluated changes in the populations of intestinal-microbiota and intestinal-mucosal gene expression in Sprague-Dawley rats (both male and female) that were gavaged orally with discrete sizes of AgNP (10, 75 and 110 nm) and silver acetate. Doses of AgNP (9, 18 and 36 mg/kg body weight/day) and silver acetate (100, 200 and 400 mg/kg body weight/day) were divided and administered to rats twice daily (∼10 h apart) for 13 weeks. The results indicate that AgNP prompted size- and dose-dependent changes to ileal-mucosal microbial populations, as well as, intestinal gene expression and induced an apparent shift in the gut microbiota toward greater proportions of Gram-negative bacteria. DNA-based analyses revealed that exposure to 10 nm AgNP and low-dose silver acetate caused a decrease in populations of Firmicutes phyla, along with a decrease in the Lactobacillus genus. Analysis of host gene expression demonstrated that smaller sizes and lower doses of AgNP exposure prompted the decreased expression of important immunomodulatory genes, including MUC3, TLR2, TLR4, GPR43 and FOXP3. Gender-specific effects to AgNP exposure were more prominent for the gut-associated immune responses. These results indicate that the oral exposure to AgNP alter mucosa-associated microbiota and modulate the gut-associated immune response and the overall homeostasis of the intestinal tract.
[Show abstract][Hide abstract] ABSTRACT: Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner's 2nd Agar or Broth (R2A or R2AB), Brain-Heart Infusion Broth (BHIB), Mueller-Hinton Broth (MHB), and Ashdown's (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.
[Show abstract][Hide abstract] ABSTRACT: Exposure of humans to antimicrobial residues in food-producing animals may alter the intestinal microbiota which could result in a potential risk to human health. To determine the effect of enrofloxacin on the human intestinal microbiota, fecal suspensions (25%) were cultured in the presence of 0.06-5 µg/ml enrofloxacin. The bacterial community was analyzed by plating on selective culture media, pyrosequencing and nuclear magnetic resonance (NMR) spectroscopy. Pyrosequencing analysis of 16S rRNA genes and viable counts on Bacteroides sp., Enterococcus sp., and Bifidobacterium sp. selection medium indicated that there were no significant changes in the bacteria numbers at the selected enrofloxacin concentrations (0.06, 0.1, and 1 µg/ml) relative to the control samples after a 48 h incubation. NMR analysis showed remarkably similar spectra in cultures treated with 0.06, 0.1, and 1 µg/ml enrofloxacin, with some slight differences in peak heights. However, hierarchical clustering analysis indicated significant differences in metabolite concentrations between the control and those samples treated with 1 µg/ml enrofloxacin. Leucine, phenylalanine, proline, and 2-oxovalerate were positively correlated with the concentration of enrofloxacin. NMR analysis is a potentially useful tool to monitor changes of the human intestinal microbiota, in addition to traditional culture methods and pyrosequencing.
Journal of Molecular Microbiology and Biotechnology 12/2012; 22(5):317-325. DOI:10.1159/000345147 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Deepwater Horizon oil spill of 2010 raised concerns that dispersant and dispersed oil, as well as crude oil itself, could contaminate shellfish and seafood habitats with hazardous residues that had potential implications for human health and the ecosystem. However, little is known about the effects of crude oil and dispersant on the human fecal microbiota. The aim of this research was to evaluate the potential effects of Deepwater Horizon crude oil, Corexit 9500 dispersant, and their combination on human fecal microbial communities, using an in vitro culture test system. Fecal specimens from healthy adult volunteers were made into suspensions, which were then treated with oil, dispersant, or oil-dispersant mixtures under anaerobic conditions in an in vitro culture test system. Perturbations of the microbial community, compared to untreated control cultures, were assessed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and pyrosequencing methods. DGGE and pyrosequencing analysis showed that oil-dispersant mixtures reduced the diversity of fecal microbiota from all individuals. Real-time PCR results indicated that the copy numbers of 16S rRNA genes in cultures treated with dispersed oil or oil alone were significantly lower than those in control incubations. The abundance of the Bacteroidetes decreased in crude oil-treated and dispersed-oil-treated cultures, while the Proteobacteria increased in cultures treated with dispersed oil. In conclusion, the human fecal microbiota was affected differently by oil and dispersed oil, and the influence of dispersed oil was significantly greater than that of either oil or dispersant alone compared to control cultures.
[Show abstract][Hide abstract] ABSTRACT: Sudan azo dyes are banned for food usage in most countries, but they are illegally used to maintain or enhance the color of food products due to low cost, bright staining, and wide availability of the dyes. In this report, we examined the toxic effects of these azo dyes and their potential reduction metabolites on 11 prevalent human intestinal bacterial strains. Among the tested bacteria, cell growth of 2, 3, 5, 5, and 1 strains was inhibited by Sudan I, II, III, IV, and Para Red, respectively. At the tested concentration of 100 μM, Sudan I and II inhibited growth of Clostridium perfringens and Lactobacillus rhamnosus with decrease of growth rates from 14 to 47%. Sudan II also affected growth of Enterococcus faecalis. Growth of Bifidobacterium catenulatum, C. perfringens, E. faecalis, Escherichia coli, and Peptostreptococcus magnus was affected by Sudan III and IV with decrease in growth rates from 11 to 67%. C. perfringens was the only strain in which growth was affected by Para Red with 47 and 26% growth decreases at 6 and 10 h, respectively. 1-Amino-2-naphthol, a common metabolite of the dyes, was capable of inhibiting growth of most of the tested bacteria with inhibition rates from 8 to 46%. However, the other metabolites of the dyes had no effect on growth of the bacterial strains. The dyes and their metabolites had less effect on cell viability than on cell growth of the tested bacterial strains. Clostridium indolis and Clostridium ramosum were the only two strains with about a 10 % decrease in cell viability in the presence of Sudan azo dyes. The present results suggested that Sudan azo dyes and their metabolites potentially affect the human intestinal bacterial ecology by selectively inhibiting some bacterial species, which may have an adverse effect on human health.
[Show abstract][Hide abstract] ABSTRACT: In this study, we obtained over 4,000 transposon mutants of Mycobacterium vanbaalenii PYR-1 and analyzed one of the mutants, 8F7, which appeared to lose its ability to degrade pyrene while still being able to degrade fluoranthene. This mutant was identified to be defective in nidA, encoding an aromatic ring-hydroxylating oxygenase (RHO), known to be involved in the initial oxidation step of pyrene degradation. When cultured with pyrene as a sole source of polycyclic aromatic hydrocarbon (PAH), high-pressure liquid chromatography analysis revealed that the nidA mutant showed a significant decrease in the rate of pyrene degradation compared to the wild-type PYR-1, although pyrene was still being degraded. However, when incubated with PAH mixtures including pyrene, phenanthrene, and fluoranthene, the pyrene degradation rate of the mutant was higher than that of the mutant previously incubated with pyrene as a sole source of PAH. There was no significant difference between wild-type PYR-1 and the mutant in the rates of phenanthrene and fluoranthene degradation. From the whole-cell proteome analysis of mutant 8F7 induced by pyrene, we identified expression of a number of RHO enzymes which are suspected to be responsible for pyrene degradation in the nidA mutant, which had no expression of NidA. Taken together, results in this study provide direct evidence for the in vivo functional role of nidA in pyrene degradation at the level of the ring-cleavage-process (RCP) functional module but also for the robustness of the PAH metabolic network (MN) to such a genetic perturbation.
[Show abstract][Hide abstract] ABSTRACT: AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ∼45° and ∼35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ℓ2 on chain B and α3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.
Archives of Biochemistry and Biophysics 02/2012; 520(2):99-107. DOI:10.1016/j.abb.2012.02.010 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The indigenous human intestinal microbiota could be disrupted by residues of antibiotics in foods as well as therapeutically administered antibiotics to humans. These disruptions may lead to adverse health outcomes. To observe the possible impact of residues of antibiotics at concentrations below therapeutic levels on human intestinal microbiota, we performed studies using in vitro cultures of fecal suspensions from three individuals with 10 different concentrations (0, 0.1, 0.5, 1, 5, 10, 15, 25, 50 and 150 μg/ml) of the fluoroquinolone, enrofloxacin. The bacterial communities of the control and enrofloxacin dosed fecal samples were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. In addition, changes of functional gene expression were analyzed by a pyrosequencing-based random whole-community mRNA sequencing method. Although each individual had a unique microbial composition, the communities of all individuals were affected by enrofloxacin. The proportions of two phyla, namely, Bacteroidetes and Proteobacteria, were significantly reduced with increasing concentrations of enrofloxacin exposure, while the proportion of Firmicutes increased. Principal Coordinate Analysis (PCoA) using the Fast UniFrac indicated that the community structures of intestinal microbiota were shifted by enrofloxacin. Most of the mRNA transcripts and the anti-microbial drug resistance genes increased with increasing concentrations of enrofloxacin. 16S rRNA gene pyrosequencing of control and enrofloxacin treated fecal suspensions provided valuable information of affected bacterial taxa down to the species level, and the community transcriptomic analyses using mRNA revealed the functional gene expression responses of the changed bacterial communities by enrofloxacin.
[Show abstract][Hide abstract] ABSTRACT: The ingestion of antimicrobial residues in foods of animal origin has the potential risk of exposing colonic bacteria to small concentrations of antibiotics and inducing resistance in the colonic bacteria. To investigate whether human intestinal contents would influence resistance development in bacteria, Escherichia coli ATCC 25922 (MIC of enrofloxacin <0.03 μg ml(-1)) was exposed to 0.01 to 1 μg ml(-1) of enrofloxacin in media supplemented with glucose, sucrose, sodium acetate or sterilized human fecal extract. In the first passage, only the medium containing sterilized fecal extract supported the growth of E. coli at an enrofloxacin concentration equal to the MIC. In the second and third passages following exposure to sub-inhibitory concentrations of the drug, the bacteria in media containing sterilized fecal extract grew at 0.1 μg ml(-1) of enrofloxacin. The efflux pump inhibitors, reserpine and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), increased the sensitivity of bacteria to 0.1 μg ml(-1) of enrofloxacin in the medium containing sucrose, but their effect was not observed in the medium supplemented with 2.5% sterilized fecal extract. The proportions of unsaturated and saturated fatty acids in E. coli grown in the medium with 2.5% sterilized fecal extract differed from those grown in the medium alone. Fecal extract may contain unknown factors that augment the ability of E. coli to grow in concentrations of enrofloxacin higher than MIC, both in the presence and absence of efflux pump inhibitors. This is the first study showing that fecal extract affects the level of sensitivity of E. coli to antimicrobial agents.
The Journal of Antibiotics 01/2012; 65(4):179-84. DOI:10.1038/ja.2012.1 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Approximately 0.7 million tons of azo dyes are synthesized each year. Azo dyes are composed of one or more R₁-N=N-R₂ linkages. Studies have shown that both mammalian and microbial azoreductases cleave the azo bonds of the dyes to form compounds that are potentially genotoxic. The human gastrointestinal tract harbors a diverse microbiota comprised of at least several thousand species. Both water-soluble and water-insoluble azo dyes can be reduced by intestinal bacteria. Some of the metabolites produced by intestinal microbiota have been shown to be carcinogenic to humans although the parent azo dyes may not be classified as being carcinogenic. Azoreductase activity is commonly found in intestinal bacteria. Three types of azoreductases have been characterized in bacteria. They are flavin dependent NADH preferred azoreductase, flavin dependent NADPH preferred azoreductase, and flavin free NADPH preferred azoreductase. This review highlights how azo dyes are metabolized by intestinal bacteria, mechanisms of azo reduction, and the potential contribution in the carcinogenesis/mutagenesis of the reduction of the azo dyes by intestinal microbiota.
Frontiers in bioscience (Elite edition) 01/2012; 4(2):568-86. DOI:10.2741/400