Silvia T. Cardona

Storage conditions of intestinal microbiota matter in metagenomic analysis

BackgroundThe structure and function of human gut microbiota is currently inferred from metagenomic and metatranscriptomic analyses. Recovery of intact DNA and RNA is therefore a critical step in these studies. Here, we evaluated how different storage conditions of fecal samples affect the quality of extracted nucleic acids and the stability of their microbial communities.ResultsWe assessed the quality of genomic DNA and total RNA by microcapillary electrophoresis and analyzed the bacterial community structure by pyrosequencing the 16S rRNA gene. DNA and RNA started to fragment when samples were kept at room temperature for more than 24 h. The use of RNAse inhibitors diminished RNA degradation but this protection was not consistent among individuals. DNA and RNA degradation also occurred when frozen samples were defrosted for a short period (1 h) before nucleic acid extraction. The same conditions that affected DNA and RNA integrity also altered the relative abundance of most taxa in the bacterial community analysis. In this case, intra-individual variability of microbial diversity was larger than inter-individual one.ConclusionsThough this preliminary work explored a very limited number of parameters, the results suggest that storage conditions of fecal samples affect the integrity of DNA and RNA and the composition of their microbial community. For optimal preservation, stool samples should be kept at room temperature and brought at the laboratory within 24 h after collection or be stored immediately at −20°C in a home freezer and transported afterwards in a freezer pack to ensure that they do not defrost at any time. Mixing the samples with RNAse inhibitors outside the laboratory is not recommended since proper homogenization of the stool is difficult to monitor.

Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci

Extracellular DNA is an adhesive component of staphylococcal biofilms. The aim of this study was to evaluate the antibiofilm activity of recombinant human DNase I (rhDNase) against Staphylococcus aureus and Staphylococcus epidermidis. Using a 96-well microtiter plate crystal-violet binding assay, we found that biofilm formation by S. aureus was efficiently inhibited by rhDNase at 1–4 μg l−1, and preformed S. aureus biofilms were efficiently detached in 2 min by rhDNase at 1 mg l−1. Pretreatment of S. aureus biofilms for 10 min with 10 mg l−1 rhDNase increased their sensitivity to biocide killing by 4–5 log units. rhDNase at 10 mg l−1 significantly inhibited biofilm formation by S. epidermidis in medium supplemented with sub-MICs of antibiotics. We also found that rhDNase significantly increased the survival of S. aureus-infected Caenorhabditis elegans nematodes treated with tobramycin compared with nematodes treated with tobramycin alone. We concluded that rhDNase exhibits potent antibiofilm and antimicrobial-sensitizing activities against S. aureus and S. epidermidis at clinically achievable concentrations. rhDNase, either alone or in combination with antimicrobial agents, may have applications in treating or preventing staphylococcal biofilm-related infections.

A Quorum-Quenching Approach To Investigate the Conservation of Quorum-Sensing-Regulated Functions within the Burkholderia cepacia Complex

ABSTRACT Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.

A Functional Phenylacetic Acid Catabolic Pathway Is Required for Full Pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans Host Model

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans.

Identification of Essential Operons with a Rhamnose-Inducible Promoter in Burkholderia cenocepacia†

ABSTRACT Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the Burkholderia cepacia complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter PrhaB for identifying essential genes and operons in Burkholderia cenocepacia. We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver PrhaB at two different distances from the site of insertion. One of these vectors places PrhaB at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e-gfp) downstream from PrhaB. This system allows us to identify essential genes and operons in B. cenocepacia and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms.

ABSTRACT We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

BackgroundMetabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.ResultsTranslational fusions of the PA degradation gene promoters with eGFP were constructed and introduced in B. cenocepacia K56-2. eGFP expression was observed when the reporter strains were grown in minimal media containing glycerol and PA or other compounds expected to proceed through the PA pathway, and in synthetic CF medium (SCFM). Addition of succinate or glucose to the PA containing medium repressed eGFP expression. To show that BCAL0210, a putative TetR-type regulator gene encodes a regulator for the PA genes in B. cenocepacia, we developed a BCAL0210 insertional mutant reporter strain. Results show that these strains exhibit fluorescence regardless of the presence of PA in the culture.ConclusionThe PA catabolic genes of B. cenocepacia K56-2 are induced by PA and other related compounds, are negatively regulated by PaaR (named herein), a TetR-type regulator, and are subjected to catabolic repression by glucose and succinate. As the PA catabolic pathway of B. cenocepacia appears to be induced during growth in synthetic cystic fibrosis medium (SCFM), further research is necessary to determine the relevance of this pathway in CF-like conditions and in other host-pathogen interactions.

The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

ABSTRACT Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.

The Glycogen-Bound Polyphosphate Kinase from Sulfolobus acidocaldarius Is Actually a Glycogen Synthase

ABSTRACT Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya,Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.