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PubMed2006

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia.

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.4) which has been suggested to be involved in reactivation of dormant mycobacteria. We have previously reported that inactivation of an identical gene (sa5k) in Mycobacterium bovis BCG causes impaired ability of the mutant strain (BCGsa5k::aph) to grow inside human macrophages. This study aimed to investigate the role of TB8.4 in the reactivation of aged cultures of BCG as well as the role of the sa5k gene in the resistance of BCG to intracellular stress conditions and adaptation to hypoxia. Although when added to aged cultures of BCG, TB8.4 caused a statistically significant increase in the number of colony-forming units, a similar effect was obtained in cultures incubated with BSA, suggesting a non-specific growth stimulation by TB8.4. Compared to parental BCG, the BCGsa5k::aph strain showed an increased susceptibility to reactive oxygen and nitrogen intermediates and to acid stress and an impaired ability to adapt to reduced O2 concentrations, when tested in the oxygen-limited Wayne culture system. These results suggest that the product of the sa5k gene (SA5K protein) has a role in both resistance of BCG to intracellular stress and in its adaptation to hypoxia.

Acids↗

PubMed2006

Role of Streptococcus sanguinis sortase A in bacterial colonization.

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.

Adhesins, Bacterial↗

PubMed2006

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Base Sequence↗

PubMed2006

Regulation of expression of atypical orphan response regulators of Helicobacter pylori.

The human gastric pathogen Helicobacter pylori exhibits a remarkably small repertoire of transcriptional regulators including three complete two-component systems as well as the orphan response regulators HP1021 and HP1043. Both HP1021 and HP1043 show atypical receiver sequences and are required for the normal cell growth of H. pylori. Recently, we demonstrated that phosphorylation of HP1021 and HP1043 according to the two-component paradigm is not a prerequisite for the cell growth-associated functions of these response regulators, raising the question of how the activity of this regulatory proteins is modulated. Here, we report that strict transcriptional control of its expression is not involved in the cell-growth associated function of HP1021. We show that expression of hp1043 is controlled both on the post-transcriptional or post-translational level and by transcriptional regulation. Furthermore, we provide evidence that hp1043 can be replaced by the orthologous gene cj0355 from Campylobacter jejuni.

Adaptation, Physiological↗

PubMed2006

Effects of Citrus sinensis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem.

Essential oils from different plant parts are known for their antimicrobial activity but the antifungal effects of essential oil from Citrus sinensis (L.) Osbeck epicarp on growth and morphogenesis of Aspergillus niger has not been observed so far. The mycelial growth was inhibited at 2.5 and 3.0 microg/ml of oil in Potato Dextrose Broth and Agar medium, respectively. These concentrations were fungicidal under the test conditions. The fungitoxicity of oil did not change even at exposure to 100 degrees C and autoclaving. The main changes observed under light and scanning electron microscopy after oil treatment were loss of cytoplasm in fungal hyphae, and budding of hyphal tip. The hyphal wall and its diameter became markedly thinner, distorted and resulted in cell wall disruption. The flattened and empty hyphal tips bifurcated into bud like structures. GC-MS studies of the oil revealed the presence of 10 chemical constituents. Limonene has been found to be major component (84.2%).

Antifungal Agents↗

PubMed2005

Construction of artificially structured microbial consortia (ASMC) using dielectrophoresis: examining bacterial interactions via metabolic intermediates within environmental biofilms.

The construction of artificial biofilms with defined internal architectures is described. Bacterial cells are suspended in a low conductivity medium, guided to specific areas in a microelectrode array by dielectrophoresis (DEP), and then immobilised using the flocculating agent poly(ethylenimine). Multispecies biofilms can be constructed by introducing different species at different times. The rapid construction of such biofilms with defined internal architectures provides, when combined with visual reporters of gene activity, a powerful new method for the investigation of the effects of the spatial organisation on interactions between bacterial species in biofilms. To demonstrate the utility of the technique as a method for investigating metabolic interactions in biofilms, aggregates were constructed from Acinetobacter sp. C6 and Pseudomonas putida::gfp. The Acinetobacter degrades benzyl alcohol, overproducing benzoate, which in turn is consumed by the Pseudomonas strain. The P. putida has a chromosomally expressed cassette encoding a gfp downstream of the promoter which controls degradation of benzoate, making the interaction between the two strains in the metabolism of benzyl alcohol visible by the production of green fluorescent protein (GFP). Microscopic observation of the biofilms, including the use of confocal laser scanning microscopy (CLSM), confirmed that metabolic exchange occurred. In addition, it was observed that the bacteria appear to have a preferred biofilm architecture, with P. putida in the bottom layer, and Acinetobacter at the top.

Acinetobacter↗

PubMed2006

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction.

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.

Animals↗

PubMed2006

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge.

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF=1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF=1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI=2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.

3T3 Cells↗

PubMed2006

Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method.

The aim of this study was to reassess the use of autofluorescence for evaluating AM colonization in mycorrhizal roots in the light of criticisms of this method that affirmed that only metabolically inactive arbuscules autofluoresce. It was also investigated whether other mycorrhizal structures, such as hyphae, vesicles and spores, could be detected by autofluorescence, and whether the autofluorescence pattern of AM fungal structures could be exploited methodologically, for example, in the detection and sorting of spores by flow cytometry. Mycorrhizal roots of the palm species Brahea armata, Chamaerops humilis, Phoenix canariensis and Phoenix dactylifera were sectioned and observed by means of fluorescence microscopy. In addition, fungal structures isolated from mycorrhizal roots of P. dactylifera were examined. The same root sections and isolated fungal structures were subjected to vital staining with nitro blue tetrazolium to determine their metabolic state (active or inactive). Moreover, spores of Glomus intraradices, and Glomus clarum were studied by epifluorescence and flow cytometry. Mycorrhizal whole roots of Medicago sativa were also assessed by autofluorescence detection. In contrast to previous reports, the results presented in this paper clearly demonstrate that all fungal structures, both intra- and extraradical, autofluoresced under blue light excitation, regardless of their state (dead or alive). Some arbuscules isolated from roots and mature spores showed further autofluorescence under green light excitation. The source of the autofluorescence was localized in the fungal cell wall. It was shown that AM spores can be detected by flow cytometry. The results support the use of autofluorescence for the evaluation of AM colonization, at least in palm species, and refute previous criticisms of the method.

Arecaceae↗

PubMed2005

Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium.

The tolerance of the lactic acid bacterium Oenococcus oeni to hostile wine conditions is essential for the success of malolactic fermentation (MLF). In this study, reverse transcription quantitative PCR (RT-qPCR) was used to quantify the transcript level of 13 genes that could play a role in adaptation of O. oeni in wine. To optimize survival and growth in wine, cells were adapted during growth at low pH (3.5) prior to inoculation into wine. The level of gene expression was analyzed after growth at pH 3.5 in a rich medium and during MLF in a wine-like medium. RT-qPCR analyses exhibited different expression ratios of stress genes. The data obtained showed that determination of mRNA levels could constitute a new approach to studying the stress response of O. oeni after adaptation at low pH and during growth in a wine-like medium.

Fermentation↗

PubMed2005

Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds.

Six Lactobacillus strains including commercial probiotic ones (L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota, L. johnsonii La1, L. plantarum ACA-DC 287 and L. rhamnosus GG) were investigated, through batch fermentations under controlled conditions, for their capacity to inhibit Salmonella enterica serovar Typhimurium SL1344. All lactobacilli displayed strong antibacterial activity toward this Gram-negative pathogen and significantly inhibited invasion of the pathogen into cultured human enterocyte-like Caco-2/TC7 cells. By studying the production kinetics of antibacterial activity and applying the appropriate acid and pH control samples during a killing assay, we were able to distinguish between the effect of lactic acid and other inhibitory compounds produced. The antibacterial activity of L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota and L. rhamnosus GG was solely due to the production of lactic acid. The antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the production of lactic acid and (an) unknown inhibitory substance(s). The latter was (were) only active in the presence of lactic acid. In addition, the lactic acid produced was responsible for significant inhibitory activity upon invasion of Salmonella into Caco-2/TC7 cells.

Caco-2 Cells↗

PubMed2006

Species identification of erythromycin-resistant Campylobacter isolates and optimization of a duplex PCR for rapid detection.

This study describes the approach used to verify the species identity of 23 erythromycin-resistant Campylobacter isolates whose identity was initially determined based mainly on the results of the rapid hippurate hydrolysis test or the results of the API-Campy identification system. Species identification of the isolates investigated was confirmed by repeating hippurate hydrolysis using a modification of the rapid hydrolysis test, in addition to performing three genetic-based assays. The original identification was verified in 69.6% of the isolates. The remaining isolates showed discrepancies in identity as determined by results of the identification assays performed. A duplex PCR assay, targeting the hipO and aspA genes, indicated the existence of mixed cultures of C. jejuni and C. coli in the frozen stocks of two of these isolates.

Anti-Bacterial Agents↗

PubMed2006

Characterization of a two-gene operon epeRA involved in multidrug resistance in Streptomyces clavuligerus.

Two genes, epeR and epeA, are located downstream of argH in the Streptomyces clavuligerus genome. EpeR belongs to the TetR family of transcriptional regulators. It is homologous to PqrA of Streptomyces coelicolor (74.3% identity) and to NfxB of Pseudomonas aeruginosa (30.9% identity). EpeA encodes a protein with 14 transmembrane spanning domains (TMS) of the major facilitator superfamily. It shares 68.9% identity to PqrB of S. coelicolor and 46.5% identity to LfrA, conferring resistance to fluoroquinolones in Mycobacterium smegmatis. Disruption of epeR results in a S. clavuligerus epeR::aph mutant which shows increased resistance to ethidium bromide and proflavine (16- and 32-fold higher than the wild type). Taking into consideration the sensitivity to drugs of different transformants carrying functional copies of either epeR or epeA, it might be concluded that both genes appear to be co-transcribed, with epeR encoding a regulatory protein which controls the expression of epeA.

Amino Acid Sequence↗

PubMed2006

Effect of bile acid on the cell membrane functionality of lactic acid bacteria for oral administration.

Lactic acid bacteria and other species dwelling in the gut must be tolerant to bile salts. This study sought to determine the effects of the bile salts taurodeoxycholate (TDCA) and deoxycholate (DCA) on Lactobacillus reuteri CRL 1098, a strain of likely probiotic value. When compared to other lactobacilli, L. reuteri showed the highest survival rate but remained sensitive to high (>3 mM) DCA concentrations. DCA produced complete permeabilization of cells, abolished glucose uptake and severely distorted the cell envelope, as shown by electron microscopy. Detailed analytical studies revealed a change in the phospholipid to glycolipid ratio, and also in lipid proportions. The C18:1 W9C form remarkably increased, possibly following a rapid adaptive response during DCA treatment. This study provides the first solid evidence of the mechanism of DCA toxicity in a lactic acid bacterium.

Cell Membrane↗

PubMed2006

Enhanced culture of Borrelia garinii and Borrelia afzelii strains on a solid BSK-based medium in anaerobic conditions.

The growth of 29 human strains from the three main pathogenic species of Borrelia burgdorferi sensu lato on a solid BSK-based medium was compared in two culture atmospheres: 3% CO(2) air and anaerobiosis. All strains grew under anaerobic conditions, whereas only 13 strains were able to grow in aerobiosis with 3% CO(2) (P<0.001). In the latter condition, 75% of the B. burgdorferi sensu stricto strains grew versus 33% of the B. garinii and B. afzelii strains. These data suggest that, especially for B. garinii and B. afzelii species, anaerobic conditions enhance growth yield and speed of low-passage Borrelia strains.

Anaerobiosis↗

PubMed2006

Lactobacillus paracasei A survives gastrointestinal passage and affects the fecal microbiota of healthy infants.

This study focuses on the potentiality of a putative probiotic strain, Lactobacillus paracasei A, to survive gastrointestinal (GI) passage and modulate the resident microbiota of healthy infants. In a placebo-controlled study, 26 children aged 12-24 months received 100 g/day of either fermented milk containing strain A or pasteurized yogurt for four weeks. Fecal samples were analyzed before starting the administration, after 1, 3 and 4 weeks of consumption and after washout. The fate of strain A was followed by means of a newly developed PCR targeting a strain-specific genomic marker. The composition and dynamics of fecal microbial communities during the study were analyzed by culturing on selective media and by the PCR-denaturing gradient gel electrophoresis (DGGE) technique using universal and group-specific (Lactobacillus and Bifidobacterium) primers. The variation in enzymatic activities in infant feces during probiotic consumption was also analyzed. Strain A survived in fecal samples in most (92%) of the infants examined after 1 week of consumption, and temporarily dominated the intestinal Lactobacillus community. The administration of L. paracasei A led to a significant increment in the Lactobacillus population, while a moderate effect upon the main bacterial groups in the GI ecosystem was observed. Strain A also affected the diversity of the Lactobacillus and Bifidobacterium populations. The fecal bacterial structure of 1 - 2-year-old infants seems to combine neonate and adult-like features. The microbiota of these subjects promptly responded to probiotic consumption, later restoring the endogenous equilibrium.

Cultured Milk Products↗

PubMed2006

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria.

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".

Bacteria, Anaerobic↗

PubMed2006

Wake up! Peptidoglycan lysis and bacterial non-growth states.

When stressed, bacteria can enter various non-dividing states, which are medically important. For example, dormancy is used by Mycobacterium tuberculosis to evade host responses. A major breakthrough has been the discovery of resuscitation-promoting factor (Rpf) from Micrococcus luteus, which is an extremely potent anti-dormancy factor. Mycobacteria have multiple proteins that contain this domain. Surprisingly, the highly conserved resuscitation-promoting factor domain has strong structural similarities to lysozyme and soluble lytic transglycosylases, and it has been demonstrated that resuscitation-promoting factors cleave peptidoglycan. This suggests that the activation of dormant cells requires peptidoglycan hydrolysis, which either alters the mechanical properties of the cell wall to facilitate cell division or releases lysis products that function as anti-dormancy signals.

Bacterial Proteins↗