Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

Aim: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. Methods and Results: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Φ3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx₂. A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Φ3538 (Δstx₂::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. Conclusions: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host’s DNA. Significance and Impact of the Study: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Properties of the neutralizing factors against T 2 and ΦX 174 phages present in normal sera

The properties of the natural neutralizing antibodies against the serologically unrelated T 2 and ΦX 174 bacteriophages were compared. It was found that the main component of the antiphage neutralizing system of normal serum against both phages is a thermostable “naturally” formed antibody. For the neutralization of the T 2 phage with normal serum a certain thermolabile serum component (complement or another serum factor) is required additionally in contrast to the bacteriophage ΦX 174, which does nob need this factor for neutralization.     

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

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.     

Environmental Isolates of Burkholderia pseudomallei in Ceará State,Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. ​(Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. ​(Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table ​(Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth

Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Teju?uoca, Ceará, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.     

Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution

The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I–IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).     

Impact of gut-associated bifidobacteria and their phages on health: two sides of the same coin?

Applied Microbiology and Biotechnology - Bifidobacteria are among the first microbial colonisers of the human infant gut post-partum. Their early appearance and dominance in the human infant gut...     

Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and φ80 transducing phages

φ80 transducing phages for the proC², phoA and phoB genes of Escherichia coli have been obtained. Two mutants have been isolated, in which the brnQ, phoA, proC, phoB (and possibly phoR) genes have been deleted. Derivatives of a phoA, phoB deletion strain which are lysogenic for a φ80phoA transducing phage make only very low levels of alkaline phosphatase activity. These results are in agreement with a positive control mechanism for the regulation of alkaline phosphatase synthesis.     

The proportional lack of archaeal pathogens: Do viruses/phages hold the key?

Although Archaea inhabit the human body and possess some characteristics of pathogens, there is a notable lack of pathogenic archaeal species identified to date. We hypothesize that the scarcity of disease-causing Archaea is due, in part, to mutually-exclusive phage and virus populations infecting Bacteria and Archaea, coupled with an association of bacterial virulence factors with phages or mobile elements. The ability of bacterial phages to infect Bacteria and then use them as a vehicle to infect eukaryotes may be difficult for archaeal viruses to evolve independently. Differences in extracellular structures between Bacteria and Archaea would make adsorption of bacterial phage particles onto Archaea (i.e. horizontal transfer of virulence) exceedingly hard. If phage and virus populations are indeed exclusive to their respective host Domains, this has important implications for both the evolution of pathogens and approaches to infectious disease control.     

IFN-γ Stimulates Autophagy-Mediated Clearance of Burkholderia cenocepacia in Human Cystic Fibrosis Macrophages

Burkholderia cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including B. cenocepacia. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of B. cenocepacia, but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with B. cenocepacia. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after B. cenocepacia infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.