Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37 degrees C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.     

ORF9p Phosphorylation by ORF47p Is Crucial for the Formation and Egress of Varicella-Zoster Virus Viral Particles

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.     

1-O-acylceramides are natural components of human and mouse epidermis

The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids, and ceramides (Cers) in an equimolar ratio. The huge class of skin Cers consists of three groups: group I, “classical” long and very long chain Cers; group II, ultra-long chain Cers; and group III, ω-esterified ultra-long chain Cers, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acylceramides as a new class of epidermal Cers in humans and mice. These Cers contain, in both the N- and 1-O-position, long to very long acyl chains. They derive from the group I of classical Cers and make up 5% of all esterified Cers. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A₂. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified Cers. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at endoplasmic reticulum-related sites.     

Maternal Diet Influences Offspring Feeding Behavior and Fearfulness in the Precocial Chicken

Background

In chicken, oils in the maternal diet confer a specific scent to the yolk. Embryos are known to perceive and memorize chemosensory signals of the surrounding environment; however, the potential impact of the maternal diet has not previously been investigated. In the present study, we hypothesized that chicken embryos memorize the chemical signals of the maternal diet and that this perceptual learning may orient subsequent feeding behavior of the hatchlings.

Methodology/Principal Findings

Laying hens were fed standard food enriched with 2% menhaden oil (MH group) or 2% soybean oil (controls). The scent of menhaden was significantly more detected in MH egg yolks than in control yolks by a human panel. We analyzed the development and behavior of offspring towards different types of food, bearing or not bearing the menhaden scent. When chicks were exposed to a 3-min choice test between the familiar food bearing the menhaden scent and the familiar food without menhaden, no effect of treatment was observed. In a 3-min choice test with unfamiliar food (mashed cereals) MH chicks showed a clear positive orientation toward the unfamiliar food bearing the menhaden scent. By contrast, control chicks showed a preference for the non-odorized unfamiliar food. MH chicks expressed higher emotional reactivity level than control chicks as expressed by food neophobia and longer immobility in a restraint test.

Conclusion/Significance

Chicks exposed in ovo to menhaden oil via the maternal diet preferentially oriented their feeding behavior towards food containing menhaden oil, but only when the food was unfamiliar. We propose that oil in the maternal diet engenders maternal effects and contributes to the development of behavioral phenotype in the offspring. In ovo chemosensory learning may have evolved to prepare precocial offspring for their environment. This suggests a common principle of embryonic chemosensory learning across vertebrate taxa.     

Phylogenetic Analysis of Invertebrate Lysozymes and the Evolution of Lysozyme Function

We isolated and sequenced the cDNAs coding for lysozymes of six bivalve species. Alignment and phylogenetic analysis showed that, together with recently described bivalve lysozymes, the leech destabilase, and a number of putative proteins from extensive genomic and cDNA analyses, they belong to the invertebrate type of lysozymes (i type), first described by Jollès and Jollès (1975). We determined the genomic structure of the gene encoding the lysozyme of Mytilus edulis, the common mussel. We provide evidence that the central exon of this gene is homologous to the second exon of the chicken lysozyme gene, belonging to the c type. We propose that the origin of this domain can be traced back in evolution to the origin of bilaterian animals. Phylogenetic analysis suggests that i-type proteins form a monophyletic family. Received: 21 May 2001 / Accepted: 22 October 2001     

Roles for kinesin and myosin during cytokinesis

Cytokinesis in higher plants involves the phragmoplast, a complex cytoplasmic structure that consists of microtubules (MTs), microfilaments (MFs) and membrane elements. Both MTs and MFs are essential for cell plate formation, although it is not clear which motor proteins are involved. Some candidate processes for motor proteins include transport of Golgi vesicles to the plane of the cell plate and the spatiotemporal organization of the cytoskeletal elements in order to achieve proper deposition and alignment of the cell plate. We have focused on the kinesin-like calmodulin binding protein (KCBP) and, more broadly, on myosins. Using an antibody that constitutively activates KCBP, we find that this MT motor, which is minus-end directed, contributes to the organization of the spindle and phragmoplast MTs. It does not participate in vesicle transport; rather, because of the orientation of the phragmoplast MTs, it is supposed that plus-end kinesins fill this role. Myosins, on the other hand, based on their inhibition with 2,3-butanedione monoxime and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), are associated with the process of post-mitotic spindle/phragmoplast alignment and with late lateral expansion of the cell plate. They are also not the principal motors involved in vesicle transport.     

Diversity in the genus Rhabdias (Nematoda,Rhabdiasidae): Evidence for cryptic speciation

Lungworms from the genus Rhabdias are common parasites of amphibians and reptiles distributed worldwide. To assess the diversity of Rhabdias spp., we performed molecular analyses of 35 specimens sampled in different regions of Brazil. Molecular analyses were based on the internal transcribed spacer (ITS), large subunit (28S) ribosomal and the cytochrome oxidase I (COI) mitochondrial genes. DNA sequence divergence was compared among ribosomal and mitochondrial genes, analyses using the general mixed Yule‐coalescent (GMYC) method based on the COI gene were used to identify possible cryptic diversity, and phylogenetic analyses using concatenated ITS and 28S ribosomal genes were used to test the monophyly of Rhabdiasidae. We revealed five morphospecies: R. cf. stenocephala, R. breviensis, R. pseudosphaerocephala and two new species, Rhabdias sp.4 and Rhabdias sp.5. DNA sequence levels of divergence among genes ITS, 28S and COI were compared, and the efficiency of the molecular markers to identify species (ITS and COI) and lineages (COI) was tested. GMYC was assigned to 17 well‐supported clades (i.e., 17 species), and cryptic diversity was detected in the Neotropical region as evidenced by the multiple lineages in R. breviensis and R. pseudosphaerocephala. In addition, our results suggest evidence for host–parasite cophylogeny in the R. pseudosphaerocephala complex and dispersal events among their populations. Phylogenetic analyses supported the monophyly of Rhabdiasidae and improved the resolution of main clades. Rhabdias breviensis is closely related to Rhabdias cf. africanus, Rhabdias cf. stenocephala, R. pseudosphaerocephala, Rhabdias sp.4 and Rhabdias sp.5 grouping together in a main clade with Neotropical‐related species. The large geographical distribution appeared to be a phylogenetic pattern among the species of Rhabdias from the neotropics.     

Biological activities of extracts from Chenopodium ambrosioides Lineu and Kielmeyera neglecta Saddi

ABSTRACT: BACKGROUND: Chenopodium ambrosioides and Kielmeyera neglecta are plants traditionally used in Brazil to treat various infectious diseases. The study of the biological activities of these plants is of great importance for the detection of biologically active compounds. METHODS: Extracts from these plants were extracted with hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH) and assessed for their antimicrobial properties, bioactivity against Artemia salina Leach and antifungal action on the cell wall of Neurospora crassa. RESULTS: Extracts from C. ambrosioides (Hex, DCM and EtOH) and K. neglecta (EtOAc and EtOH) showed high bioactivity against A. salina (LD50 < 1000 ug/mL), which might be associated with cytotoxic activity against cancer cells. C. ambrosioides Hex and DCM showed specific activity against yeasts, highlighting the activity of hexanic extract against Candida krusei (MIC = 100 ug/mL). By comparing the inhibitory concentration of 50% growth (IC 50%) with the growth control, extracts from K. neglecta EtOAc and EtOH have shown activities against multidrug-resistant bacteria (Enterococcus faecalis ATCC 51299 and Staphylococcus aureus ATCC 43300), with IC 50% of 12.5 ug/mL The assay carried out on N. crassa allowed defining that extracts with antifungal activity do not have action through inhibition of cell wall synthesis. CONCLUSIONS: Generally speaking, extracts from C. ambrosioides and K. neglecta showed biological activities that have made the search for bioactive substances in these plants more attractive, illustrating the success of their use in the Brazilian folk medicine.