Fallen Branches as Part of Leaf-Cutting Ant Trails: Their Role in Resource Discovery and Leaf Transport Rates in Atta cephalotes

Fallen branches, logs, and exposed roots (fallen branches hereafter) commonly form part of the trunk trail system of leaf-cutting ants that inhabit the tropical rain forest. We studied the role of fallen branches on resource discovering and on leaf transport rates in Atta cephalotes . Fallen branches were common components of the A. cephalotes trail system; they were present in all the nests, and in the majority of the trunk trails examined (13/16). A field experiment revealed that, at the beginning of their foraging activity, ants discovered food sources located at the end of fallen branches earlier than those located on the leaf litter. Additionally, laden ants walked faster along a fallen branch than along soil tracks of the trunk trails. This increment in speed was higher in slow-walking ants ( e.g. , with larger loads) than in fast-walking ants ( e.g. , with smaller loads). These results suggest that the presence of fallen branches may direct the searching effort of leaf-cutters and increase the foraging speed of laden ants when these structures are part of the trunk trail system. The advantages of using fallen branches as part of a trail system, and their potential consequences in the spatial foraging pattern of leaf-cutting ants, are discussed.     

Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.     

Enterococcus faecalis pCF10‐encoded surface proteins PrgA,PrgB (aggregation substance) and PrgC contribute to plasmid transfer,biofilm formation and virulence

Enterococcus faecalis pCF10 transfers at high frequencies upon pheromone induction of the prgQ transfer operon. This operon codes for three cell wall‐anchored proteins – PrgA, PrgB (aggregation substance) and PrgC – and a type IV secretion system through which the plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to plasmid transfer, biofilm formation and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 transfer at wild‐type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone‐responsive, conjugative plasmids of E. faecalis have retained Prg‐like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation and widespread lateral gene transfer.     

Clerodane-type diterpenes from the seed pods of Hymenaea courbaril var. stilbocarpa.

Three known and two new diterpenes were isolated from the ethyl acetate extract of Hymenaea courbaril var. stilbocarpa seed pods. One of the compounds was elucidated as (5R*,8S*,9S*,10R*)-cleroda-3,13E-dien-15-oic acid and the other was elucidated, after treatment with diazomethane, as methyl (5S*,8S*,9S*,10R*)-cleroda-3,13E-dien-15-oate. The known diterpenes were identified as (-)-ozic acid, (-)-isoozic acid and (-)-kovalenic acid which were characterized as their methyl ester derivatives.     

Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria

Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.     

Identification of Peptidoglycan-Associated Proteins as Vaccine Candidates for Enterococcal Infections

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.     

Relapses after multibacillary leprosy treatment

Leprosy relapses are mainly due to bacillary persistence and diamino-diphenyl-sulphone (DDS) monotherapy. Case histories were examined for 33 patients with lepromatous leprosy (LL), diagnosed 7-48 years before the relapse and treated only with DDS during 4 to 38 years. Twenty-eight patients received irregular non-supervised polychemotherapy (PCT) since 1983. Five patients received only DDS, and presented relapses 13-20 years after the treatment was stopped. Relapses were diagnosed by clinical methods, including the reappearance of lesions or presence of new anesthetic areas. All cases were confirmed by bacilloscopy, and a subset of 20 cases by skin biopsy. Four patients presented indeterminate leprosy (IL) and one patient borderline tuberculoid leprosy (BT) in the biopsy. The latter 5 demonstrated presence of intraneural bacilli; the remainder were LL. Two patients relapsed even with PCT treatment. The others were cured with supervised PCT. Predisposing factors for relapses were as follows: DDS monotherapy, irregular PCT with inadequate dosage, unsupervised treatment, treatment uncompliance, and inadequate relationship between the patient and the health staff. Inspections for relapse in leprosy is recommended for in all multibacillary patients that were treated with DDS. The clinical appearance of new lesions or new anesthetic zones, the bacilloscopy and skin biopsy, used together, are effective in establishing the presence of relapses.     

Desiccation resistance in two species of Drosophila

Three populations of Drosophila pseudoobscura, each one represented by 12 isofemale lines, and one laboratory strain of D. melanogaster were tested for desiccation resistance at two time periods. Except in the case of one population of D. pseudoobscura, the ability to withstand drying was significantly greater in females than in the corresponding males. The males of the three populations of D. pseudoobscura differed significantly among themselves in their resistance to desiccation, as did the females. The females of D. melanogaster exhibited a consistently higher survival rate than those of D. pseudoobscura, but not the males. These results are discussed with reference to the third chromosome inversion polymorphism of D. pseudoobscura and the cosmopolitan distribution of D. melanogaster.     

Intra- and interspecies genomic transfer of the Enterococcus faecalis pathogenicity island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.