Helminths associated with Norops fuscoauratus (Squamata, Dactyloidae) in highland marshes of the Brazilian semi-arid.

Helminthological studies may contribute with valuable information on host biology and conservation. Herein, we provide new data on helminths infecting the lizard Norops fuscoauratus, testing one of the factors considered most important in parasitic ecology: host size. We analysed 25 specimens of N. fuscoauratus from three highland marshes in the Brazilian semi-arid. Eight taxa of helminths belonging to Nematoda, Trematoda and Acanthocephala were found. Physaloptera sp. showed the higher prevalence (40%), with a mean intensity of infection of 3.3 ± 1.46 (1-16) and mean abundance 1.32 ± 0.65 (0-16). Norops fuscoauratus represents four new host records for the helminths Cyrtosomum sp., Pharyngodon travassosi, Strongyloides sp. and Centrorhynchus sp. There is no relationship of host body size (P = 0.79) and mass (P = 0.50) with parasite richness. In addition, the present study contributes to the knowledge of the parasitic fauna of N. fuscoauratus and the Neotropical region.

Molecular characterisation of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus.

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30-31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus.

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators, pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS257 was not involved in the recombination events which gave rise to pRJ29.

Antimicrobial substances produced by Staphylococcus aureus strains isolated from cattle in Brazil.

Among 46 isolates of Staphylococcus aureus obtained from cattle in the State of Paraíba, Brazil, four were shown to produce antimicrobial substances (AMS). The two best AMS producers carried single plasmids of about 8.0 kbp and 50 kbp, respectively, which were designated pRJ34 and pRJ35. Curing experiments and molecular analysis associated the AMS production with the presence of these plasmids in the cells. The biochemical properties exhibited by the AMS suggested that they might be bacteriocins (Bac). The bacteriocin encoded by pRJ34 showed properties identical to those of the bacteriocins encoded by other small staphylococcal Bac plasmids. However, the bacteriocin encoded by the large plasmid pRJ35 has shown some properties which distinguish it from the other bacteriocins of Staph. aureus described so far, suggesting it may be a new member of the staphylococcal bacteriocin family.

Staphylococcal strains involved in bovine mastitis are inhibited by Staphylococcus aureus antimicrobial peptides.

The inhibitory activity of five bacteriocin (Bac)-producer strains of Staphylococcus aureus was tested against bacteria pathogenic for cattle. Sixty-five epidemiologically unrelated strains of Staph. aureus involved in bovine mastitis were used as indicators in an agar diffusion test. Bacteriocins produced by four strains could inhibit only a limited number of test organisms. However, all 65 indicator strains proved to be susceptible to the combined action of both bacteriocins encoded by pRJ9, a Bac plasmid found in strain A53. Therefore, the bacteriocins produced by this strain may represent new antimicrobial peptides with potential applications in the prevention and treatment of bovine mastitis.

Genetic analysis of the bacteriocin-encoding plasmids pRJ6 and pRJ9 of Staphylococcus aureus by transposon mutagenesis and cloning of genes involved in bacteriocin production.

pRJ6 and pRJ9, small Staphylococcus aureus plasmids which code for bacteriocins, exhibited a bactericidal activity against several lactic acid bacteria and strains of Listeria monocytogenes, an important food-borne pathogen. Filter-mating experiments using plasmid derivatives tagged with either Tn551 or Tn917-lac showed that pRJ6, but not pRJ9, could be mobilized by staphylococcal conjugative plasmids. Transposon mutagenesis of both plasmids was also performed. The bacteriocin and immunity structural genes of pRJ6 are part of the same operon, which is located around co-ordinate 4.0, being transcribed from right to left. However, gene cloning experiments using a staphylococcal vector showed some evidence for the involvement of additional functions of pRJ6 in bacteriocin expression. One function involved in pRJ6 mobilization mapped around co-ordinate 5.2, and it appears to be transcribed from left to right. The bactericidal action exerted by strains harbouring pRJ9 appears to reflect the activity of at least two bacteriocins, whose combined action results in a broader spectrum of activity and in a higher antagonistic activity. Gene cloning experiments also supported these assumptions.

Cloning of the replication region on the bacteriocinogenic plasmid pRJ9 of Staphylococcus aureus.

A 5.8-kb ClaI fragment of pRJ9, a bacteriocinogenic plasmid of Staphylococcus aureus, was cloned in the unique ClaI site of pRJ5. The recombinant plasmid obtained, pRJ23, failed to confer bacteriocin production and immunity to bacteriocin on host cells. The cloned fragment was shown to contain the complete replicon of pRJ9. Attempts to clone the 4.4-kb ClaI fragment of pRJ9 were unsuccessful, apparently due to the inactivation of the basic replicon of the cloning vector. Therefore, plasmid pRJ5 cut at its ClaI site appears to be a suitable vector for cloning replication regions of plasmids that can replicate in S. aureus.