Olinda Cabral da Silva Santos

Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil.

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with alpha-Proteobacteria (Pseudovibrio sp.) and four with gamma-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.

Resistance to bacteriocins produced by Gram-positive bacteria

Bacteriocins are prokaryotic proteins or peptides with antimicrobial activity. Most of them exhibit a broad spectrum of activity, inhibiting micro-organisms belonging to different genera and species, including many bacterial pathogens which cause human, animal or plant infections. Therefore, these substances have potential biotechnological applications in either food preservation or prevention and control of bacterial infectious diseases. However, there is concern that continuous exposure of bacteria to bacteriocins may select cells resistant to them, as observed for conventional antimicrobials. Based on the models already investigated, bacteriocin resistance may be either innate or acquired and seems to be a complex phenomenon, arising at different frequencies (generally from 10(-9) to 10(-2)) and by different mechanisms, even amongst strains of the same bacterial species. In the present review, we discuss the prevalence, development and molecular mechanisms involved in resistance to bacteriocins produced by Gram-positive bacteria. These mechanisms generally involve changes in the bacterial cell envelope, which result in (i) reduction or loss of bacteriocin binding or insertion, (ii) bacteriocin sequestering, (iii) bacteriocin efflux pumping (export) and (iv) bacteriocin degradation, amongst others. Strategies that can be used to overcome this resistance are also addressed.

Investigation of biotechnological potential of sponge‐associated bacteria collected in Brazilian coast

Marine bacteria are a rich source of structurally unique natural compounds, several of which have shown a wide variety of biological activities. In this study, the metabolites present in the culture supernatants of the eight sponge‐associated bacteria were extracted using ethyl acetate, and all extracts showed activity against Staphylococcus aureus. Subsequently, the extracts of the Pseudomonas fluorescens H40 and H41, and Pseudomonas aeruginosa H51 were subjected to solvent partitioning, and the active fractions were submitted to chromatographic separation. Three different active fractions were obtained, one of which was identified as diketopiperazine cyclo‐(L‐Leu‐L‐Pro). This substance was bactericidal for Staph. aureus and Ps. aeruginosa and showed cytotoxic activity against HEp‐2 tumour cells. Putative gene fragments coding for the type I polyketide synthase (PKS‐I) and nonribosomal peptide synthetase (NRPS) domains were PCR‐amplified from five and three strains, respectively. The results suggest that sponge‐associated bacteria analysed in this study may represent a potential source for production of antimicrobial substances against bacterial pathogens of medical importance.

Immunity to the Staphylococcus aureus leaderless four-peptide bacteriocin aureocin A70 is conferred by AurI, an integral membrane protein.

Aureocin A70, which is produced by Staphylococcus aureus A70, is the only four-component bacteriocin described thus far. The genetic determinants responsible for its production are arranged as three transcriptional units encoded by the 7.9-kb plasmid pRJ6. While the transcriptional unit formed by the genes aurABCD encodes the bacteriocin structural peptides, a second divergent gene, aurT, codes for an ABC transporter involved in bacteriocin externalization. The third transcriptional unit is composed of two genes, orfAB, whose functions were hitherto unknown. RT-PCR analysis of orfAB expression revealed that they are arranged as an operon. When orfAB, either with or without the transcriptional terminator found downstream of orfB, was expressed in two different S. aureus strains sensitive to aureocin A70, all strains became immune to this bacteriocin. Cloning of orfB alone, with or without the transcriptional terminator, confirmed orfB participation in immunity, although full immunity was not observed. An increase in immunity was achieved when two copies of orfB were cloned oriented with the exogenous Plac promoter present in the expression vector pT181mcs. orfB (here referred to as aurI) was shown to be responsible for aureocin A70 immunity, but the full immunity phenotype seems to depend on translational coupling involving orfA, which encodes a putative transcriptional regulator, and aurI.

Antimicrobial activity of marine sponges against coagulase-negative staphylococci isolated from bovine mastitis.

Bovine mastitis remains worldwide a major challenge for the dairy industry despite the widespread implementation of control strategies. The increasing number of coagulase-negative staphylococci (CNS) causing mastitis and of bacteria resistant to conventional antibiotics has become a serious problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. In the present study, 49 CNS strains were isolated from bovine mastitis cases from 21 different dairy herds kept at farms in Southeast Brazil. Strains were analyzed for antimicrobial susceptibility and mecA gene detection. Fifty-nine percent of the CNS strains were resistant to at least one of the drugs tested and 12.2% were classified as multiresistant. Three strains carried the mecA gene, confering resistance to the beta-lactamic antibiotics. In addition, the CNS strains were submitted to in vitro screening for antimicrobial activities of extracts from marine sponges. Extracts from the sponge species Cinachyrella sp., Haliclona sp. and Petromica citrina showed antibacterial activity against 61% of the CNS strains, including strains resistant to conventional antibiotics. Extracts from P. citrina showed the largest spectrum of inhibitory activity. The aqueous extract inhibited 51% of the CNS strains and presented a bactericidal effect over susceptible and multiresistant-bacteria at a minimal inhibitory concentration of 1.024μg/ml. This study shows the potential of marine sponges as new sources of antibiotics and disinfectants for the control of CNS involved in bovine mastitis.

The antimicrobial peptide aureocin A53 as an alternative agent for biopreservation of dairy products.

The aim of this study was to investigate the potential of aureocin A53, a staphylococcal antimicrobial peptide, for improving food safety.

Reliable identification of clinically prevalent species and subspecies of staphylococci by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis

We identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 10 species and 5 subspecies of Staphylococcus among 139 clinical isolates and compared it with conventional tests. All isolates showed species-specific whole-cell protein profiles, even atypical strains, with up to 60% and at least 73.5% of interspecies and intraspecies similarity, respectively. Except for 5 isolates that presented biochemical profiles of Staphylococcus hominis subsp. hominis, but were identified as S. hominis subsp. novobiosepticus by SDS-PAGE, there was 100% accordance between the methods used. Comparison with the partial 16S-rDNA sequences confirmed by SDS-PAGE showed the high accuracy of this method to identify staphylococci subspecies and species.

The four-component aureocin A70 as a promising agent for food biopreservation

Aureocin A70 is the only four-component bacteriocin described to date. As it inhibits the growth of a wide range of Gram-positive bacteria, including Listeria monocytogenes strains isolated from food, its potential for improving food safety was investigated in this study. Aureocin A70 (10,240AU/mL) proved to be bactericidal, but not extensively lytic, against listerial strains. The antibacterial activity of aureocin A70 (16AU/mL) was then tested in UHT-treated skimmed milk inoculated with the food-associated L. monocytogenes L12 strain (4-log CFU/mL) during storage at 4°C for one week. Aureocin A70 caused a time-dependent reduction in the listerial viable cell counts (5.51-log units) up to 7days of incubation. Aureocin A70 was neither toxic to the Vero and the L-929 cell lines nor exhibited a hemolytic activity against sheep red blood cells. Aureocin A70 proved to be completely stable for one month at 25°C, 16weeks at 4°C and 20weeks at -20°C. Aureocin A70 exhibited a time-dependent susceptibility to simulated gastric juice and bile salts mimicking gastrointestinal conditions. The entrapment of aureocin A70 in an alginate/gelatin matrix revealed that this bacteriocin can be released from this matrix. Moreover, it remained adsorbed to and active on a low-density polyethylene plastic surface suggesting that aureocin A70 may be employed in bioactive packaging to control the growth of undesirable bacteria. Taken together these results suggest that aureocin A70 is a promising alternative to be used in food applications.

Draft Genome Sequence of the Aureocin A53–Producing Strain Staphylococcus aureus A53

ABSTRACT Here, we present the 2,658,363-bp draft genome sequence of the aureocin A53–producing strain Staphylococcus aureus A53. This genome information may contribute to the optimal and rational exploitation of aureocin A53 as an antimicrobial agent and to its production in large scale.