Andréa Oliveira Barros Ribon

An association between milk and slime increases biofilm production by bovine Staphylococcus aureus

BackgroundStaphylococcus aureus is associated with chronic mastitis in cattle, and disease manifestation is usually refractory to antibiotic therapy. Biofilm production is a key element of S. aureus pathogenesis and may contribute to the treatment failure that is consistently reported by veterinarians. Minas Gerais State is the largest milk-producing state in Brazil, and the characterization of bacterial isolates is an important aspect of disease control for dairy farmers. Here, we investigated the potential of S. aureus isolated from bovine mastitis to produce slime and biofilm in a skim-milk medium and classified the isolates according to their agr type.ResultsSlime was detected using the Congo Red agar (CRA) test in 35.18% (19/54) of the strains; however, 87.04% (47/54) of the strains were considered biofilm-positive based on crystal violet staining. Compared to TSB supplemented with 0.25% glucose, skim milk significantly increased the production of biofilm, but this effect was only observed in slime-producing strains. The bacteria belonged to agr groups I (12/54), II (34/54), III (6/54), and IV (2/54), and bacteria in agr group III were found to be stronger biofilm producers than those in groups I and II. Again, milk had a significant influence only on slime-positive agr I and II isolates, revealing an association between milk and slime.ConclusionsThe present study demonstrated that skim-milk medium and slime production are two factors that together influence biofilm formation by bovine strains of S. aureus. A predominance of bacteria belonging to agr group II was observed, and bacteria from agr group III showed the highest proportion of biofilm producers. The majority of bacteria characterized in this study formed biofilm in milk, which suggests that biofilm formation has an important role in the virulence of S. aureus isolated from bovine intramammary infections.

Staphylococcus aureus of bovine origin: Genetic diversity, prevalence and the expression of adhesin-encoding genes

Staphylococcus aureus is a well-armed pathogen that is a leading cause of bovine mastitis. Attempts to define a set of bacterial proteins that are crucial for infection have failed. The identification of these proteins is important to define biomarkers that can be used for diagnostic purposes and to identify potential vaccine targets. In this study, seven genes that encode virulence factors were analyzed in 85 bacterial isolates that were derived from animals with bovine mastitis. The clfB, spa, sdrCDE and fnBP genes were detected in 91.8%, 85.9%, 85.9% and 63.5% of the isolates, respectively. At least one gene was present in all of the strains, while the most prevalent combination was clfB and sdrCDE (82.4%). The genetic diversity of the isolates was high and allowed for clustering into more than 40 groups, with each group containing bacteria collected from different locations. The gene expression of the four most prevalent adhesins was examined in nine genetically distinct strains. No common pattern of expression was observed for the genes, suggesting that the capacity of S. aureus to cause infection may rely on differential expression of the virulence factors in different isolates. Our results conclude that using only one antigen is unlikely to provide effective protection against bovine mastitis and suggest that a combination of at least three adhesins may be more suitable for developing preventive therapies. We also conclude that the characterization of isolates distributed worldwide is necessary to improve our understanding of pathogenesis in the natural populations of S. aureus.

Screening of medicinal plants for antibacterial activities on Staphylococcus aureus strains isolated from bovine mastitis

Staphylococcus aureus is the main causative agent of bovine mastitis. The activity of several extracts from ten medicinal plants traditionally used in Brazil as antiseptic was investigated against fifteen strains of Staphylococcus aureus isolated from animals with mastitis manifestation by the disc diffusion method and broth microdilution assay. The interference of the extracts on cell in the form of adherent colonies was also evaluated. MIC values ranged from 0.5 mg/mL to 1.0 mg/mL and biofilm inhibitory concentration (BIC) were between 0.25 mg/mL and 0.8 mg/mL. Results revealed the potential of extracts of Senna macranthera, Artemisia absinthium, Cymbopogon nardus and Baccharis dracunculifolia as antibacterial agents against S. aureus strains isolated from bovine mastitis and support the possible use of these phytotherapic agents in the clinical management of the disease.

Pleiotropic impact of endosymbiont load and co-occurrence in the maize weevil Sitophilus zeamais.

Individual traits vary among and within populations, and the co-occurrence of different endosymbiont species within a host may take place under varying endosymbiont loads in each individual host. This makes the recognition of the potential impact of such endosymbiont associations in insect species difficult, particularly in insect pest species. The maize weevil, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), a key pest species of stored cereal grains, exhibits associations with two endosymbiotic bacteria: the obligatory endosymbiont SZPE (“Sitophilus zeamais Primary Endosymbiont”) and the facultative endosymbiont Wolbachia. The impact of the lack of SZPE in maize weevil physiology is the impairment of nutrient acquisition and energy metabolism, while Wolbachia is an important factor in reproductive incompatibility. However, the role of endosymbiont load and co-occurrence in insect behavior, grain consumption, body mass and subsequent reproductive factors has not yet been explored. Here we report on the impacts of co-occurrence and varying endosymbiont loads achieved via thermal treatment and antibiotic provision via ingested water in the maize weevil. SZPE exhibited strong effects on respiration rate, grain consumption and weevil body mass, with observed effects on weevil behavior, particularly flight activity, and potential consequences for the management of this pest species. Wolbachia directly favored weevil fertility and exhibited only mild indirect effects, usually enhancing the SZPE effect. SZPE suppression delayed weevil emergence, which reduced the insect population growth rate, and the thermal inactivation of both symbionts prevented insect reproduction. Such findings are likely important for strain divergences reported in the maize weevil and their control, aspects still deserving future attention.

Changes in the Salmonella enterica Enteritidis phenotypes in presence of acyl homoserine lactone quorum sensing signals.

Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI‐1, acyl homoserine lactone (AHL) common to gram‐negative bacteria; however, it has a receptor for AI‐1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs‐1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12‐HSL, increased biofilm formation and promoted expression of biofilm formation genes (lpfA, fimF, fliF, glgC) and virulence genes (hilA, invA, invF). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis.

A C-Type Lectin from Bothrops jararacussu Venom disrupts staphylococcal biofilms

Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.

Differential expression of polygalacturonase-encoding genes from Penicillium griseoroseum in different carbon sources.

A second polygalacturonase-encoding gene (pgg2) of Penicillium griseoroseum was cloned and consists of an opening reading frame of 1107 bp after removal of two introns. The gene encodes a protein of 369 amino acids with a predicted molecular mass of 38.3 kDa. The deduced protein sequence exhibited high homology with other fungal endopolygalacturonases. A polymerase chain reaction (PCR)-based strategy was used to study the expression patterns of pgg1 and pgg2 genes under different culture conditions and our results show that both genes are regulated by the carbon source at the transcriptional level. The pgg1 transcript was detected at 76 h of fungal growth in pectin while the pgg2 transcript was also induced by sucrose. The addition of yeast extract to glucose medium abolished the repressive effect of glucose, suggesting that the transcription of these genes is controlled by different mechanisms. Journal of Industrial Microbiology & Biotechnology (2002) 29, 145–148 doi:10.1038/sj.jim.7000296

Structural organization of polygalacturonase-encoding genes from Penicillium griseoroseum

The pectinolytic system of Penicillium griseoroseum has been studied as a model to investigate aspects of gene organization in filamentous fungi. Here we show that the endopolygalacturonase-coding genes previously isolated exist as single copies in the fungus genome. DNA blot analysis revealed the presence of corresponding genes in other Penicillium species, although only one or two genes were found in opposition to the endoPG gene family reported for other filamentous fungi. The nucleotide and amino acid sequences of Penicillium PG genes of retrieved from data banks were compared for intron length and number, codon usage, and consensus sequences for translation initiation sites. The introns are conserved in the same position, although there was no conservation of their nucleotide sequences. Other sequence features resemble those seen in Aspergillus and Neurospora genes.

Moving towards the immunodiagnosis of staphylococcal intramammary infections

Bovine mastitis is the primary disease of dairy cattle that has a great impact on the dairy industry. It is estimated that worldwide economic losses due to mastitis range between US

Quorum sensing regulated phenotypes in Aeromonas hydrophila ATCC 7966 deficient in AHL production

82 and US