Hilário Cuquetto Mantovani

Nisin Resistance of Streptococcus bovis

ABSTRACT The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 μM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 μM nisin was added, but resistant cells retained potassium even after addition of 10 μM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.

Bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and alpha-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2.0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.

Biofilm formation and acyl homoserine lactone production in Hafnia alvei isolated from raw milk

The objective of this study was to detect the presence of acyl homoserine lactones (AHLs), signal molecules of the quorum sensing system in biofilm formed by Hafnia alvei strains. It also evaluated the effect of synthetic quorum sensing inhibitors in biofilm formation. AHLs were assayed using well diffusion techniques, thin layer chromatography (TLC) and detection directly in biofilm with biomonitors. The extracts obtained from planktonic and sessile cell of H. alvei induced at least two of three monitor strains evaluated. The presence of AHLs with up to six carbon atoms was confirmed by TLC. Biofilm formation by H. alvei was inhibited by furanone, as demonstrated by 96-well assay of crystal violet in microtitre plates and by scanning electron microscopy. The H. alvei 071 hall mutant was deficient in biofilm formation. All these results showed that the quorum sensing system is probably involved in the regulation of biofilm formation by H. alvei.

Degradação in vitro da fibra em detergente neutro de forragem tropical de baixa qualidade em função de suplementação com proteína e/ou carboidratos

The effect of the protein and (or) carbohydrates supplementation on the rumen degradation of neutral detergent fiber (NDF) from low quality forage was evaluated. The experiment simulated the supplementation of finishing cattle grazing low quality Brachiaria decumbens pasture during dry season (70:30 forage to concentrate ration, %DM). The concentrate referring to the basal treatment was formulated to contain 30% CP, using starch as the energetic source and casein as the protein source. The treatments were established by omission of protein and (or) carbohydrates sources, associated with substitution of starch by pectin. In that way, six treatments were evaluated: 1. Forage, 2. Forage plus Starch, 3. Forage plus Pectin, 4.Forage plus Casein, 5. Forage plus casein plus starch and 6. Forage plus casein plus pectin. The treatments were evaluated under rumen environment, simulated by in vitro incubation, where the experimental diets were submitted to different incubation periods: 0, 3, 6, 9, 12, 24, 36, 48, 72, and 96 hours. The incubation procedure was repeated four times in a way that four evaluations within each incubation time were done for each treatment. The incubation residues were evaluated according to its contents of NDF and interpreted using a non-linear logistic model. It was observed that the degradation rate of potentially degradable NDF (pdNDF) was increased almost 46% with casein supplementation, resulting in an increment of 14.6% on the effective degraded fraction. A minor effect was observed with the inclusion or substitution of the supplemental carbohydrate source. The starch supplementation resulted in reduction on pdNDF degradation rate, whereas the supplementation with pectin did not affected this parameter, when compared to no carbohydrate supplementation. In the presence of casein, the starch supplementation raised the discrete lag time for NDF degradation.

Role of Lipid II and Membrane Thickness in the Mechanism of Action of the Lantibiotic Bovicin HC5

ABSTRACT Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well-known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [Ka] = 3.1 × 106 M−1), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.

Bovicin HC5 and nisin reduce Staphylococcus aureus adhesion to polystyrene and change the hydrophobicity profile and Gibbs free energy of adhesion

Staphylococcus aureus is an opportunistic pathogen often multidrug-resistant that not only causes a variety of human diseases, but also is able to survive on biotic and abiotic surfaces through biofilm communities. The best way to inhibit biofilm establishment is to prevent cell adhesion. In the present study, subinhibitory concentrations of the bacteriocins bovicin HC5 and nisin were tested for their capability to interfere with the adhesion of S. aureus to polystyrene. Subinhibitory dosages of the bacteriocins reduced cell adhesion and this occurred probably due to changes in the hydrophobicity of the bacterial cell and polystyrene surfaces. After treatment with bovicin HC5 and nisin, the surfaces became more hydrophilic and the free energy of adhesion (∆G(adhesion)) between bacteria and the polystyrene surface was unfavorable. The transcriptional level of selected genes was assessed by RT-qPCR approach, revealing that the bacteriocins affected the expression of some important biofilm associated genes (icaD, fnbA, and clfB) and rnaIII, which is involved in the quorum sensing mechanism. The conditioning of food-contact surfaces with bacteriocins can be an innovative and powerful strategy to prevent biofilms in the food industry. The results are relevant for food safety as they indicate that bovicin HC5 and nisin can inhibit bacterial adhesion and consequent biofilm establishment, since cell adhesion precedes biofilm formation.

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp

Aims:  To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp.

Bactericidal effect of bovicin HC5 and nisin against Clostridium tyrobutyricum isolated from spoiled mango pulp.

Aims:  To test the effect of bovicin HC5 – a bacteriocin from Streptococcus bovis HC5 – against the strains of Clostridium tyrobutyricum isolated from canned spoiled mango pulp.

Use of phages against antibiotic-resistant Staphylococcus aureus isolated from bovine mastitis1

Bovine mastitis is the primary disease of dairy cattle worldwide and it causes large economic losses. Among several microorganisms that are the causative agents of this disease, Staphylococcus aureus is the most prevalent. Although antibiotic therapy is still the most widely used procedure for the treatment of bovine mastitis, alternative means of treatment are necessary due to the presence of antibiotic residues in milk, which is a growing concern because of its interference with the production of milk derivatives and the selection of resistant bacterial strains. The use of bacteriophages as a tool for the control of pathogens is an alternative treatment to antibiotic therapy. In this work, to obtain phages with the potential for use in phage therapy as a treatment for mastitis, we isolated and identified the bacteria from the milk of mastitis-positive cows. A total of 19% of the animals from small and medium farms of the Zona da Mata Mineira, Brazil, was positive for bovine mastitis, and bacteria of the genus Staphylococcus were the most prevalent pathogens. The majority of the S. aureus isolates tested was resistant to penicillin and ampicillin. In parallel, we isolated 10 bacteriophages able to infect some of these S. aureus isolates. We determined that these phages contained DNA genomes of approximately 175 kb in length, and the protein profiles indicated the presence of 4 major proteins. Electron microscopy revealed that the phages are caudate and belong to the Myoviridae family. The isolates exhibited interesting features for their use in phage therapy such as a high lytic potential, a wide range of hosts, and thermostability, all of which favor their use in the field.

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp

Aims:  To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp.