Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

AIM: The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. METHODS AND RESULTS: The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. CONCLUSION: The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

The quorum sensing molecule C12-HSL promotes biofilm formation and increases adrA expression in Salmonella Enteritidis under anaerobic conditions.

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2″)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2″)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Protein expression profiling of Staphylococcus aureus in response to the bacteriocin bovicin HC5.

Alternative strategies to antibiotic treatment are required to inhibit pathogens, including Staphylococcus aureus. Bacteriocins, such as the lantibiotic bovicin HC5, have shown potential to control pathogens. This study aims to evaluate the stress response of S. aureus to bovicin HC5 using a proteomic approach. Sublethal concentrations of the bacteriocin repressed the synthesis of 62 cytoplasmic proteins, whereas 42 proteins were induced in S. aureus COL. Specifically, synthesis of several proteins involved in amino acid biosynthesis, mainly products of ilv-leu operon, and DNA metabolism, such as DNA polymerase I, decreased following bovicin treatment while proteins involved in catabolism, mainly tricarboxylic acid cycle metabolism, and chaperones were over-expressed. The levels of CodY and CcpA, important regulators involved in the stationary phase adaptation and catabolite repression, respectively, also increased in the presence of the bacteriocin. These results indicate that stress caused by the sublethal concentration of bovicin HC5 in the cell membrane results in growth reduction, reduced protein synthesis, and, at the same time, enhanced the levels of chaperones and enzymes involved in energy-efficient catabolism in an attempt to restore energy and cell homeostasis. These results bring relevant information to amplify the knowledge concerning the bacterial physiological changes in response to the stress caused by the cell exposition to bovicin HC5. New potential targets for controlling this pathogen can also be determined from the new protein expression pattern presented. KEY POINTS: • Bovicin HC5 changed the synthesis of cytoplasmic proteins of S. aureus. • Bovicin HC5 interfered in the synthesis of proteins of amino acids biosynthesis. • Synthesis of chaperones enhanced in the presence of sublethal dosage of bovicin HC5.

Morphology and composition of the midgut bacterial community of Scaptocoris castanea Perty, 1830 (Hemiptera: Cydnidae).

The burrower bug Scaptocoris castanea is an important soybean and pasture pest in Brazil, with an underground habit feeding directly on the sap of the roots. Underground habit hinders control and knowledge of the biology and physiology of this pest. This study describes the anatomy, histology, ultrastructure and symbionts of the midgut of S. castanea. The midgut of S. castanea is anatomically divided into five regions (ventricles). Ventricles 1-3 are similar between males and females, with cells specialized in digestion and absorption of nutrients, water transport and homeostasis. Ventricle 4 has squamous epithelium forming crypts and harboring bacteria in the lumen. Ventricle 5 of males is small with cells containing apical microvilli and broad basal folds with many openings for hemocoel, while in females, this region of the midgut is well developed and colonized by intracellular bacteria, characterizing bacteriocytes. The main bacteria are Gammaproteobacteria. The results show sexual dimorphism in ventricle 5 of the midgut of S. castanea, with formation of bacteriocytes in the females, while the other regions are involved in digestive processes in both sexes.

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products.

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.

Acyl homoserine lactone-based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions.

Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.

Biochemical and genetic diversity of carbohydrate-fermenting and obligate amino acid-fermenting hyper-ammonia-producing bacteria from Nellore steers fed tropical forages and supplemented with casein.

BACKGROUND: Dietary protein plays a major role in ruminant nutrition, and protein supplementation is a widespread practice among farmers in the tropics. Ruminal bacteria are the main agents of dietary protein and amino acid degradation, yet few studies have focused on the isolation and characterization of hyper-ammonia-producing bacteria in animals fed tropical diets or supplemented with rumen-degradable proteins. This work investigated the bacterial community diversity of the rumen of Nellore steers fed tropical forages, with or without casein supplementation. We also isolated and characterized ruminal bacteria showing high levels of ammonia production. RESULTS: Polymerase chain reaction-denaturing gradient gel electrophoresis analysis indicated no differences in the ruminal bacterial community composition between the control and supplemented animals. Amino acid-fermenting bacteria (n = 250) were isolated from crossbred Nellore steers fed Tifton 85 (Cynodon sp.) using trypticase as the sole carbon and organic nitrogen source in the enrichment and isolation media. The deamination rates in isolates obtained from steers supplemented with casein showed a higher incidence of deamination rates >350 nmol NH3 mg protein(-1) min(-1) (P < 0.05), whereas isolates obtained from steers without supplementation showed deamination rates <200 nmol NH3 mg protein(-1) min(-1). Although most isolates (84%) could ferment carbohydrates, none could hydrolyze proteins or use urea to sustain growth. All isolates were sensitive to lasalocid and monensin (1 µmol l(-1)), and similarity analysis of the 16S rRNA sequences indicated a predominance of bacteria from the order Clostridiales, with variable homology (73-99%) to known bacterial species. CONCLUSIONS: These results expand what is known about the biochemical and genetic diversity of hyper-ammonia-producing bacteria, and emphasize the role of carbohydrate-fermenting bacteria in ammonia production in the rumen.

A central composite rotatable design (CCRD) approach to study the combined effect of antimicrobial agents against bacterial pathogens.

The combination of antimicrobial agents has been proposed as a therapeutic strategy to control bacterial diseases and to reduce the emergence of antibiotic-resistant strains in clinical environments. In this study, the interaction between the lantibiotic bovicin HC5 with chloramphenicol, gentamicin, nisin, lysostaphin and hydrogen peroxide against Staphylococcus aureus O46 was evaluated by MIC assays. The central composite rotatable design (CCRD), a robust and economic statistical design, was used to combine concentration levels of different antimicrobials agents with distinct mechanisms of action and the presence of significant interactions among the antimicrobials was determined by regression analysis. According to the adjusted model, there were no significant interactions between bovicin HC5 and gentamicin, lysostaphin, nisin or hydrogen peroxide. However, bovicin HC5 showed a significant interaction (P < 0.02) with chloramphenicol. This is the first study applying the CCRD approach to evaluate the combined effect of antimicrobials against S. aureus. Based on our results, this approach is an effective strategy to determine synergistic interactions between antimicrobial agents applied in human and veterinary medicine against bacterial pathogens.

Nisin variants: What makes them different and unique?

Nisin A is a lantibiotic bacteriocin typically produced by strains of Lactococcus lactis. This bacteriocin has been approved as a natural food preservative since the late 1980 s and shows antimicrobial activity against a range of food-borne spoilage and pathogenic microorganisms. The therapeutic potential of nisin A has also been explored increasingly both in human and veterinary medicine. Nisin has been shown to be effective in treating bovine mastitis, dental caries, cancer, and skin infections. Recently, it was demonstrated that nisin has an affinity for the same receptor used by SARS-CoV-2 to enter human cells and was proposed as a blocker of the viral infection. Several nisin variants produced by distinct bacterial strains or modified by bioengineering have been described since the discovery of nisin A. These variants present modifications in the peptide structure, biosynthesis, mode of action, and spectrum of activity. Given the importance of nisin for industrial and therapeutic applications, the objective of this study was to describe the characteristics of the nisin variants, highlighting the main differences between these molecules and their potential applications. This review will be useful to researchers interested in studying the specifics of nisin A and its variants.

Comparison of ruminal microbiota, IL-1ß gene variation, and tick incidence between Holstein × Gyr and Holstein heifers in grazing system.

Introduction: The variation in bacterial communities among breeds has been previously reported and may be one of the reasons why Holstein × Gyr dairy heifers have better development in grazing systems in tropical conditions. This study aimed to explore the ruminal microbiota composition, the IL-1ß gene variation, tick incidence, and blood parameters of Holstein × Gyr (½ Holstein × ½ Gyr) and Holstein heifers grazing intensely managed Guinea grass (Panicum maximum Jacq. cv. Mombaça). Methods: Sixteen heifers were divided into two groups consisting of 8 Holstein × Gyr and 8 Holstein heifers. The experimental period was comprised of 3 periods of 21 days. Ruminal samples were taken via the stomach tube technique. The sequencing of the V4 hypervariable region of the 16S rRNA gene was performed using the Illumina MiSeq platform. Counting and collection of ticks were conducted each 21 days. Blood and skeletal muscle tissue biopsies were performed at the end of the experiment. Results: Firmicutes were the most abundant phyla present in both breed rumen samples and Bacteroidota showed differences in relative abundance between breed groups, with greater values for Holstein heifers (p < 0.05 with FDR correction). The 10 most abundant unique OTUs identified in each breed included several OTUs of the genus Prevotella. Holstein heifers had a greater tick count and weight (9.8 ticks/animal and 1.6 g/animal, respectively) than Holstein × Gyr (2.56 ticks/animal and 0.4 g/animal, respectively). We found nucleotide substitutions in the IL-1ß gene that might be related to adaptation and resistance phenotypes to tick infestation in Holstein × Gyr heifers. Blood concentrations of urea, albumin, insulin-like growth factor 1, triiodothyronine, and thyroxine were greater in Holstein × Gyr than in Holstein heifers. Conclusion: Adaptations in Holstein × Gyr heifers such as ruminal microbiota, tick resistance, nucleotide substitutions in IL-1ß gene, and hormone concentration suggest a better energy metabolism and thermoregulation resulting in better performance in tropical grazing systems.

Intestinal microbial diversity of swines fed with different sources of lithium.

A drug that is widely used in the treatment of psychiatric disorder is lithium (Li) salts. The people who make therapeutic use of this drug develop a series of side effects. Through metataxonomic data, this study assessed the impacts of lithium, as Li carbonate or Li-enriched mushrooms, on the microbial composition of the ileum, colon, and feces of piglets. Employing Bray-Curtis metric, no differences were observed among the treatments evaluated. Nevertheless, the alpha diversity indices showed differences in the Simpson, Shannon, and Chao-1 indices in the colon and Chao-1 in the feces in the diets with Li compared with the diets without Li. The taxa with the highest relative abundance varied among the ileum, colon, and feces, with a predominance of the phyla Firmicutes, Bacteroidota, and Proteobacteria in diets with Li. Many groups of microorganisms that are important for the health of the host (e.g., Lactobacillus, Ruminococcaceae, Enterorhabdus, Muribaculaceae, and Coprococcus) had their relative abundance increased in animals that received diets with the recommended dose of lithium. Furthermore, there was an increase in the abundance of Prevotellaceae and Bacteroidales (in the diet with Li-enriched mushroom) and Clostridia, Ruminococcus, Burkholderia, and Bacteroidales (diets with Li carbonate) at the recommended dosages. This is the first study to show the effects of Li carbonate and Li-enriched mushrooms on the intestinal microbiota of piglets. Thus, the effects of lithium on the body may be related to its ability to change the composition of the intestinal microbiota. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03938-3.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Safety evaluation of the antimicrobial peptide bovicin HC5 orally administered to a murine model.

BACKGROUND: Bovicin HC5 is an antimicrobial peptide that shows a broad spectrum of activity and potential for biotechnological and therapeutic applications. To gain insight about the safety of bovicin HC5 application, the histological and immunostimulatory effects of orally administrated bovicin HC5 to BALB/c mice were evaluated. BALB/c mice were divided into three groups: negative control (NC group); mice given purified bovicin HC5 (Bov group); mice given ovalbumin (positive control, PC group; a murine model of enteropathy). The mice were initially pre-sensitized, and PBS, bovicin HC5 or ovalbumin were administered for 30 days by daily gavages. Histological and morphometric analysis were performed and the relative expression of cytokines was analyzed by real-time RT-PCR. RESULTS: The oral administration of bovicin HC5 to BALB/c mice reduced weight gain and caused alterations in the small intestine, although absorptive changes have not been detected. The number of total goblet cells and the mucopolysaccharides production were not affected by bovicin HC5 administration. A hypertrophy of Paneth cells and an increase in the number of mitotic cells were observed in Bov group, while the number of mast cells remained unaltered. Increased expression of TNF-α, INF-γ and IL-12 was observed in the small intestine upon bovicin HC5 administration. CONCLUSION: Bovicin HC5 has only minor effects on intestinal permeability and did not elicit an allergenic response upon oral administration to animal models. Considering the low in vivo toxicity of bovicin HC5, it might be a good candidate for enteral applications.

Exopolysaccharides produced by Bacillus spp. inhibit biofilm formation by Staphylococcus aureus strains associated with bovine mastitis.

Bovine mastitis is a costly disease in the dairy sector worldwide. Here the objective was to identify and characterize anti-biofilm compounds produced by Bacillus spp. against S. aureus associated with bovine mastitis. Results showed that cell-free supernatants of three Bacillus strains (out of 33 analysed) reduced S. aureus biofilm formation by approximately 40 % without affecting bacterial growth. The anti-biofilm activity was associated with exopolysaccharides (EPS) secreted by Bacillus spp. The EPS decreased S. aureus biofilm formation in a dose-dependent manner, inhibiting biofilm formation by 83 % at 1 mg/mL. The EPS also showed some biofilm disruption activity (up to 36.4 %), which may be partially mediated by increased expression of the aur gene. The characterization of EPS produced by Bacillus velezensis 87 and B. velezensis TR47II revealed macromolecules with molecular weights of 31.2 and 33.7 kDa, respectively. These macromolecules were composed mainly of glucose (mean = 218.5 µg/mg) and mannose (mean = 241.5 µg/mg) and had similar functional groups (pyranose ring, beta-type glycosidic linkage, and alkynes) as revealed by FT-IR. In conclusion, this study shows the potential applications of EPS produced by B. velezensis as an anti-biofilm compound that could contribute to the treatment of bovine mastitis caused by S. aureus.

Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae.

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Interaction with lipid II induces conformational changes in bovicin HC5 structure.

Bovicin HC5 is a lantibiotic produced by Streptococcus bovis HC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

AiiA quorum-sensing quenching controls proteolytic activity and biofilm formation by Enterobacter cloacae.

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48 h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.

Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows.

Staphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0-0.5 mg ml(-1)) decreased growth of S. aureus in BHI media and 1 mg ml(-1) was bactericidal against washed cell suspensions (10(7) CFU ml(-1)). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml(-1)). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.