RESUMO
Subclinical mastitis represents one of the most contagious diseases affecting animals involved in dairy production systems. Although coagulase-negative staphylococci (CoNSs) have been considered minor pathogens for many years, they have recently emerged as opportunistic pathogens in mastitis disorders. The objectives of this work were to assess the antimicrobial resistance profile and the ability to produce a biofilm in comparison with a reference strain and to search for genes related to biofilm production, antimicrobial resistance, and enterotoxins in 18 isolates of Staphylococcus species from the milk of sheep with subclinical mastitis, collected from different Sicilian farms. This knowledge is essential to provide basic information on the pathogenicity and virulence of staphylococcal species and their impact on animal health. All isolates were resistant to ampicillin, 88.8% to streptomycin, 77.7% to gentamicin, 44.4% to chloramphenicol, 27.7% to erythromycin, and 11.1% to tetracycline, and two isolates were strong biofilm producers. Antibiotic resistance gene profiling showed that 16.6% of isolates possess the blaZ gene, whereas the search of biofilm-associated genes revealed the occurrence of the sasC gene in 33.3% of isolates, the ica gene in 27.7%, and bap and agr (accessory gene regulator) genes in 16.6% of isolates. Altogether, the results of this study indicate that CoNSs can acquire virulence genes and could have a role as pathogens in subclinical mastitis.
Assuntos
Mastite Bovina , Infecções Estafilocócicas , Feminino , Animais , Ovinos , Humanos , Bovinos , Staphylococcus , Enterotoxinas/genética , Leite , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/epidemiologia , Antibacterianos/farmacologia , Biofilmes , Resistência Microbiana a Medicamentos , Mastite Bovina/epidemiologiaRESUMO
Microbial communities provide essential information about host ecology and could be helpful as a tool to improve species conservation efforts. However, microbes can also infect and compromise the host development process and viability. Caretta caretta is the most widespread marine turtle species in the Mediterranean basin and is the only species of sea turtle nesting along the Italian coasts. Little is known about the microbiota composition of the nest of sea turtles and its correlation with hatching failures. In this study, the microbial composition of two nests of C. caretta featuring different rates of hatching success from a nesting beach in Lampedusa (Italy) was analyzed and compared. The bacterial community was determined using culture-dependent methods and next-generation sequencing based on 16S rRNA gene metabarcoding analysis. Our results showed five dominant bacterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes) and indicated different bacterial families (Pseudomonadaceae and Brucellaceae) as likely causes of hatching failures. Besides, our findings demonstrated the nests' active role in modulating the sand's bacterial communities. This study suggests microbiological analysis could be a valuable tool in monitoring nests to take preventive actions and reduce hatching failures.
Assuntos
Microbiota , Tartarugas , Animais , RNA Ribossômico 16S/genética , Ecologia , Bactérias/genéticaRESUMO
Combining biologically active compounds with nanocarriers is an emerging and promising strategy for enhancing the activities of molecules while reducing their levels of toxicity. Green nanomaterials have recently gained momentum in developing protocols for treating and preserving artifacts. In this study, we designed a functional biohybrid material by incorporating tributyltin(IV) ferulate (TBT-F) into halloysite nanotubes (HNTs), generating a new formulation called HNT/TBT-F. The primary objective was to develop a formulation with robust antimicrobial properties and reinforcing features for treating paper with artistic and historical value. To characterize HNT/TBT-F, assess the HNT's loading capacity, and investigate the TBT-F release kinetics from the nanotubes, various analytical techniques, including UV-Vis and infrared spectroscopies, thermogravimetry, and microscopy analysis, were employed. Furthermore, we evaluated the antimicrobial potential of TBT-F and HNT/TBT-F against Kocuria rhizophila, a bacterial strain known for its opportunistic behavior and a cause of artifact biodeterioration. HNT/TBT-F exhibited a significantly stronger bactericidal effect than TBT-F alone against K. rhizophila cells growing planktonically or those forming a biofilm. This enhanced performance could relate to the confinement of TBT-F within the nanotubes, which likely improved its physical-chemical stability and increased the local concentration of TBT-F upon contact with the bacterial cells. Additionally, we evaluated the mechanical properties of a paper treated with HNT/TBT-F, assessing any potential alterations in its color. The findings of this study highlight the favorable attributes of the HNT/TBT-F formulation and its potential for developing protocols aimed at consolidating and preserving culturally significant paper objects.
Assuntos
Anti-Infecciosos , Desinfetantes , Nanotubos , Argila/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Nanotubos/químicaRESUMO
The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO32-) is the most soluble, bioavailable, and toxic Te-species. On the other hand, tellurium is a rare metalloid element whose natural supply will end shortly with possible economic and technological effects. Thus, Te-containing waste represents the source from which Te should be recycled and recovered. Among the explored strategies, the microbial TeO32- biotransformation into less toxic Te-species is the most appropriate concerning the circular economy. Actinomycetes are ideal candidates in environmental biotechnology. However, their exploration in TeO32- biotransformation is scarce due to limited knowledge regarding oxyanion microbial processing. Here, this gap was filled by investigating the cell tolerance, adaptation, and response to TeO32- of a Micromonospora strain isolated from a metal(loid)-rich environment. To this aim, an integrated biological, physical-chemical, and statistical approach combining physiological and biochemical assays with confocal or scanning electron (SEM) microscopy and Fourier-transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR) was designed. Micromonospora cells exposed to TeO32- under different physiological states revealed a series of striking cell responses, such as cell morphology changes, extracellular polymeric substance production, cell membrane damages and modifications, oxidative stress burst, protein aggregation and phosphorylation, and superoxide dismutase induction. These results highlight this Micromonospora strain as an asset for biotechnological purposes.
Assuntos
Micromonospora , Telúrio , Telúrio/química , Micromonospora/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Agregados Proteicos , Superóxido DismutaseRESUMO
Two mononuclear Pd(II) complexes [PdCl2(pfptp)] (1) and [PdCl2(pfhtp)] (2), with ligands 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp) and 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), were synthesized and structurally characterized. The two complexes showed a bidentate coordination of the ligand occurring through N atom of pyridine ring and N4 atom of 1,2,4-triazole. Both complexes showed antimicrobial activity when tested against both Gram-negative and Gram-positive bacterial strains.
Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Fluorocarbonos/síntese química , Fluorocarbonos/farmacologia , Compostos Heterocíclicos/síntese química , Paládio/química , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , DNA/genética , Fluorocarbonos/química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria InfravermelhoRESUMO
An HS-SPME GC-MS analysis of the volatile compounds adsorbed at the outer surface of lemon and grapefruit pectins obtained via the hydrodynamic cavitation of industrial waste streams of lemon and grapefruit peels in water suggests important new findings en route to understanding the powerful and broad biological activity of these new pectic materials. In agreement with the ultralow degree of esterification of these pectins, the high amount of highly bioactive α-terpineol and terpinen-4-ol points to limonene (and linalool) decomposition catalyzed by residual citric acid in the citrus waste peel residue of the juice industrial production.
Assuntos
Citrus paradisi/química , Citrus/química , Compostos Orgânicos Voláteis/química , Vias Biossintéticas , Citrus/metabolismo , Citrus paradisi/metabolismo , Frutas/química , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/química , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismoRESUMO
Drug resistance is a major obstacle in antibiotic and antitumor chemotherapy. In response to the necessity to find new therapeutic strategies, plant secondary metabolites including essential oils (EOs) may represent one of the best sources. EOs in plants act as constitutive defenses against biotic and abiotic stress, and they play an important role in the pharmacology for their low toxicity, good pharmacokinetic and multitarget activity. In this context, natural products such as EOs are one of the most important sources of drugs used in pharmaceutical therapeutics. The aim of this paper was to identify the chemical composition of the essential oil of Alluaudia procera leaves, obtained by hydrodistillation and analysed by gas chromatography-mass spectrometry, and to verify its biological activities on acute myeloid leukemia cancer cell HL60 and its multidrugresistant variant HL60R and the Gram-positive Staphylococcus aureus exhibiting multi-antibiotic resistance. We speculate that cytotoxic and antibiotic effects observed in the tested resistant models may be due to the coordinate activities of forty compounds detected or to the C16 macrocyclic lactones which are the major ones (30%). Our data confirm the possibility of using EOs as therapeutic strategies in resistant models is due to the heterogeneous composition of the oils themselves.
Assuntos
Resistência a Medicamentos , Magnoliopsida/química , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Análise EspectralRESUMO
This paper deals with the synthesis and characterization of iron-hexacyanocobaltate (FeHCC) and its antibacterial properties. The nanoparticles were prepared by a facile co-precipitation technique. Crystal structure, particle morphology, and elemental composition were determined using X-ray Powder Diffraction, X-ray fluorescence spectroscopy, Transmission Electron Microscopy (TEM), and Infrared Spectroscopy (IR). The antibacterial activity of the FeHCC nanoparticles was tested against Escherichia coli and Staphylococcus aureus as models for Gram-negative and Gram-positive bacteria, respectively, by bacterial counting method and microscopic visualization (TEM, FEG-SEM, and fluorescence microscopy). The results showed that the FeHCC nanoparticles bind to the bacterial cells, inhibit bacterial growth in a dose- and time-dependent manner, inducing a loss of the membrane potential, the production of reactive oxygen species and the release of macromolecules (nucleic acids and proteins) in the extracellular environment. To the best of our knowledge, this is the first study reporting the antimicrobial effects of metal-hexacyanometallates suggesting practical uses of these materials in different areas, such as self-cleaning surfaces or food packaging.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Cobalto/química , Cianetos/química , Ferro/química , Nanopartículas Metálicas/química , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Staphylococcus aureus is the major cause of foodborne diseases worldwide. In this retrospective study, 84 S. aureus strains were characterized. The collection comprises 78 strains isolated during 1998 and 2014 from dairy products and tissue samples from livestock bred for dairy production in Sicily. One isolate was obtained from a pet (dog), one from an exotic animal (a circus elephant), and four human isolates were obtained during a severe food poisoning outbreak that occurred in Sicily in 2015. All the strains were characterized by pulsed-field gel electrophoresis (PFGE), for antibiotic resistance and presence of toxin genes. PFGE results showed 10 different pulsotypes, with three relatively frequent and three unique. The antibiotic resistance profiling showed that penicillin G (35.7%) and tetracycline (20.2%) resistance is largely spread. Most isolates contained at least one toxin gene making them a potential threat for public health. Enterotoxin sec gene was observed in 28.6% and seg in 23.8% of the strains, respectively; the human isolates were the only ones to concurrently harbor both seg and sei genes. In addition, 24 isolates were randomly selected and analyzed by multilocus sequence typing. Interestingly, the analysis showed the presence of 12 sequence types (STs), of which 6 were novel. One of them, ST700, was detected in 29% of the isolates and was found to be spread throughout Sicily. ST700 has been present in the island for almost 16 years (1998-2014) and it shows no host preference since it was isolated from different ruminant species. Four human isolates shared both the pulsotype (PT10) and the sequence type (ST9), as well as the virulence genes (seg-sei); this observation suggests that the isolates originated from a single clone, although they were obtained from two different individuals.
Assuntos
Farmacorresistência Bacteriana , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Virulência/genética , Animais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/genética , Doenças Transmitidas por Alimentos/epidemiologia , Genótipo , Humanos , Gado , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Sicília/epidemiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidadeRESUMO
Mono- and binuclear Pt(II) and Pd(II) complexes with 2,2'-dithiobis(benzothiazole) (DTBTA) ligand are reported. [Pt(DTBTA)(DMSO)Cl]ClâCHCl3 (1) and [Pd2(µ-Cl)2(DTBTA)2]Cl2 (2) have been synthesized and structurally characterized by elemental analysis, IR, 1H and 13C NMR spectroscopy, MS spectrometry and the content of platinum and palladium was determined using a flame atomic spectrometer. Two different coordination modes of 1 and 2 complexes were found; in both complexes, the coordination of Pt(II) and Pd(II) ions involves the N(3) atoms of the ligand but the binuclear complex 2, is a cis-chloro-bridged palladium complex. Evaluation of their in vitro antitumor activity against two human tumor cell lines human breast cancer (MCF-7) and hepatocellular carcinoma (HepG2); and their antimicrobial activity against Escherichia coli and Kokuria rhizophila was performed. Only complex 1 showed a dose- and time-dependent cytotoxic activity against the two tumor cell lines, associated to apoptosis and accumulation of treated cells in G0/G1 phase of cell cycle, while both 1 and 2 exhibited antimicrobial activity with complex 1 much more potent. The study on intracellular uptake in both MCF-7 and HepG2 cell lines revealed that only platinum of complex 1 is present inside the cells, suggesting a different mode of action of the two compounds. This was also in agreement with the results obtained for the antitumor and antibacterial activity.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Benzotiazóis/farmacologia , Paládio/química , Paládio/farmacologia , Compostos de Platina/química , Compostos de Platina/farmacologia , Antibacterianos/síntese química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/efeitos dos fármacos , Humanos , Ligantes , Testes de Sensibilidade Microbiana , Compostos de Platina/síntese química , Análise EspectralRESUMO
Three polyaminocyclodextrin materials, obtained by direct reaction between heptakis(6-deoxy-6-iodo)-ß-cyclodextrin and the proper linear polyamines, were investigated for their binding properties, in order to assess their potential applications in biological systems, such as vectors for simultaneous drug and gene cellular uptake or alternatively for the protection of macromolecules. In particular, we exploited polarimetry to test their interaction with some model p-nitroaniline derivatives, chosen as probe guests. The data obtained indicate that binding inside the host cavity is mainly affected by interplay between Coulomb interactions and conformational restraints. Moreover, simultaneous interaction of the cationic polyamine pendant bush at the primary rim was positively assessed. Insights on quantitative aspects of the interaction between our materials and polyanions were investigated by studying the binding with sodium alginate. Finally, the complexation abilities of the same materials towards polynucleotides were assessed by studying their interaction with the model plasmid pUC19. Our results positively highlight the ability of our materials to exploit both the cavity and the polycationic branches, thus functioning as bimodal ligands.
RESUMO
BACKGROUND: The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. RESULTS: Microbispora ATCC-PTA-5024 cultivations in a complex medium were characterized by stages of biomass accumulation (A) followed by biomass yield decline (D). NAI-107 production started at 90 h (A stage), reached a maximum at 140 h (D stage) and decreased thereafter. To reveal patterns of differentially represented proteins associated with NAI-107 production onset and maintenance, differential proteomic analyses were carried-out on biomass samples collected: i) before (66 h) and during (90 h) NAI-107 production at A stage; ii) during three time-points (117, 140, and 162 h) at D stage characterized by different profiles of NAI-107 yield accumulation (117 and 140 h) and decrement (162 h). Regulatory, metabolic and unknown-function proteins, were identified and functionally clustered, revealing that nutritional signals, regulatory cascades and primary metabolism shift-down trigger the accumulation of protein components involved in nitrogen and phosphate metabolism, cell wall biosynthesis/maturation, lipid metabolism, osmotic stress response, multi-drug resistance, and NAI-107 transport. The stimulating role on physiological differentiation of a TetR-like regulator, originally identified in this study, was confirmed by the construction of an over-expressing strain. Finally, the possible role of cellular response to membrane stability alterations and of multi-drug resistance ABC transporters as additional self-resistance mechanisms toward the lantibiotic was confirmed by proteomic and confocal microscopy experiments on a Microbispora ATCC-PTA-5024 lantibiotic-null producer strain which was exposed to an externally-added amount of NAI-107 during growth. CONCLUSION: This study provides a net contribution to the elucidation of the regulatory, metabolic and molecular patterns controlling physiological differentiation in Microbispora ATCC-PTA-5024, supporting the relevance of proteomics in revealing protein players of antibiotic biosynthesis in actinomycetes.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Resistência a Múltiplos Medicamentos/genética , Peptídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinobacteria/química , Actinobacteria/metabolismo , Antibacterianos/química , Bacteriocinas/química , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Peptídeos/química , ProteômicaRESUMO
Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.
Assuntos
Bacteriófagos/genética , Vetores Genéticos , Genética Microbiana/métodos , Biologia Molecular/métodos , Streptomyces/genética , Streptomyces/virologia , Escherichia coli/genética , Fenótipo , Plasmídeos , Recombinação GenéticaRESUMO
UNLABELLED: The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, and dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, and dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCE: This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.
Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Teicoplanina/análogos & derivados , Actinomycetales/genética , Proteínas de Bactérias/genética , Estrutura Molecular , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Teicoplanina/biossíntese , Transcrição GênicaRESUMO
A case of staphylococcal food poisoning was observed in two individuals of the same family after consumption of primosale, a semiripened sheep cheese produced in Sicily. Staphylococcus aureus isolated from the cheese produced enterotoxin C (SEC) and carried both the enterotoxin C (sec) and the toxic shock syndrome toxin (tsst-1) gene. Following this case, an extensive survey was conducted on 971 food samples (raw milk, cheese, meat, and food preparations). S. aureus was detected in 102 of 971 food samples, from all types of food with the exception of ricotta cheese. The tsst-1 gene was present in 42% of the strains, either alone or in combination with other toxin genes. The enterotoxin C gene was the most represented enterotoxin, but it was only found in dairy products. Six S. aureus isolates carried the sea gene alone, two isolates carried both sea and seb, and one isolate carried both sea and sec. A significant percentage (46%) of all isolates carried a toxin gene, creating significant concern that virulent S. aureus can be transmitted through food in Sicily.
Assuntos
Toxinas Bacterianas/genética , Enterotoxinas/genética , Intoxicação Alimentar Estafilocócica/microbiologia , Staphylococcus aureus/isolamento & purificação , Superantígenos/genética , Animais , Qualidade de Produtos para o Consumidor , DNA Bacteriano/isolamento & purificação , Laticínios/microbiologia , Microbiologia de Alimentos , Carne/microbiologia , Reação em Cadeia da Polimerase , Sicília , Staphylococcus aureus/genéticaRESUMO
BACKGROUND: NAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides. The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes. RESULTS: After having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production. CONCLUSION: These results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use.
Assuntos
Actinobacteria/crescimento & desenvolvimento , Bacteriocinas/biossíntese , Fosfatos/farmacologia , HumanosRESUMO
In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.
Assuntos
Biofilmes , Queijo , Cabras , Leite , Queijo/microbiologia , Animais , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Leite/microbiologia , Madeira/microbiologia , Microbiologia de Alimentos , Sicília , Lactobacillales/genética , Lactobacillales/fisiologia , Lactobacillales/metabolismo , RNA Ribossômico 16S/genética , Antibacterianos/farmacologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/genéticaRESUMO
Volatile fatty acids (VFA) from sewage sludge represent an excellent recovered resource from wastewater treatment. This study investigated four sludge pre-treatments (namely, potassium permanganate - KMnO4, initial pH = 10, initial pH = 2.5 and low-temperature thermal hydrolysis) by operating batch reactors under acidogenic fermentation conditions. Results revealed that 0.1 g KMnO4/g of total suspended solids represents the best pre-treatment obtaining up to 2713 mgCOD L-1 and 452 mgCOD/g of volatile suspended solids. These results also paralleled metataxonomic analysis highlighting changes in prokaryotic microbial structures of sewage sludge of the batch fermentations subjected to the different pre-treatments.
Assuntos
Reatores Biológicos , Esgotos , Fermentação , Esgotos/química , Ácidos Graxos Voláteis , Hidrólise , Concentração de Íons de HidrogênioRESUMO
The effects of oxyanions selenite (SeO32-) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized.
Assuntos
Actinobacteria , Selênio , Ácido Selenioso/toxicidade , Selenito de Sódio/metabolismo , Selenito de Sódio/toxicidade , Actinobacteria/genética , Actinobacteria/metabolismo , Bactérias/metabolismo , Selênio/metabolismo , OxirreduçãoRESUMO
Bovine tuberculosis and paratuberculosis are endemic in many areas worldwide. This work aims to study cytokines production and gene expression profiles of bovine macrophages infected with Mycobacterium bovis and Mycobacterium paratuberculosis subsp. avium (MAP) strains to identify potential diagnostic biomarkers. Bovine bone marrow stem cells were differentiated into macrophages and subsequently infected in vitro with different spoligotypes of M. bovis and MAP field strains (as single infections and coinfections), using different multiplicity of infection. Supernatant and cell pellets were collected 24 h, 48 h, and one week post-infection. Preliminarily, gene expression on cell pellets of IL-1ß, IL-2, INFγ, IL-6, IL-10, IL-12, and TNFα was assessed by qRT-PCR one week p.i. Subsequently, IL-1ß and IL-6 were measured by ELISA and qRT-PCR to investigated their production retrospectively 24 h and 48 h p.i. A variability in macrophages response related to the concentration of mycobacteria, the coinfection with MAP, and M. bovis spoligotypes was identified. An early and constant IL-6 increase was observed in the M. bovis infection. A lower increase in IL-1ß was also detected at the highest concentration of the two M. bovis spoligotypes one week post-infection. IL-6 and IL-1 ß production was reduced and differently expressed in the MAP infection. IL-6 appeared to be the earliest cytokines produced by bovine macrophages infected with M. bovis.