Specific Antimicrobial Activity Research Articles

COMPARATIVE ELECTRON-MICROSCOPIC AND MICROBIOLOGICAL INVESTIGATION OF SILVER PROTEINATE PREPARATIONS

The purpose of this work was a comparative study of the particle sizes of commercial preparations of colloidal silver by the method of electron microscopy, in connection with the characteristics of specific antimicrobial activity of the preparations under the same conditions. Four commercial preparations of colloidal silver were investigated: Vitargol Forte (manufactured by NPC «Elusan»), Protalor (manufactured by OOO «Esko-Farm», Armenia), Sialor (manufactured by PFK «Obnovlenie»). Protargol (prescription drugstore) served as the drug of comparison. Material and methods. The silver particle sizes in the preparations were determined by high-resolution transmission electron microscopy using a JEM 2010 electron microscope (JEOL, Japan) with a resolution of 0.14 nm at an accelerating voltage of 200 kV. The antimicrobial activity of the silver protein preparations was studied by a standard serial dilution method in liquid nutrient media and diffusion method using bacterial cultures of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Results and discussion. Vitargol Forte, manufactured using advanced technology, is the most fine-dispersed and monodisperse preparation. The preparation of standard classical Protargol, prepared in pharmacy conditions, was the most coarsely dispersed and polydispersed. The conclusion. The dependence of antimicrobial activity of the preparation on the size of its silver particles was shown. Of the four investigated preparations of silver proteinate, the smallest particle size was in the Vitargol Fort preparation. The same drug showed the highest bactericidal and bacteriostatic activity.

First-In-Class Inhibitor of Ribosomal RNA Synthesis with Antimicrobial Activity against Staphylococcus aureus

We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

Probiotic Characteristics of Lactobacillus curvatus DN317, a Strain Isolated from Chicken Ceca.

The probiotic characteristics of Lactobacillus curvatus DN317, a strain isolated from chicken ceca, were evaluated. This strain was selected for study from the isolated Lactobacillus strains because it has specific anti-microbial activity against Campylobacter jejuni ATCC 33560, Camp. jejuni NCTC 11168, Listeria monocytogenes ATCC 7644, and Bacillus subtilis ATCC 8633. Lact. curvatus DN317 showed an auto-aggregation percentage of 72% and presented the highest co-aggregation with Lact. monocytogenes ATCC 7644 (68%) compared to B. subtilis ATCC 8633 (45%), Camp. jejuni ATCC 33560 (36%), and Camp. jejuni NCTC 11168 (35%). The data revealed that Lact. curvatus DN317 could survive at 0.25% bile, maintain viability at pH 2.5 for 30min, produce biosurfactants, and adhere to Caco-2 cells. Quantification of IL-6, IL-8, IL-10, and β-defensin 2 levels shows that Lact. curvatus DN317 induces an increase in IL-8 and β-defensin 2 secretion, while the levels of IL-6 and IL-10 do not change. Lact. curvatus DN317 showed high levels of esterase and cysteine arylamidase activities (5); moderate levels of esterase lipase, β-galactosidase, and α-galactosidase activities (4, 3); and weak levels of leucine arylamidase, valine arylamidase, and acid phosphatase activity (1). Various activities were obtained of α-chymotrypsin, β-glucuronidase, β-glucosidase, and N-acetyl-β-glucosaminidase, which have been associated with intestinal diseases. Lact. curvatus DN317 lowered the cholesterol level in MRS with and without bile. Antibiotic susceptibility tests indicated that DN317 was sensitive to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, clindamycin, erythromycin, and vancomycin but was resistant to chloramphenicol and ciprofloxacin. These results suggest that Lact. curvatus DN317 could potentially function as a probiotic.

Halocin HA1: An archaeocin produced by the haloarchaeon Haloferax larsenii HA1

Abstract An effective activity-guided purification strategy was applied to purify halocin HA1 from cell-free supernatant (CFS) of Haloferax larsenii HA1. The yield of the purified halocin was 12.8%, with specific antimicrobial activity of 12.16 AU μg−1. Halocin activity was stable up to 121 °C and pH 4.0–12.0 but reduced in the presence of proteinase K and trypsin, suggesting the proteinaceous nature of the compound. It was halocidal against H. larsenii HA10 and caused cellular deformation and release of cell contents leading to cell death. The halocidal activity was also found against the other strains of haloarchaea tested. The molecular mass of halocin HA1 was ∼14 kDa, as determined by tricine SDS-PAGE. MALDI-TOF MS/MS and N-terminal sequencing indicated no apparent homology with known halocins, suggesting that this is a new compound. These properties of halocin HA1 may be useful for the preservation of salted foods and leather hides in food and leather industries.

Preparation, characterization, antimicrobial and cytotoxicity studies of copper/zinc- loaded montmorillonite

BackgroundA series of modified montmorillonites (Mt) including zinc-loaded Mt (Zn-Mt), copper-loaded Mt (Cu-Mt), copper/zinc-loaded Mt with different Cu/Zn ratio (Cu/Zn-Mt-1, Cu/Zn-Mt-2, Cu/Zn-Mt-3) were prepared by an ion-exchange reaction, and characterized using X-ray diffraction (XRD), fourier transformed infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The specific surface areas, antimicrobial activity and cytotoxicity of the modified Mt were investigated.ResultsIn the modified Mt, hydrated Cu ions and Zn ions were exchanged in the interlayer space of Mt and the particles were irregular shapes. The results showed that Cu/Zn-Mt enhanced antibacterial and antifungal activity compared with Zn-Mt and Cu-Mt possibly due to the synergistic effect between Cu and Zn. Among the Cu/Zn-Mt with different Cu/Zn raitos, Cu/Zn-Mt with a Cu/Zn ratio of 0.98 or 0.51 showed higher antimicrobial activity against gram-negative bacteria (Escherichia coli), gram-positive bacteria (Staphylococcus aureus), fungi (Candida albicans). Moreover, the antimicrobial activity of Cu/Zn-Mt was correlated with its specific surface area. Cytotoxicity studies on IPEC-J2 cell showed a slight cytotoxicity of Cu/Zn-Mt.ConclusionsThe current data provide clear evidence that in terms of its antimicrobial activity and relatively low toxicity, the Cu/Zn-Mt holds great promise for applications in animal husbandry.

Any use for alternative lock solutions in the prevention of catheter-related blood stream infections?

The prevention of catheter-related blood stream infections (CRBSI) in hemodialysis (HD) patients remains a challenge because of high morbidity and mortality associated to CRBSI. Alternative locking solutions (ALS) containing an antithrombotic substance with additional antimicrobial or antibiofilm properties (citrate, ethylenediaminetetraacetic acid [EDTA], 70% ethanol, thrombolytics) with or without the addition of molecules with specific antimicrobial activity (antibiotics, taurolidine, paraben-methylene-blue) has been proposed with the aim to prevent or eradicate intraluminal biofilm colonization and subsequent CRBSI. In this review, we examine the available evidence concerning their efficacy and potential side effects, in order to determine whether ALS should be implemented widely or only in selected cases.

Preparation and Biological Activity of New Collagen Composites Part II: Collagen/Reduced Graphene Oxide Composites

: With the idea of exploring the biological activity of some newly synthetized chemical compounds and their combinations for development of novel antimicrobial collagen biomaterials, a serial investigation was initiated, starting with the preparation and biological activity study of Collagen/ZnTiO3 nano-composites. This serial investigation continued with the preparation and biological activity study of new collagen-based composites in which self-prepared reduced graphene oxide (RGO) sheets were included as an antimicrobial agent. The new porous collagen/RGO composites demonstrated specific antimicrobial activity to different types microbial species; well pronounced activity against Gram-positive microorganisms (Listeria innocua and Bacillus cereus, both bacteria with typical chains forming, large size cells, and Candida lusitaniae, fungus with specific micelle organization) and lack of activity against Gram-negative bacteria (Pseudomonas putida, Salmonella enterica, Pseudomonas aeruginosa, and Escherichia coli; all bacteria with small size cells) combined with lack of cytotoxicity to eukaryotic cells. For the first time, well-pronounced antifungal activity of collagen/RGO composites, depending on the RGO concentration was observed. Sterile zone of 17 mm was measured for C. lusitaniae on collagen/RGO composite, 2:1 wt/wt. The possible mechanism of the biological activity of the new collagen/RGO composites was correlated with their characteristics and the specific cell morphology and size of the test microorganisms. The results of this investigation demonstrated that with their specific and adjustable bioactivity, the new collagen/RGO composites are promising antimicrobial biomaterial for variety of biomedical applications, including tissue engineering.

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.

Antimicrobial proteins and peptides (AMPs) are valuable as leads in the pharmaceutical industry for the development of novel anti-infective drugs. Here we describe the efficient heterologous expression and basic characterization of a Gloverin-family AMP derived from the greater wax moth Galleria mellonella. Highly productive single-cell clones prepared by limiting dilution achieved a 100% increase in productivity compared to the original polyclonal Drosophila melanogaster S2 cell line. Comprehensive screening for suitable expression conditions using statistical experimental designs revealed that optimal induction was achieved using 600µM CuSO4 at the mid-exponential growth phase. Under these conditions, 25mg/L of the AMP was expressed at the 1-L bioreactor scale, with optimal induction and harvest times ensured by dielectric spectroscopy and the online measurement of optical density. Gloverin was purified from the supernatant by immobilized metal ion affinity chromatography followed by dialysis. In growth assays, the purified protein showed specific antimicrobial activity against two different strains of Escherichia coli.

One-step surface grafting of organic nanoparticles: in situ deposition of antimicrobial agents vanillin and chitosan on polyethylene packaging films.

Nanoparticles of natural antimicrobial agents, volatile vanillin and non-volatile chitosan, were deposited in situ from an aqueous/ethanol solution onto a polyethylene (PE) surface using the ultrasonic method. The modified PE films were comprehensively characterized in terms of their microscopic, spectroscopic, mechanical and physical properties, and the presence of stable organic nanoparticles on the polymer surface was established. The nanoparticle-grafted films showed specific antimicrobial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The vanillin nanoparticles led to a total inhibition of E. coli and the chitosan nanoparticles led to a total inhibition of S. aureus. The antimicrobial effect of the prepared active PE films was also examined on a food model, fresh-cut watermelons. Contact with the active film significantly inhibits the fruit microbial spoilage, especially in the case of the vanillin nanoparticles on PE. As they are surface-grafted in a nanoparticle form, the active agents are fully utilized, allowing for a significant enhancement in their effectivity and a reduction in the amount required. The presented method is of general interest as a facile technique for the surface deposition of organic nanoparticles, and can potentially be applied as a feasible approach for the incorporation of active agents into polymer matrices.

Tetrabromobisphenol A (TBBPA) exhibits specific antimicrobial activity against Gram-positive bacteria without detectable resistance.

We report here the antimicrobial ability of tetrabromobisphenol A (TBBPA) against Gram-positive bacteria without detectable resistance. Microscopy imaging and gene analysis reveal that the bacterial cell wall is the target of TBBPA. Structure-activity studies of TBBPA and its analogues relate the observed antimicrobial activity to the halogen substitution neighboring the phenolic hydroxyl group.

Antimicrobial Properties of an Immunomodulator - 15 kDa Human Granulysin.

Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against Pseudomonas aeruginosa within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by P. aeruginosa. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.

Investigation of antimicrobial activity of silver and copper citrate for the development of pharmaceuticals preparations

Быстрое формирование устойчивости микроорганизмов к антибиотикам вызывает необходимость поиска новых, альтернативных антимикробных лекарственных средств. С целью фармацевтической разработки препаратов изучена антимикробная активность различных концентраций растворов цитрата серебра и меди стандартным методом серийных разведений на тест-штаммах микроорганизмов. Наиболее чувствительным к раствору цитрата серебра оказался штамм Escherichia coli ATCC 25922 (F-50) и Pseudomonas aeruginosae АТСС 9027 при концентрации серебра в растворе 10 мкг/мл. Чувствительность Staphylococcus aureus 6538 Р проявилась в концентрации серебра 25 мкг/ мл. Полученные в ходе эксперимента данные указывают на то, что минимальная ингибирующая концентрация цитрата серебра (содержание серебра 500 мкг/мл) по отношению к тест-штамму Staphylococcus aureus 6538 Р составляет 20 мкг/мл, по отношению к Escherichia coli ATCC 25922 – 6,7 мкг/мл, а к Pseudomonas aeruginosae АТСС 9027 –10 мкг/мл соответственно. Staphylococcus aureus является чувствительным к раствору серебра и меди цитрата в разведении 1/20 при содержании металлов по 12,5 мкг/мл. Штамм Escherichia coli ATCC 25922 был чувствительным к исследуемой композиции в разведении 1/50, что соответствует содержанию металлов серебра и меди по 5 мкг/мл. Установлено, что исследуемые растворы проявляют выраженное антимикробное действие по отношению к грамположительным и грамотрицательным микроорганизмам. Это свидетельствует о перспективности использования соединений серебра и меди в составе антимикробных препаратов мягкой и жидкой формы выпуска.

Simultaneous Assessment of Acidogenesis-Mitigation and Specific Bacterial Growth-Inhibition by Dentifrices.

Dentifrices can augment oral hygiene by inactivating bacteria and at sub-lethal concentrations may affect bacterial metabolism, potentially inhibiting acidogenesis, the main cause of caries. Reported herein is the development of a rapid method to simultaneously measure group-specific bactericidal and acidogenesis-mitigation effects of dentifrices on oral bacteria. Saliva was incubated aerobically and anaerobically in Tryptone Soya Broth, Wilkins-Chalgren Broth with mucin, or artificial saliva and was exposed to dentifrices containing triclosan/copolymer (TD); sodium fluoride (FD); stannous fluoride and zinc lactate (SFD1); or stannous fluoride, zinc lactate and stannous chloride (SFD2). Minimum inhibitory concentrations (MIC) were determined turbidometrically whilst group-specific minimum bactericidal concentrations (MBC) were assessed using growth media and conditions selective for total aerobes, total anaerobes, streptococci and Gram-negative anaerobes. Minimum acid neutralization concentration (MNC) was defined as the lowest concentration of dentifrice at which acidification was inhibited. Differences between MIC and MNC were calculated and normalized with respect to MIC to derive the combined inhibitory and neutralizing capacity (CINC), a cumulative measure of acidogenesis-mitigation and growth inhibition. The overall rank order for growth inhibition potency (MIC) under aerobic and anaerobic conditions was: TD> SFD2> SFD1> FD. Acidogenesis-mitigation (MNC) was ordered; TD> FD> SFD2> SFD1. CINC was ordered TD> FD> SFD2> SFD1 aerobically and TD> FD> SFD1> SFD2 anaerobically. With respect to group-specific bactericidal activity, TD generally exhibited the greatest potency, particularly against total aerobes, total anaerobes and streptococci. This approach enables the rapid simultaneous evaluation of acidity mitigation, growth inhibition and specific antimicrobial activity by dentifrices.

Immunomodulatory and Antimicrobial Activity of Babassu Mesocarp Improves the Survival in Lethal Sepsis.

Attalea speciosa syn Orbignya phalerata Mart. (babassu) has been used in the treatment of inflammatory and infectious diseases. Aim of the study. To investigate the antimicrobial and immunological activity of babassu mesocarp extract (EE). Material and Methods. The in vitro antimicrobial activity was evaluated by disk diffusion assay and by determination of the minimum inhibitory concentration (MIC) to Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The flavonoids and phenolic acids content were determined by chromatography. The in vivo assays were performed in Swiss mice submitted to sepsis by cecal ligation and puncture (CLP). The mice received EE subcutaneously (125 or 250 mg/Kg), 6 hours after the CLP. The number of lymphoid cells was quantified and the cytokines production was determined by ELISA after 12 h. Results. EE was effective as antimicrobial to E. faecalis, S. aureus, and MRSA. EE is rich in phenolic acids, a class of compounds with antimicrobial and immunological activity. An increased survival can be observed in those groups, possibly due to a significant inhibition of TNF-α and IL-6. Conclusions. The EE showed specific antimicrobial activity in vitro and an important antiseptic effect in vivo possibly due to the antimicrobial and immunomodulatory activity.

Cytotoxic and Antimicrobial Diterpenes Isolated from Hyptis dilatata

Diterpenes 7-ethoxyrosmanol (1) and carnosol (2) were isolated and identified from the extracts of Hyptis dilatata collected in the Orinoco region, Colombia, used traditionally in this region for the treatment of wound infections of cattle. Their stereostructures were elucidated by 1D and 2D NMR experiments including 1H, 13C, COSY, HSQC, HMBC and NOESY. Although compound 1 has been reported previously, its NMR assignments were revised, completed and corrected. These compounds showed a specific antimicrobial activity against Gram (+) strains. The cytotoxic activity of compounds 1 and 2 was evaluated against a panel of three cancer cell lines (MCF7, HeLa and HT29) using curcumin as positive control. Carnosol (2) showed cytotoxic activity against all three cell lines having a similar response as the positive control with an IC50 approximately of 15-20 µg/mL in each case. On the other hand, 7-ethoxyrosmanol (1) showed lower activity than carnosol, with an IC50 approx. of 20-30 µg/mL for the three cancer cell lines. This is the first report on cytotoxic activity against MCF7, HeLa and HT29 cancer cell lines and the antimicrobial activity of compound 1, and the first report on cytotoxic activity against HeLa of compound 2. In addition, the main volatile components of the essential oil of H. dilatata which displayed quorum sensing inhibitory activity were identified by GC/MS analysis. Finally, these results let us to propose H. dilatata as a potential resource of antimicrobial and cytotoxic compounds. Keywords: 7-ethoxyrosmanol, antimicrobial activity, carnosol, cytotoxic activity, Hyptis dilatata, quorum sensing inhibition.

Mechanism study on a new antimicrobial peptide Sphistin derived from the N-terminus of crab histone H2A identified in haemolymphs of Scylla paramamosain.

Histone H2A is known to participate in host immune defense through generating special antimicrobial peptides (AMPs), for which it has been an interesting research focus to characterize this kind of peptides in vertebrates and invertebrates. Although thousands of AMPs have been reported in variety of life species, only several AMPs are known in crabs and in particular no H2A-derived AMP has yet been reported. In the present study, a 38-amino acid peptide with antimicrobial activity was determined based on the sequence analysis of a histone H2A identified from the mud crab Scylla paramamosain. The histone H2A derived peptide was an AMP-like molecule and designated as Sphistin. Sphistin showed typical features of AMPs such as amphiphilic α-helical second structrue and positive charge net. The synthetic Sphistin exerted high antimicrobial activity against Gram-positive, Gram-negative bacteria and yeast, among which Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas stutzeri are important aquatic pathogens. Leakage of the cell content and disruption of the cell surface were observed in bacterial cells treated with Sphistin using scanning electron microscopy. It was proved that the increasing cytoplasmic membrane permeability of Escherichia coli was caused by Sphistin. Further observation under confocal microscopy showed that Sphistin could combine onto the membrane of Staphylococcus aureus, E. coli MC1061 and Pichia pastoris but not translocate into the cytoplasm. Moreover, the affinity of Sphistin with either LPS or LTA was also testified that there was an interaction between Sphistin and cell membrane. Thus, the antimicrobial mechanism of this peptide likely exerted via adsorption and subsequently permeabilization of the bacterial cell membranes other than penetrating cell membrane. In addition, synthetic Sphistin exhibited no cytotoxicity to primary cultured crab haemolymphs and mammalian cells even at a high concentration of 100 μg/mL for 24 h. This is the first report of a histone-derived Sphistin identified from S. paramamosain with a specific antimicrobial activity and mechanism, which could be a new candidate for future application in aquaculture and veterinary medicine.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475.

BackgroundBacteriocins produced by lactic acid bacteria (LAB) attract considerable interest as natural and nontoxic food preservatives and as therapeutics whereas the bacteriocin-producing LAB are considered potential probiotics for food, human and veterinary applications, and in the animal production field. Within LAB the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins, and for production of bioactive peptides. Research efforts for production of bacteriocins in heterologous hosts should be performed if the use of bacteriocins and the LAB bacteriocin-producers is ever to meet the high expectations deposited in these antimicrobial peptides. The recombinant production and functional expression of bacteriocins by lactobacilli would have an additive effect on their probiotic functionality.ResultsThe heterologous production of the bacteriocin enterocin A (EntA) was evaluated in different Lactobacillus spp. after fusion of the versatile Sec-dependent signal peptide (SPusp45) to mature EntA plus the EntA immunity gene (entA + entiA) (fragment UAI), and their cloning into plasmid vectors that permitted their inducible (pSIP409 and pSIP411) or constitutive (pMG36c) production. The amount, antimicrobial activity (AA) and specific antimicrobial activity (SAA) of the EntA produced by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI varied depending of the expression vector and the host strain. The Lb. casei CECT475 recombinant strains produced the largest amounts of EntA, with the highest AA and SAA. Supernatants from Lb. casei CECT (pSIP411UAI) showed a 4.9-fold higher production of EntA with a 22.8-fold higher AA and 4.7-fold higher SAA than those from Enterococcus faecium T136, the natural producer of EntA. Moreover, supernatants from Lb. casei CECT475 (pSIP411UAI) showed a 15.7- to 59.2-fold higher AA against Listeria spp. than those from E. faecium T136.ConclusionLb. casei CECT457 (pSIP411UAI) may be considered a promising recombinant host and cell factory for the production and functional expression of the antilisterial bacteriocin EntA.

Antimicrobial analysis of Triphala and comparison with its individual constituents

Triphala, a well known ayurvedic formulation is used against number of ailments since ancient times. It consists of Emblica officinalis, Terminalia chebula and Terminalia bellerica in equal proportion. Triphala as a whole and its three individual constituents show specific antimicrobial activity against certain bacteria and fungi. Antimicrobial activity of aqueous extract of triphala and its constituents was studied against P.aeruginasa, E.coli , B.subtilis, K. pneumoniae and S.aureus by cup-plate method. Triphala was found strongly bactericidal against P. aeruginosa with 1.8 cm of inhibitory zone. This was on account of T. chebula which showed 1.2 cm of inhibitory zone against the same pathogen, followed by E. coli and other two Gram positive bacteria. T. bellerica however showed maximum inhibitory activity against B.subtilis by showing 2.2 cm of inhibitory zone. It was confirmed that antimicrobial activity against Gram negative bacteria is due to T. chebula and E. officinalis while antimicrobial activity against Gram positive bacteria is on account of T. bellerica. Antifungal activity of Triphala and its constituents was studied against two pathogenic fungi viz. Aspergillus niger and Candida albicans by the same method. Triphala was found more efficient against A. niger, this was on account of T. bellerica which showed nearly 2 cm zone of inhibition. T. bellerica and Triphala showed more than 2 cm inhibitory zone against C. albicans. Inhibitory zone of Emblica officinalis was observed to be of 1.5-2 cm against both the fungi, whereas T. chebula did not show inhibitory activity against C. albicans but showed nearly 1 cm of inhibitory zone against A. niger. This confirms that the antifungal activity of Triphala is primarily due to T. bellerica and E. offiicinalis.

Molecular characterization of the genes involved in the secretion and immunity of lactococcin Q, a two-peptide bacteriocin produced by Lactococcus lactis QU 4.

Lactococcin Q is a two-peptide (Qα and Qβ) bacteriocin produced by Lactococcus lactis QU 4, which exhibits specific antimicrobial activity against L. lactis strains. The lactococcin Q gene cluster (approximately 4.5 kb) was sequenced and found to include genes encoding lactococcin Q immunity (laqC), an ATP-binding cassette transporter (laqD) and a transport accessory protein (laqE), downstream of the lactococcin Q structural genes (laqA and laqB). In addition, the gene cluster showed high sequence identity with that of a lactococcin Q homologue bacteriocin, lactococcin G. Heterologous expression studies showed that LaqD was responsible for lactococcin Q secretion in a manner dependent on LaqE expression, and that LaqC conferred self-immunity to lactococcin Q and cross-immunity to lactococcin G. Amino acid alignment of both lactococcin transporters revealed that LaqD contains an insertion (160-168 residues) that is essential for lactococcin Q secretion, as L. lactis cells that expressed LaqDΔ160-168 were devoid of this function. Additional experiments demonstrated that the LaqDΔ160-168 mutant was, however, able to secrete lactococcin G, suggesting that the insertion is necessary only for the lactococcin Q secretion by LaqD. This report demonstrates the biosynthetic mechanism of lactococcin Q/G-type bacteriocins and the complementarity of the genes responsible for the secretion of lactococcins Q and G.