Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Total Synthesis and Antibacterial Study of Cyclohexapeptides Desotamide B,Wollamide B and Their Analogs

As natural‐product‐derived antibiotics, desotamides A – D and wollamides exhibit growth inhibitory activity against Gram‐posivite bacteria (IC₅₀ 0.6 – 7 μm ) and are noncytotoxic to mammalian cells (IC₅₀ > 30 μm ). Herein we firstly report the total synthesis of above two cyclohexapeptides as well as a series of structural variants through solid phase peptide synthesis, of which 3 displayed a 2‐fold increase of antibacterial activity when compared with the original peptide 1 . This strategy may offer good improvements for the synthesis of other cyclic peptides.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Overexpression of Jatropha curcas Defensin (JcDef) Enhances Sheath Blight Disease Resistance in Tobacco

Plant defensins are small, basic, cysteine‐rich peptides, belonging to the antimicrobial peptide superfamily, commonly found in the plant kingdom. In this study, we cloned and characterized a plant defensin gene from Jatropha curcas (JcDef). JcDef carried conserved receptor binding sites and a cysteine motif, and it was phylogenetically grouped together with defensin Ec‐AMP‐D2‐like in Elaeis guineensis. JcDef is localized to cytoplasm and highly expressed in young tissues with fast metabolism such as cotyledons and stem apexes. Transgenic expression of JcDef in tobacco showed enhanced resistance against sheath blight disease caused by R. solani, indicating the antibacterial function.     

Cytotoxic Triterpenoids from the Stalks of Microtropis triflora

Bioassay‐guided phytochemical investigation of the stalks of Microtropis triflora Merr . & F.L. Freeman led to the isolation of ten triterpenes 1  –  10 , including one novel compound 3,24‐epoxy‐2α,24‐dihydroxyfriedelan‐29‐oic acid ( 1 ). Their chemical structures were identified on the basis of spectroscopic analysis, including HR‐ESI mass spectrometry, 1D‐ and 2D‐NMR (¹H, ¹³C, ¹H,¹H‐COSY, HSQC, HMBC, and NOESY), and by comparison with the data reported. The cytotoxicities of compounds 1  –  10 against a panel of cultured human tumor cell lines (Bcap37, SMMC7721, HeLa, CNE) were evaluated. The new compound 1 showed moderate anti‐tumor activities with IC₅₀ values of 39.22, 29.24, 23.28, and 68.81 μm /ml, respectively. These results might be helpful for explaining the use of M. triflora in traditional medicine. Triterpenes are characteristic of Microtropis genus and could be useful as potential chemotaxonomic markers.     

Penetration by Botryosphaeriaceae species in avocado,guava and persimmon fruit during postharvest

Botryosphaeriaceae species have a wide host range and a worldwide distribution. These fungal species can colonize several plant organs, such as the trunk, leaves and fruit. Some Botryosphaeriaceae species cause important diseases on persimmon, avocado and guava fruit. However, there is a lack of information regarding the mechanisms of penetration by Botryosphaeriaceae species on these tropical and subtropical fruits. This study aimed to better understand the mechanisms involved in fungal penetration, host specificity and aggressiveness of Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae and Neofusicoccum parvum on avocado (Persea americana), guava (Psidium guajava) and persimmon (Diospyros kaki) fruit. Scanning electron microscopy (SEM) image analysis showed that in avocado fruit, the three studied Botryosphaeriaceae species penetrated through lenticels. In guava fruit, penetration through stomata was verified for Botryosphaeria dothidea and Neofusicoccum parvum. In persimmon fruit, an appressoria-like structure was observed for B. dothidea, which suggests direct penetration. Disease incidence in wounded fruit was 24% higher than in non-wounded fruit. L. pseudotheobromae and N. parvum showed differences in aggressiveness in guava fruit. The longest incubation period was observed for N. parvum inoculated on guava, with an average of 4.5 days, and the shortest incubation period was verified for B. dothidea inoculated on avocado, with an average of 2.8 days. The area under the disease progress curve (AUDPC) did not differ between Botryosphaeriaceae species on avocado, whereas on guava and persimmon fruit, the AUDPC was lower for B. dothidea. The information regarding penetration mechanisms and aggressiveness is important to improve postharvest disease control strategies.     

Halocyntin and papillosin,two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram‐positive and Gram‐negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.     

Chemical Composition and Activity of Essential Oils of Carissa macrocarpa (Eckl.) A.DC. Cultivated in Tunisia and Its Anatomical Features

This is the first study investigating the chemical composition of essential oils (EOs) isolated from different tissues of Carissa macrocarpa (Eckl .) A.DC., their antimicrobial activity and the anatomical characters of the aerial organs and the fruits. The main EO components were pentadecanal and tetradecan‐1‐ol (31.9 and 16.5% in fresh leaf EO, respectively), (E)‐nerolidol and caryophyllene oxide (27.3 and 15.0% in fruit EO, respectively), linalool and hexahydrofarnesyl acetone (30.9 and 24.9% in stem EO, respectively), benzyl benzoate (24.3% in flower EO). The fruit EO was more active against Candida albicans (MIC = 0.46 mg/mL) compared to the reference antibiotic (17.66 mg/mL). Furthermore, at this concentration it inhibited all the Gram‐positive bacteria. Concerning the anatomical features, it is noteworthy to mention the presence of a large cluster of calcium oxalate crystals inside some parenchymatous cells. Large ducts corresponding to non articulated laticifers were identified in the cortex of leaf, stem and fruit pericarp. The laticifers categories and their distribution are taxonomically important to discriminate this species from others acclimated in different countries. Considering the obtained results, EOs of C. macrocarpa can be a good source of antimicrobial compounds, contributing to solve the problem of microbial resistance to antibiotics.     

Volatiles in perennial ryegrass infected with strains of endophytic fungus: impact on African black beetle host selection

Perennial ryegrass (Lolium perenne) is often infected with the fungal‐endophyte Neotyphodium lolii. In addition to the ‘wild‐type’ strain (E^WT), several ‘selected’ strains of N. lolii are now being marketed as AR1 (E^AR1) and AR37 (E^AR37). Each of these strains impact positively on L. perenne's resistance against many insects, including the African black beetles (Heteronychus arator). The impact of volatile oils produced specifically by each strain in the endophyte–grass association in enhancing the grass's resistance to insects is still largely unknown. Keeping these in view, we determined the volatile oil profiles produced by L. perenne infected with either E^WT or E^AR1 or E^AR37 and determined the impacts of these volatiles on the host‐selection behaviour of H. arator adults. In the absence of endophyte infection (E^–), L. perenne produced 18 different volatile oils. In L. perenne E^WT, quantities of 2‐ethyl‐1‐hexanol acetate (Rt = 14.5 min), (Z)‐2‐octen‐1‐ol (Rt = 22.2 min), and butylated hydroxyl toluene (Rt = 23.2 min) were 24, 16 and 26%, respectively, greater than L. perenne E^–. The strains E^AR1 and E^AR37 affected differently the quantities of the volatile compounds but not their identity. In the four‐choice bioassay, males and females of H. arator were equally attracted to each strain. In Y‐tube olfactometer, compared against E^–, H. arator adults were less attracted to L. perenne E^WT and E^AR1. The attractiveness of E^AR37 was similar in effect to E^– to H. arator. The results indicate that each strain of N. lolii alters the profile of volatile oils in L. perenne differently and that alteration can influence H. arator adult‐host selection.     

Chemical Composition and Allelopathic,Antibacterial, Antifungal,and Antiacetylcholinesterase Activity of Fish‐mint (Houttuynia cordataThunb.) from India

Fish‐mint (Houttuynia cordataThunb .), belonging to family Saururaceae, has long been used as food and traditional herbal medicine. The present study was framed to assess the changes occurring in the essential‐oil composition of H. cordata during annual growth and to evaluate allelopathic, antibacterial, antifungal, and antiacetylcholinesterase activities. The essential‐oil content ranged from 0.06 – 0.14% and 0.08 – 0.16% in aerial parts and underground stem, respectively. The essential oils were analysed by GC‐FID, GC/MS, and NMR (¹H and ¹³C). Major constituents of aerial‐parts oil was 2‐undecanone (19.4 – 56.3%), myrcene (2.6 – 44.3%), ethyl decanoate (0.0 – 10.6%), ethyl dodecanoate (1.1 – 8.6%), 2‐tridecanone (0.5 – 8.3%), and decanal (1.1 – 6.9%). However, major constituents of underground‐stem oil were 2‐undecanone (29.5 – 42.3%), myrcene (14.4 – 20.8%), sabinene (6.0 – 11.1%), 2‐tridecanone (1.8 – 10.5%), β‐pinene (5.3 – 10.0%), and ethyl dodecanoate (0.8 – 7.3%). Cluster analysis revealed that essential‐oil composition varied substantially due to the plant parts and season of collection. The oils exhibited significant allelopathic (inhibition: 77.8 – 88.8%; LD₅₀: 2.45 – 3.05 μl/plate), antibacterial (MIC: 0.52 – 2.08 μl/ml; MBC: bacteriostatic) and antifungal (MIC: 2.08 – 33.33 μl/ml; MFC: 4.16 – 33.33 μl/ml) activities. The results indicate that the essential oil from H. cordata has a significant potential to allow future exploration and exploitation as a natural antimicrobial and allelopathic agent.     

Antifungal properties of durancins isolated from Enterococcus durans A5‐11 and of its synthetic fragments

The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5‐11 and of their chemically synthesized fragments. Enterococcus durans A5‐11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5‐11a and durancin A5‐11b, which have similar antimicrobial properties. The whole durancins A5‐11a and A5‐11b, as well as their N‐ and C‐terminal fragments were synthesized, and their antifungal properties were studied. C‐terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l^?1 of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra‐structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N‐terminal peptides show activities against both bacterial and fungal strains tested. C‐terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C‐terminal fragment enhances the activity of the N‐terminal fragment in the whole bacteriocins against bacteria.

Significance and Impact of the Study

Antifungal properties of durancins isolated from Enterococcus durans A5‐11 and of their chemically synthesized fragments were determined. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l^?1 of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra‐structure of the yeast cells. This work contributes to improve understanding of molecular causes of antimicrobial activities of bacteriocins and their fragments. It may be proposed that the studied peptides affect all the yeast cellular and intramembranes including cytoplasmatic reticulum and nuclear and vacuolar membranes.     

Septicemia in an Indian Rhesus Macaque (Macaca mulatta) associated with Providencia stuartii

Providencia stuartii (P. stuartii) is an opportunistic pathogen and major concern in urinary catheter‐related infections in human medicine. Here we report P. stuartii‐induced septicemia in an eighteen‐year‐old, female India‐origin Rhesus macaque with multiple traumatic wounds. The animal had neutrophilic leukocytosis, necrosuppurative meningoencephalitis, hypophysitis and bronchopneumonia with vasculitis, thrombosis, and clusters of extracellular Gram‐negative bacilli. P. stuartii was isolated from the lesions of the brain and lung and confirmed by PCR and sequencing. To the authors’ knowledge, this is the first case of septicemia associated with P. stuartii in a non‐human primate.     

Evaluation of Clove Essential Oil as a Mycobiocide Against Rhizopus stolonifer and Fusarium solani,Tuber Rot Causing Fungi in Yam (Dioscorea rotundata Poir.)

To exploit natural products for plant disease control, the essential oil of Syzygium aromaticum (L.) Merr. & Perr. (clove) was investigated for its antifungal activity against Rhizopus stolonifer and Fusarium solani, the postharvested yam tuber rot pathogens. The essential oil was obtained by hydrodistillation using a Clevenger‐type apparatus. The chemical composition of the oil was determined by gas chromatography and gas chromatography coupled with mass spectrometry. Antifungal activities of the oil were tested in vitro against mycelia growth and spores germination. In situ tests were conducted on healthy yam tubers, and necrosis symptoms were assessed. Results showed that eugenol (79.4%), eugenylacetate (9.2%) and isocaryophyllene (7.0%) were the major components. The oil exerted antifungal activities with total inhibition (TI) of the mycelia growth of R. stolonifer and F. solani was recorded at 200 and 300 ppm, respectively, while TI of spores germination was recorded at 31.25 and 250 ppm, respectively. For the standard fungicide (Ridomil^®), TI value of mycelia growth was 1600 ppm for the both pathogens, while TI of spores germination were 200 ppm and 1600 ppm, respectively, for Rhizopus and Fusarium. In situ tests showed complete inhibition of yam tuber rot when the essential oil was applied at 2000 ppm for preventive tests. This oil also reduced significantly (P ≤ 0.05) necrosis development on yam tuber for curative test at the same concentration. Total inhibition of the necrosis by Ridomil (3000 ppm) was observed only for Rhizopus on preventive test. There were positive correlations between the oil concentration and the reduction of necrosis cause by R. stolonifer and F. solani. These findings showed that clove essential oil may serve as environmental friendly bio‐fungicide for the management of postharvest yam tuber rot.     

SNPs in SNCA,MCCC1, DLG2, GBF1 and MBNL2 are associated with Parkinson's disease in southern Chinese population

Numerous single nucleotide polymorphisms (SNPs), which have been identified as susceptibility factors for Parkinson's disease (PD) as per genome‐wide association studies, have not been fully characterized for PD patients in China. This study aimed to replicate the relationship between 12 novel SNPs of 12 genes and PD risk in southern Chinese population. Twelve SNPs of 12 genes were detected in 231 PD patients and 249 controls, using the SNaPshot technique. Meta‐analysis was used to assess heterogeneity of effect sizes between this study and published data. The impact of SNPs on gene expression was investigated by analysing the SNP‐gene association in the expression quantitative trait loci (eQTL) data sets. rs8180209 of SNCA (allele model: P = .047, OR = 0.77; additive model: P = .047, OR = 0.77), rs2270968 of MCCC1 (dominant model: P = .024, OR = 1.52), rs7479949 of DLG2 (recessive model; P = .019, OR = 1.52), rs10748818 of GBF1 (additive model: P < .001, OR = 0.37), and rs4771268 of MBNL2 (recessive model: P = .003, OR = 0.48) were replicated to be significantly associated with the increased risk of PD. Noteworthy, a meta‐analysis of previous studies suggested rs8180209, rs2270968, rs7479949 and rs4771268 were in line with those of our cohort. Our study replicated five novel functional SNPs in SNCA, MCCC1, DLG2, GBF1 and MBNL2 could be associated with increased risk of PD in southern Chinese population.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.