Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Assessment of biological activities of chitosan Schiff base tagged with medicinal plants

A chitosan Schiff base with an aromatic aldehyde was synthesized and characterized by FTIR and NMR spectroscopies. Furthermore, the degree of substitution was calculated based on the ratios of the area of the proton of the imine (A_imine) and the area of the peak of the proton of the pyranose ring (A_H-2). The antimicrobial activities were determined against bacterial and fungal strains, as well as multiple drug-resistant (MDR) bacteria. The chitosan Schiff base was also tagged with medicinal plants, for example, Curcuma longa, Peganum harmala, Lepidium sativam, and cruciferous vegetables, and the biological activities determined against pathogenic bacterial and fungal strains. The chitosan Schiff base showed maximum zone of inhibition of 22 mm against Staphylococcus aureus with a minimum zone of inhibition of 15 mm against Bacillus cereus. The chitosan Schiff base was fused with C longa, isothiocyanates and a combined mixture of P harmala and L sativam that has shown activities against Escherichia coli with a zone of inhibition of 28, 24, and 30 mm, respectively. The Schiff base of chitosan fused with medicinal plants also showed significant inhibitory activities against MDR bacteria.     

MODIFICATION OF LYSOZYME WITH OLEOYL CHLORIDE FOR BROADENING THE ANTIMICROBIAL SPECIFICITY

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.     

Synthesis and in vitro microbiological evaluation of novel diethyl 6,6′-(1,4-phenylene)bis(4-aryl-2-oxo-cyclohex-3-enecarboxylates)

Novel bis cyclohexenone ester derivatives 14–19 were synthesized and characterized by their spectral data. In vitro microbiological evaluations were carried out for all the novel compounds 14–19 against clinically isolated bacterial and fungal strains. Compounds 15, 16, 18 against Staphylococcus aureus, 14, 15 against β-Haemolytic streptococcus, 15, 19 against Micrococcus luteus, 17, 18 against Salmonella typhii, 14, 17 against Shigella flexneri, 15 against Escherichia coli, 16 against Pseudomonas aeruginosa, 15, 18, 19 against Klebsiella pneumonia exhibited potent antibacterial activity at an minimum inhibitory concentration (MIC) value of 6.25 μg/ml, whereas compound 16 against Aspergillus flavus, 17 against A. niger, 16, 18 against Mucor indicus, 15, 17–19 against Microsporum gypseum revealed excellent antifungal activity at an MIC value of 6.25 μg/ml.     

RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

Insecticidal activity of Origanum majorana L. essential oil as anti‐cholinergic agent

The present study was focused on exploring the presence of active compounds in Origanum majorana essential oil (OmEO), and its various knock‐down effects against the rice moth, Corcyra cephalonica. GC–MS analysis detected the existence of major compounds such as monoterpenes, cis‐β‐terpineol and terpinen‐4‐ol with the total proportion of 52.16%. Fumigant toxicity against adult and larvae was calculated with an LC₅₀ value of 11.31 and 49.83 μL/L air, respectively. The contact toxicity against adult, pupa, larvae and eggs was observed with LC₅₀ value 2.54, 0.95, 2.78, and 0.49 μL/L, respectively. Furthermore, the influential repellent behavior against adults has been observed. Acetylesterase (AChE) inhibition activity of OmEO was observed against adult and larvae of C. cephalonica with an IC₅₀ value of 35.89 and 118.54 μL/mL, respectively. Moreover, computational docking study revealed the binding affinity of Cis‐β‐terpineol and terpinen‐4‐ol towards the active binding sites of AChE. On the other hand, Fluorescence‐assisted cytometry and comet assay confirmed the cytotoxic and genotoxic effect of OmEO at various concentrations on C. cephalonica. Altogether, the results showed the knock‐down effect of OmEO against C. cephalonica, and it could be a potential biocontrol measure against the stored product pest.     

Absence of suppressible alleles at the his 1 locus of yeast

Summary No suppressible alleles have been found at the his1 locus of Saccharomyces cerevisiae. Seventeen noncomplementing alleles have been tested against the strong ochre suppressor SUP2. These alleles, plus an additional 34 complementing alleles, were previously tested against a weaker suppressor SUP11. These results, which are in marked contrast to experience with other yeast loci where suppressible alleles are frequent, may be explained if the his1 product is transcribed from a polycistronic message so that nonsense mutations lead to loss of a second nonsupplementable function, or if the his1 protein serves as an essential subunit for an unknown enzyme complex.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Anticandidal properties of N-acylpeptides containing an inhibitor of glucosamine-6-phosphate synthase

A series of N-acyl peptides 1–9, containing an inhibitor of glucosamine-6-phosphate synthase have been synthesised and tested against Candida strains. N-Acylated peptides inhibit glucosamine-6-phosphate synthase in cell free extracts from Candida albicans. Antifungal activities of the tested compounds correlated with their lipophilic properties. Peptides acylated with decanoic acid were found to be the most potent in the series. N-decanoylpeptides also showed activity against Candida albicans Gu5 resistant mutant with Cdr1 and Cdr2 drug extrusion proteins that causes MDR by an active efflux mechanism.     

Antimicrobial bioassay of colchicum luteum baker

The methanolic extract of the corms of Colchicum luteum Baker (Liliaceae) and its subsequent fractions in different solvent systems were screened for antibacterial and antifungal activities. The crude extract and all the fractions demonstrated moderate to excellent antifungal activities against tested pathogens in antifungal bioassay. Excellent antifungal activity was shown against trichophyton longifusus, up to 75%, and microsporum canis, up to 85%, while the crude extract and subsequent fractions showed mild to moderate activities in an antibacterial bioassay with maximum antibacterial activity 58% against Bacillus subtilis.     

New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit micromolar EC₅₀ values and against T. gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Biological Control of Postharvest Diseases of Peaches and Nectarines by Yeasts

Abstract Biocontrol activities of a total of 103 yeast isolates were tested against postharvest diseases of peaches. Seven isolates, with the best efficacy in reducing the number of infected wounds or lesion sizes of Penicillium expansum and Botrytis cinerea, were selected for further large‐scale experiments. In large‐scale experiments, all selected isolates were significantly effective (P ≤ 0.05) in reducing the number of infected wounds and the diameter of lesions caused by P. expansum, B. cinerea, and Monilinia fructicola. DR52 was significantly superior to all the other yeasts in effectiveness against all three pathogens. The efficacy of the other yeast antagonists against B. cinerea and P. expansum was almost equal, while the control of M. fructicola was inferior. DR52 was selected for further storage experiment because its efficacy was higher against the three pathogens and it also showed a different random amplified polymorphic DNA‐polymerase chain reaction pattern compared with other isolates. DR52 was identified by Centraalbureau voor Schimmeelcultures (Baarn, The Netherlands) as Kloeckera apiculata. K. apiculata completely controlled both pathogens after 30 days of storage. Its efficacy declined to an 83.4% reduction in B. cinerea incidence and an 87.5% reduction in P. expansum incidence after 45 days of storage.     

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis

Abstract

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024?μg?ml^?1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.     

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes–Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI–BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI–BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.     

Synthesis of oxalamide and triazine derivatives as a novel class of hybrid 4-aminoquinoline with potent antiplasmodial activity

Frequency of malaria and its resistance to chemotherapeutic options are emerging rapidly. To counter this problem, a series of 4-aminoquinolines having oxalamide and triazine functionalities in the side chain were synthesized and screened for their antimalarial activities. Triazine derivative 48 found to be the most active against CQ sensitive strain 3D7 of Plasmodium falciparum in an in vitro assay with an IC₅₀ of 5.23 ng/mL and oxalamide derivative 13 showed an in vivo suppression of 70.45% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.     

Antibacterial activity of the leaf essential oil of <Emphasis Type="Italic">Blumea mollis</Emphasis> (D. Don) Merr.

The antibacterial activity of the leaf essential oil of Blumea mollis was assayed against 14 clinically isolated bacterial strains on Muller–Hinton Agar medium and Muller–Hinton Agar medium with 5% sheep blood. The essential oil had promising antibacterial activity against all the bacterial strains tested. The highest mean zone of inhibition and lowest values of minimum inhibitory concentration were recorded against methicillin-resistant Staphylococcus aureus followed by beta hemolytic Streptococcus pyogenes. The Gram-positive bacteria were more sensitive than Gram-negative bacteria. Among the bacterial strains tested, Psudomonas aeruginosa was resistant to the essential oil. The results of the present study suggest that the essential oil of B. mollis is one of the new medicinal resources as an antibacterial agent against the bacterial strains tested.     

Evolutionary costs and benefits of infection with diverse strains of Spiroplasma in pea aphids*

The heritable endosymbiont Spiroplasma infects many insects and has repeatedly evolved the ability to protect its hosts against different parasites. Defenses do not come for free to the host, and theory predicts that more costly symbionts need to provide stronger benefits to persist in host populations. We investigated the costs and benefits of Spiroplasma infections in pea aphids (Acyrthosiphon pisum), testing 12 bacterial strains from three different clades. Virtually all strains decreased aphid lifespan and reproduction, but only two had a (weak) protective effect against the parasitoid Aphidius ervi, an important natural enemy of pea aphids. Spiroplasma‐induced fitness costs were variable, with strains from the most slowly evolving clade reaching higher titers and curtailing aphid lifespan more strongly than other strains. Some Spiroplasma strains shared their host with a second endosymbiont, Regiella insecticola. Although the result of an unfortunate handling error, these co‐infections proved instructive, because they showed that the cost of infection with Spiroplasma may be attenuated in the presence of Regiella. These results suggest that mechanisms other than protection against A. ervi maintain pea aphid infections with diverse strains of Spiroplasma, and that studying them in isolation will not provide a complete picture of their effects on host fitness.     

Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea

Detached leaf disc bioassays were conducted against cucumber powdery mildew and three species of aphid with three entomopathogenic species of Lecanicillium; Lecanicillium longisporum (Vertalec^®), Lecanicillium attenuatum (CS625), and an unidentified isolate (DAOM198499). The three Lecanicillium species had high virulence against the aphids Myzus persicae, Macrosiphum euphorbiae and Aulacorthum solani with the exception of DAOM 198499, which demonstrated reduced virulence to A. solani with an LT₅₀ of 6.4 days. Otherwise, LT₅₀ ranged between two and four days. Suspensions of conidia and blastospores of the Lecanicillium species were also applied onto 15 mm leaf discs dissected from cucumber plants previously inoculated with Sphaerotheca fuliginea. Powdery mildew did not develop when the Lecanicillium applications were made one and eight days after S. fuliginea inoculations. When Lecanicillium was applied to highly infected leaf discs 11 and 15 days after S. fuliginea inoculation, the application suppressed subsequent production of S. fuliginea spores as compared to the controls. These results suggest the potential of a dual role for Lecanicillium spp. as microbial control agents against aphids and powdery mildew.