Antiadhesive Properties of Imidazolium Ionic Liquids Based on (−)-Menthol against Candida spp.

Infections with Candida spp. are commonly found in long-time denture wearers, and when under immunosuppression can lead to stomatitis. Imidazolium ionic liquids with an alkyl or alkyloxymethyl chain and a natural (1R,2S,5R)-(−)-menthol substituent possess high antifungal and antiadhesive properties towards C. albicans, C. parapsilosis, C. glabrata and C. krusei. We tested three compounds and found they disturbed fungal plasma membranes, with no significant hemolytic properties. In the smallest hemolytic concentrations, all compounds inhibited C. albicans biofilm formation on acrylic, and partially on porcelain and alloy dentures. Biofilm eradication may result from hyphae inhibition (for alkyl derivatives) or cell wall lysis and reduction of adhesins level (for alkyloxymethyl derivative). Thus, we propose the compounds presented herein as potential anti-fungal denture cleaners or denture fixatives, especially due to their low toxicity towards mammalian erythrocytes after short-term exposure.

Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity

O3 dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O3 was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O3 (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O3 remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.

Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

Characterization of a novel endopeptidase Cwl0971 that hydrolyzes peptidoglycan of Clostridioides difficile involved in pleiotropic cellular processes

SummaryClostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from intracell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (endopeptidase, CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary / late phase of cell growth. Moreover, biofilm formation, germination, and sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the depletion of Cwl097 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which can affect several phenotypes of R20291. Our data indicate that the endopeptidase Cwl0971 is an attractive target for C. difficile infection therapeutics and prophylactics.

THE EVIDENCE OF NON N-GLYCAN LINKED MANNOSE IN EXOCHITINASE (42 kDa) FROM Trichoderma harzianum BIO10671 GLYCOSYLATION

Chitinase 42 kDa produced by Trichoderma harzianum has been proven as a prime compound to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction. This finally initiate the process of the host cell becomes empty of cytoplasm, disintegrates and shows a rapid collapse. This study investigates the existence of N-glycan linked mannose in chitinase 42 kDa produced by the Malaysian T. harzianum strain BIO10671. The chitinase 42 kDa from T. harzianum BIO10671 was initially purified using anion exchange chromatography prior to a series of experiments such as immunoblotting against the chitinase 42 kDa antibody, lectin staining for detecting any terminal linked mannose, and galactofuranose detection to determine the presence of galatofuranose components in glycoproteins. The enzyme purification harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671 with strong indication of the presence chitinase 42 kDa presence on SDS-Page. This was confirmed by immunoblotting with a strong response around 42 kDa after overnight incubation in chitinase 42 kDa antibody suggesting that the gene for chitinase 42 kDa was greatly expressed in this strain. There are no intervation of galatofuranose on any of the terminal mannose in chitinase 42 kDa as shown by negative results on samples treated with or without endoglycosidase-H and lectin staining. Therefore, it can be concludeed that glycosylation occurred in the chitinase 42 kDa from T. harzianum 42 kDa was not in the form of N-glycan linked mannose as expected.

Kemampuan Kitinase Streptomyces RKt5 sebagai Antijamur terhadap Patogen Fusarium oxysporum

The purpose of the reasearch is to determine of antifungal activity from chitinase from Streptomyces RKt5 to inhibite growth of Fusariumoxysporum. The chitinase of Streptomyces RKt5 produced in liquid chitin medium with optimum conditions (inoculum concentration, pHand incubation time) and then partially purified with ammonium sulphate. The enzyme products were tested the antifungal activity againstF.oxysporum. The results showed that mycelial growth of F.oxysporum can be inhibited by Streptomyces RKt 5 in dual culture test. Thepartial purified chitinase enzyme couldn’t inhibit the fungal growth. But if the mycellium fragmented, the enzyme could degrade the fungalcell wall in incubation time. The frequency of fungal cell wall lysis and levels of N-acetylglucosamine released that have been increasingalong with the length of incubation time.

Daptomycin Produces an Enhanced Bactericidal Activity Compared to Ceftriaxone, Measured by [3H]Choline Release in the Cerebrospinal Fluid, in Experimental Meningitis Due to a Penicillin-Resistant Pneumococcal Strain without Lysing Its Cell Wall

ABSTRACT Daptomycin monotherapy was superior to ceftriaxone monotherapy and was highly efficacious in experimental pneumococcal meningitis, sterilizing the cerebrospinal fluid (CSF) of three of three rabbits after 4 to 6 h. With daptomycin therapy only a negligible release of [3H]choline as marker of cell wall lysis was detectable in the CSF, peaking around 250 cpm/min after 4 h, compared to a peak of around 2,400 cpm/min after 4 to 6 h for the ceftriaxone-treated rabbits.

Methanobrevibacter millerae sp. nov. and Methanobrevibacter olleyae sp. nov., methanogens from the ovine and bovine rumen that can utilize formate for growth

Four formate-utilizing methanogens were isolated from ovine (strain KM1H5-1PT) and bovine (strains AK-87, OCP and ZA-10T) rumen contents. Based on 16S rRNA gene sequence analysis, the methanogen strains were found to belong to the order Methanobacteriales in the genus Methanobrevibacter. Strains ZA-10T and KM1H5-1PT gained energy for growth by the reduction of CO2 to CH4 using H2 or formate exclusively as electron donors. Increasing formate concentrations to 220 mM in batch cultures increased the growth of strain KM1H5-1PT but did not affect the growth of strain ZA-10T. Substrate specificity and resistance to cell-wall lysis supported the affiliation of the strains to the genus Methanobrevibacter. Strains ZA-10T and KM1H5-1PT showed 16S rRNA gene sequence similarity of 98.0 and 98.6 % to their closest recognized relatives, Methanobrevibacter thaueri CWT and Methanobrevibacter ruminantium M1T, respectively. DNA–DNA hybridization experiments indicated that the strains were not affiliated at the species level to their closest recognized relatives, with DNA reassociation values of only 28 % between strains ZA-10T and Methanobrevibacter thaueri CWT and <25 % between strains KM1H5-1PT and Methanobrevibacter ruminantium M1T. Based on the data presented, the new strains are considered to represent two novel species of the genus Methanobrevibacter, for which the names Methanobrevibacter millerae sp. nov. (type strain ZA-10T=DSM 16643T=OCM 820T) and Methanobrevibacter olleyae sp. nov. (type strain KM1H5-1PT=DSM 16632T=OCM 841T) are proposed.

An endophytic chitinase-producing isolate of Actinoplanes missouriensis, with potential for biological control of root rot of lupin caused by Plectosporium tabacinum

Twenty-one streptomycete and 15 non-streptomycete actinomycetes were isolated from surface-disinfested lupin roots and evaluated for their potential to produce chitinase and to inhibit the growth of Plectosporium tabacinum, the causal agent of lupin root rot in Egypt. The most inhibitory isolate was identified as Actinoplanes missouriensis which produced relatively high levels of chitinase and degraded the hyphae of P.�tabacinum in vitro, causing extensive plasmolysis and cell-wall lysis. A crude culture filtrate of A. missouriensis exhibited antifungal activity and significantly (P < 0.05) reduced spore germination and germ-tube growth of the pathogen. The antagonist was recovered from inside the root at all samplings up to 8 weeks after inoculation, indicating that the roots of healthy lupin may be a habitat for the endophyte. A. missouriensis significantly (P < 0.05) reduced the severity of root rot under glasshouse conditions. An endophytic isolate of Actinoplanes italicus incapable of producing chitinase and a mutant strain of A. missouriensis that did not produce detectable levels of chitinase, did not lyse hyphae of P. tabacinum or reduce root rot in the glasshouse experiments, although colonisation of the lupin root by both these isolates was similar to that of the chitinase-producing wild-type isolate of A. missouriensis. This study is the first record of control of a soil-borne plant pathogen by a chitinolytic actinomycete, endophytic in plant roots.