Determining the dispersion time in Staphylococcus epidermidis biofilm using physical and molecular approaches.

Despite being an innocuous commensal of human skin and mucous membranes, Staphylococcus epidermidis, infects surgical wounds and causes infections through biofilm formation. This study evaluates, in a time-dependent experiment, the self-dispersion of S. epidermidis CIP 444 biofilm when formed on borosilicate glass (hydrophilic) and polystyrene (hydrophobic) surfaces, using physical and molecular approaches. During a seven-day period of incubation, absorbance measurement revealed a drop in biofilm optical density on both studied surfaces on day 4 (0.043-0.035 nm/cm2, polystyrene), (0.06-0.053 nm/cm2, borosilicate glass). Absorbance results were correlated with crystal violet staining that showed a clear detachment from day 4. The blue color increases again on day 7, with an increase in biofilm optical density indicating the regeneration of the biofilm. Changes in gene expression in the S. epidermidis biofilm were assessed using a real-time reverse transcription-polymerase chain reaction. High expression of agr genes was detected on days 4 and 5, confirming our supposition of dispersion in this period, autolysin genes like atlE1 and aae were upregulated from day 3 until day 6 and the genes responsible for slime production and biofilm accumulation, were upregulated on days 4, 5, and 6 (ica ADBC) and on days 5, 6 and 7 (aap), indicating a dual process taking place. These findings suggest that S. epidermidis CIP 444 biofilms disperse at day 4 and reform at day 7. Over the course of the seven-day investigation, 2-ΔΔCt results showed that some genes in the biofilm were dramatically enhanced while others were significantly decreased as compared to planktonic ones.

Staphylococcus epidermidis biofilm assembly and self-dispersion: bacteria and matrix dynamics.

Staphylococcus epidermidis, despite being a commensal of human skin and mucosa, is a major nosocomial pathogen implicated in device-associated infections. The dissemination of infection to other body sites is related to biofilm dispersal. This study focused on the dispersion stage of S. epidermidis CIP 444 biofilm, with the assessment of biofilm matrix composition in a time-dependent experiment (7 days extended) with 3 independent repetitions, using confocal laser scanning microcopy (CLSM) in association with ZEN 3.4 blue edition, COMSTAT, and ImageJ software. SYTO-9, propidium iodide (PI), DID'OIL, FITC, and calcofluor white M2R (CFW) were used to stain biofilm components. The results indicated that the biomass of dead cells increased from 15.18 ± 1.81 µm3/µm2 (day 3) to 23.15 ± 6.075 µm3/µm2 (day 4), along with a decrease in alive cells' biomass from 22.75 ± 2.968 µm3/µm2 (day 3) to 18.95 ± 5.713 µm3/µm2 (day 4). When the intensities were measured after marking the biofilm components, in a 24-h-old biofilm, polysaccharide made up the majority of the investigated components (52%), followed by protein (18.9%). Lipids make up just 11.6% of the mature biofilm. Protein makes up the largest portion (48%) of a 4-day-old biofilm, followed by polysaccharides (37.8%) and lipids (7.27%). According to our findings, S. epidermidis CIP 444 dispersion occurred on day 4 of incubation, and new establishment of the biofilm occurred on day 7. Remarkable changes in biofilm composition will pave the way for a new approach to understanding bacterial strategies inside biofilms and finding solutions to their impacts in the medical field.

Identification of beneficial Lebanese Trichoderma spp. wheat endophytes.

Wheat is one of the most important crops in the world. Its production can be influenced by a diversity of beneficial and pathogenic rhizospheric microbes, including fungi. Amongst them, beneficial Trichoderma spp. can be used as alternatives to chemical fertilizers, as they are cheap and harmless to the environment. Our study aimed to isolate, identify, and characterize Trichoderma spp. from Lebanon associated with wheat. Two Trichoderma strains belonging to T. afroharzianum, and T. guizhouense species, were isolated and found to be endophytes, enhancing root growth and producing Indole-3-acetic acid. Inoculation also improved seedling development, and increased plant growth and yield. Furthermore, the two strains inhibit Fusarium growth in vitro. These Trichoderma spp. have thus the capacity to be used as organic fertilizers for wheat.

Screening for antibacterial and antibiofilm activities in Astragalus angulosus.

AIM: In a search for finding novel therapeutic agents, extracts from an endemic Lebanese plant, Astragalus angulosus, were evaluated for their potential in-vitro antibacterial and antibiofilm activities against three Gram-positive bacterial strains; Staphylococcus epidermidis (CIP444), Staphylococcus aureus (ATCC25923), and Enterococcus faecalis (ATCC29212); in addition to two Gram-negative strains, Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853). MATERIALS AND METHODS: The plant was collected in April of 2013 and divided into several different portions, then its extracts were obtained by maceration using two different solvents. Extract analysis followed directly where microtiter broth dilution method was employed to assess antibacterial activity, while antibiofilm potential was tested using colorimetric method. RESULTS: Whole plant ethanolic extract showed the highest bacteriostatic effect at a concentration of 12.78 mg/ml and also was the most versatile exerting its effect against 3 different strains. Other extracts also exhibited an effect but at higher concentrations and each against a single strain. Regarding antibiofilm activity, the majority of the extracts were able to eradicate >50% of S. epidermidis preformed biofilm, where the highest activity was obtained with flower fraction extracted in water, achieving 67.7% biofilm eradication at 0.2 mg/ml. CONCLUSIONS: This plant possesses a promising potential in regard to eradicating bacteria and their biofilms and it is the first contributing step of establishing a library for the endemic Lebanese plants in this domain.

Consensus mapping of major resistance genes and independent QTL for quantitative resistance to sunflower downy mildew.

Major gene resistance to sunflower downy mildew (Plasmopara halstedii) races 304 and 314 was found to segregate independently from the resistance to races 334, 307 and 304 determined by the gene Pl2, already positioned on Linkage Group (LG) 8 of sunflower molecular maps. Using a consensus SSR-SNP map constructed from the INEDI RIL population and a new RIL population FU × PAZ2, the positions of Pl2 and Pl5 were confirmed and the new gene, denoted Pl21, was mapped on LG13, at 8 cM from Pl5. The two RIL populations were observed for their quantitative resistance to downy mildew in the field and both indicated the existence of a QTL on LG8 at 20-40 cM from the major resistance gene cluster. In addition, for the INEDI population, a strong QTL on LG10, reported previously, was confirmed and a third QTL was mapped on LG7. A growth chamber test methodology, significantly correlated with field results, also revealed the major QTL on LG10, explaining 65 % of variability. This QTL mapped in the same area as a gene involved in stomatal opening and root growth, which may be suggested as a possible candidate to explain the control of this character. These results indicate that it should be possible to combine major genes and other resistance mechanisms, a strategy that could help to improve durability of sunflower resistance to downy mildew.

Transcriptomic analysis of the interaction between Helianthus annuus and its obligate parasite Plasmopara halstedii shows single nucleotide polymorphisms in CRN sequences.

BACKGROUND: Downy mildew in sunflowers (Helianthus annuus L.) is caused by the oomycete Plasmopara halstedii (Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between H. annuus and P. halstedii have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of P. halstedii. RESULTS: A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of P. halstedii, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using in silico filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms. CONCLUSIONS: This study identified a large number of genes that are expressed during H. annuus/P. halstedii compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in P. halstedii similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of P. halstedii. This work therefore provides valuable tools for further discoveries regarding the H. annuus/P. halstedii pathosystem.