Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Prolonged inhibitory effects against planktonic growth,adherence, and biofilm formation of pathogens causing ventilator-associated pneumonia using a novel polyamide/silver nanoparticle composite-coated endotracheal tube

Abstract

Microbial cells can rapidly form biofilm on endotracheal tubes (ETT) causing ventilator-associated pneumonia, a serious complication in patients receiving mechanical ventilation. A novel polyamide with a good balance of hydrophilic/hydrophobic moieties was used for the embedment of green-reduction silver nanoparticles (AgNPs) for the composite-coated ETT. The films were conformal with a thickness of ～ 17?±?3?µm accommodating high loading of 60?±?35?nm spherical-shaped AgNPs. The coated ETT resulted in a significant difference in reducing both planktonic growth and microbial adhesion of single and mixed-species cultures, compared with uncoated ETT (p?<?0.05). A time-kill assay demonstrated rapid bactericidal effects of the coating on bacterial growth and cell adhesion to ETT surface. Biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus, commonly encountered pathogens, was inhibited by > 96% after incubation for 72?h. Polyamide/AgNP composite-coated ETT provided a broad-spectrum activity against both Gram-positive and Gram-negative bacteria as well as Candida albicans and prolonged antimicrobial activity.     

Using the F/R-ratio for an evaluation of the ability of the demosponge Halichondria panicea to nourish solely on phytoplankton versus free-living bacteria in the sea

The F/R-ratio (litres of water filtered per ml of oxygen respired) was determined for the filter-feeding demosponge Halichondria panicea to be 15.5?l?H₂O?(ml O₂)^?1 which was used to evaluate the potential of the sponge to nourish solely on nano- (2–20?µm) and micro- (20–200?µm) phytoplankton cells in the sea. It was estimated that in order to balance the maintenance requirement of H. panicea the necessary content of suspended particulate organic carbon must be at least 0.03?mg?C?l^?1, which may be compared with actually reported values of 0.04 to 0.2?mg?C?l^?1 thus implying that H. panicea may be able to nourish on a sole diet of phytoplankton in nature. However, the amount of carbon represented by free-living heterotrophic bacteria, cyanobacteria and other small (0.2–2?µm) picoplankton which are also accessible to the sponge lies in the range of 0.05–0.10?mg?C?l^?1, and therefore bacteria seem to be an important, although in many cases apparently a somewhat insufficient food source relative to phytoplankton. Video-microscope observations of the osculum cross-sectional area (OSA) and simultaneous measurement of the filtration rate of H. panicea showed that the filtration rate varied considerably over time concurrently with often pronounced variations in the OSA caused by disturbance when the aquarium through-flow was stopped during filtration rate measurements in the laboratory. It is concluded that the optimal and undisturbed filtration rate may be considerably higher than measured here, i.e. 6.1?ml water (ml sponge)^?1 min^?1, thus increasing the F/R-ratio to > 15.5?l?H₂O (ml O₂)^?1, which is comparable to values for more advanced eumetazoan filter-feeding marine invertebrates grazing on phytoplankton.     

Improving operational stability of thermostable Pythium myriotylum secretory serine protease by preparation of cross-linked enzyme aggregates (CLEAs)

Abstract

Present study was undertaken to develop cross-linked enzyme aggregate (CLEA)of alkaline serine proteases (sp) from Pythium myriotylum (Pm), a necrotrophic oomycete reported to considerably secrete serine proteases. Among various precipitants screened for spPm1-CLEA preparation, ammonium sulfate at 80% saturation (w/v) yielded 100% activity recovery and retention of spherical morphology as observed by SEM analysis. Addition of glutaraldehyde as cross-linker at 1% (v/v) concentration with optimized ammonium sulfate concentration for 1?hour at 100?rpm yielded 100% activity recovery of spPm1-CLEA from 8-day old P. myriotylum culture filtrate. Addition of BSA (10?mg/ml) to CLEA cross-linking reaction mix reduced CLEA size from the range of 1.82–1.19?µm to 394–647?nm. spPm1-CLEA preparations retained 100% activity at temperature of 80?°C and pH 12.0 signifying their potential commercial applications. In terms of kinetic parameters, present process enhanced kinetic parameters as revealed by 1.67?U.mg^?1 specific activity, K_m of 0.062?mM and V_max of 0.145?µmol.min^?1.mg^?1 for the spPm1-CLEA compared to 0.288?U.mg^?1 specific activity, K_m of 0.060?mM and V_max of 0.20?µmol.min^?1.mg^?1 determined for the free spPm1 enzyme. Study has successfully demonstrated the concept of CLEA in enhancing spPm1 stability and the results so generated can be translated in future towards development of robust biocatalysts.     

Morphological analysis of pore size and connectivity in a thick mixed‐culture biofilm

Morphological parameters are commonly used to predict transport and metabolic kinetics in biofilms. Yet, quantification of biofilm morphology remains challenging because of imaging technology limitations and lack of robust analytical approaches. We present a novel set of imaging and image analysis techniques to estimate internal porosity, pore size distributions, and pore network connectivity to a depth of 1 mm at a resolution of 10 µm in a biofilm exhibiting both heterotrophic and nitrifying activities. Optical coherence tomography (OCT) scans revealed an extensive pore network with diameters as large as 110 µm directly connected to the biofilm surface and surrounding fluid. Thin‐section fluorescence in situ hybridization microscopy revealed that ammonia‐oxidizing bacteria (AOB) distributed through the entire thickness of the biofilm. AOB were particularly concentrated in the biofilm around internal pores. Areal porosity values estimated from OCT scans were consistently lower than those estimated from multiphoton laser scanning microscopy, though the two imaging modalities showed a statistically significant correlation (r = 0.49, p < 0.0001). Estimates of areal porosity were moderately sensitive to gray‐level threshold selection, though several automated thresholding algorithms yielded similar values to those obtained by manually thresholding performed by a panel of environmental engineering researchers (±25% relative error). These findings advance our ability to quantitatively describe the geometry of biofilm internal pore networks at length scales relevant to engineered biofilm reactors and suggest that internal pore structures provide crucial habitat for nitrifier growth.     

Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacteria isolated from saline desert

Indole-3-acetic acid (IAA)-producing bacteria Kocuria turfanensis strain 2M4 was isolated from the rhizospheric soil of halotolerant plant Suaeda fruticosa from a unique saline desert of Little Rann of Kutch, Gujarat, India. Rhizobacteria was bright orange pigmented, gram-positive, coccoid, non-endospore forming, and aerobic in nature. 16S rRNA gene sequence analysis showed that 2M4 isolate matched best with type strain of K. turfanensis HO-9042^T. Isolate optimally produced 38 µg ml^?1 IAA when growth medium was supplemented with 600 µg ml^?1 of L-tryptophan. Thin layer chromatography and Fourier transform infrared spectroscopy analysis were performed to corroborate IAA production. To characterize rhizobacterial isolate as a plant growth-promoting bacteria, it was tested for phosphate solubilization where it solubilized maximum 12 µg ml^?1 phosphate in presence of fructose, produced 53% siderophore units under iron-free minimal MM9 medium and produced 1.8 µmol ml^?1 ammonia in peptone water broth. Plant growth promotion by test isolate was studied on groundnut (Arachis hypogaea L.) under non-saline and saline soil. There was increase by 18% in total plant length and 30% in fresh biomass observed under non-saline control soil. Under saline soil, test isolate showed 17% increase in total length of the plant and 13% increase in fresh biomass.     

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor was investigated over a 10-month period in a hotel. Planktonic and sessile numbers of sulphate reducing bacteria (SRB) and heterotrophic bacteria were monitored. The corrosion rate was determined by the weight loss method. The corrosion products were analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction. A mineralized, heterogeneous biofilm was observed on the coupons. Although a biocide and a corrosion inhibitor were regularly added to the cooling water, the results showed that microorganisms, such as SRB in the mixed species biofilm, caused corrosion of galvanized steel. It was observed that Zn layers on the test coupons were completely depleted after 3?months. The Fe concentrations in the biofilm showed significant correlations with the weight loss and carbohydrate concentration (respectively, p?<?0.01 and p?<?0.01).     

Optimization of indole acetic acid production by isolated bacteria from Stevia rebaudiana rhizosphere and its effects on plant growth

The ability to synthesize Indole-3-acetic acid (IAA) is widely associated with the plant growth promoting rhizobacteria (PGPR). The present work deals with isolation and characterization of such bacteria from the rhizosphere of medicinal plant Stevia rebaudiana and optimization of IAA production from its isolates. The optimization of IAA production was carried out at different pH and temperature with varied carbon and nitrogen sources of culture media. Out of different isolates obtained, three of them were screened as efficient PGPRs on the basis of different plant growth promoting attributes. Isolates CA1001 and CA2004 showed better production of IAA at pH 9 (91.7?µg?ml^?1) and at temperature 37?°C (81.7?µg?ml^?1). Dextrose (1%) was found to be the best carbon source for isolate CA1001 with 104?µg?ml^?1 IAA production. Isolate CA 2004 showed best production of IAA 36?µg?ml^?1 and 34?µg?ml^?1 at 1.5% and 1% Beef extract as nitrogen source respectively. Isolate CA 1001 showed 32?µg?ml^?1 IAA production at 0.5% nicotinic acid concentration. From the current study, CA1001 and CA2004 emerged as noble alternatives for IAA production further which also resulted in root and shoot biomass generation in crop plants, hence can be further used as bio-inoculants for plant growth promotion.     

Evaluating the Adsorption Potential of Alachlor and Its Subsequent Removal from Soils via Activated Carbon

Alachlor, a globally used aniline herbicide, has great agronomic interest for controlling the development of broadleaved weeds and grasses. This research aspires to evaluate the sorption attributes of Alachlor through batch equilibrium method and its successive removal through biomass based activated carbon prepared from Sawdust (Cedrus deodara). Six soil samples were collected from selected regions of Pakistan to assess the adsorption and removal phenomena. Adsorption capacity for Alachlor varied in soils depending upon their physicochemical properties. Adsorption coefficient (K_d) values ranged from 12 to 31 µg ml^?1 with the highest K_d value observed in soil sample with highest organic content (1.4%) and least pH (5.62). The Gibbs free energy values ranged from ?17 to ?20 kJ mol^?1 proposing physio-sorption and exothermic interaction with soils. Values of R² (0.96–0.99) exhibited the best fit to linear adsorption model. Adsorption coefficient displayed a negative correlation (r = ?0.97) with soil pH and positive correlation with organic matter (r = 0.87). The effect of contact time and pesticide concentration on the removal efficiency by activated carbon was investigated. The highest removal percentages observed through activated carbon were 66% and 64% at concentrations of 5 and 7.5 ppm respectively. Activated carbon from sawdust (Cedrus deodara) was investigated as a suitable adsorbent for the removal of Alachor from selected soils. Biomass based activated carbon can prove to be an effective and a sustainable mean to remove pesticides from soil.     

Determination of S-phenylmercapturic acid by GC-MS and ELISA: a comparison of the two methods

Abstract

S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1?ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection?=?0.1?µg?l^?1), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5?µg?l^?1, GC-MS <5?µg?l^?1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.     

Adaptations of tropical marine microphytobenthic assemblages along a gradient of light and nutrient availability in Suva Lagoon,Fiji

How are microphytobenthic biofilms adapted to the high incident irradiances and temperatures, low inorganic nutrient concentrations and high desiccation stresses on intertidal flats present in tropical environments? This study investigated biofilms subject to different environmental conditions in a range of tropical sites in Suva lagoon, Fiji. PAM fluorescence was used to measure photophysiological responses to the light climate. Biofilm colloidal carbohydrate, extracellular polymeric substances (EPS) and low molecular weight (MW) carbohydrate concentrations and diel carbohydrate production patterns were measured. Average biomass (Chl a) ranged from 15 to 36?mg?m^?2, and was highest in seagrass bed sediments, but biomass was not correlated with water column or sediment porewater nutrient concentrations. Biofilm photophysiology differed significantly along a combined gradient of light and nutrient availability, with F _v/F _m, relative ETR_max and E _k of biofilms highest in mangrove and intertidal main island sites and lowest in subtidal coral reef flats. Subtidal biofilms showed photoinhibition at irradiances > 1000?µmol?m^?2. Significant correlations between Chl a and colloidal carbohydrate concentrations were present (except on intertidal sandflats), and tropical biofilms had higher ratios of colloidal carbohydrate and EPS to Chl a than temperate estuarine biofilms, probably due to a combination of high irradiance and low nutrient availability leading to the production of excess photoassimilates. The percentage of EPS present in the colloidal fraction was highest in coral sand biofilms (42%), which had the lowest nutrient concentrations, compared with other sites (25–32%). Intertidal biofilms predominantly consisted of large motile taxa and showed strong rhythms of vertical migration. During tidal emersion, high sediment temperatures (41?°C), irradiance (>2300?µmol?m^?2?s^?1) and salinity (49‰) stimulated downward migration. In silty sediments, migration resulted in a reduction in photosynthetic activity during the midday period but, in sands with high light penetration (to a depth of > 1700?µm), high production rates of EPS (18.2?µg carbo. µg Chl a^?1 h^?1) and low MW carbohydrate exudates (40.2?µg carbo. µg Chl a^?1 h^?1) occurred. Vertical migration, high E _k and high rates of photoassimilate dumping are all adaptations to living in the tropical intertidal zone. Seagrass and reef flat biofilms consisted of a diverse non-migratory flora of motile and non-motile taxa that were not subject to such extreme temperature and irradiance conditions. Low values of photosynthetic parameters and high colloidal and EPS content indicated that these biofilms were nutrient-limited.     

Growth and biochemical profiling of artificially associated micropropagated oil palm plantlets with Herbaspirillum seropedicae

The challenges of various biotic and abiotic stresses can imperil the growth of micropropagated plantlets either direct or indirectly. Hence, in this study, a mutual relationship was established between diazotrophs and micropropagated plantlets to enhance plant growth and development. Artificial symbiosis was created for different inoculums of Herbaspirillum seropedicae (Z78), namely sonicated cells, broth culture, and pellet cells with micropropagated oil palm plantlets Elaeis guineensis Jacq. Results reveal significant differences on root volume, total protein content, and Brix value for Z78 broth culture treatment compared with plantlets treated with 25% N. High nitrogenase enzyme activities (6.7?×?10^?4?µmol?C₂H₄ g^?1?h^?1) and indole-3-acetic acid production (205.21?µmol (g?FW)^?1) were also detected on roots of plantlets treated with Z78 broth culture. These beneficial traits reviewed that the application of diazotrophs (Z78) in associative manner for micropropagated plantlets hold vast potential for promoting plant growth and plant’s healthiness.     

Temporal variability of algicidal and growth-inhibiting bacteria at an eelgrass bed in the Ariake Sea,Japan

Abstract

Temporal changes of algicidal and growth-inhibiting bacteria on the fish-killing raphidophyte flagellate, Chattonella antiqua, at an eelgrass (Zostera marina) bed in southern Ariake Sea, Japan in 2011 was investigated. The maximum value (5.1?×?10⁷ CFU g^?1 wet leaf) of algicidal bacteria (AB) was detected from a biofilm formed on Z. marina on August 1 when AB in the adjacent seawater had also peaked (1.2?×?10⁴ CFU mL^?1). Two causative bacteria isolated from the biofilm and seawater on August 1 were both identified to be of the genus Alteromonas (γ-proteobacteria). AB and growth-inhibiting bacteria (GIB) were present from the beginning of sampling (May 20) to August 26, fluctuating between 8.6?×?10² and 1.2?×?10⁴, 1.2?×?10³ and 9.3?×?10³ CFU mL^?1, respectively. The highest phytoplankton density observed was 6423 cells mL^?1 on September 29 and was comprised of centric diatoms such as Chaetoceros, Skeletonema, and Thalassiosira and coincided with the absence of AB and GIB where the decline of Z. marina was also observed. These findings provide a new ecological insight on AB and GIB associated with Z. marina beds, indicating eelgrass beds have the important role as the nursery of those bacteria that can be utilized as mitigation measures of harmful algal blooms (HABs) in the future.     

Induction of non-protein thiols and phytochelatins by cadmium in Eichhornia crassipes

Abstract

Impact of root Cd concentration on production of cysteine, non-protein thiols (NP-SH), glutathione (GSH), reduced glutathione (GSSG), and phytochelatins (PCs) in Eichhornia crassipes exposed to different dilutions of brass and electroplating industry effluent (25%, 50%, and 75%), and synthetic metal solutions of Cd alone (1, 2.5, and 3.5?ppm) and with Cr (1?ppm Cd + 1?ppm Cr, 2.5?ppm Cd + 3?ppm Cr, and 3.5?ppm Cd + 4?ppm Cr) was assessed in a 45?days study. Different treatments were used to understand and compare differential antioxidant defense response of plant under practical drainage (effluent) and experimental synthetic solutions. The production of NP-SH and cysteine was maximum under 2.5?ppm Cd + 3?ppm Cr treatments i.e., 1.78?µmol/g fw and 288?nmol/g fw, respectively. The content of GSH declined whereas that of GSSG increased progressively with exposure duration in all treatments. HPLC chromatograms revealed that the concentrations of PC2, PC3, and PC4 (248, 250, and 288?nmol-SH equiv.g^?1 fw, respectively) were maximum under 1?ppm Cd, 1?ppm Cd + 1?ppm Cr, and 2.5?ppm Cd + 3?ppm Cr treatments, respectively. PC2, PC3, and PC4 concentrations increased with Cd accumulation in the range 812–1354?µg/g dry wt, 1354–2032?µg/g dry wt and 2032–3200?µg/g dry wt, respectively. Thus, the study establishes a direct proportionality relationship between concentration/length of phytochelatins and root Cd concentrations, upto threshold limits, in E. crassipes.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Spermiogenesis and spermatozoa ultrastructure of Aonides oxycephala (Annelida: Spionidae) from the Sea of Japan

Adults of Aonides oxycephala, common inhabitants of shallow boreal waters in the Atlantic and Pacific Oceans, release gametes into the water where fertilization and lecithotrophic larval development occur. During spermiogenesis, the acrosomal vesicle migrates from the posterior to the anterior end of the spermatid and the number of mitochondria reduces from six in early spermatids to four in mature spermatozoa. Each spermatozoon has an ovoid head with the acrosome 1.4?±?0.1?µm long and 1.6?±?0.1?µm wide and the nucleus 1.7?±?0.1?µm long and 2.3?±?0.1?µm in diameter, four spherical mitochondria, two centrioles oriented perpendicular to each other, putative glycogen in the shape of dense granules in the midpiece, and a flagellum with 9?×?2?+?2 organization of microtubules. The acrosome is a complex heterogeneous structure with five ordered layers of different electron densities, lying in a shallow depression on the anterior end of the nucleus. The nucleus is barrel-shaped (truncated ovoid) with the centriolar fossa housing the distal and proximal centrioles. Spermiogenesis and ultrastructure of spermatozoa of A. oxycephala are similar to those of another free spawning spionid, Marenzelleria viridis. Aonides and Marenzelleria have not, however, been considered as closely related taxa; thus, similarity in the morphology of their sperm might result from convergence or parallelism.     

Reducing the microcapsule diameter by micro-emulsion to improve the insecticidal activity of Bacillus thuringiensis encapsulated formulations

The emulsion/internal gelation method is highly effective to produce microcapsules of Bacillus thuringiensis (Bt) in a short time; however, it has the limitation to produce microcapsules within a wide range of diameters (1–1000?µm). The aim of this study was to reduce the range of small microcapsule diameters by using a water/corn-oil (W/CO) micro-emulsion as the dispersing medium and the mixture Tween 80–Span 80 as the surfactant. It involved the development of the W/CO micro-emulsion and the determination of the suitable agitation time to disperse the gelling medium (sodium alginate) through the micro-emulsion. A micro-emulsion formulation that allowed reduction of the microcapsule diameter was composed of 82% corn oil, 12% alginate solution and 6% surfactant mixture Tween80–Span80 (31:69). Evaluation of four dispersing times showed that 45 min was suitable to produce 75% of microcapsules of an average diameter of 3.1?±?1?µm containing the spore–protein complex (SPC) produced by Bt. Bioassays carried out at low concentrations of microencapsulated formulations of cry proteins allowed determination of how its insecticidal effect increased if the range of microcapsule diameters was reduced in the range 1–9?µm. Furthermore, the SPC formulation in alginate microcapsules showed high resistance to extreme irradiation (2.9?±?0.5?×?10⁸ erg) of a long wavelength (365?nm), which made the microencapsulated formulation profitable and of high yield since repeated applications of the biopesticide during the same harvest period may not be necessary.