Silvia Elena Murialdo

Analysis of bacterial chemotactic response using dynamic laser speckle

Chemotaxis has a meaningful role in several fields, such as microbial physiology, medicine and biotechnology. We present a new application of dynamic laser speckle (or biospeckle) to detect different degrees of bacterial motility during chemotactic response experiments. Encouraging results showed different bacterial dynamic responses due to differences in the hardness of the support in the swarming plates. We compare this method to a conventional technique that uses white light. Both methods showed to be analogous and, in some cases, complementary. The results suggest that biospeckle processed images can be used as an alternative method to evaluate bacterial chemotactic response and can supply additional information about the bacterial motility in different areas of the swarm plate assay that might be useful for biological analysis.

Achromobacter marplatensis sp. nov., isolated from a pentachlorophenol-contaminated soil

A polyphasic taxonomic approach was applied to the study of a Gram-negative bacterium (B2(T)) isolated from soil by selective enrichment with pentachlorophenol. 16S rRNA gene sequence analysis of strain B2(T) showed that the strain belongs to the genus Achromobacter within the Betaproteobacteria. The 16S rRNA gene sequence displayed more than 99 % similarity to the sequences of the type strains of all species of Achromobacter, with the highest sequence similarity to those of Achromobacter spanius CCM 7183(T) and A. piechaudii CCM 2986(T) (99.8 %). On the basis of phylogenetic analysis, genomic DNA-DNA relatedness and phenotypic characteristics, including chemotaxonomic (cellular fatty acid profile) analysis, a novel species is proposed, Achromobacter marplatensis sp. nov., with the type strain B2(T) ( = CCM 7608(T)  = CCUG 56371(T)  = CECT 7342(T)).

Hydrocarbon biodegradation and dynamic laser speckle for detecting chemotactic responses at low bacterial concentration

We report on the biodegradation of pure hydrocarbons and chemotaxis towards these compounds by an isolated chlorophenol degrader, Pseudomonas strain H. The biochemical and phylogenetic analysis of the 16S rDNA sequence identified Pseudomonas strain H as having 99.56% similarity with P. aeruginosa PA01. This strain was able to degrade n-hexadecane, 1-undecene, 1-nonene, 1-decene, 1-dodecene and kerosene. It grew in the presence of 1-octene, while this hydrocarbons is toxic to other hydrocarbons degraders. Pseudomonas strain H was also chemotactic towards n-hexadecane, kerosene, 1-undecene and 1-dodecene. These results show that this Pseudomonas strain H is an attractive candidate for hydrocarbon-containing wastewater bioremediation in controlled environments. Since the classical standard techniques for detecting chemotaxis are not efficient at low bacterial concentrations, we demonstrate the use of the dynamic speckle laser method, which is simple and inexpensive, to confirm bacterial chemotaxis at low cell concentrations (less than 10(5) colony-forming unit per millilitre (CFU/mL)) when hydrocarbons are the attractants.

Biospeckle image stack process based on artificial neural networks

This paper proposes the identification of regions of interest in biospeckle patterns using unsupervised neural networks of the type Self-Organizing Maps. Segmented images are obtained from the acquisition and processing of laser speckle sequences. The dynamic speckle is a phenomenon that occurs when a beam of coherent light illuminates a sample in which there is some type of activity, not visible, which results in a variable pattern over time. In this particular case the method is applied to the evaluation of bacterial chemotaxis. Image stacks provided by a set of experiments are processed to extract features of the intensity dynamics. A Self-Organizing Map is trained and its cells are colored according to a criterion of similarity. During the recall stage the features of patterns belonging to a new biospeckle sample impact on the map, generating a new image using the color of the map cells impacted by the sample patterns. It is considered that this method has shown better performance to identify regions of interest than those that use a single descriptor. To test the method a chemotaxis assay experiment was performed, where regions were differentiated according to the bacterial motility within the sample.

Dynamic laser speckle and fuzzy mathematical morphology applied to studies of chemotaxis towards hydrocarbons

The movement of the microorganisms towards a higher concentration of the chemical attractant is called positive chemotaxis and is involved in the efficiency of chemical degradation. Several studies are focused in this field related to genomics, and towards demonstrating chemotactic responses by bacteria, but there is little information related to the activity and morphology of their response. In this work, we use a recently reported dynamic speckle laser method, to process images and to distinguish motile surface patterns per area of colonisation by applying image processing techniques called fuzzy mathematical morphology (FMM). The images of bacterial colonies are usually surfaced, with vague edges and non-homogeneous grey levels. Hence, conventional image processing methods for shape analysis cannot be applied in these cases. In this paper, we propose the application FMM to solve this problem. The approach given was effective to segment, detect and also to describe colonisation patterns.

Application of a laser speckle method for determining chemotactic responses of Pseudomonas aeruginosa toward attractants

Dynamic speckle images are useful tools to characterize the activity of biological tissues. In this paper, this technique was applied to determine chemotaxis responses of Pseudomonas aeruginosa towards attractants. Generalized weighted differences, wavelet entropy and spectral bands decomposition algorithms were used to characterize the speckle activity. Experimental results show regions with different bacterial activity. Dynamic speckle method exhibits a good performance for this application.

Monitoring the characteristics of cultivable halophilic microbial community during salted-ripened anchovy (Engraulis anchoita) production

The halophilic microbial community of the salted-ripened anchovy process was studied. Samples from raw materials (salt and fresh anchovies) and from the stages of brining and ripening were collected and analyzed for their bacterial counts at 15 and 20% NaCl. No halophilic colonies were found in fresh anchovy and counts of about 103 CFU/g were determined in salt samples. A fluctuation of bacterial counts during the process was found. At the end of brining, ~104 CFU/g were determined in anchovy samples and this value was reduced to not detectable counts at the beginning of the ripening stage. After one month, counts increased to ~104 CFU/g and remained stable until the end of the process. From each sample, colonies having different morphotypes were isolated and submitted to a macro and microscopic characterization, a study of salt requirement for growth, and biochemical and phenotypic tests. The results were submitted to Univariate, Bivariate and Multiple Correspondence Factorial Analysis (MCFA). A total of 79 colonies were isolated during the salting-ripening anchovy process. Among the isolates, about 40-50% was positive for indole production and lipolytic activity and a 25% showed ability to produce H2S and proteolytic capacity. Proteolytic and lipolytic activities were well balanced along the process and resulted independent from the isolation stage, which is a desirable condition due to the contribution of microbial proteolysis and lipolysis to the development of texture and final aroma, respectively. H2S and indole producers practically were not detected during ripening. This fact is important because indole and H2S are associated with the development of off-flavors and spoilage in salted fish products. MFCA and Cluster Analyses complemented the Bivariate Analyses. The factor map showed proximity between the isolates from salt samples and from ripening. Isolates were statistically clustered in two groups. Cluster 1 grouped non-desirable activities (H2S and indole production) with cultures proceeding from brining whereas Cluster 2 related isolates mainly from salt samples and during ripening with some desirable microbial capacities (Cytochrome oxidase activity and non-H2S and non-indole production). These results would indicate that during the ripening process of salted anchovies, a natural selection of beneficial microorganisms for the development of the typical product sensory attributes occurred.

New Findings on Aromatic Compounds’ Degradation and Their Metabolic Pathways, the Biosurfactant Production and Motility of the Halophilic Bacterium Halomonas sp. KHS3

The study of the aromatic compounds’ degrading ability by halophilic bacteria became an interesting research topic, because of the increasing use of halophiles in bioremediation of saline habitats and effluents. In this work, we focused on the study of aromatic compounds’ degradation potential of Halomonas sp. KHS3, a moderately halophilic bacterium isolated from hydrocarbon-contaminated seawater of the Mar del Plata harbour. We demonstrated that H. sp. KHS3 is able to grow using different monoaromatic (salicylic acid, benzoic acid, 4-hydroxybenzoic acid, phthalate) and polyaromatic (naphthalene, fluorene, and phenanthrene) substrates. The ability to degrade benzoic acid and 4-hydroxybenzoic acid was analytically corroborated, and Monod kinetic parameters and yield coefficients for degradation were estimated. Strategies that may enhance substrate bioavailability such as surfactant production and chemotactic responses toward aromatic compounds were confirmed. Genomic sequence analysis of this strain allowed us to identify several genes putatively related to the metabolism of aromatic compounds, being the catechol and protocatechuate branches of β-ketoadipate pathway completely represented. These features suggest that the broad-spectrum xenobiotic degrader H. sp. KHS3 could be employed as a useful biotechnological tool for the cleanup of aromatic compounds-polluted saline habitats or effluents.

Responses of Phalaris canariensis L. Exposed to Commercial Fuels during Growth

The growth behavior of canary grass (Phalaris canariensis L) when cultivated in presence of farming fuels is reported in this work. P. canariensis L. is relevant in several countries. It is an emergent plant for phytoremediation and biofuel activities. The following variables: root length, stem length, total plant weight, green tissue weight (tiller, leaf), and total chlorophyll and chlorophyll a/b ratio, were monitored during the growth in presence of commercial fuels (premium grade, regular grade, diesel, and kerosene) at different concentrations. We applied a comprehensive statistical analysis to understand the results: Univariate analysis, factorial analysis of variance, and subsequent Tukey test were applied to the variables to assess the significance of the differences found. The normality of these variables was analyzed with the Shapiro Wilk test. All parameters were affected by all type and concentrations of fuels and its interaction. This is one of the first reported cases which describe the growth parameters responses from canary grass when cultivated in presence of an essentially constant concentration of farming fuels.