Screening, isolation, and purification of active compounds against Klebsiella pneumoniae

The spread of multi-resistant bacteria is a contemporary real threat that constitutes a public health risk. Resistance to antibiotics has increased progressively since their discovery due to their misuse and abuse. Therefore, finding new antimicrobial compounds has become a worldwide task.By screening a culture collection, this study aimed to find bacteria and fungi able to inhibit the growth of Klebsiella pneumoniae. Replica plating, SPE-X columns, disk- diffusion assays, and agar-well diffusion assays are some of the techniques that have been used to isolate and purify the active compounds.Of all microorganisms tested, Epicoccum nigrum (MES 1587) showed the most promising results. It was able to inhibit not only K. pneumoniae, but also S. aureus, P. aeruginosa, B. subtilis, E. faecalis, and E. coli in both disk-diffusion assays and agar- well diffusion assays. It was also shown that its inhibition started upon 8 days of culture and increased over time.Regarding the rest of the microorganisms that inhibited K. pneumoniae in replica plating, it was impossible to reproduce the positive results after SPE-X purification.<br /

Scientific Symposium “Small Solution for Big Water-Related Problems: Innovative Microarrays and Small Sensors to Cope with Water Quality and Food Security”

This issue presents the conclusive results of two European Commission funded Projects, namely Universal Microarrays for the Evaluation of Fresh-water Quality Based on Detection of Pathogens and their Toxins (MicroAQUA) and Rationally Designed Aquatic Receptors (RADAR). These projects focused their activities on the quality of drinking water as an extremely important factor for public health of humans and animals. The MicroAQUA Project aimed at developing a universal microarray chip for the detection of various pathogens (cyanobacteria, bacteria, viruses and parasitic protozoa) and their toxins in waters. In addition, the project included the detection of select species of diatoms, which represent reliable bio-indicators to assess overall water quality. Large numbers of compounds are released into the environment; some of these are toxins such as endocrine disrupting compounds (EDCs) and can affect the endocrine, immune and nervous systems of a wide range of animals causing alterations such as reproductive disorders and cancer. Detection of these contaminants in water systems is important to protect sensitive environmental sites and reduce the risk of toxins entering the food chain. A modular platform for monitoring toxins in water and food production facilities, using biosensors derived from aquatic organisms, was the main goal of RADAR Project

Molecular detection of a potentially toxic diatom species

A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels

Draft Genome Sequence of Streptomyces sp. Strain AM-2504, Identified by 16S rRNA Comparative Analysis as a Streptomyces kasugaensis Strain

We report here the draft genome sequence of Streptomyces sp. strain AM-2504, a microorganism producing a broad range of biotechnologically relevant molecules. The comparative analysis of its 16S rRNA sequence allowed the assignment of this strain to the Streptomyces kasugaensis species, thus fostering functional characterization of the secondary metabolites produced by this microorganism

Characterization of the Self-Resistance Mechanism to Dityromycin in the Streptomyces Producer Strain

Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain.IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers' microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic's target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli, thereby eliciting antibiotic resistance in this human commensal bacterium

Screening an archetypal collection of microorganisms for the presence of unexplored antimicrobial compounds.

WHO estimates that more than 700.000 people die every year as a result of drug-resistant infections. To tackle this problem and change the current trend, it is necessary to design advanced strategies for drug discovery and to promote early-stage research activities finalized at the development of new drugs. In this study, we present the results of the preliminary screening of a library of microorganisms, collected from different environmental settings. Approximately 300 strains of the culture collection were tested on solid medium for inhibition of growth of three tester species, namely Bacillus subtilis, Escherichia coli and Staphylococcus aureus. One of the active strains, MES18, was classified as a Bacillus spp. by means of small-subunit rRNA gene sequencing. To identify the compound(s) responsible for this inhibitory activity, MES18 cells were grown in liquid medium at 30°C and samples were taken at different time points over a period of 12 days. The supernatants obtained from the fermentation media were subjected to fractionation by chromatography on reversed-phase column and all the eluted compounds were assayed for their ability to repress the growth of tester strains. This approach allowed us to identify the fractions containing the bioactive compound(s) and to establish that the production of these secondary metabolites reached a maximum during the idiophase of the cell culture, when cell growth and replication decline. Further analyses to identify the physical-chemical features of the compound(s) produced by this strain using HPLC coupled to mass-spectrometry are currently ongoing

Molecular tools for the selective detection of nine diatom species biomarkers of various water quality levels

Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed