Compounds from Natural Sources for New Diagnostics and Drugs against Biofilm Infections

Biofilm infections represent a new medical challenge that drives towards the discovery of new diagnostics and new drugs specifically designed for this purpose. All living organisms offer a huge source of compounds which represent the biochemical substrate of the biological competition on the Earth and can be used to this aim. We describe an innovative diagnostic tool to early diagnose medical device infections sustained by Staphylococci; then we list new compounds that modulate bacterial phenotype and reduce virulence without affecting bacterial viability so as to avoid the emergence of genetic resistances. These compounds are all derived from natural sources: prokaryotes, plants, and human body. From prokaryotes we studied new compounds extracted from different environmental bacterial species, including Antarctic species growing in extreme environments. We describe also the anti-biofilm properties of extracts obtained from plants well known since centuries in folk medicine. The humoral immune response is the source of the last anti-biofilm compound: transferrin (Tf), a protein derived from human plasma involved in inflammation and natural immunity. All these compounds can be used as scaffolds for the design of new drugs active on the sessile form of pathogens prevalent in human biofilm infections

Recombinant protein expression system in cold loving microorganisms

Soluble and functional proteins are of high demand in modern biotechnology. Although many recombinant proteins have been successfully obtained from common prokaryotic and eukaryotic hosts, these systems result to be often unproductive due to the peculiar properties of the protein to be produced. Incorrect folding of the nascent polypeptide chains is one of the main problems occurring during heterologous protein production in bacteria. Since formation of inclusion bodies often impairs the recombinant production of valuable proteins, many experimental approaches have been explored to minimize this undesirable effect [1, 2]. Expression of "difficult" proteins has also been carried out by lowering the temperature at the physiological limit allowed for the growth of mesophilic host organisms (between 15 and 18°C for Escherichia coli). Lowering the temperature, in fact, has a pleiotropic effect on the folding process, destabilising the hydrophobic interactions needed for intermediates aggregation [3]. On the basis of the above considerations, a rational alternative to mesophilic organisms is the use of naturally cold-adapted bacteria as hosts for protein production at low temperature (even at around 0°C)

Antimicrobial and antibiofilm activity and machine learning classification analysis of essential oils from different mediterranean plants against pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous organism and opportunistic pathogen that can cause persistent infections due to its peculiar antibiotic resistance mechanisms and to its ability to adhere and form biofilm. The interest in the development of new approaches for the prevention and treatment of biofilm formation has recently increased. The aim of this study was to seek new non-biocidal agents able to inhibit biofilm formation, in order to counteract virulence rather than bacterial growth and avoid the selection of escape mutants. Herein, different essential oils extracted from Mediterranean plants were analyzed for their activity againstP. aeruginosa. Results show that they were able to destabilize biofilm at very low concentration without impairing bacterial viability. Since the action is not related to a bacteriostatic/bactericidal activity onP. aeruginosa, the biofilm change of growth in presence of the essential oils was possibly due to a modulation of the phenotype. To this aim, application of machine learning algorithms led to the development of quantitative activity-composition relationships classification models that allowed to direct point out those essential oil chemical components more involved in the inhibition of biofilm production. The action of selected essential oils on sessile phenotype make them particularly interesting for possible applications such as prevention of bacterial contamination in the community and in healthcare environments in order to prevent human infections. We assayed 89 samples of different essential oils asP. aeruginosaanti-biofilm. Many samples inhibitedP. aeruginosabiofilm at concentrations as low as 48.8 µg/mL. Classification of the models was developed through machine learning algorithms

Essential oils against bacterial isolates from cystic fibrosis patients by means of antimicrobial and unsupervised machine learning approaches

Recurrent and chronic respiratory tract infections in cystic fibrosis (CF) patients result in progressive lung damage and represent the primary cause of morbidity and mortality. Staphylococcus aureus (S. aureus) is one of the earliest bacteria in CF infants and children. Starting from early adolescence, patients become chronically infected with Gram-negative non-fermenting bacteria, and Pseudomonas aeruginosa (P. aeruginosa) is the most relevant and recurring. Intensive use of antimicrobial drugs to fight lung infections inevitably leads to the onset of antibiotic resistant bacterial strains. New antimicrobial compounds should be identified to overcome antibiotic resistance in these patients. Recently interesting data were reported in literature on the use of natural derived compounds that inhibited in vitro S. aureus and P. aeruginosa bacterial growth. Essential oils, among these, seemed to be the most promising. In this work is reported an extensive study on 61 essential oils (EOs) against a panel of 40 clinical strains isolated from CF patients. To reduce the in vitro procedure and render the investigation as convergent as possible, machine learning clusterization algorithms were firstly applied to pick-up a fewer number of representative strains among the panel of 40. This approach allowed us to easily identify three EOs able to strongly inhibit bacterial growth of all bacterial strains. Interestingly, the EOs antibacterial activity is completely unrelated to the antibiotic resistance profile of each strain. Taking into account the results obtained, a clinical use of EOs could be suggested

Microscopy methods for biofilm imaging: focus on SEM and VP-SEM pros and cons

Several imaging methodologies have been used in biofilm studies, contributing to deep- ening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron mi- croscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preserva- tion. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared

Antarctic Marine Bacteria as a Source of Anti-Biofilm Molecules to Combat ESKAPE Pathogens

: The ESKAPE pathogens, including bacteria such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, pose a global health threat due to their ability to resist antimicrobial drugs and evade the immune system. These pathogens are responsible for hospital-acquired infections, especially in intensive care units, and contribute to the growing problem of multi-drug resistance. In this study, researchers focused on exploring the potential of Antarctic marine bacteria as a source of anti-biofilm molecules to combat ESKAPE pathogens. Four Antarctic bacterial strains were selected, and their cell-free supernatants were tested against 60 clinical ESKAPE isolates. The results showed that the supernatants did not exhibit antimicrobial activity but effectively prevented biofilm formation and dispersed mature biofilms. This research highlights the promising potential of Antarctic bacteria in producing compounds that can counteract biofilms formed by clinically significant bacterial species. These findings contribute to the development of new strategies for preventing and controlling infections caused by ESKAPE pathogens

Essential oils biofilm modulation activity, chemical and machine learning analysis. Application on staphylococcus aureus isolates from cystic fibrosis patients

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus (S. aureus) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and “green alternatives” to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients’ isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed dierent biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis

Characterization of Scardovia wiggsiae biofilm by original scanning electron microscopy protocol

Early childhood caries (ECC) is a severe manifestation of carious pathology with rapid and disruptive progression. The ECC microbiota includes a wide variety of bacterial species, among which is an anaerobic newly named species, Scardovia wiggsiae, a previously unidentified Bifidobacterium. Our aim was to provide the first ultrastructural characterization of S. wiggsiae and its biofilm by scanning electron microscopy (SEM) using a protocol that faithfully preserved the biofilm architecture and allowed an investigation at very high magnifications (order of nanometers) and with the appropriate resolution. To accomplish this task, we analyzed Streptococcus mutans’ biofilm by conventional SEM and VP-SEM protocols, in addition, we developed an original procedure, named OsO4-RR-TA-IL, which avoids dehydration, drying and sputter coating. This innovative protocol allowed high-resolution and high-magnification imaging (from 10000× to 35000×) in high-vacuum and high-voltage conditions. After comparing three methods, we chose OsO4-RR-TA-IL to investigate S. wiggsiae. It appeared as a fusiform elongated bacterium, without surface specialization, arranged in clusters and submerged in a rich biofilm matrix, which showed a well-developed micro-canalicular system. Our results provide the basis for the development of innovative strategies to quantify the effects of different treatments, in order to establish the best option to counteract ECC in pediatric patients

Effect of different soil treatments on production and chemical composition of essential oils extracted from Foeniculum vulgare Mill., Origanum vulgare L. and Thymus vulgaris L

The aim of the present study was to investigate how essential oil production and associated chemical composition and related biological activity could be influenced by different cultivation treatments and distillation method. Foeniculum vulgare Mill. (fennel), Origanum vulgare L. (oregano), and Thymus vulgaris L. (thyme) were cultivated in absence of any fertilizer (control) and in presence of three different fertilizers: a chemical one with augmented of mineral phosphorus and potassium, a second added with hydrolysed organic substance and mineral phosphorus and potassium (organic-mineral) and a third one treated with high content of organic nitrogen of protein origin (organic). The plants were subjected to steam distillation using two modalities: recycled and continuous to obtain 32 essential oil samples. Chemical composition analysis was performed by gas chromatography-mass spectrometry; in vitro antimicrobial activity was evaluated by broth microdilution method. In general, the recycled distillation method appeared to have a slightly higher yield than the continuous method. The "mineral" and "organic-mineral" treatments resulted in the higher yield compared to the "organic" or "control" treatments, and this was particularly evident in the recycled method. The "control" plants had a lower yield of essential oils. Anethole (13.9-59.5%) and estragole (13.4-52.2%) were the main constituents of fennel oils, p-cymene and its derivatives carvacrol and thymol were the main constituents of oregano and thyme samples. The antimicrobial activity of thyme oils on Staphylococcus aureus ranged from 0.31 to 0.16% (v/v); a lower effect of oregano samples and no activity of fennel samples were observed. The essential oils failed to inhibit the growth of Pseudomonas aeruginosa strains