Muramic and dipicolinic acids in atmospheric particulate matter as biomarkers of bacteria and bacterial spores.

Airborne bacteria are components of the atmospheric aerosol particles and can be responsible of allergic disease, regardless of their viability. In this paper, we report a method for the determination of total (viable and nonviable) bacterial content in airborne particles, using muramic and dipicolinic acids as biomarkers of bacteria and bacterial spores, respectively. The analytical procedure was optimized with bacteria and spores of Bacillus subtilis. After extraction and purification, the two biomarkers were analyzed by HPLC-ESI-MS/MS and their percentage was evaluated to be used as conversion factor. The present method for the determination of the total bacterial content was then applied to environmental samples, after a proper collection in an urban site. Thanks to the use of a low pressure impactor, capable of fractionating particles into the range of 0.03-10 µm, it was also possible to study the bacterial content in ultrafine, fine, and coarse particulate matter. The results from this study showed that muramic and dipicolinic acids can be determined together in one chromatographic run in reversed phase ion pair chromatography. Bacteria were more abundant than bacterial spores in the urban atmosphere, both showing a higher concentration in the coarse fraction of particles, although bacteria and bacterial spore amounts per unit mass of ultrafine particles were higher than in fine and coarse particles.

Evaluation of the antibacterial power and biocompatibility of zinc oxide nanorods decorated graphene nanoplatelets: new perspectives for antibiodeteriorative approaches.

BACKGROUND: Nanotechnologies are currently revolutionizing the world around us, improving the quality of our lives thanks to a multitude of applications in several areas including the environmental preservation, with the biodeterioration phenomenon representing one of the major concerns. RESULTS: In this study, an innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was tested for the ability to inhibit two different pathogens belonging to bacterial genera frequently associated with nosocomial infections as well as biodeterioration phenomenon: the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. A time- and dose-dependent bactericidal effect in cell viability was highlighted against both bacteria, demonstrating a strong antimicrobial potential of ZNGs. Furthermore, the analysis of bacterial surfaces through Field emission scanning electron microscopy (FESEM) revealed ZNGs mechanical interaction at cell wall level. ZNGs induced in those bacteria deep physical damages not compatible with life as a result of nanoneedle-like action of this nanomaterial together with its nanoblade effect. Cell injuries were confirmed by Fourier transform infrared spectroscopy, revealing that ZNGs antimicrobial effect was related to protein and phospholipid changes as well as a decrease in extracellular polymeric substances; this was also supported by a reduction in biofilm formation of both bacteria. The antibacterial properties of ZNGs applied on building-related materials make them a promising tool for the conservation of indoor/outdoor surfaces. Finally, ZNGs nanotoxicity was assessed in vivo by exploiting the soil free living nematode Caenorhabditis elegans. Notably, no harmful effects of ZNGs on larval development, lifespan, fertility as well as neuromuscular functionality were highlighted in this excellent model for environmental nanotoxicology. CONCLUSIONS: Overall, ZNGs represent a promising candidate for developing biocompatible materials that can be exploitable in antimicrobial applications without releasing toxic compounds, harmful to the environment.

Graphene-based dental adhesive with anti-biofilm activity.

BACKGROUND: Secondary caries are considered the main cause of dental restoration failure. In this context, anti-biofilm and bactericidal properties are desired in dental materials against pathogens such as Streptococcus mutans. To this purpose, graphene based materials can be used as fillers of polymer dental adhesives. In this work, we investigated the possibility to use as filler of dental adhesives, graphene nanoplatelets (GNP), a non toxic hydrophobic nanomaterial with antimicrobial and anti-biofilm properties. RESULTS: Graphene nanoplatelets have been produced starting from graphite intercalated compounds through a process consisting of thermal expansion and liquid exfoliation. Then, a dental adhesive filled with GNPs at different volume fractions has been produced through a solvent evaporation method. The rheological properties of the new experimental adhesives have been assessed experimentally. The adhesive properties have been tested using microtensile bond strength measurements (µ-TBS). Biocidal activity has been studied using the colony forming units count (CFU) method. The anti-biofilm properties have been demonstrated through FE-SEM imaging of the biofilm development after 3 and 24 h of growth. CONCLUSIONS: A significantly lower vitality of S. mutans cells has been demonstrated when in contact with the GNP filled dental adhesives. Biofilm growth on adhesive-covered dentine tissues demonstrated anti-adhesion properties of the produced materials. µ-TBS results demonstrated no significant difference in µ-TBS between the experimental and the control adhesive. The rheology tests highlighted the necessity to avoid low shear rate regimes during adhesive processing and application in clinical protocol, and confirmed that the adhesive containing the 0.2%wt of GNPs possess mechanical properties comparable with the ones of the control adhesive.

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production.

BACKGROUND: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. RESULTS: The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (µ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous ß-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous ß-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). CONCLUSIONS: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.

Graphite nanoplatelets and Caenorhabditis elegans: insights from an in vivo model.

We evaluated the toxicity of graphite nanoplatelets (GNPs) in the model organism Caenorhabditis elegans. The GNPs resulted nontoxic by measuring longevity as well as reproductive capability end points. An imaging technique based on Fourier transform infrared spectroscopy (FT-IR) mapping was also developed to analyze the GNPs spatial distribution inside the nematodes. Conflicting reports on the in vitro antimicrobial properties of graphene-based nanomaterials prompted us to challenge the host-pathogen system C. elegans-Pseudomonas aeruginosa to assess these findings through an in vivo model.

KlMID1, a relevant key player between endoplasmic reticulum homeostasis and mitochondrial dysfunction in Kluyveromyces lactis.

The interplay between calcium metabolism and glycosylation in yeast is largely unknown. In order to clarify this relationship, the effect of a mutation in the KlOCH1 gene, encoding the Golgi α-1,6-mannosyltransferase, on calcium homeostasis was studied in the yeast Kluyveromyces lactis. In particular, the role of the KlMID1 gene, encoding one of the components of the plasma membrane calcium channel (Cch1-Mid1), was investigated. Almost complete suppression of the phenotypes occurring in the mutant strain, ranging from oxidative stress to cell wall alteration, was observed by increased dosage of KlMID1. In addition, the N-glycosylation mutant showed increased calcium accumulation and decreased transcription of KlMID1 and KlCCH1. Moreover, the calcium alterations included an increased expressional profile for the KlPMC1 gene, encoding the vacuolar calcium ion pump. Furthermore, perturbation of endoplasmic reticulum (ER) homeostasis was observed in Kloch1-1 cells. Similarly, down-modulation of calcium signalling genes as well as altered mitochondrial functionality were induced in wild-type cells after treatment with DTT. However, no mitochondrial alteration occurred in the treated cells when KlMID1 was overexpressed. Our results suggest that the ER stress taking place in Kloch1-1 cells appears to be the primary cause of the KlMID1 down-modulation and its resulting effects on the expression of calcium homeostasis genes.

Anti-Pseudomonas activity of frog skin antimicrobial peptides in a Caenorhabditis elegans infection model: a plausible mode of action in vitro and in vivo.

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which are hard to eradicate, especially in patients with a compromised immune system. Naturally occurring membrane-active cationic antimicrobial peptides (CAMPs) serve as attractive candidates for the development of new therapeutic agents. Amphibian skin is one of the richest sources for such peptides, but only a few studies on their in vivo activities and modes of action have been reported. We investigated (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on an MDR clinical isolate of P. aeruginosa and (ii) their in vivo antibacterial activities and modes of action, using the minihost model of Caenorhabditis elegans. Our data revealed that in vivo, both temporin-1Tb and esculentin(1-18) were highly active in promoting the survival of Pseudomonas-infected nematodes, although temporin-1Tb did not show significant activity in vitro under the experimental conditions used. Importantly, esculentin(1-18) permeated the membrane of Pseudomonas cells within the infected nematode. To the best of our knowledge, this is the first report showing the ability of a CAMP to permeate the microbial membrane within a living organism. Besides shedding light on a plausible mode of action of frog skin CAMPs in vivo, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections.

The Golgi alpha-1,6 mannosyltransferase KlOch1p of Kluyveromyces lactis is required for Ca2+/calmodulin-based signaling and for proper mitochondrial functionality.

BACKGROUND: Protein N-glycosylation is a relevant metabolic pathway in eukaryotes and plays key roles in cell processes. In yeasts, outer chain branching is initiated in the Golgi apparatus by the alpha-1,6-mannosyltransferase Och1p. RESULTS: Here we report that, in Kluyveromyces lactis, this glycosyltransferase is also required to maintain functional mitochondria and calcium homeostasis. Cells carrying a mutation in KlOCH1 gene showed altered mitochondrial morphology, increased accumulation of ROS and reduced expression of calcium signalling genes such as calmodulin and calcineurin. Intracellular calcium concentration was also reduced in the mutant cells with respect to the wild type counterparts.Phenotypes that occur in cells lacking the alpha-1,6-mannosyltransferase, including oxidative stress and impaired mitochondria functionality, were suppressed by increased dosage of KlCmd1p. This, in turn, acts through the action of calcineurin. CONCLUSIONS: Proper functioning of the alpha-1,6-mannosyltransferase in the N-glycosylation pathway of K. lactis is required for maintaining normal calcium homeostasis; this is necessary for physiological mitochondria dynamics and functionality.

Virulence behavior of uropathogenic Escherichia coli strains in the host model Caenorhabditis elegans.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Although a number of bacteria can cause UTIs, most cases are due to infection by uropathogenic Escherichia coli (UPEC). UPEC are a genetically heterogeneous group that exhibit several virulence factors associated with colonization and persistence of bacteria in the urinary tract. Caenorhabditis elegans is a tiny, free-living nematode found worldwide. Because many biological pathways are conserved in C. elegans and humans, the nematode has been increasingly used as a model organism to study virulence mechanisms of microbial infections and innate immunity. The virulence of UPEC strains, characterized for antimicrobial resistance, pathogenicity-related genes associated with virulence and phylogenetic group belonging was evaluated by measuring the survival of C. elegans exposed to pure cultures of these strains. Our results showed that urinary strains can kill the nematode and that the clinical isolate ECP110 was able to efficiently colonize the gut and to inhibit the host oxidative response to infection. Our data support that C. elegans, a free-living nematode found worldwide, could serve as an in vivo model to distinguish, among uropathogenic E. coli, different virulence behavior.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Zinc Oxide Nanorods-Decorated Graphene Nanoplatelets: A Promising Antimicrobial Agent against the Cariogenic Bacterium Streptococcus mutans.

Nanomaterials are revolutionizing the field of medicine to improve the quality of life due to the myriad of applications stemming from their unique properties, including the antimicrobial activity against pathogens. In this study, the antimicrobial and antibiofilm properties of a novel nanomaterial composed by zinc oxide nanorods-decorated graphene nanoplatelets (ZNGs) are investigated. ZNGs were produced by hydrothermal method and characterized through field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) techniques. The antimicrobial activity of ZNGs was evaluated against Streptococcus mutans, the main bacteriological agent in the etiology of dental caries. Cell viability assay demonstrated that ZNGs exerted a strikingly high killing effect on S. mutans cells in a dose-dependent manner. Moreover, FE-SEM analysis revealed relevant mechanical damages exerted by ZNGs at the cell surface of this dental pathogen rather than reactive oxygen species (ROS) generation. In addition, inductively coupled plasma mass spectrometry (ICP-MS) measurements showed negligible zinc dissolution, demonstrating that zinc ion release in the suspension is not associated with the high cell mortality rate. Finally, our data indicated that also S. mutans biofilm formation was affected by the presence of graphene-zinc oxide (ZnO) based material, as witnessed by the safranin staining and growth curve analysis. Therefore, ZNGs can be a remarkable nanobactericide against one of the main dental pathogens. The potential applications in dental care and therapy are very promising.

Impact of a Complex Food Microbiota on Energy Metabolism in the Model Organism Caenorhabditis elegans.

The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism.

ER stress induced by the OCH1 mutation triggers changes in lipid homeostasis in Kluyveromyces lactis.

In Kluyveromyces lactis yeast, OCH1 encodes for the α-1,6-mannosyltrasferase that adds the initial α-1,6-mannose to the outer-chains of N-glycoproteins. Kloch1-1 mutant cells showed altered calcium homeostasis and endoplasmic reticulum (ER) stress. Since ER plays a major role in lipid biosynthesis and lipid droplet (LD) formation, herein the impact of Och1p depletion on lipid homeostasis was investigated. Transcriptional profiles of genes involved in biosynthesis of fatty acids, their amount and composition changed in mutant cells. An increased amount of ergosterol was determined in these cells. Enhanced transcription of genes involved in both synthesis and mobilization of LDs was also found in Kloch1-1 cells, accompanied by a reduced amount of LDs. We provide evidence that ER alterations, determined by protein misfolding as a result of reduced N-glycosylation, induced altered lipid homeostasis in Kloch1-1 cells. Chemical chaperone 4-phenyl butyrate (4-PBA) slightly alleviated the LD phenotype in cells depleted of Och1p. Remarkably, complete suppression of ER stress, via increased expression of plasma membrane calcium channel subunit Mid1, fully restored lipid homeostasis in mutant cells. To further reinforce this finding, low numbers of LDs were observed in wild type cells when ER stress was triggered by DTT treatment.

Inhibition of microbial growth by carbon nanotube networks.

In the last years carbon nanotubes have attracted increasing attention for their potential applications in the biomedical field as diagnostic and therapeutic nano tools. Here we investigate the antimicrobial activity of different fully characterized carbon nanotube types (single walled, double walled and multi walled) on representative pathogen species: Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa and the opportunistic fungus Candida albicans. Our results show that all the carbon nanotube types possess a highly significant antimicrobial capacity, even though they have a colony forming unit capacity and induction of oxidative stress in all the microbial species to a different extent. Moreover, scanning electron microscopy analysis revealed that the microbial cells were wrapped or entrapped by carbon nanotube networks. Our data taken together suggest that the reduced capacity of microbial cells to forming colonies and their oxidative response could be related to the cellular stress induced by the interactions of pathogens with the CNT network.

Extension of Chronological Lifespan by Hexokinase Mutation in Kluyveromyces lactis Involves Increased Level of the Mitochondrial Chaperonin Hsp60.

Oxidative damage, mitochondrial dysfunction, genomic instability, and telomere shortening represent all molecular processes proposed as causal factors in aging. Lifespan can be increased by metabolism through an influence on such processes. Glucose reduction extends chronological lifespan (CLS) of Saccharomyces cerevisiae through metabolic adaptation to respiration. To answer the question if the reduced CLS could be ascribed to glucose per se or to glucose repression of respiratory enzymes, we used the Kluyveromyces lactis yeast, where glucose repression does not affect the respiratory function. We identified the unique hexokinase, encoded by RAG5 gene, as an important player in influencing yeast lifespan by modulating mitochondrial functionality and the level of the mitochondrial chaperonin Hsp60. In this context, this hexokinase might have a regulatory role in the influence of CLS, shedding new light on the complex regulation played by hexokinases.