Quantitative Evaluation of Bacteria Adherent and in Biofilm on Single-Wall Carbon Nanotube-Coated Surfaces

Biofilm is a common bacterial lifestyle, and it plays a crucial role in human health, causing biofilm-mediated infections. Recently, to counteract biofilm development, new nano-structured biomaterials have been proposed. However, data about the antibacterial properties of nano-structured surfaces are fragmentary and controversial, and, in particular, the susceptibility of nano-structured materials to colonization and biofilm formation by bacterial pathogens has not been yet thoroughly considered. Here, the ability of the pathogenic Streptococcus mutans and Pseudomonas aeruginosa to adhere and form biofilm on surfaces coated with single-wall carbon nanotubes (SWCNTs) was analyzed. Our results showed that the surfaces of SWCNTs-coated glass beads (SWCNTs-GBs) were colonized at the same extent of uncoated GBs both by S. mutans and P. aeruginosa. In conclusion, our results demonstrate that single wall SWCNTs-coated surfaces are not suitable to counteract bacterial adhesion and biofilm development

Microbiological quality control of single-walled carbon-nanotubes-coated surfaces experimentally contaminated

The emergence of new nanotechnologies involves the spreading of nanoparticles in various fields of human life. Nanoparticles in general and, more specifically, carbon nanotubes have been adopted for many practical approaches i.e.: coatings for medical devices, food process industry and drug delivery. Humans will be increasingly exposed to nanoparticles but the susceptibility of nanostructured materials to microbial colonization in process of manufacturing and storage has not been thoroughly considered. Therefore, the microbiological quality control of nanoparticles plays a pivotal role. Different analytical methods have been attempted for detecting bacterial population contaminating a surface, but no one can be considered fully appropriate. Here, BioTimer Assay (BTA) and conventional sonication followed by colony forming units method (S-CFU) were applied for microbiological quality control of single-walled carbon nanotubes (SWCNTs)-coated surfaces experimentally contaminated with Streptococcus mutans and Pseudomonas aeruginosa. Our results demonstrated that S-CFU is unreliable to actually determine the number of bacteria, contaminating abiotic surfaces, as it does not detach all adherent bacteria and kills part of the bacterial population. Instead, BTA is a reliable method to enumerate bacteria colonizing SWCNTs-coated surfaces and can be considered a useful tool for microbiological quality control of nanomaterials for human use. © Società Italiana di Fisica

Body iron delocalization: the serious drawback in iron disorders in both developing and developed countries

Over 2 billion people in both developing as well as developed countries - over 30% of the world's population - are anaemic. With the classical preconception that oral iron administration or the intake of foods rich in iron increase haemoglobin concentration and reduce the prevalence of anaemia, specific programs have been designed, but iron supplementations have been less effective than expected. Of note, this hazardous simplification on iron status neglects its distribution in the body. The correct balance of iron, defined iron homeostasis, involves a physiological ratio of iron between tissues/secretions and blood, thus avoiding its delocalization as iron accumulation in tissues/secretions and iron deficiency in blood. Changes in iron status can affect the inflammatory response in multiple ways, particularly in the context of infection, an idea that is worth remembering when considering the value of iron supplementation in areas of the world where infections are highly prevalent. The enhanced availability of free iron can increase susceptibility and severity of microbial and parasitic infections. The discovery of the hepcidin-ferroportin (Fpn) complex, which greatly clarified the enigmatic mechanism that supervises the iron homeostasis, should prompt to a critical review on iron supplementation, ineffective on the expression of the most important proteins of iron metabolism. Therefore, it is imperative to consider new safe and efficient therapeutic interventions to cure iron deficiency (ID) and ID anaemia (IDA) associated or not to the inflammation. In this respect, lactoferrin (Lf) is emerging as an important regulator of both iron and inflammatory homeostasis. Oral administration of Lf in subjects suffering of ID and IDA is safe and effective in significantly increasing haematological parameters and contemporary decreasing serum IL-6 levels, thus restoring iron localization through the direct or indirect modulation of hepcidin and ferroportin synthesis. Of note, the nuclear localization of Lf suggests that this molecule may be involved in the transcriptional regulation of some genes of host inflammatory response. We recently also reported that combined administration of oral and intravaginal Lf on ID and IDA pregnant women with preterm delivery threat, significantly increased haematological parameters, reduced IL-6 levels in both serum and cervicovaginal fluid, cervicovaginal prostaglandin PGF2 alpha, and suppressed uterine contractility. Moreover, Lf combined administration blocked further the shortening of cervical length and the increase of foetal fibronectin, thus prolonging the length of pregnancy until the 37th-38th week of gestation. These new Lf functions effective in curing ID and IDA through the restoring of iron and inflammatory homeostasis and in preventing preterm delivery, could have a great relevance in developing countries, where ID and IDA and inflammation-associated anaemia represent the major risk factors of preterm delivery and maternal and neonatal death

Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.

Biofilm is a bacterial lifestyle widespread in microbial world and represents a concern in health care. Despite the great life expectancy related to advanced health care, the increasing numbers of biofilm-mediated infections remain a significant public health challenge. Moreover, the problem of biofilm-mediated infections becomes much more severe when biofilm colonizes medical devices and biomaterials. The public health risk due to microbial biofilm-related infections is a concern that requires full attention. However, the complexity of biofilm makes difficult its exhaustive analysis. Although biofilm represents a major challenge in both microbiological and hygiene areas, at now methods aimed to analyse biofilm formation and development are not standardized yet. Different methods have been employed to qualitatively and quantitatively evaluate biofilm each of which is useful to estimate a peculiar aspect of biofilm lifestyle. In the present review, fifteen assays for the qualitative and quantitative evaluation of bacterial biofilm colonizing abiotic substrates, such as medical devices, prosthesis or surfaces for food production together with advantages and limitations of each method were described and compared. Some methods are suited to quantify biofilm matrix while others are capable to evaluate both living and dead cells or quantify exclusively viable cells in biofilm. In particular, colorimetric methods to evaluate biofilm matrix (crystal violet; 1,9-dimethyl methylen blue and fluorescein-di-acetate methods) or viable cells (LIVE/DEAD BacLight, BioTimer Assay, resazurin, tetrazolium hydroxide salt methods) and genetic methods to estimate the bacterial population (PCR and FISH) are reported. Moreover, a section is dedicated to examine the performances of advanced microscopic techniques employed to study microbial biofilms (mass spectrometry; confocal laser scanning microscopy; Raman spectroscopy and electron microscopy). Because of its complexity, an exhaustive study of biofilm requires a combination of different experimental approaches as biochemical, genetic or physical ones

BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BidTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to deter-mine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm. Published by Elsevier B.V

Antiviral Properties of Lactoferrin—A Natural Immunity Molecule

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LACTOFERRIN DECREASES INFLAMMATORY RESPONSE BY CYSTIC FIBROSIS BRONCHIAL CELLS INVADED WITH BURKHOLDERIA CENOCEPACIA IRON-MODULATED BIOFILM

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported. bLf did not significantly affect invasion efficacy by biofilm-forming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1 beta and increased the anti-inflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein

STREPTOCOCCUS MUTANS AND STREPTOCOCCUS SOBRINUS ARE ABLE TO ADHERE AND INVADE HUMAN GINGIVAL FIBROBLAST CELL LINE

Streptococcus mutans and Streptococcus sobrinus, the principal etiologic agents of caries decay of teeth, are generally acquired in oral cavity at the moment of tooth eruption. However, as S. mutans has been detected in oral cavity of predentate children, the eruption of teeth seems not to be a necessary prerequisite, suggesting that this species may be not confined to dental plaque. Here, we evaluate the ability of S. mutans and S. sobrinus in planktonic and biofilm lifestyle to adhere, invade and survive within human gingival fibroblast (HGF-1) cells. Planktonic and biofilm streptococci adhered and invaded host cells to different extents, showing higher efficiencies of biofilm than planktonic counterparts. Moreover, planktonic and biofilm streptococci showed the same percentage of survival within host cells. Transmission electron and confocal microscopy observations confirmed intracellular localization of planktonic and biofilm bacteria. The adhesion, invasion and survival abilities within human oral cells may be considered S. mutans and S. sobrinus virulence mechanisms to colonize and persist in the oral cavity in the absence of tooth surface