Antibacterial, anti-invasive, and anti-inflammatory activity of bovine lactoferrin extracted from milk or colostrum versus whole colostrum.

Lactoferrin (Lf), a multifunctional cationic glycoprotein extracted from milk or colostrum, is able to chelate two ferric ions per molecule, inhibit the formation of reactive oxygen species, interact with the anionic components of bacteria or host cells, and enter inside host cell nucleus, thereby exerting antibacterial, anti-invasive, and anti-inflammatory activities. By virtue of Lf presence, bovine colostrum is expected to perform analogous functions to pure Lf, along with additional activities attributable to other bioactive constituents. The present research aims to compare the antibacterial, anti-invasive, and anti-inflammatory activities of bovine Lf purified from milk (mbLf) and colostrum (cbLf) in comparison to those exhibited by whole bovine colostrum (wbc). The results demonstrated a major efficacy of mbLf in inhibiting pathogenic bacteria and in exerting anti-invasive and anti-survival activities with respect to cbLf and wbc. Furthermore, mbLf lowered IL-6 levels to those of uninfected cells, while a less evident decrease was observed upon cbLf treatment. Conversely, wbc managed to slightly lower IL-6 levels compared to those synthesized by infected cells. These data demonstrate that, to obtain maximum effectiveness in such activities, Lf should be formulated/used without addition of other substances and should be sourced from bovine milk rather than colostrum.

Gentamicin loaded niosomes against intracellular uropathogenic Escherichia coli strains.

Urinary tract infections (UTIs) are the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the main etiological agent of UTIs. UPEC can persist in bladder cells protected by immunological defenses and antibiotics and intracellular behavior leads to difficulty in eradicating the infection. The aim of this paper is to design, prepare and characterize surfactant-based nanocarriers (niosomes) able to entrap antimicrobial drug and potentially to delivery and release antibiotics into UPEC-infected cells. In order to validate the proposed drug delivery system, gentamicin, was chosen as "active model drug" due to its poor cellular penetration. The niosomes physical-chemical characterization was performed combining different techniques: Dynamic Light Scattering Fluorescence Spectroscopy, Transmission Electron Microscopy. Empty and loaded niosomes were characterized in terms of size, ζ-potential, bilayer features and stability. Moreover, Gentamicin entrapped amount was evaluated, and the release study was also carried out. In addition, the effect of empty and loaded niosomes was studied on the invasion ability of UPEC strains in T24 bladder cell monolayers by Gentamicin Protection Assay and Confocal Microscopy. The observed decrease in UPEC invasion rate leads us to hypothesize a release of antibiotic from niosomes inside the cells. The optimization of the proposed drug delivery system could represent a promising strategy to significatively enhance the internalization of antimicrobial drugs.

Merkel Cell Polyomavirus in the Context of Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders.

Despite recent advances in prevention, detection and treatment, oral squamous cell carcinoma (OSCC) remains a global health concern, strongly associated with environmental and lifestyle risk factors and infection with oncogenic viruses. Merkel Cell Polyomavirus (MCPyV), well known to be the causative agent of Merkel Cell Carcinoma (MCC) has been found in OSCC, suggesting its potential role as a co-factor in the development of oral cavity cancers. To improve our understanding about MCPyV in oral cavities, the detection and analysis of MCPyV DNA, transcripts and miRNA were performed on OSCCs and oral potentially malignant disorders (OPMDs). In addition, the cellular miR-375, known to be deregulated in tumors, was examined. MCPyV DNA was found in 3 out of 11 OSCC and 4 out of 12 OPMD samples, with a viral mean value of 1.49 × 102 copies/mL. Viral integration was not observed and LTAg and VP1 transcripts were detected. Viral miRNAs were not detected whereas the cellular miR-375 was found over expressed in all MCPyV positive oral specimens. Our results reported evidence of MCPyV replication in both OSCC and OPMD suggesting the oral cavity as a site of replicative MCPyV infection, therefore underscoring an active role of this virus in the occurrence of oral lesions.

Women Skin Microbiota Modifications during Pregnancy.

Several studies have shown fluctuations in the maternal microbiota at various body sites (gut, oral cavity, and vagina). The skin microbiota plays an important role in our health, but studies on the changes during pregnancy are limited. Quantitative and qualitative variations in the skin microbiota in pregnant woman could indeed play important roles in modifying the immune and inflammatory responses of the host. These alterations could induce inflammatory disorders affecting the individual's dermal properties, and could potentially predict infant skin disorder in the unborn. The present study aimed to characterize skin microbiota modifications during pregnancy. For this purpose, skin samples were collected from 52 pregnant women in the first, second, and third trimester of non-complicated pregnancies and from 17 age- and sex-matched healthy controls. The skin microbiota composition was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial rRNA 16S. Our results indicate that from the first to the third trimester of pregnancy, changes occur in the composition of the skin microbiota, microbial interactions, and various metabolic pathways. These changes could play a role in creating more advantageous conditions for fetal growth.

Atopic dermatitis-derived Staphylococcus aureus strains: what makes them special in the interplay with the host.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin condition whose pathogenesis involves genetic predisposition, epidermal barrier dysfunction, alterations in the immune responses and microbial dysbiosis. Clinical studies have shown a link between Staphylococcus aureus and the pathogenesis of AD, although the origins and genetic diversity of S. aureus colonizing patients with AD is poorly understood. The aim of the study was to investigate if specific clones might be associated with the disease. Methods: WGS analyses were performed on 38 S. aureus strains, deriving from AD patients and healthy carriers. Genotypes (i.e. MLST, spa-, agr- and SCCmec-typing), genomic content (e.g. virulome and resistome), and the pan-genome structure of strains have been investigated. Phenotypic analyses were performed to determine the antibiotic susceptibility, the biofilm production and the invasiveness within the investigated S. aureus population. Results: Strains isolated from AD patients revealed a high degree of genetic heterogeneity and a shared set of virulence factors and antimicrobial resistance genes, suggesting that no genotype and genomic content are uniquely associated with AD. The same strains were characterized by a lower variability in terms of gene content, indicating that the inflammatory conditions could exert a selective pressure leading to the optimization of the gene repertoire. Furthermore, genes related to specific mechanisms, like post-translational modification, protein turnover and chaperones as well as intracellular trafficking, secretion and vesicular transport, were significantly more enriched in AD strains. Phenotypic analysis revealed that all of our AD strains were strong or moderate biofilm producers, while less than half showed invasive capabilities. Conclusions: We conclude that in AD skin, the functional role played by S. aureus may depend on differential gene expression patterns and/or on post-translational modification mechanisms rather than being associated with peculiar genetic features.

Genotypic and Phenotypic Characterization of Escherichia coli Isolates Recovered from the Uterus of Mares with Fertility Problems.

Escherichia coli is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 E. coli strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) E. coli strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with fimH detected in all strains, and followed by kpsMTII (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates versus strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into E. coli strains associated with mares' infertility. These results broaden the knowledge of E. coli and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares.

Effect of bovine lactoferrin on recurrent urinary tract infections: in vitro and in vivo evidences.

Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections (UTIs). UPEC strains are able to invade, multiply and persisting in host cells. Therefore, UPEC strains are associated to recurrent UTIs requiring long-term antibiotic therapy. However, this therapy is suboptimal due to the increase of multidrug-resistant UPEC. The use of non-antibiotic treatments for managing UTIs is required. Among these, bovine lactoferrin (bLf), a multifunctional cationic glycoprotein, could be a promising tool because inhibits the entry into the host cells of several intracellular bacteria. Here, we demonstrate that 100 µg/ml bLf hinders the invasion of 2.0 ± 0.5 × 104 CFU/ml E. coli CFT073, prototype of UPEC, infecting 2.0 ± 0.5 × 105 cells/ml urinary bladder T24 epithelial cells. The highest protection (100%) is due to the bLf binding with host surface components even if an additional binding to bacterial surface components cannot be excluded. Of note, in the absence of bLf, UPEC survives and multiplies, while bLf significantly decreases bacterial intracellular survival. After these encouraging results, an observational survey on thirty-three patients affected by recurrent cystitis was performed. The treatment consisted in the oral administration of bLf alone or in combination with antibiotics and/or probiotics. After the observation period, a marked reduction of cystitis episodes was observed (p < 0.001) in all patients compared to the episodes occurred during the 6 months preceding the bLf-treatment. Twenty-nine patients did not report cystitis episodes (87.9%) whereas the remaining four (12.1%) experienced only one episode, indicating that bLf could be a worthwhile and safe treatment in counteracting recurrent cystitis.

Effect of Ciprofloxacin-Loaded Niosomes on Escherichia coli and Staphylococcus aureus Biofilm Formation.

Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli, Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation.

Extraintestinal Pathogenic Escherichia coli: Beta-Lactam Antibiotic and Heavy Metal Resistance.

Multiple-antibiotic-resistant (MAR) extra-intestinal pathogenic Escherichia coli (ExPEC) represents one of the most frequent causes of human nosocomial and community-acquired infections, whose eradication is of major concern for clinicians. ExPECs may inhabit indefinitely as commensal the gut of humans and other animals; from the intestine, they may move to colonize other tissues, where they are responsible for a number of diseases, including recurrent and uncomplicated UTIs, sepsis and neonatal meningitis. In the pre-antibiotic era, heavy metals were largely used as chemotherapeutics and/or as antimicrobials in human and animal healthcare. As with antibiotics, the global incidence of heavy metal tolerance in commensal, as well as in ExPEC, has increased following the ban in several countries of antibiotics as promoters of animal growth. Furthermore, it is believed that extensive bacterial exposure to heavy metals present in soil and water might have favored the increase in heavy-metal-tolerant microorganisms. The isolation of ExPEC strains with combined resistance to both antibiotics and heavy metals has become quite common and, remarkably, it has been recently shown that heavy metal resistance genes may co-select antibiotic-resistance genes. Despite their clinical relevance, the mechanisms underlining the development and spread of heavy metal tolerance have not been fully elucidated. The aim of this review is to present data regarding the development and spread of resistance to first-line antibiotics, such as beta-lactams, as well as tolerance to heavy metals in ExPEC strains.

Resveratrol-Loaded Nanoemulsions: In Vitro Activity on Human T24 Bladder Cancer Cells.

The chemopreventive potential of Resveratrol (RV) against bladder cancer and its mechanism of action have been widely demonstrated. The physicochemical properties of RV, particularly its high reactivity and low solubility in aqueous phase, have been limiting factors for its bioavailability and in vivo efficacy. In order to overcome these limitations, its inclusion in drug delivery systems needs to be taken into account. In particular, oil-in-water (O/W) nanoemulsions (NEs) have been considered ideal candidates for RV encapsulation. Since surfactant and oil composition can strongly influence NE features and their application field, a ternary phase diagram was constructed and evaluated to select a suitable surfactant/oil/water ratio. The selected sample was deeply characterized in terms of physical chemical features, stability, release capability and cytotoxic activity. Results showed a significant decrease in cell viability after the incubation of bladder T24 cancer cells with RV-loaded NEs, compared to free RV. The selected NE formulation was able to preserve and improve RV cytotoxic activity by a more rapid drug uptake into the cells. O/W NEs represent an effective approach to improve RV bioavailability.

Nanoemulsions of Satureja montana Essential Oil: Antimicrobial and Antibiofilm Activity against Avian Escherichia coli Strains.

Satureja montana essential oil (SEO) presents a wide range of biological activities due to its high content of active phytochemicals. In order to improve the essential oil's (EO) properties, oil in water nanoemulsions (NEs) composed of SEO and Tween-80 were prepared, characterized, and their antimicrobial and antibiofilm properties assayed against Escherichia coli strains isolated from healthy chicken. Since surfactant and oil composition can strongly influence NE features and their application field, a ternary phase diagram was constructed and evaluated to select a suitable surfactant/oil/water ratio. Minimal inhibitory concentration and minimal bactericidal concentration of NEs, evaluated by the microdilution method, showed that the SEO NE formulation exhibited higher inhibitory effects against planktonic E. coli than SEO alone. The quantification of biofilm production in the presence of NEs, assessed by crystal violet staining and scanning electron microscopy, evidenced that sub-MIC concentrations of SEO NEs enable an efficient reduction of biofilm production by the strong producer strains. The optimized nanoemulsion formulation could ensure food safety quality, and counteract the antibiotic resistance of poultry associated E. coli, if applied/aerosolized in poultry farms.

16S Metagenomics Reveals Dysbiosis of Nasal Core Microbiota in Children With Chronic Nasal Inflammation: Role of Adenoid Hypertrophy and Allergic Rhinitis.

Allergic rhinitis (AR) and adenoid hypertrophy (AH) are, in children, the main cause of partial or complete upper airway obstruction and reduction in airflow. However, limited data exist about the impact of the increased resistance to airflow, on the nasal microbial composition of children with AR end AH. Allergic rhinitis (AR) as well as adenoid hypertrophy (AH), represent extremely common pathologies in this population. Their known inflammatory obstruction is amplified when both pathologies coexist. In our study, the microbiota of anterior nares of 75 pediatric subjects with AR, AH or both conditions, was explored by 16S rRNA-based metagenomic approach. Our data show for the first time, that in children, the inflammatory state is associated to similar changes in the microbiota composition of AR and AH subjects respect to the healthy condition. Together with such alterations, we observed a reduced variability in the between-subject biodiversity on the other hand, these same alterations resulted amplified by the nasal obstruction that could constitute a secondary risk factor for dysbiosis. Significant differences in the relative abundance of specific microbial groups were found between diseased phenotypes and the controls. Most of these taxa belonged to a stable and quantitatively dominating component of the nasal microbiota and showed marked potentials in discriminating the controls from diseased subjects. A pauperization of the nasal microbial network was observed in diseased status in respect to the number of involved taxa and connectivity. Finally, while stable co-occurrence relationships were observed within both control- and diseases-associated microbial groups, only negative correlations were present between them, suggesting that microbial subgroups potentially act as maintainer of the eubiosis state in the nasal ecosystem. In the nasal ecosystem, inflammation-associated shifts seem to impact the more intimate component of the microbiota rather than representing the mere loss of microbial diversity. The discriminatory potential showed by differentially abundant taxa provide a starting point for future research with the potential to improve patient outcomes. Overall, our results underline the association of AH and AR with the impairment of the microbial interplay leading to unbalanced ecosystems.

Bovine Lactoferrin Pre-Treatment Induces Intracellular Killing of AIEC LF82 and Reduces Bacteria-Induced DNA Damage in Differentiated Human Enterocytes.

LF82, a prototype of adherent-invasive E. coli (AIEC), is able to adhere to, invade, survive and replicate into intestinal epithelial cells. LF82 is able to enhance either its adhesion and invasion by up-regulating carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6), the main cell surface molecule for bacterial adhesion, and its intracellular survival by inducing host DNA damage, thus blocking the cellular cycle. Lactoferrin (Lf) is a multifunctional cationic glycoprotein of natural immunity, exerting an anti-invasive activity against LF82 when added to Caco-2 cells at the moment of infection. Here, the infection of 12 h Lf pre-treated Caco-2 cells was carried out at a time of 0 or 3 or 10 h after Lf removal from culture medium. The effect of Lf pre-treatment on LF82 invasiveness, survival, cell DNA damage, CEACAM-6 expression, apoptosis induction, as well as on Lf subcellular localization, has been evaluated. Lf, even if removed from culture medium, reduced LF82 invasion and survival as well as bacteria-induced DNA damage in Caco-2 cells independently from induction of apoptosis, modulation of CEACAM-6 expression and Lf sub-cellular localization. At our knowledge, this is the first study showing that the sole Lf pre-treatment can activate protective intracellular pathways, reducing LF82 invasiveness, intracellular survival and cell-DNA damages.

Features of uropathogenic Escherichia coli able to invade a prostate cell line.

RWPE-1 normal prostate cells were tested as an experimental model for adhesion/invasion assays by genotypically and phenotypically characterized community uropathogenic strains of Escherichia coli (UPEC), a frequent cause of urinary tract infections (UTIs) and significant etiologic agent also in bacterial prostatitis. Adhesive ability and strong biofilm production was significantly associated with the bacterial invasive phenotype. Invasive strains derived mainly from male and pediatric patients. This study suggests that such a cell model could usefully integrate other available methods of urovirulence analysis, to deepen knowledge on the bacterial interaction with host cells.

Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn's disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features. RESULTS: A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ2 = 6.167 P = 0.013), kpsMT II (χ2 = 6.167 P = 0.013), fyuA (χ2 = 6.167 P = 0.013), and ibeA (χ2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls. CONCLUSIONS: The identification of AIEC strains in both CD and non-IBD controls, confirmed the "pathobiont" nature of AIEC strains. The finding that AIEC-like isolates were more abundant in CD patients, indicates that a close association of these strains with CD may also exists in pediatric patients.