Innovative Phospholipid Carriers: A Viable Strategy to Counteract Antimicrobial Resistance.

The overuse and misuse of antibiotics have led to the emergence and spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) bacteria strains, usually associated with poorer patient outcomes and higher costs. In order to preserve the usefulness of these life-saving drugs, it is crucial to use them appropriately, as also recommended by the WHO. Moreover, innovative, safe, and more effective approaches are being investigated, aiming to revise drug treatments to improve their pharmacokinetics and distribution and to reduce the onset of drug resistance. Globally, to reduce the burden of antimicrobial resistance (AMR), guidelines and indications have been developed over time, aimed at narrowing the use and diminishing the environmental spread of these life-saving molecules by optimizing prescriptions, dosage, and times of use, as well as investing resources into obtaining innovative formulations with better pharmacokinetics, pharmacodynamics, and therapeutic results. This has led to the development of new nano-formulations as drug delivery vehicles, characterized by unique structural properties, biocompatible natures, and targeted activities such as state-of-the-art phospholipid particles generally grouped as liposomes, virosomes, and functionalized exosomes, which represent an attractive and innovative delivery approach. Liposomes and virosomes are chemically synthesized carriers that utilize phospholipids whose nature is predetermined based on their use, with a long track record as drug delivery systems. Exosomes are vesicles naturally released by cells, which utilize the lipids present in their cellular membranes only, and therefore, are highly biocompatible, with investigations as a delivery system having a more recent origin. This review will summarize the state of the art on microvesicle research, liposomes, virosomes, and exosomes, as useful and effective tools to tackle the threat of antibiotic resistance.

Efficacy and Microbiota Modulation Induced by LimpiAL 2.5%, a New Medical Device for the Inverse Psoriasis Treatment.

(1) Inverse psoriasis (IP), also known as intertriginous, typically affects the groin, armpits, navel, intergluteal fissure, and external genitalia. Skin lesions are erythematous plaques of inflammatory nature, smooth, well-delimited, non-scaly, and non-infiltrated. Lesions may be accompanied by itching, pain, or burning sensation. The aim of this study is both to investigate the modulation of the skin microbiota induced by IP and, on the other hand, to test the effectiveness of the new biotechnological product LimpiAL 2.5%. (2) Patients affected by IP were recruited in a private practice and treated for 4 weeks with LimpiAL 2.5% exclusively. The clinical effects on the lesion skin were evaluated, and the skin microbiotas before and after treatment were compared. (3) The clinical outcomes reveled a significant beneficial effect of the tested product. At the same time, LimpiAL increased the biological diversity of the skin microbiota and exerted a significant decrease of some Corynebacterium species, and the increase of some Staphylococcus species. (4) Together, the clinical outcomes and the microbiota analysis suggest that LimpiAL treatment improves the skin condition of affected patients, basically restoring the eubiosis conditions of the affected sites and modulating the bacterial composition of the resident microbiota.

A Wall Fragment of Cutibacterium acnes Preserves Junctional Integrity Altered by Staphylococcus aureus in an Ex Vivo Porcine Skin Model.

(1) Background alteration of the skin microbiota, dysbiosis, causes skin barrier impairment resulting in disease development. Staphylococcus aureus, the main pathogen associated with dysbiosis, secretes several virulence factors, including α-toxin that damages tight junctions and compromises the integrity of the skin barrier. The use of members of the resident microbiota to restore the skin barrier, bacteriotherapy, represents a safe treatment for skin conditions among innovative options. The aim of this study is the evaluation of a wall fragment derived from a patented strain of Cutibacterium acnes DSM28251 (c40) alone and conjugated to a mucopolysaccharide carrier (HAc40) in counteracting S. aureus pathogenic action on two tight junction proteins (Claudin-1 and ZO-1) in an ex vivo porcine skin infection model. Methods: skin biopsies were infected with live S. aureus strains ATCC29213 and DSM20491. Tissue was pre-incubated or co-incubated with c40 and HAc40. (3) Results: c40 and HAc40 prevent and counteract Claudin-1 and Zo-1 damage (4) Conclusions: c40 and the functional ingredient HAc40 represent a potential non-pharmacological treatment of skin diseases associated with cutaneous dysbiosis of S. aureus. These findings offer numerous avenues for new research.

Emerging Evidence on Tenebrio molitor Immunity: A Focus on Gene Expression Involved in Microbial Infection for Host-Pathogen Interaction Studies.

In recent years, the scientific community's interest in T. molitor as an insect model to investigate immunity and host-pathogen interactions has considerably increased. The reasons for this growing interest could be explained by the peculiar features of this beetle, which offers various advantages compared to other invertebrates models commonly used in laboratory studies. Thus, this review aimed at providing a broad view of the T. molitor immune system in light of the new scientific evidence on the developmental/tissue-specific gene expression studies related to microbial infection. In addition to the well-known cellular component and humoral response process, several studies investigating the factors associated with T. molitor immune response or deepening of those already known have been reported. However, various aspects remain still less understood, namely the possible crosstalk between the immune deficiency protein and Toll pathways and the role exerted by T. molitor apolipoprotein III in the expression of the antimicrobial peptides. Therefore, further research is required for T. molitor to be recommended as an alternative insect model for pathogen-host interaction and immunity studies.

The new invasive mosquito species Aedes koreicus as vector-borne diseases in the European area, a focus on Italian region: What we know from the scientific literature.

The increased mobility of goods, people, and animals worldwide has caused the spread of several arthropod vectors, leading to an increased risk of animal and human infections. Aedes koreicus is a common species in South Korea, China, Japan, and Russia. Due to its cold-resistant dormant eggs, the adults last from the late summer until the autumn seasons. For these reasons, it seems to be better adapted to colder temperatures, favoring its colonization of hilly and pre-alpine areas. Its first appearance in Europe was in 2008 in Belgium, where it is currently established. The species was subsequently detected in Italy in 2011, European Russia, Germany, the Swiss-Italian border region, Hungary, Slovenia, Crimea, Austria, the Republic of Kazakhstan, and the Netherlands. The role of A. koreicus in the transmission of vector-borne pathogens remains unclear. The available scientific evidence is very old, often not available in English or not indexed in international databases, and therefore difficult to find. According to the literature reviewed, A. koreicus can be considered a new invasive mosquito species in Europe, establishing populations on the European continent. In addition, experimental evidence demonstrated its vector competence for both Dirofilaria immitis and Chikungunya and is relatively low for ZIKA but not for Western Nile Virus. On the other hand, even if the field evidence does not confirm the experimental findings, it is currently not possible to exclude with absolute certainty the potential involvement of this species in the spread, emergence, or re-emergence of these vector-borne disease agents.

Anti-Candidal Activity of the Parasitic Plant Orobanche crenata Forssk.

Candida albicans (C. albicans) and Candida glabrata (C. glabrata) are part of the human microbiome. However, they possess numerous virulence factors, which confer them the ability to cause both local and systemic infections. Candidiasis can involve multiple organs, including the eye. In the present study, we investigated the anti-candidal activity and the re-epithelizing effect of Orobanche crenata leaf extract (OCLE). By the microdilution method, we demonstrated an inhibitory effect of OCLE on both C. albicans and C. glabrata growth. By crystal violet and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we showed the ability of OCLE to inhibit the biofilm formation and the viability of yeast cells, respectively. By germ tube and adhesion assays, we proved the capacity of OCLE to affect the morphological transition of C. albicans and the adhesion of both pathogens to human retinal pigment epithelial cells (ARPE-19), respectively. Besides, by MTT and wound healing assay, we evaluated the cytotoxic and re-epithelizing effects of OCLE on ARPE-19. Finally, the Folin-Ciocalteu and the ultra-performance liquid chromatography-tandem mass spectrometry revealed a high content of phenols and the presence of several bioactive molecules in the extract. Our results highlighted new properties of O. crenata, useful in the control of Candida infections.

Potential Mucosal Irritation Discrimination of Surface Disinfectants Employed against SARS-CoV-2 by Limacus flavus Slug Mucosal Irritation Assay.

Preventive measures have proven to be the most effective strategy to counteract the spread of the SARS-CoV-2 virus. Among these, disinfection is strongly suggested by international health organizations' official guidelines. As a consequence, the increase of disinfectants handling is going to expose people to the risk of eyes, mouth, nose, and mucous membranes accidental irritation. To assess mucosal irritation, previous studies employed the snail Arion lusitanicus as the mucosal model in Slug Mucosal Irritation (SMI) assay. The obtained results confirmed snails as a suitable experimental model for their anatomical characteristics superimposable to the human mucosae and the different easily observed readouts. Another terrestrial gastropod, Limacus flavus, also known as " Yellow slug ", due to its larger size and greater longevity, has already been proposed as an SMI assay alternative model. In this study, for the first time, in addition to the standard parameters recorded in the SMI test, the production of yellow pigment in response to irritants, unique to the snail L. flavus, was evaluated. Our results showed that this species would be a promising model for mucosal irritation studies. The study conducted testing among all those chemical solutions most commonly recommended against the SARS-CoV-2 virus.

Atopic Dermatitis as a Multifactorial Skin Disorder. Can the Analysis of Pathophysiological Targets Represent the Winning Therapeutic Strategy?

Atopic dermatitis (AD) is a pathological skin condition with complex aetiological mechanisms that are difficult to fully understand. Scientific evidence suggests that of all the causes, the impairment of the skin barrier and cutaneous dysbiosis together with immunological dysfunction can be considered as the two main factors involved in this pathological skin condition. The loss of the skin barrier function is often linked to dysbiosis and immunological dysfunction, with an imbalance in the ratio between the pathogen Staphylococcus aureus and/or other microorganisms residing in the skin. The bibliographic research was conducted on PubMed, using the following keywords: 'atopic dermatitis', 'bacterial therapy', 'drug delivery system' and 'alternative therapy'. The main studies concerning microbial therapy, such as the use of bacteria and/or part thereof with microbiota transplantation, and drug delivery systems to recover skin barrier function have been summarized. The studies examined show great potential in the development of effective therapeutic strategies for AD and AD-like symptoms. Despite this promise, however, future investigative efforts should focus both on the replication of some of these studies on a larger scale, with clinical and demographic characteristics that reflect the general AD population, and on the process of standardisation, in order to produce reliable data.

In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model.

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Comparison of Cell Culture with Three Conventional Polymerase Chain Reactions for Detecting Chlamydophila pneumoniae in Adult's Pharyngotonsillitis.

Chlamydophila pneumoniae is an intracellular pathogen responsible for respiratory tract infections. The isolation of the microorganism from clinical specimens is essential for a diagnosis. However, the identification of C. pneumoniae by cell cultures is very difficult besides strongly depending on the sample conditions. The study aimed to investigate, in adult patients with pharyngotonsillitis, the frequency of Chlamydophila pneumoniae detection by cell cultures and three conventional PCRs (a conventional PCR targeting the 16S rRNA gene and two nested PCRs, targeting the 16S rRNA gene and the ompA gene, respectively). The presence of chlamydial inclusion in cell cultures was observed in 11/94 samples (11.70%) by IFA. C. pneumoniae DNA was detected in 12/94 (12.76%) specimens by the 16S rRNA gene nested PCR, 4/94 (4.26%) by ompA gene nested PCR, and in 2/94 (2.13%) by 16S rRNA single-step PCR. Our data show poor agreement between the three applied DNA-amplification methods; in fact, only 16S rRNA gene nested PCR showed a statistically significant difference. Moreover, this result allowed us to achieve a definitive confirmation of the previous finding and to avoid the risk of an overestimation of the C. pneumoniae as a pathogen in pharyngotonsillitis.

Preliminary in Vitro Studies on Corynebacterium urealyticum Pathogenetic Mechanisms, a Possible Candidate for Chronic Idiopathic Prostatitis?

Corynebacterium urealyticum is a well-known opportunistic uropathogen that can occur with cystitis, pyelonephritis, and urinary sepsis. Although a wide variety of coryneform bacteria have been found from the male genital tract of prostatitis patients, only one clinical case of prostatitis caused by C. urealyticum has been reported. The aim of this study was to evaluate the in vitro tropism of C. urealyticum towards LNCaP (lymph node carcinoma of the prostate) human cells line and the influence of acetohydroxamic acid as an irreversible urease inhibitor on different aspects of its pathogenicity by means of several in vitro tests, such as the determination and analysis of growth curves, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the production of biofilms, and adhesion to LNCaP and HeLa cell lines. Results have brought new pieces of evidence on the in vitro tropism of C. urealyticum for the human prostate cell line LNCaP and the therapeutic use of the irreversible urease inhibitors such as acetohydroxamic acid (AHA), not only as enzyme blockers to facilitate the removal of encrustations but also as modulators of some pathogenic mechanisms. These interesting preliminary data allow us to assert that there is a real possibility that C. urealyticum is a new candidate for chronic idiopathic prostatitis.

Do Ocular Fluids Represent a Transmission Route of SARS-CoV-2 Infection?

The spread of the new SARS-CoV-2 is marked by a short timeline. In this scenario, explaining or excluding the possible transmission routes is mandatory to contain and manage the spread of the disease in the community. In the recent pandemic, it is still unclear how coronavirus can end up in ocular fluids. Nevertheless, eye redness and irritation in COVID-19 patients have been reported, suggesting that a possible ocular manifestation of SARS-CoV-2 infection may be conjunctivitis. On the basis of epidemiological data provided by previous SARS-Cove infection, numerous theories have been proposed: (1) conjunctiva as the site of direct inoculation by infected droplets; (2) the nasolacrimal duct as a migration route of the virus to the upper respiratory tract, or (3) haematogenic infection of the tear gland. The demand for further investigations to verify ocular involvement in COVID-19 infection came out from the results of recent meta-analysis studies, so the eye cannot be completely excluded as a transmission route of the infection. Thus, healthcare personnel and all the people that enter in contact with infected or suspected patients must always use the prescribed protective equipment.

Galleria mellonella as a consolidated in vivo model hosts: New developments in antibacterial strategies and novel drug testing.

A greater ethical conscience, new global rules and a modified perception of ethical consciousness entail a more rigorous control on utilizations of vertebrates for in vivo studies. To cope with this new scenario, numerous alternatives to rodents have been proposed. Among these, the greater wax moth Galleria mellonella had a preponderant role, especially in the microbiological field, as demonstrated by the growing number of recent scientific publications. The reasons for its success must be sought in its peculiar characteristics such as the innate immune response mechanisms and the ability to grow at a temperature of 37°C. This review aims to describe the most relevant features of G. mellonella in microbiology, highlighting the most recent and relevant research on antibacterial strategies, novel drug tests and toxicological studies. Although solutions for some limitations are required, G. mellonella has all the necessary host features to be a consolidated in vivo model host.

Antioxidant and antimicrobial properties of Casteanea sativa Miller chestnut honey produced on Mount Etna (Sicily).

The aim of this study was the evaluation of antibacterial and antioxidant properties of Monofloral Etna Castanea sativa Miller honeys. Escherichia coli ATCC 25,922, Pseudomonas aeruginosa ATCC 27,853, Enterococcus faecalis ATCC 29,211 and Staphylococcus aureus ATCC 29,213 were investigated for their susceptibilities to two different honeys. Antioxidant activity was evaluated by ORAC, NO scavenger assays, FRAP and DPPH. Antioxidant activity and antibacterial properties were compared with chestnut honeys from different geographical areas and with Manuka honey. UPLC-MS/MS was used for major components characteri sation.

A Method for Efficient Loading of Ciprofloxacin Hydrochloride in Cationic Solid Lipid Nanoparticles: Formulation and Microbiological Evaluation.

The aim of the study was the production of solid lipid nanoparticles (SLN) loaded with ciprofloxacin (CIP) through two different production techniques, quasi-emulsion solvent diffusion (QESD) and solvent injection (SI). In order to efficaciously entrap the commercial salt form (hydrochloride) of the antibiotic in these lipid systems, a conversion of CIP hydrochloride to the free base was realized in situ, through the addition of triethylamine. To ensure physical stability to the carriers over time and ameliorate the interaction with bacterial cell membranes, positively charged SLN were produced by addition of the cationic lipid didecyldimethylammonium bromide (DDAB). Homogeneous SLN populations with a mean particle sizes of 250⁻350 nm were produced by both methods; drug encapsulation was over 85% for most samples. The SLN were physically stable for up to nine months both at 4 °C and 25 °C, although the former condition appears more suitable to guarantee the maintenance of the initial particle size distribution. As expected, CIP encapsulation efficiency underwent a slight reduction after nine months of storage, although the initial high drug content values would ensure a residual concentration of the antibiotic in the SLN still appropriate to exert an acceptable antibacterial activity. Selected SLN formulations were subjected to an in vitro microbiological assay against different bacterial strains, to verify the effect of nanoencapsulation on the cell growth inhibitory activity of CIP. In general, CIP-SLN produced without DDAB showed MIC values for CIP comparable to those of the free drug. Conversely, addition of increasing percentages of the cationic lipid, reflected by a progressive increase of the positive value of the Zeta potential, showed a variety of MIC values against the various bacterial strains, but with values 2⁻4 order of dilution lower than free CIP. An hypothesis of the effect of the cationic lipid upon the increased antibacterial activity of CIP in the nanocarriers is also formulated.

Vitamin E for Prevention of Biofilm-caused Healthcare-associated Infections.

The healthcare-associated infections (HCAIs) occur in patients both in nosocomial environments and in community. More often HCAIs are associated to the use of medical devices and bacterial biofilm development on these equipments. Due to the clinical and economic relevance of this topic, new strategies for the treatment of infections caused by biofilm proliferation are unceasingly searched by scientists. The present study investigated the role of vitamin E to reduce the biofilm formation for a larger panel of human pathogens, including strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Pseudomonas aeruginosa and Pseudomonas putida. This potential activity was tested by placing a preparation of vitamin E (α-Tocopheryl acetate) as interface between the bacterial culture and the polystyrene walls of a 96 well plate at different concentrations of glucose, used as a biofilm enhancer. The Staphylococcus genus was further investigated by spreading the vitamin E on a silicone catheter lumen and evaluating its influence on the bacterial colonization. From our results, vitamin E has been able to interfere with bacterial biofilm and prevent in vitro biofilm formation. Furthermore, the ability of Staphylococcus aureus and Staphylococcus epidermidis to colonize the catheter surface decreased as a result of vitamin E application.

Antimicrobial and Anti-Proliferative Effects of Skin Mucus Derived from Dasyatis pastinaca (Linnaeus, 1758).

Resistance to chemotherapy occurs in various diseases (i.e., cancer and infection), and for this reason, both are very difficult to treat. Therefore, novel antimicrobial and chemotherapic drugs are needed for effective antibiotic therapy. The aim of the present study was to assess the antimicrobial and anti-proliferative effects of skin mucus derived from Dasyatis pastinaca (Linnaeus, 1758). Our results showed that skin mucus exhibited a significant and specific antibacterial activity against Gram-negative bacteria but not against Gram-positive bacteria. Furthermore, we also observed a significant antifungal activity against some strains of Candida spp. Concerning anti-proliferative activity, we showed that fish mucus was specifically toxic for acute leukemia cells (HL60) with an inhibition of proliferation in a dose dependent manner (about 52% at 1000 µg/mL of fish skin mucous, FSM). Moreover, we did not observe effects in healthy cells, in neuroblastoma cells (SH-SY5Y), and multiple myeloma cell lines (MM1, U266). Finally, it exhibited strong expression and activity of chitinase which may be responsible, at least in part, for the aforementioned results.

Key Roles of Human Polymorphonuclear Cells and Ciprofloxacin in Lactobacillus Species Infection Control.

Lactobacilli have the potential to act as reservoirs of antibiotic resistance genes similar to those found in human pathogens, with the risk of transferring these genes to many pathogenic bacteria. In this study, we investigated the role of human polymorphonuclear cells (PMNs) against Lactobacillus spp. both resistant and susceptible to ciprofloxacin (a fluoroquinolone) and the effect of ciprofloxacin on the interaction between PMNs and three Lactobacillus spp. with different patterns of susceptibility to this drug. Hence, the primary functions of PMNs, such as phagocytosis and bacterial intracellular killing, against lactobacilli were investigated. The rate of PMN phagocytosis was high for ciprofloxacin-sensitive and ciprofloxacin-resistant strains. The patterns of intracellular killing of ciprofloxacin-sensitive and ciprofloxacin-resistant strains by PMNs underline that PMNs alone were able to kill lactobacilli. The addition of ciprofloxacin to PMNs did not result in a significant increase in the bacterial uptake by phagocytes. On the contrary, ciprofloxacin had a marked effect on PMN intracellular killing, resulting in increased numbers of killed ciprofloxacin-sensitive bacteria in comparison with antibiotic-free controls. Our data show that by itself, the profile of antibiotic resistance does not constitute an intrinsic factor of greater or lesser pathogenicity toward the host. The ability of PMNs to kill a diverse array of bacterial pathogens is essential for human innate host defense, primarily in immunocompromised patients.