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Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure., (© 2024. The Author(s).)

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns., Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae , a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation., Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species., Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 La Rosa, Varotto-Boccazzi, Saresella, Marventano, Cattaneo, Hernis, Piancone, Otranto, Epis, Bandi and Clerici.)

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigate the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses reveal a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets reveals only a partial habitat separation of the clusters. Globally, two clusters only show a separate gene composition coherent with ecological adaptations. These results suggest that genetic isolation has preceded ecological adaptations in the shaping of the species diversity, an evolutionary scenario coherent with the Evolutionary Extended Synthesis., (© 2024. The Author(s).)

Both viral infection and vaccination affect the antibody repertoire of a person. Here, we demonstrate that the analysis of serum antibodies generates information not only on the virus type that caused the infection but also on the specific virus variant. We developed a rapid multiplex assay providing a fingerprint of serum antibodies against five different SARS-CoV-2 variants based on a microarray of virus antigens immobilized on the surface of a label-free reflectometric biosensor. We analyzed serum from the plasma of convalescent subjects and vaccinated volunteers and extracted individual antibody profiles of both total immunoglobulin Ig and IgA fractions. We found that Ig level profiles were strongly correlated with the specific variant of infection or vaccination and that vaccinated subjects displayed a larger quantity of total Ig and a lower fraction of IgA relative to the population of convalescent unvaccinated subjects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Carzaniga, Casiraghi, Nava, Zanchetta, Inzani, Chiari, Bollati, Epis, Bandi, Lai, Zehender, Bellini and Buscaglia.)

Here, we present a computational protocol to perform a spatiotemporal reconstruction of an epidemic. We describe steps for using epidemiological data to depict how the epidemic changes over time and for employing clustering analysis to group geographical units that exhibit similar temporal epidemic progression. We then detail procedures for analyzing the temporal and spatial dynamics of the epidemic within each cluster. This protocol has been developed to be used on historical data but could also be applied to modern epidemiological data. For complete details on the use and execution of this protocol, please refer to Galli et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mosquito control is of paramount importance, in particular, in light of the major environmental alterations associated with human activities, from climate change to the altered distribution of pathogens, including those transmitted by Arthropods. Here, we used the common house mosquito, Culex pipiens to test the efficacy of MosChito raft, a novel tool for mosquito larval control. MosChito raft is a floating hydrogel matrix, composed of chitosan, genipin and yeast cells, as bio-attractants, developed for the delivery of a Bacillus thuringiensis israeliensis (Bti)-based bioinsecticide to mosquito larvae. To this aim, larvae of Cx. pipiens were collected in field in Northern Italy and a novel colony of mosquito species (hereafter: Trescore strain) was established. MosChito rafts, containing the Bti-based formulation, were tested on Cx. pipiens larvae from the Trescore strain to determine the doses to be used in successive experiments. Thus, bioassays with MosChito rafts were carried out under semi-field conditions, both on larvae from the Trescore strain and on pools of larvae collected from the field, at different developmental stages. Our results showed that MosChito raft is effective against Cx. pipiens. In particular, the observed mortality was over 50% after two days exposure of the larvae to MosChito rafts, and over 70-80% at days three to four, in both laboratory and wild larvae. In conclusion, our results point to the MosChito raft as a promising tool for the eco-friendly control of a mosquito species that is not only a nuisance insect but is also an important vector of diseases affecting humans and animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Negri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Mosquito bite is normally associated with mild allergic responses, but severe localized or systemic reactions are also possible. Reliable tools for the diagnosis of mosquito allergy are still unavailable. Here, we investigated the IgE response to 3 potential salivary allergens identified in the saliva of the tiger mosquito Aedes albopictus ., Methods: Serum from 55 adult individuals (28 controls and 27 allergic people), were analysed using an in-house Enzyme Linked ImmunoSorbent Assay (ELISA) against the Salivary Gland Extract (SGE) and the recombinant proteins alb D7l2 (Aed al 2), alb Antigen5-3 (Aed al 13) and alb LIPS-2 (Aed al 14)., Results: Fifteen of the 27 (56%) individuals having hypersensitive reactions to mosquito bites had IgE serum levels recognizing SGE. Negative sera did not show detectable levels of IgE targeting the SGE from the most common sympatric mosquito Culex pipiens. Among the positive individuals, 2 subjects displayed IgE targeting Aed al 2 (13%), while IgE recognizing Aed al 13 and Aed al 14 were detected in ten (67%) and seven (47%) individuals, respectively. Two sera from non-hypersensitive subjects had detectable levels of IgE targeting Aed al 13, suggesting possible cross-reaction with the homologue salivary proteins of multiple mosquito species or, more generally, of hematophagous insects., Conclusions: Our results indicate that Aed al 13 and Aed al 14 hold the potential to be developed as tools for the diagnosis of allergy to Ae. albopictus bites. Such tools would facilitate epidemiological studies on tiger mosquito allergy in humans and might foster the development of further protein-based assays to investigate cross-species allergies., Competing Interests: The authors report no competing interests., (© 2023 The Author(s).)

Background: Visceral leishmaniosis caused by infection with the zoonotic protozoan Leishmania infantum is a life-threatening disease affecting dogs and humans. The sympatric occurrence of L. infantum and Leishmania tarentolae in an area of southern Italy endemic for canine leishmaniosis, where dogs are also exposed to the latter species, suggests the persistence of herpetophilic L. tarentolae in a non-permissive host, therefore raising questions about the performance of serological diagnostic tests routinely employed., Methods: The diagnostic performance of serological tests such as the immunofluorescence antibody test (IFAT), two commercial immunoenzymatic assays (i.e. NovaTec VetLine Leishmania ELISA® and rK39 ICT®) and an in-house enzyme-linked immunosorbent assay (ELISA) was evaluated in healthy dogs seropositive to L. infantum, whereas the only IFAT available was used to detect antibodies to L. tarentolae., Results: With the IFAT, out of a total of 104 dogs tested, 15 were seronegative for L. infantum of which three were L. tarentolae seropositive' and 89 were L. infantum seropositive. Of the latter 89 dogs, representing the highest proportion of seropositive animals (85.6%) detected by IFAT' 66 were also seropositive for L. tarentolae. Cohen's kappa (κ) agreement coefficient between the IFAT results and those of all the other tests was very low, and the IFAT results were significantly different from those of all the other serological tests as calculated by Cochran's Q-test. Analysis using the Bayesian latent class (Bayes-LCA) showed that the in-house ELISA and IFAT contributed the most towards identifying infected and non-infected dogs, respectively. The IFAT test showed low positive predictive value (59.5%), but high negative predictive value (100%)., Conclusions: These results demonstrate that the IFAT for L. infantum, although highly sensitive, may not be considered a useful diagnostic test due to its low specificity. Therefore, an accurate serological tool with high specificity is mandatory for avoiding cross-reaction in epidemiological contexts where the two species of Leishmania occur in sympatry., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The genus Leptoconops Skuse (Diptera: Ceratopogonidae) are blood-sucking midges known to pester humans and domestic animals. In certain Mediterranean areas, midges occur in large numbers during summer and limit the use of recreational areas, also raising serious health and social concerns. Despite such impact, the diversity and distribution of Leptoconops in Maremma Regional Park (Tuscany Region, Italy), a heavily infested area, is not well known, and neither molecular nor detailed morphological studies exist. We sampled adult midge females in six areas and used high-resolution digital stereomicroscopy and scanning electron microscopy to identify species and investigate the morphology of structures involved in host searching/recognition (antennae and maxillary palps) and host attack (mouthparts). We also performed energy-dispersive X-ray spectroscopy to characterize the elemental composition of mouthparts. Finally, the cytochrome c oxidase subunit 1 ( cox 1) gene was amplified and sequenced, to confirm species identification of collected specimens. We identified two species: Leptoconops ( L .) irritans Noé and Leptoconops ( L. ) noei Clastrier & Coluzzi, with the former being more frequently sampled than the latter and closer to sea coast and rivers. The antennal segments appeared slightly more globular in L. noei than in L. irritans . Five types of trichoid, basiconic and chaetic sensilla were found on the antennae, with some differences between the two species. Mouthparts had the labellum visibly larger in L. noei compared with L. irritans . The maxillary palps possessed a pit filled with bulb-shaped sensilla, which appeared denser in L. noei than in L. irritans . Mouthpart cuticle included Calcium (Ca) and Aluminum (Al) at small but significant concentrations (0.3-1.0%) in both species. Our results suggest that the limited but appreciable differences in sensory system between the studied species of Leptoconops and other Ceratopogonidae may reflect different host or habitat preferences, a scenario potentially suggested also by preliminarily data on their distribution in the studied area. The presence of Ca and Al in the cuticle of mouthparts may help host skin drilling during bite activity. Finally, the gene sequences obtained in this study provide a first reference for future investigations on the taxonomy and dispersal patterns of Leptoconops spp. in the Mediterranean area., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Summary: Bacterial Healthcare-Associated Infections (HAIs) are a major threat worldwide, which can be counteracted by establishing effective infection control measures, guided by constant surveillance and timely epidemiological investigations. Genomics is crucial in modern epidemiology but lacks standard methods and user-friendly software, accessible to users without a strong bioinformatics proficiency. To overcome these issues we developed P-DOR, a novel tool for rapid bacterial outbreak characterization. P-DOR accepts genome assemblies as input, it automatically selects a background of publicly available genomes using k-mer distances and adds it to the analysis dataset before inferring a Single-Nucleotide Polymorphism (SNP)-based phylogeny. Epidemiological clusters are identified considering the phylogenetic tree topology and SNP distances. By analyzing the SNP-distance distribution, the user can gauge the correct threshold. Patient metadata can be inputted as well, to provide a spatio-temporal representation of the outbreak. The entire pipeline is fast and scalable and can be also run on low-end computers., Availability and Implementation: P-DOR is implemented in Python3 and R and can be installed using conda environments. It is available from GitHub https://github.com/SteMIDIfactory/P-DOR under the GPL-3.0 license., (© The Author(s) 2023. Published by Oxford University Press.)

The mucosal immune system plays a pivotal role in the control of infections, as it represents the first line of defense against most pathogens, from respiratory viruses to intestinal parasites. Mucosal vaccination is thus regarded as a promising strategy to protect animals, including humans, from infections that are acquired by ingestion, inhalation or through the urogenital system. In addition, antigens delivered at the mucosal level can also elicit systemic immune responses. Therefore, mucosal vaccination is potentially effective also against systemic infections acquired through non-mucosal routes, for example, through the bite of hematophagous insects, as in the case of leishmaniasis, a widespread disease that affects humans and dogs. Here, we explored the potential of antigen rectal administration for the generation of anti- Leishmania immunity. Mice were immunized through rectal administration of whole cells of the model parasite Leishmania tarentolae (using a clone engineered to express the spike protein of the SARS-CoV-2 virus generated in a previous study). A specific anti- Leishmania IgG antibody response was detected. In addition, the recorded IgG2a/IgG1 ratio was higher than that of animals injected subcutaneously; therefore, suggesting a shift to a Th1-biased immune response. Considering the importance of a Th1 polarization as a protective response against Leishmania infections, we suggest that further investigation should be focused on the development of novel types of vaccines against these parasites based on rectal immunization.

Background: Aedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions., Methods: Available raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers., Results: Mosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples., Conclusions: The results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas., (© 2023. The Author(s).)

Currently, the mechanisms underlying sensory perception and sensory performance in children with food allergies are far from being understood. As well, only recently, single research afforded the oral host-commensal milieu, addressing oral microbial communities in children with peanut allergies. To bridge the current gaps in knowledge both in the sensory and microbial fields, a psychophysiological case-control study was performed in allergic children (n = 29) and a healthy sex-age-matched control group (n = 30). Taste perception, food neophobia, and liking were compared in allergic and non-allergic children. The same subjects were characterized for their oral microbiota composition by addressing saliva to assess whether specific profiles were associated with the loss of oral tolerance in children with food allergies. Our study evidenced an impaired ability to correctly identify taste qualities in the allergic group compared to controls. These results were also consistent with anatomical data related to the fungiform papillae on the tongue, which are lower in number in the allergic group. Furthermore, distinct oral microbial profiles were associated with allergic disease, with significant down-representations of the phylum Firmicutes and of the genera Veillonella spp., Streptococcus spp., Prevotella spp., and Neisseria spp. For the first time, this study emphasizes the link between sensory perception and food allergy, which is a novel and whole-organism view of this pathology. Our data indicated that an impaired taste perception, as regards both functionality and physiologically, was associated with food allergy, which marginally influences the food neophobia attitude. It is also accompanied by compositional shifts in oral microbiota, which is, in turn, another actor of this complex interplay and is deeply interconnected with mucosal immunity. This multidisciplinary research will likely open exciting new approaches to therapeutic interventions., (© 2023. The Author(s).)

In 1630, a devastating plague epidemic struck Milan, one of the most important Italian cities of that time, deeply affecting its demography and economy for decades. The lack of digitized historical data strongly limits our comprehension of that important event. In this work, we digitized and analyzed the Milan death registers of 1630. The study revealed that the epidemic evolved differently among the areas of the city. Indeed, we were able to group the parishes of the city (comparable with modern neighborhoods) in two groups based on their epidemiological curves. These different epidemiological progressions could reflect socio-economical and/or demographic features specific of the neighborhoods, opening questions about the relationship between these features and the evolution of epidemics in the pre-modern period. The study of historical records, like the one presented here, can help us to better understand European history and pre-modern epidemics., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Wolbachia is a Gram-negative endosymbiont associated with several species of arthropods and filarioid nematodes, including Dirofilaria immitis. This endosymbiont may elicit a Th1 response, which is a component of the immunity against Leishmania infantum., Methods: To investigate the interactions between Wolbachia of D. immitis and L. infantum in naturally infected dogs and cytokine circulation, dogs without clinical signs (n = 187) were selected. Dogs were tested for microfilariae (mfs) by Knott, for female antigens of D. immitis by SNAP, and for anti-L. infantum antibodies by IFAT and assigned to four groups. Dogs of group 1 (G1) and 2 (G2) were positive for D. immitis and positive or negative to L. infantum, respectively. Dogs of group 3 (G3) and 4 (G4) were negative to D. immitis and positive or negative to L. infantum, respectively. Wolbachia and L. infantum DNA was quantified by real-time PCR (qPCR) in dog blood samples. A subset of dogs (n = 65) was examined to assess pro- and anti-inflammatory cytokine production using an ELISA test., Results: Of 93 dogs positive to D. immitis with circulating mfs, 85% were positive to Wolbachia, with the highest amount of DNA detected in G1 and the lowest in dogs with low mfs load in G1 and G2. Among dogs positive to L. infantum, 66% from G1 showed low antibody titer, while 48.9% from G3 had the highest antibody titer. Of 37 dogs positive to Wolbachia from G1, 26 (70.3%) had low antibody titers to L. infantum (1:160). Among cytokines, TNFα showed the highest mean concentration in G1 (246.5 pg/ml), IFNγ being the one most represented (64.3%). IL-10 (1809.5 pg/ml) and IL-6 (123.5 pg/ml) showed the highest mean concentration in dogs from G1. A lower percentage of dogs producing IL-4 was observed in all groups examined, with the highest mean concentration (2794 pg/ml) recorded in G2., Conclusion: Results show the association of D. immitis and Wolbachia with the lower antibody titers of L. infantum in co-infected dogs, suggesting the hypothesis that the endosymbiont may affect the development of the patent leishmaniosis. However, due to the limitations associated with the heterogeneity of naturally infected dogs in field conditions, results should be validated by investigation on experimental models., (© 2023. The Author(s).)

Adult mosquito females, through their bites, are responsible for the transmission of different zoonotic pathogens. Although adult control represents a pillar for the prevention of disease spread, larval control is also crucial. Herein we characterized the effectiveness of a suitable tool, named "MosChito raft", for the aquatic delivery of a Bacillus thuringiensis var. israelensis (Bti) formulate, a bioinsecticide active by ingestion against mosquito larvae. MosChito raft is a floating tool composed by chitosan cross-linked with genipin in which a Bti-based formulate and an attractant have been included. MosChito rafts (i) resulted attractive for the larvae of the Asian tiger mosquito Aedes albopictus, (ii) induced larval mortality within a few hours of exposure and, more importantly, (iii) protected the Bti-based formulate, whose insecticidal activity was maintained for more than one month in comparison to the few days residual activity of the commercial product. The delivery method was effective in both laboratory and semi-field conditions, demonstrating that MosChito rafts may represent an original, eco-based and user-friendly solution for larval control in domestic and peri-domestic aquatic habitats such as saucers and artificial containers in residential or urban environments., (© 2023. The Author(s).)

Parasites of the genus Leishmania are unusual unicellular microorganisms in that they are characterized by the capability to subvert in their favor the immune response of mammalian phagocytes, including dendritic cells. Thus, in overt leishmaniasis, dendritic cells and macrophages are converted into a niche for Leishmania spp. in which the parasite, rather than being inactivated and disassembled, survives and replicates. In addition, Leishmania parasites hitchhike onto phagocytic cells, exploiting them as a mode of transport to lymphoid tissues where other phagocytic cells are potentially amenable to parasite colonization. This propensity of Leishmania spp. to target dendritic cells has led some researchers to consider the possibility that the non-pathogenic, reptile-associated Leishmania tarentolae could be exploited as a vaccine platform and vehicle for the production of antigens from different viruses and for the delivery of the antigens to dendritic cells and lymph nodes. In addition, as L. tarentolae can also be regarded as a surrogate of pathogenic Leishmania parasites, this parasite of reptiles could possibly be developed into a vaccine against human and canine leishmaniases, exploiting its immunological cross-reactivity with other Leishmania species, or, after its engineering, for the expression of antigens from pathogenic species. In this article we review published studies on the use of L. tarentolae as a vaccine platform and vehicle, mainly in the areas of leishmaniases and viral infections. In addition, a short summary of available knowledge on the biology of L. tarentolae is presented, together with information on the use of this microorganism as a micro-factory to produce antigens suitable for the serodiagnosis of viral and parasitic infections., (© 2023. The Author(s).)

The aim of this review was to provide a general overview to the topic of this special issue. In this study the available categories of biotics were defined (i.e., probiotics, prebiotics, postobiotics and synbiotics) as first actors of therapies that target the gut flora, with the aim to modify it in a specific direction, generally with the goal of controlling inflammatory phenomena. The points that must be considered when evaluating the results of these interventions, and, specifically, the changes in gut microbiota following the assumption of biotics were analyzed. This context typically represented the one of clinical trials aimed at inducing or maintaining remission in pediatric autoimmune inflammatory diseases, that often yield conflicting results. We finally attempted to draft possible research developments for the next years.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation., Competing Interests: Declaration of Competing Interest The candidate vaccine Lt-spike and its potential application have been described in the PCT/IB2022/051585 (23 February 2022). The antigen Lt-RBD and its potential application have been described in the patent application no. IT 02021000004160 (23 February 2021). A.M., F.D., M.M., and I.R. are employed by VisMederi Srl; M.L. is employed by VisMederi Research Srl; E.M. is the President and Founder of VisMederi Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Insecticide resistance is a major threat challenging the control of harmful insect species. The study of resistant phenotypes is, therefore, pivotal to understand molecular mechanisms underpinning insecticide resistance and plan effective control and resistance management strategies. Here, we further analysed the diflubenzuron (DFB)-resistant phenotype due to the point-mutation I1043M in the chitin-synthase 1 gene ( chs 1) in the mosquito Culex pipiens . By comparing susceptible and resistant strains of Cx. pipiens through DFB bioassays, molecular analyses and scanning electron microscopy, we showed that the I1043M-resistant mosquitoes have: (i) a striking level of DFB resistance (i.e., resistance ratio: 9006); (ii) a constitutive 11-fold over-expression of the chs 1 gene; (iii) enhanced cuticle thickness and cuticular chitin content. Culex pipiens is one of the most important vector species in Europe and the rapid spread of DFB resistance can threaten its control. Our results, by adding new data about the DFB-resistant phenotype, provide important information for the control and management of insecticide resistance., Competing Interests: The authors declare no conflicts of interest.

Puberty is a critical phase of life associated with physiological changes related to sexual maturation, and represents a complex process regulated by multiple endocrine and genetic controls. Puberty is driven by hormones, and it can impact the gut microbiome (GM). GM differences between sex emerge at puberty onset, confirming a relationship between microbiota and sex hormones. In this narrative review, we present an overview of precocious pubertal development and the changes in the GM in precocious puberty (PP) in order to consider the role of the sex hormone-gut microbiome axis from the perspective of pediatric endocrinology. Bidirectional interactions between the GM and sex hormones have been proposed in different studies. Although the evidence on the interaction between microbiota and sex hormones remains limited in pediatric patients, the evidence that GM alterations may occur in girls with central precocious puberty (CPP) represents an interesting finding for the prediction and prevention of PP. Deepening the understanding of the connection between the sex hormones and the role of microbiota changes can lead to the implementation of microbiota-targeted therapies in pubertal disorders by offering a pediatric endocrinology perspective., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Calcaterra, Rossi, Massini, Regalbuto, Hruby, Panelli, Bandi and Zuccotti.)

During 2021, we collected blood and serum samples from 135 persons exposed to tick bites in southern Italy. We serologically and molecularly screened for zoonotic tickborne pathogens and only molecularly screened for Candidatus Midichloria mitochondrii. Overall, 62 (45.9%) persons tested positive for tickborne pathogens. Coxiella burnetii was detected most frequently (27.4%), along with Rickettsia spp. (21.5%) and Borrelia spp. (10.4%). We detected Candidatus M. mitochondrii DNA in 46 (34.1%) participants who had statistically significant associations to tickborne pathogens (p<0.0001). Phylogenetic analysis of Candidatus M. mitochondrii sequences revealed 5 clades and 8 human sequence types that correlated with vertebrates, Ixodes spp. ticks, and countries in Europe. These data demonstrated a high circulation of tickborne pathogens and Candidatus M. mitochondrii DNA in persons participating in outdoor activities in southern Italy. Our study shows how coordinated surveillance among patients, clinicians, and veterinarians could inform a One Health approach for monitoring and controlling the circulation of tickborne pathogens.

Leishmaniasis (or the leishmaniases), classified as a neglected tropical parasitic disease, is found in parts of the tropics, subtropics and southern Europe. Leishmania parasites are transmitted by the bite of phlebotomine sand flies and million cases of human infection occur annually. Leishmania tarentolae has been historically considered a non-pathogenic protozoan of reptiles, which has been studied mainly for its potential biotechnological applications. However, some strains of L. tarentolae appear to be transiently infective to mammals. In areas where leishmaniasis is endemic, recent molecular diagnostics and serological positivity to L. tarentolae in humans and dogs have spurred interest in the interactions between these mammalian hosts, reptiles and Leishmania infantum, the main aetiologic agent of human and canine leishmaniasis. In this review, we discuss the systematics and biology of L. tarentolae in the insect vectors and the vertebrate hosts and address questions about evolution of reptilian leishmaniae. Furthermore, we discuss the possible usefulness of L. tarentolae for new vaccination strategies., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Biological methods for mosquito larvae control are completely biodegradable and have null or limited effects on nontarget organisms. However, commercially available products have a low residual activity, with the consequent need for multiple applications that inevitably increase costs and the risk of resistance phenomena insurgence. Smart delivery systems made of hydrogels proved their efficacy in increasing the action duration of biolarvicides up to several months, but the lack of an efficient baiting mechanism to strongly attract the target pest remains a problem in practical applications. In this work, we investigated two novel hydrogel-based formulations of completely natural composition for baiting and killing larvae of Aedes albopictus mosquitos. The proposed materials consist of charged crosslinked polysaccharides (chitosan and cellulose) and are specifically manufactured to float in water, simulating organic matter usually present at breeding sites. Within the hydrogels' matrix, yeast colonies of Saccharomyces cerevisiae were embedded as phagostimulants alongside a biolarvicide ( Bacillus thuringiensis var. israelensis ( Bti )). Despite the similar chemical nature and structure, chitosan-based hydrogels exhibited a markedly superior baiting potential compared to those made of cellulose and also succeeded in efficiently killing mosquito larvae just after a few hours from administration. We are confident that the proposed smart delivery hydrogel made of chitosan can be an enabling tool to attract mosquito larvae towards biopesticides of different nature without delocalizing active ingredients away from the breeding site and to simultaneously increase their residual activity, thus holding the potential of minimizing environmental pollution related to pest control and vector-borne disease prevention.

The mosquito proboscis is an efficient microelectromechanical system, which allows the insect to feed on vertebrate blood quickly and painlessly. Its efficiency is further enhanced by the insect saliva, although through unclear mechanisms. Here, we describe the initial trigger of an unprecedented feedback signaling pathway in Aedes mosquitoes affecting feeding behavior. We identified LIPS proteins in the saliva of Aedes mosquitoes that promote feeding in the vertebrate skin. LIPS show a new all-helical protein fold constituted by two domains. The N-terminal domain interacts with a cuticular protein (Cp19) located at the tip of the mosquito labrum. Upon interaction, the morphology of the labral cuticle changes, and this modification is most likely sensed by proprioceptive neurons. Our study identifies an additional role of mosquito saliva and underlines that the external cuticle is a possible site of key molecular interactions affecting the insect biology and its vector competence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the last decade, Aedes koreicus and Aedes japonicus japonicus mosquitoes, which are competent vectors for various arboviruses of public health relevance, colonised Italy and other European countries. Nevertheless, information about their current and potential distribution is partial. Accordingly, in this study four regions of Northern Italy (Lombardy, Liguria, Piedmont and Aosta Valley) were surveyed during 2021 for the presence of eggs, larvae and pupae of these two invasive species. We found evidence for a widespread presence of Ae. koreicus in pre-Alpine territories of Lombardy and Piedmont. Larvae from the invasive subspecies of Ae. j. japonicus were also collected in the same geographic areas, though they were less frequent. Occurrence data from this study and results from previous monitoring campaigns were used to generate a Maxent model for the prediction of habitat suitability for Ae. koreicus mosquitoes in Northern Italy and the rest of Europe. Peri-urban areas located in proximity to forests, pastures and vineyards were revealed as highly suitable environments for colonisation by this invasive species. Maps of the potential distribution also suggest the presence of further suitable areas in currently uncolonized countries. We conclude that this invasive mosquito species has the potential for a broad expansion at the European level in the coming decades., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were T>G and G>T transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans., (Copyright © 2022. Published by Elsevier B.V.)

The guidelines on ventilator-associated pneumonia (VAP) recommend an empiric therapy against methicillin-resistant Staphylococcus aureus (MRSA) according to its prevalence rate. Considering the MRSA and MSSA VAP prevalence over the last 9 years in our tertiary care hospital, we assessed the clinical value of the MRSA nasal-swab screening in either predicting or ruling out MRSA VAP. We extracted the data of 1461 patients with positive bronchoalveolar lavage (BAL). Regarding the MRSA nasal-swab screening, 170 patients were positive for MRSA or MSSA. Overall, MRSA had a high prevalence in our ICU. Despite the COVID-19 pandemic, there was a significant downward trend in MRSA prevalence, while MSSA remained steady over time. Having VAP due to MRSA did not have any impact on LOS and mortality. Finally, the MRSA nasal-swab testing demonstrated a very high negative predictive value for MRSA VAP. Our results suggested the potential value of a patient-centered approach to improve antibiotic stewardship.

Severe neurological impairment (NI) is often accompanied by the need for artificial nutritional support, normally provided enterally (enteral nutrition [EN]) to ensure growth, counteract morbidity and mortality, and improve quality of life. On the other hand, long-term EN (LTEN) may contribute to the establishment, or exacerbation, of gastrointestinal disorders that may lead to malnutrition, which in turn is associated with alterations in gut microbiota (GM) composition and functional capacities. To the best of our knowledge, we investigated, for the first time in this study, the consequences of LTEN in a pediatric population in this pathological context. Using amplicon sequencing, we compared the fecal microbiota of a pediatric population suffering from severe NI and under LTEN to that of sex- and age-matched controls. The two groups presented evident differences in GM composition and a consistent differential clustering. In general, the taxonomic picture in NI children under LTEN seemed to mirror a profound dysbiotic condition, in which anti-inflammatory taxa appear severely depleted (among others, the Clostridiales families of Lachnospiraceae and Ruminococcaceae , and, within the latter, Faecalibacterium spp. and Gemmiger spp.), while known pathobionts ( Gammaproteobacteria and Klebsiella ) or emerging pathogens (e.g., Synergistales, Cloacibacillus , and Fusobacterium ) were significantly enriched. Our data suggest that LTEN has a significant impact on the GM taxonomic composition in NI children. Even if other factors are probably at work, such as the bidirectional interaction between gastrointestinal impairment/immaturity and the central nervous system (CNS), the assumption of drugs, and physical inactivity, these data define possible routes and targets to try to alleviate this dysbiosis, with a view to better management of these patients and an improvement in their quality of life., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Panelli, Calcaterra, Verduci, Comandatore, Pelizzo, Borghi, Bandi and Zuccotti.)

Background: Protozoa of the genus Leishmania are characterized by their capacity to target macrophages and Dendritic Cells (DCs). These microorganisms could thus be exploited for the delivery of antigens to immune cells. Leishmania tarentolae is regarded as a non-pathogenic species; it was previously used as a biofactory for protein production and has been considered as a candidate vaccine or as an antigen delivery platform. However, results on the type of immune polarization determined by L. tarentolae are still inconclusive., Methods: DCs were derived from human monocytes and exposed to live L. tarentolae , using both the non-engineered P10 strain, and the same strain engineered for expression of the spike protein from SARS-CoV-2. We then determined: (i) parasite internalization in the DCs; and (ii) the capacity of the assayed strains to activate DCs and the type of immune polarization., Results: Protozoan parasites from both strains were effectively engulfed by DCs, which displayed a full pattern of maturation, in terms of MHC class II and costimulatory molecule expression. In addition, after parasite infection, a limited release of Th1 cytokines was observed., Conclusions: Our results indicate that L. tarentolae could be used as a vehicle for antigen delivery to DCs and to induce the maturation of these cells. The limited cytokine release suggests L. tarentolae as a neutral vaccine vehicle that could be administered in association with appropriate immune-modulating molecules.

The emerging distribution of new alien mosquito species was recently described in Europe. In addition to the invasion of Aedes albopictus , several studies have focused on monitoring and controlling other invasive Aedes species, as Aedes koreicus and Aedes japonicus . Considering the increasing development of insecticide resistance in Aedes mosquitoes, new control strategies, including the use of bacterial host symbionts, are proposed. However, little is known about the bacterial communities associated with these species, thus the identification of possible candidates for Symbiotic Control is currently limited. The characterization of the natural microbiota of field-collected Ae. koreicus mosquitoes from North-East Italy through PCR screening, identified native infections of Wolbachia in this species that is also largely colonized by Asaia bacteria. Since Asaia and Wolbachia are proposed as novel tools for Symbiotic Control, our study supports their use for innovative control strategies against new invasive species. Although the presence of Asaia was previously characterized in Ae. koreicus , our study characterized this Wolbachia strain, also inferring its phylogenetic position. The co-presence of Wolbachia and Asaia may provide additional information about microbial competition in mosquito, and to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction in Ae. koreicus .

Since 1996, the Geneva Arthroplasty Registry at the University Hospitals of Geneva (HUG) has been collecting, archiving and disseminating relevant high-quality information on primary total hip and knee arthroplasties and revision procedures performed at the Division of Orthopaedics and Traumatology. Patients are followed throughout their lifetime with the prosthesis. The registry has been essential to better understand and subsequently improve the care of patients with hip and knee replacements. It will continue to fulfil its mission and to work towards an even more effective transfer of the knowledge obtained to all stakeholders and as well as towards its implementation., Competing Interests: Les auteurs n’ont déclaré aucun conflit d’intérêts en relation avec cet article.

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories., Competing Interests: The antigen Lt-RBD and its potential application have been described in the patent application no. IT 02021000004160; publication date 23 February 2021. AM, FD, and EM were employed by VisMederi Srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Varotto-Boccazzi, Manenti, Dapporto, Gourlay, Bisaglia, Gabrieli, Forneris, Faravelli, Bollati, Rubolini, Zuccotti, Montomoli, Epis and Bandi.)

Background: Aedes koreicus is a mosquito species characterized by marked anthropophilic behavior, and a potential vector of nematodes and viruses. It is native to East Asia, but its presence has recently been reported in many regions of Europe. In Italy, these mosquitoes had been detected in the northeast since 2011 and are now spreading towards the southwest of the country., Methods: In 2020, during a surveillance program for invasive mosquito species in the district of Bergamo (Lombardy Region, Italy), about 6000 mosquito larvae were collected. Emerged adults were assigned to mosquito species according to morphological analyses, followed by amplification and sequencing of genetic markers (COI, ND4, ITS2 and D2)., Results: According to the morphological and genetic data, about 50 individuals belonged to the species Ae. koreicus., Conclusion: We report the presence of Ae. koreicus in the district of Bergamo, which confirms the spread of this species in the north of Italy and raises concerns about its possible role as a vector of diseases in the Alpine area., (© 2021. The Author(s).)

The human skin microbiota is essential for maintaining homeostasis and ensuring barrier functions. Over the years, the characterization of its composition and taxonomic diversity has reached outstanding goals, with more than 10 million bacterial genes collected and cataloged. Nevertheless, the study of the skin microbiota presents specific challenges that need to be addressed in study design. Benchmarking procedures and reproducible and robust analysis workflows for increasing comparability among studies are required. For various reasons and because of specific technical problems, these issues have been investigated in gut microbiota studies, but they have been largely overlooked for skin microbiota. After a short description of the skin microbiota, the review tackles methodological aspects and their pitfalls, covering NGS approaches and high throughput culture-based techniques. Recent insights into the "core" and "transient" types of skin microbiota and how the manipulation of these communities can prevent or combat skin diseases are also covered. Finally, this review includes an overview of the main dermatological diseases, the changes in the microbiota composition associated with them, and the recommended skin sampling procedures. The last section focuses on topical and oral probiotics to improve and maintain skin health, considering their possible applications for skin diseases.

Dogs are biologically predisposed to communicate with humans in cooperative contexts. They show individual differences in dog-human communication and inhibition, potentially enhanced by life experience, e.g., Animal-Assisted Interventions. This study aimed to investigate whether dogs' personality, defined by biologically meaningful neural circuits described in the Reinforcement Sensitivity Theory of Personality (RST), predicted dogs' communication, task orientation, emotional state, and approach of an unsolvable task. We also investigated the differences between dogs experienced in Animal-Assisted Interventions (AAI) and inexperienced dogs. The results indicated that a high sensitivity to the RST personality trait related to managing uncertainty (Behavioural Inhibition System, BIS) predicted fewer task orientations but increased referential and non-referential looking, which we interpreted as a way to obtain directions from the owner. Conversely, a high sensitivity to the traits reflecting tendencies to approach rewards (Behavioural Approach System, BAS) and avoid punishment (Fight-Flight-Freeze System, FFFS) predicted lower looking. High sensitivity to the FFFS also predicted more frequent task orientations, which we interpreted as frustration. Finally, the dogs in the AAI program looked more at their owner and were less oriented towards the task. These results provide empirical evidence of individual differences tied to the psychobiological personality traits in canine cognitive skills. Understanding such cognitive profiles may have an enormous impact on activities that rely upon dog-human collaborative interaction.

" Candidatus Midichloria mitochondrii" is a Gram-negative bacterium that lives in strict intracellular symbiosis with the hard tick Ixodes ricinus, forming one of the most intriguing endosymbiosis described to date. The bacterium is capable of durably colonizing the host mitochondria, a peculiar tropism that makes " Ca . Midichloria mitochondrii" a very interesting tool to study the physiology of these cellular organelles. The interaction between the symbiont and the organelle has, however, been difficult to characterize. A parallelism with the predatory bacterium Bdellovibrio bacteriovorus has been drawn, suggesting the hypothesis that " Ca . Midichloria mitochondrii" could prey on mitochondria and consume them to multiply. We studied the life cycle of the bacterium within the host oocytes using a multidisciplinary approach, including electron microscopy, molecular biology, statistics, and systems biology. Our results were not coherent with a predatory-like behavior by " Ca . Midichloria mitochondrii" leading us to propose a novel hypothesis for its life cycle. Based on our results, we here present a novel model called the "mitochondrion-to-mitochondrion hypothesis." Under this model, the bacterium would be able to move from mitochondrion to mitochondrion, possibly within a mitochondrial network. We show that this model presents a good fit with quantitative electron microscopy data. IMPORTANCE Our results suggest that " Candidatus Midichloria mitochondrii," the intramitochondrial bacterium, does not invade mitochondria like predatory bacteria do but instead moves from mitochondrion to mitochondrion within the oocytes of Ixodes ricinus. A better understanding of the lifestyle of " Ca . Midichloria mitochondrii" will allow us to better define the role of this bacterial symbiont in the host physiology.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata , an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia , strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia , also considering the widespread production of pyrethrins by several plants. IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia , despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi , for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides., (Copyright © 2021 Comandatore et al.)

To assess the effect of a fermented rice-flour obtained from Lactobacillus paracasei CBA L74 in managing infants with moderate to severe atopic dermatitis. Infants with moderate to severe atopic dermatitis, aged 6-36 months, were randomly assigned to receive once-daily consumption of rice flour containing heat-killed probiotic Lactobacillus paracasei CBA L74 or placebo for 12 weeks as supplementary approach to topical treatment. Primary outcome was SCORAD index change from baseline to 12 weeks; secondary outcomes were gut microbiota composition, as evaluated by the analysis of fecal samples, and serum cytokines at baseline and at the end of the intervention period in both groups, and steroid usage over the treatment period and one month after stopping it. V3-V4 region of the 16S ribosomal RNA gene was sequenced to evaluate changes in the gut microbiota. SCORAD index decreased over the treatment period in both groups. The difference in the SCORAD change was -2.1 (-5.5 to 1.3; p = 0.223) for the experimental vs. the placebo group, not reaching the minimal clinical difference of 8.7 units. The use of topical steroids, measured as finger tips units, decreased from 4 to 16 weeks, in both groups; the reduction was significantly higher in experimental than in placebo group (p value from Wilcoxon rank sum test = 0.031). No significant differences were observed for cytokines levels between groups. The composition of gut microbiota at the phylum and class taxonomic levels resulted very similar, at baseline and after intervention, in both groups. Similarly, no significant differences were observed in the relative abundance of bacterial genera between groups. In conclusion, though the heat-killed Lactobacillus paracaseiwas not proved to be effective in reducing the severity of atopic dermatitis, it showed a steroid sparing effect the value of which needs to be further investigated., (Copyright © 2020. Published by Elsevier Ltd.)

Phytophagous stink bugs typically harbor nutritional symbiotic bacteria in their midgut, to integrate their unbalanced diet. In the Pentatomidae, most symbionts are affiliated to the genus Pantoea, and are polyphyletic. This suggests a scenario of an ancestral establishment of symbiosis, followed by multiple symbiont replacement events by akin environmental bacteria in different host lineages. In this study, a novel Pantoeaspecies ('CandidatusPantoea persica') was characterized from the gut of the pentatomid Acrosternum arabicum, and shown to be highly abundant in a specific portion of the gut and necessary for the host development. The genome of the symbiont (2.9 Mb), while presenting putative host-supportive metabolic pathways, including those for amino acids and vitamin synthesis, showed a high level of pseudogenization, indicating ongoing genome reduction. Comparative analyses with other free-living and symbiotic Pantoea highlighted a convergent pattern of genome reduction in symbionts of pentatomids, putatively following the typical phases modelized in obligate nutritional symbionts of insects. Additionally, this system has distinctive traits, as hosts are closely related, and symbionts originated multiple independent times from closely related free-living bacteria, displaying convergent and independent conspicuous genome reduction. Due to such peculiarities, this may become an ideal model to study genome evolutionary processes in insect symbionts., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Taxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as "holobionts". We illustrate this approach through the description of the ciliate Euplotes vanleeuwenhoeki sp. nov. and its bacterial endosymbiont "Candidatus Pinguicoccus supinus" gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of Asaia WSP , a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with Asaia WSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, Asaia WSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to Asaia WSP . In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea, especially in hospitalized elderly patients, representing a global public health concern. Clinical presentations vary from mild diarrhea to severe pseudomembranous colitis that may progress to toxic megacolon or intestinal perforation. Antibiotic therapy is recognized as a risk factor and exacerbates dysbiosis of the intestinal microbiota, whose role in CDI is increasingly acknowledged. A clinically challenging complication is the development of recurrent disease (rCDI). In this study, using amplicon metagenomics, we compared the fecal microbiota of CDI and rCDI patients (sampled at initial and recurrent episode) and of non-infected controls. We also investigated whether CDI severity relates to specific microbiota compositions. rCDI patients showed a significantly decreased bacterial diversity as compared to controls ( p < 0.01). The taxonomic composition presented significant shifts: both CDI and rCDI patients displayed significantly increased frequencies of Firmicutes , Peptostreptococcaceae , Clostridium XI , Clostridium XVIII , and Enterococcaceae . Porphyromonadaceae and, within it, Parabacteroides displayed opposite behaviors in CDI and rCDI, appearing discriminant between the two. Finally, the second episode of rCDI was characterized by significant shifts of unclassified Clostridiales , Escherichia/Shigella and Veillonella . No peculiar taxa composition correlated with the severity of infection, likely reflecting the role of host-related factors in determining severity.

This review tackles the concept of the evolutionary mismatch, in relation with the reduction of the prevalence of the so-called "dirty old friends". These formed the variegated community of parasites and microorganisms, either prokaryotic or eukaryotic, that, over long evolutionary times, co-evolved with humans and their ancestors, inhabiting their digestive tracts, and other body districts. This community of microbial symbionts and metazoan parasites is thought to have evolved a complex network of inter-independence with the host, in particular in relation with their immune stimulating capacity, and with the consequent adaptation of the host immune response to this chronic stimulation. Strictly related to this evolutionary mismatch, the hygiene hypothesis, proposed by David Strachan in 1989, foresees that the increase in the incidence of inflammatory and autoimmune disorders during the twentieth century has been caused by the reduced exposure to parasites and microorganisms, especially in industrialized countries. Among these pathologies, inflammatory bowel diseases (IBDs) occupy a prominent role. From these premises, this review summarizes current knowledge on how variations in the composition of the gut bacterial microbiota, as well as its interactions with fungal communities, influence the overall immune balance, favouring or counteracting gut inflammation in IBDs. Additionally, the effect of worm parasites, either directly on the immune balance, or indirectly, through the modulation of bacterial and fungal microbiota, will be addressed. Finally, we will review a series of studies related to the use of molecules derived from parasitic worms and fungi, which hold the potential to be developed as postbiotics for the treatment of IBDs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

There is great concern regarding the rapid emergence and spread of drug-resistance in Plasmodium falciparum, the parasite responsible for the most severe form of human malaria. Parasite populations resistant to some or all the currently available antimalarial treatments are present in different world regions. Considering the need for novel and integrated approaches to control malaria, combinations of drugs were tested on P. falciparum. The primary focus was on doxycycline, an antibiotic that specifically targets the apicoplast of the parasite. In combination with doxycycline, three different drugs known to inhibit efflux pumps (verapamil, elacridar and ivermectin) were tested, with the assumption that they could increase the intracellular concentration of the antibiotic and consequently its efficacy against P. falciparum. We emphasize that elacridar is a third-generation ABC transporters inhibitor, never tested before on malaria parasites. In vitro experiments were performed on asexual stages of two strains of P. falciparum, chloroquine-sensitive (D10) and chloroquine-resistant (W2). Incubation times on asynchronous or synchronous cultures were 72h or 96h, respectively. The antiplasmodial effect (i.e. the IC50) was determined by measuring the activity of the parasite lactate dehydrogenase, while the interaction between drugs was determined through combination index (CI) analyses. Elacridar achieved an IC50 concentration comparable to that of ivermectin, approx. 10-fold lower than that of verapamil, the other tested ABC transporter inhibitor. CI results showed synergistic effect of verapamil plus doxycycline, which is coherent with the starting hypothesis, i.e. that ABC transporters represent potential targets, worth of further investigations, towards the development of companion molecules useful to enhance the efficacy of antimalarial drugs. At the same time, the observed antagonistic effect of doxycycline in combination with ivermectin or elacridar highlighted the importance of drug testing, to avoid the de-facto generation of a sub-dosage, a condition that facilitates the development of drug resistance., Competing Interests: The authors have declared that no competing interests exist.

Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (Asaia WSP ). Asaia WSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make Asaia WSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.

In recent years, Serratia marcescens has emerged as an important agent of hospital-acquired infections, such as pneumonia, urinary tract infection, septicaemia and meningitis, particularly in vulnerable patients. Compared to Klebsiella pneumoniae and Escherichia coli , S. marcescens is less commonly associated with bla KPC genes, yet few cases of plasmid transmission at the gastrointestinal level from K. pneumoniae carbapenemase (KPC)-producing Enterobacterales to S. marcescens have been described. Here we report a case of in vivo acquisition, during a 3-month period of hospitalization in the intensive care unit, of a bla KPC-3 gene carried by a pKpQIL-IT plasmid, and its probable transmission at the bronchial level among different species of Enterobacterales , including K. pneumoniae and S. marcescens . By using whole genome sequence analyses we were able provide insight into the dynamics of carbapenem-resistance determinants acquisition in the lower respiratory tract, a novel anatomical region for such plasmid transmission events, that usually involve the gastrointestinal tract. The co-presence at the same time of both wild-type and resistant Enterobacterales could have been the critical factor leading to the spread of plasmids harbouring carbapenem-resistance genes, of particular importance during surveillance screenings. The possibility of such an event may have significant consequences in terms of antimicrobial treatment, with a potential limitation of therapeutic options, thereby further complicating the clinical management of high-risk critically ill patients.

The circulation of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant problem worldwide. In this work we characterize the isolates and reconstruct the spread of a multi-clone epidemic event that occurred in an Intensive Care Unit in a hospital in Northern Italy. The event took place from August 2015 to May 2016 and involved 23 patients. Twelve of these patients were colonized by CRKP at the gastrointestinal level, while the other 11 were infected in various body districts. We retrospectively collected data on the inpatients and characterized a subset of the CRKP isolates using antibiotic resistance profiling and whole genome sequencing. A SNP-based phylogenetic approach was used to depict the evolutionary context of the obtained genomes, showing that 26 of the 32 isolates belong to three genome clusters, while the remaining six were classified as sporadic. The first genome cluster was composed of multi-resistant isolates of sequence type (ST) 512. Among those, two were resistant to colistin, one of which indicating the insurgence of resistance during an infection. One patient hospitalized in this period was colonized by two strains of CRKP, both carrying the blaKPC gene (variant KPC-3). The analysis of the genome contig containing the bla KPC locus indicates that the gene was not transmitted between the two isolates. The second infection cluster comprised four other genomes of ST512, while the third one (ST258) colonized 12 patients, causing five clinical infections and resulting in seven deaths. This cluster presented the highest level of antibiotic resistance, including colistin resistance in all 17 analyzed isolates. The three outbreaking clones did not present more virulence genes than the sporadic isolates and had different patterns of antibiotic resistance, however, were clearly distinct from the sporadic ones in terms of infection status, being the only ones causing overt infections., (Copyright © 2019 Ferrari, Corbella, Gaiarsa, Comandatore, Scaltriti, Bandi, Cambieri, Marone and Sassera.)