Search

Abstract Background Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. Graphical abstract

Abstract 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.

Abstract 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.

IntroductionA common thought is that extensive and organic breeding systems are associated with lower prevalence of infections in livestock animals, compared to intensive ones. In addition, organic systems limit the use of anti-microbial drugs, which may lead to lower emergence of antimicrobial resistances (AMR).MethodsTo examine these issues, avoiding any a priori bias, we carried out a systematic literature search on dairy cattle breeding. Search was targeted to publications that compared different types of livestock farming (intensive, extensive, conventional, organic) in terms of the circulation of infectious diseases and AMR.Results and discussionA total of 101 papers were finally selected. These papers did not show any trend in the circulation of the infections in the four types of breeding systems. However, AMR was more prevalent on conventional dairy farms compared to organic ones. The prevalence of specific pathogens and types of resistances were frequently associated with specific risk factors that were not strictly related to the type of farming system. In conclusion, we did not find any evidence suggesting that extensive and organic dairy farming bears any advantage over the intensive and conventional ones, in terms of the circulation of infectious agents.

BackgroundInnate 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.MethodsTo 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.ResultsL. 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.ConclusionThe 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.

Abstract 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. Graphical Abstract

Abstract 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. Graphical Abstract

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.

Mosquito control, which is not always easily accomplished, is further complicated by the spread of invasive species. This is the case of Aedes koreicus, a mosquito native to East Asia, whose presence has been recorded in several European countries, including Italy. This mosquito found suitable ecological conditions in central Europe in general, and in northern Italy in particular, as shown by the ongoing expansion of its distribution. While basic knowledge on feeding habits of Ae. koreicus have already been acquired, information on its vectorial competence is scarce. Therefore, active monitoring on the presence of this mosquito, and the pre-planning of future control actions, are of paramount importance. Currently, there are no specific guidelines for controlling this mosquito, both in its native regions and in invaded countries. Here we present the first study on the efficacy of a bioinsecticide based on Bacillus thuringiensis on Ae. koreicus larvae, with a comparison with results obtained on the tiger mosquito Aedes albopictus. Our results proved that this bioinsecticide is effective on Ae. koreicus, both dissolved in water and incorporated into MosChito raft, a hydrogel-based matrix that has recently been developed for the delivery of insecticides to other mosquito species and suitable for safe and eco-compatible applications.

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract 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.

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.

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 albD7l2 (Aed al 2), albAntigen5-3 (Aed al 13) and albLIPS-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.

Abstract 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. Graphical Abstract

Abstract 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.

Abstract 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. Graphical Abstract

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

Summary: 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.

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 (cox1) 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.

Bacterial populations inhabiting a variety of natural and human-associated niches have the ability to grow in the form of biofilms. A large part of pathological chronic conditions, and essentially all the bacterial infections associated with implanted medical devices or prosthetics, are caused by microorganisms embedded in a matrix made of polysaccharides, proteins, and nucleic acids. Biofilm infections are generally characterized by a slow onset, mild symptoms, tendency to chronicity, and refractory response to antibiotic therapy. Even though the molecular mechanisms responsible for resistance to antimicrobial agents and host defenses have been deeply clarified, effective means to fight biofilms are still required. Lactic acid bacteria (LAB), used as probiotics, are emerging as powerful weapons to prevent adhesion, biofilm formation, and control overgrowth of pathogens. Hence, using probiotics or their metabolites to quench and interrupt bacterial communication and aggregation, and to interfere with biofilm formation and stability, might represent a new frontier in clinical microbiology and a valid alternative to antibiotic therapies. This review summarizes the current knowledge on the experimental and therapeutic applications of LAB to interfere with biofilm formation or disrupt the stability of pathogenic biofilms.

Abstract 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. Graphical Abstract

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.

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.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), ranging from asymptomatic conditions to severe/fatal lung injury and multi-organ failure. Growing evidence shows that the nasopharyngeal microbiota composition may predict the severity of respiratory infections and may play a role in the protection from viral entry and the regulation of the immune response to the infection. In the present study, we have characterized the nasopharyngeal bacterial microbiota (BNM) composition and have performed factor analysis in a group of 54 asymptomatic/paucisymptomatic subjects who tested positive for nasopharyngeal swab SARS-CoV-2 RNA and/or showed anti-RBD-IgG positive serology at the enrolment. We investigated whether BNM was associated with SARS-CoV-2 RNA positivity and serum anti-RBD-IgG antibody development/maintenance 20–28 weeks after the enrolment. Shannon’s entropy α-diversity index [odds ratio (OR) = 5.75, p = 0.0107] and the BNM Factor1 (OR = 2.64, p = 0.0370) were positively associated with serum anti-RBD-IgG antibody maintenance. The present results suggest that BNM composition may influence the immunological memory against SARS-CoV-2 infections. To the best of our knowledge, this is the first study investigating the link between BNM and specific IgG antibody maintenance. Further studies are needed to unveil the mechanisms through which the BNM influences the adaptive immune response against viral infections.

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.

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.

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 (chs1) 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 chs1 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.

Our results suggest that Midichloria mitochondriiIxodes ricinusM. mitochondrii

Abstract Background Insecticides are still at the core of insect pest and vector control programmes. Several lines of evidence indicate that ABC transporters are involved in detoxification processes against insecticides, including permethrin and other pyrethroids. In particular, the ABCG4 gene, a member of the G subfamily, has consistently been shown to be up-regulated in response to insecticide treatments in the mosquito malaria vector Anopheles stephensi (both adults and larvae). Methods To verify the actual involvement of this transmembrane protein in the detoxification process of permethrin, bioassays on larvae of An. stephensi, combining the insecticide with a siRNA, specifically designed for the inhibition of ABCG4 gene expression were performed. Administration to larvae of the same siRNA, labeled with a fluorescent molecule, was effected to investigate the systemic distribution of the inhibitory RNA into the larval bodies. Based on siRNA results, similar experiments using antisense Vivo-Morpholinos (Vivo-MOs) were effected. These molecules, compared to siRNA, are expected to guarantee a higher stability in environmental conditions and in the insect gut, and present thus a higher potential for future in-field applications. Results Bioassays using two different concentrations of siRNA, associated with permethrin, led to an increase of larval mortality, compared with results with permethrin alone. These outcomes confirm that ABCG4 transporter plays a role in the detoxification process against the selected insecticide. Moreover, after fluorescent labelling, it was shown the systemic dissemination of siRNA in different body districts of An. stephensi larvae, which suggest a potential systemic effect of the molecule. At the same time, results of Vivo-MO experiments were congruent with those obtained using siRNA, thus confirming the potential of ABCG4 inhibition as a strategy to increase permethrin susceptibility in mosquitoes. For the first time, Vivo-MOs were administered in water to larvae, with evidence for a biological effect. Conclusions Targeting ABCG4 gene for silencing through both techniques resulted in an increased pyrethroid efficacy. These results open the way toward the possibility to exploit ABCG4 inhibition in the context of integrated programmes for the control An. stephensi mosquitoes and malaria transmission.

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.

We have studied genome reduction within several strains of the insect symbiont AsaiaAsaiaAsaia

Objectives In Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the ‘UNIversity against CORoNavirus study’) to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020.Participants The study population included 2023 subjects asymptomatic at the enrolment.Primary outcome measures A nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected.Results Subjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes.Conclusions Results from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.

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 following document is based on the current knowledge of SARS-CoV-2 and the related disease (COVID-19). The Italian Institute of Health (ISS) is monitoring the virus spread throughout the country through daily reports sent by individual regions and regional reference laboratories. The diagnosis of infection is based on the use of standardized and validated molecular tests for the search for viral RNA in different sets of biological samples, from patients suspected of having contracted the infection. This document provides a specific guidance for the collection and storage of biological samples from deceased persons. It also provides recommendations on safety practices to be adopted during both collection and handling of specimens, and during autopsy procedures. With regard autopsy, it is recommended to follow the procedures for the execution of diagnostic findings in patients died with SARS-CoV-2 infection issued by ISS Working Group on Causes of Death from COVID-19, published on March 27, 2020.

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.

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.

Abstract Background Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 μM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment. Results Cell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 μM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 μM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 μM (P < 0.01). Conclusions The adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.

Abstract Background Ticks are obligate haematophagous ectoparasites of vertebrates and frequently parasitize avian species that can carry them across continents during their long-distance migrations. Ticks may have detrimental effects on the health state of their avian hosts, which can be either directly caused by blood-draining or mediated by microbial pathogens transmitted during the blood meal. Indeed, ticks host complex microbial communities, including bacterial pathogens and symbionts. Midichloria bacteria (Rickettsiales) are widespread tick endosymbionts that can be transmitted to vertebrate hosts during the tick bite, inducing an antibody response. Their actual role as infectious/pathogenic agents is, however, unclear. Methods We screened for Midichloria DNA African ticks and blood samples collected from trans-Saharan migratory songbirds at their arrival in Europe during spring migration. Results Tick infestation rate was 5.7%, with most ticks belonging to the Hyalomma marginatum species complex. Over 90% of Hyalomma ticks harboured DNA of Midichloria bacteria belonging to the monophylum associated with ticks. Midichloria DNA was detected in 43% of blood samples of avian hosts. Tick-infested adult birds were significantly more likely to test positive to the presence of Midichloria DNA than non-infested adults and second-year individuals, suggesting a long-term persistence of these bacteria within avian hosts. Tick parasitism was associated with a significantly delayed timing of spring migration of avian hosts but had no significant effects on body condition, whereas blood Midichloria DNA presence negatively affected fat deposits of tick-infested avian hosts. Conclusions Our results show that ticks effectively transfer Midichloria bacteria to avian hosts, supporting the hypothesis that they are infectious to vertebrates. Bird infection likely enhances the horizontal spread of these bacteria across haematophagous ectoparasite populations. Moreover, we showed that Midichloria and tick parasitism have detrimental non-independent effects on avian host health during migration, highlighting the complexity of interactions involving ticks, their vertebrate hosts, and tick-borne bacteria.

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.

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 blaKPC 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.

Klebsiella oxytoca causes opportunistic human infections and post-antibiotic haemorrhagic diarrhea. This Enterobacteriaceae species is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4 and Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from, Klebsiella michiganensis (Ko1), K. oxytoca (Ko2), K. huaxiensis (Ko8), and K. grimontii (Ko6). The average nucleotide identity (ANI) of Ko3 and Ko4 were 90.7% with K. huaxiensis and 95.5% with K. grimontii, respectively. In addition, three strains of K. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human feces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4, and Ko8 from the other K. oxytoca species. Based on these results, we propose the names Klebsiella spallanzanii for the Ko3 phylogroup, with SPARK_775_C1T (CIP 111695T and DSM 109531T) as type strain, and Klebsiella pasteurii for Ko4, with SPARK_836_C1T (CIP 111696T and DSM 109530T) as type strain. Strains of K. spallanzanii were isolated from human urine, cow feces, and farm surfaces, while strains of K. pasteurii were found in fecal carriage from humans, cows, and turtles.

In recent years, the advent of NGS technology has made genome sequencing much cheaper than in the past; the high parallelization capability and the possibility to sequence more than one organism at once have opened the door to processing whole symbiotic consortia. However, this approach needs the development of specific bioinformatics tools able to analyze these data. In this work, we describe SeqDex, a tool that starts from a preliminary assembly obtained from sequencing a mixture of DNA from different organisms, to identify the contigs coming from one organism of interest. SeqDex is a fully automated machine learning–based tool exploiting partial taxonomic affiliations and compositional analysis to predict the taxonomic affiliations of contigs in an assembly. In literature, there are few methods able to deconvolve host–symbiont datasets, and most of them heavily rely on user curation and are therefore time consuming. The problem has strong similarities with metagenomic studies, where mixed samples are sequenced and the bioinformatics challenge is trying to separate contigs on the basis of their source organism; however, in symbiotic systems, additional information can be exploited to improve the output. To assess the ability of SeqDex to deconvolve host–symbiont datasets, we compared it to state-of-the-art methods for metagenomic binning and for host–symbiont deconvolution on three study cases. The results point out the good performances of the presented tool that, in addition to the ease of use and customization potential, make SeqDex a useful tool for rapid identification of endosymbiont sequences.

Obesity in childhood and adolescence is considered the most prevalent nutritional disorder, in which eating behaviours represent one important factors of influence. Many aspects influence eating behaviours, but taste is considered the main predictor. However, data concerning correlations of obesity, taste sensitivity and behavioural attitudes, such as food neophobia, in children and adolescents are inconsistent. Moreover, it has been suggested that oral bacteria could have a possible role in obesity development and, also, in taste perception. In this context, the present study focused on host related factors with a proposed link to weight gain. To this purpose, taste sensitivity, salivary microbiota composition and food neophobia were compared between children and adolescents with and without obesity in a cross-sectional study. Results showed that children with obesity presented a significantly lower ability in correctly identifying taste qualities and were characterized by a lesser number of Fungiform Papillae (reported as FP/cm2) compared to normal-weight subjects. Differences in the ecological indexes of microbial alpha-diversity was found between subjects with obesity and normal-weight ones. Moreover, independently from nutritional status, some bacterial genera seemed to differ between subjects with different sensitivity. The potentiality of this multidisciplinary approach could help to better understand and deepen the sensory-driven and microbiological factors related to weight gain.

Abstract Background Cannibalism has been observed in a wide range of animal taxa and its importance in persistence and stability of populations has been documented. In anopheline malaria vectors the inter-instar cannibalism between fourth- and first-instar larvae (L4-L1) has been shown in several species, while intra-instar cannibalism remains poorly investigated. In this study we tested the occurrence of intra-instar cannibalism within larvae of second-, third- and fourth-instar (L2, L3 and L4) of Anopheles gambiae (s.s.) and An. stephensi. Experiments were set up under laboratory conditions and the effects of larval density, duration of the contact period among larvae and the presence of an older larva (i.e. a potential cannibal of bigger size) on cannibalism rate were analysed. Cannibalism was assessed by computing the number of missing larvae after 24 and 48 h from the beginning of the experiments and further documented by records with a GoPro videocamera. Results Intra-instar cannibalism was observed in all larval instars of both species with higher frequency in An. gambiae (s.s.) than in An. stephensi. In both species the total number of cannibalistic events increased from 0–24 to 0–48 h. The density affected the cannibalism rate, but its effect was related to the larval instar and to the presence of older larvae. Interestingly, the lower cannibalism rate between L4 larvae was observed at the highest density and the cannibalism rate between L3 larvae decreased when one L4 was added. Conclusions The present study provides experimental evidence of intra-instar cannibalism in the malaria vectors An. gambiae (s.s.) and An. stephensi and highlights the possible occurrence of complex interactions between all larval instars potentially present in the breeding sites. We hypothesize that the high density and the presence of a potential cannibal of bigger size could affect the readiness to attack conspecifics, resulting into low risk larval behavior and lower cannibalism rate. The understanding of cannibalistic behavior and the factors affecting it is of utmost importance for malaria vectors, as nutrition during larval development can strongly affect the fitness of adult female mosquitoes and ultimately their vector ability.

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.

At its core, the work of clinical microbiologists consists in the retrieving of a few bytes of information (species identification; metabolic capacities; staining and antigenic properties; antibiotic resistance profiles, etc.) from pathogenic agents. The development of next generation sequencing technologies (NGS), and the possibility to determine the entire genome for bacterial pathogens, fungi and protozoans will likely introduce a breakthrough in the amount of information generated by clinical microbiology laboratories: from bytes to Megabytes of information, for a single isolate. In parallel, the development of novel informatics tools, designed for the management and analysis of the so-called Big Data, offers the possibility to search for patterns in databases collecting genomic and microbiological information on the pathogens, as well as epidemiological data and information on the clinical parameters of the patients. Nosocomial infections and antibiotic resistance will likely represent major challenges for clinical microbiologists, in the next decades. In this paper, we describe how bacterial genomics based on NGS, integrated with novel informatic tools, could contribute to the control of hospital infections and multi-drug resistant pathogens.

Abstract Background Proteins from the ABC family (ATP-binding cassette) represent the largest known group of efflux pumps, responsible for transporting specific molecules across lipid membranes in both prokaryotic and eukaryotic organisms. In arthropods they have been shown to play a role in insecticide defense/resistance. The presence of ABC transporters and their possible association with insecticide transport have not yet been investigated in the mosquito Anopheles stephensi, the major vector of human malaria in the Middle East and South Asian regions. Here we investigated the presence and role of ABCs in transport of permethrin insecticide in a susceptible strain of this mosquito species. Methods To identify ABC transporter genes we obtained a transcriptome from untreated larvae of An. stephensi and then compared it with the annotated transcriptome of Anopheles gambiae. To analyse the association between ABC transporters and permethrin we conducted bioassays with permethrin alone and in combination with an ABC inhibitor, and then we investigated expression profiles of the identified genes in larvae exposed to permethrin. Results Bioassays showed an increased mortality of mosquitoes when permethrin was used in combination with the ABC-transporter inhibitor. Genes for ABC transporters were detected in the transcriptome, and five were selected (Anst ABCB2, Anst ABCB3, Anst ABCB4, Anst ABCmember6 and Anst ABCG4). An increased expression in one of them (Anst ABCG4) was observed in larvae exposed to the LD50 dose of permethrin. Contrary to what was found in other insect species, no up-regulation was observed in the Anst ABCB genes. Conclusions Our results show for the first time the involvement of ABC transporters in larval defense against permethrin in An. stephensi and, more in general, confirm the role of ABC transporters in insecticide defense. The differences observed with previous studies highlight the need of further research as, despite the growing number of studies on ABC transporters in insects, the heterogeneity of the results available at present does not allow us to infer general trends in ABC transporter-insecticide interactions.

The isolation and characterization from the sand fly Phlebotomus perniciosus of a Wickerhamomyces anomalus yeast strain (Wa1F1) displaying the killer phenotype was recently reported. In the present work, the killer toxin (KT) produced by Wa1F1 was purified and characterized, and its antimicrobial activity in vitro was investigated against fluconazole- susceptible and -resistant clinical isolates and laboratory strains of Candida albicans and C. glabrata displaying known mutations. Wa1F1-KT showed a differential killing ability against different mutant strains of the same species. The results may be useful for the design of therapeutic molecules based on Wa1F1-KT and the study of yeast resistance mechanisms.