First imported case of Candida auris infection in Milan, Italy: genomic characterisation.

PURPOSE: Candida auris, an emerging multidrug-resistant yeast, has been reported worldwide. In Italy, the first case was reported in 2019. We describe the first case of C. auris, imported from Greece, in Milan, using whole genome sequencing to characterise mutations associated with antifungal resistance. CASE PRESENTATION: On October 2022 an 80-year-old Italian man was hospitalised in Greece. In the absence of clinical improvement, the patient was transferred to our hospital, in Italy, where blood culture resulted positive for C. auris. Despite therapy, the patient died of septic shock. In a phylogenetic analysis the genome was assigned to Clade I with strains from Kenya, United Arab Emirates and India. D1/D2 region resulted identical to a Greek strain, as for many other strains from different World regions, highlighting the diffusion of this strain. CONCLUSION: Importation of C. auris from abroad has been previously described. We report the first case of C. auris imported into Italy from Greece, according to phylogenetic analysis. This case reinforces the need for monitoring critically ill hospitalised patients also for fungi and addresses the need for the standardisation of susceptibility testing and strategies for diagnosis and therapy.

Genetic barriers more than environmental associations explain Serratia marcescens population structure.

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.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

P-DOR, an easy-to-use pipeline to reconstruct bacterial outbreaks using genomics.

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.

Computational protocol to perform a spatiotemporal reconstruction of an epidemic.

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.

The evolution of intramitochondriality in Midichloria bacteria.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

A spatiotemporal reconstruction of the 1630 plague epidemic in Milan.

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.

Alteration of taste perception, food neophobia and oral microbiota composition in children with food allergy.

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.

Spatial and Temporal Analyses of the Event of Death for 1480 in Milan Using the Data Contained in the Sforza's Registers of the Dead.

Historical death registration was conducted primarily to assess the presence of plague. The Liber Mortuorum of Milan was one of Europe's first registers with many socio-demographical details. In this work, we consider 1480 to make spatial and temporal analyses of the event of death to find possible explanations about the events' distribution and the events' trend over time. The spatial analyses involved Moran's I, the LISA, and the heatmaps; the temporal analysis applied the Durbin-Watson test. All the analyses were conducted separately on all subjects (1813), children (765), and adults (1046). Contrade (districts) were considered for spatial analysis. Moran's I and the Durbin Watson test were significant on all subjects and children's analyses, and the LISA showed the same results for those groups. Children may significantly impact the distribution of death and the trend over time. At least half of the children were 0 years old, and survival in the very first childhood period was closely linked to the family, so that it could be a proxy of the conditions of an area.

The Bio-Diversity and the Role of Gut Microbiota in Postmenopausal Women with Luminal Breast Cancer Treated with Aromatase Inhibitors: An Observational Cohort Study.

The interactions between aromatase inhibitors (AI) in breast cancer (BC) and gut microbiota (GM) have not been completely established yet. The aim of the study is to evaluate the bio-diversity of GM and the relationship between GM, inflammation and tumor-infiltrating lymphocytes (TILs) in postmenopausal women with BC during adjuvant AI treatment compared to women with disease relapse during or after one year of AI therapy ("endocrine-resistant"). We conducted a monocenter observational case-control study. Eighty-four women with BC (8 cases, 76 controls) were enrolled from 2019 to 2021. We observed a significant difference in the mean microbial abundance between the two groups for the taxonomic rank of order (p 0.035) and family (p 0.029); specifically, the case group showed higher diversity than the control group. Veillonella reached its maximum abundance in cases (p 0.022). Cytokine levels were compared among the groups created considering the TILs levels. We obtained a statistically significant difference (p 0.045) in IL-17 levels among the groups, with patients with low TILs levels showing a higher median value for IL-17 (0.15 vs. 0.08 pg/mL). Further studies about the bio-diversity in women with BC may lead to the development of new biomarkers and targeted interventions.

A large-scale genomic snapshot of Klebsiella spp. isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings.

The Klebsiella group, found in humans, livestock, plants, soil, water and wild animals, is genetically and ecologically diverse. Many species are opportunistic pathogens and can harbour diverse classes of antimicrobial resistance genes. Healthcare-associated Klebsiella pneumoniae clones that are non-susceptible to carbapenems can spread rapidly, representing a high public health burden. Here we report an analysis of 3,482 genome sequences representing 15 Klebsiella species sampled over a 17-month period from a wide range of clinical, community, animal and environmental settings in and around the Italian city of Pavia. Northern Italy is a hotspot for hospital-acquired carbapenem non-susceptible Klebsiella and thus a pertinent setting to examine the overlap between isolates in clinical and non-clinical settings. We found no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside the clinical environment. Although we noted occasional transmission between clinical and non-clinical settings, our data point to a limited role of animal and environmental reservoirs in the human acquisition of Klebsiella spp. We also provide a detailed genus-wide view of genomic diversity and population structure, including the identification of new groups.

The red thread between methylation and mutation in bacterial antibiotic resistance: How third-generation sequencing can help to unravel this relationship.

DNA methylation is an important mechanism involved in bacteria limiting foreign DNA acquisition, maintenance of mobile genetic elements, DNA mismatch repair, and gene expression. Changes in DNA methylation pattern are observed in bacteria under stress conditions, including exposure to antimicrobial compounds. These changes can result in transient and fast-appearing adaptive antibiotic resistance (AdR) phenotypes, e.g., strain overexpressing efflux pumps. DNA methylation can be related to DNA mutation rate, because it is involved in DNA mismatch repair systems and because methylated bases are well-known mutational hotspots. The AdR process can be the first important step in the selection of antibiotic-resistant strains, allowing the survival of the bacterial population until more efficient resistant mutants emerge. Epigenetic modifications can be investigated by third-generation sequencing platforms that allow us to simultaneously detect all the methylated bases along with the DNA sequencing. In this scenario, this sequencing technology enables the study of epigenetic modifications in link with antibiotic resistance and will help to investigate the relationship between methylation and mutation in the development of stable mechanisms of resistance.

The Role of Virulence Factors in Neonatal Sepsis Caused by Enterobacterales: A Systematic Review.

Neonatal sepsis is a life-threatening condition with high mortality. Virulence determinants relevant in causing Gram-negative (GN) neonatal sepsis are still poorly characterized. A better understanding of virulence factors (VFs) associated with GN neonatal sepsis could offer new targets for therapeutic interventions. The aim of this review was to assess the role of GN VFs in neonatal sepsis. We primarily aimed to investigate the main VFs leading to adverse outcome and second to evaluate VFs associated with increased invasiveness/pathogenicity in neonates. MEDLINE, Embase, and Cochrane Library were systematically searched for studies reporting data on the role of virulome/VFs in bloodstream infections caused by Enterobacterales among neonates and infants aged 0-90 days. Twenty studies fulfilled the inclusion criteria. Only 4 studies reported data on the association between pathogen virulence determinants and neonatal mortality, whereas 16 studies were included in the secondary analyses. The quality of reporting was suboptimal in the great majority of the published studies. No consistent association between virulence determinants and GN strains causing neonatal sepsis was identified. Considerable heterogeneity was found in terms of VFs analysed and reported, included population and microbiological methods, with the included studies often showing conflicting data. This variability hampered the comparison of the results. In conclusions, pathogens responsible for neonatal sepsis are widely heterogenous and can use different pathways to develop invasive disease. The recent genome-wide approach needs to include multicentre studies with larger sample sizes, analyses of VF gene profiles instead of single VF genes, alongside a comprehensive collection of clinical information. A better understanding of the roles of virulence genes in neonatal GN bacteraemia may offer new vaccine targets and new markers of highly virulent strains. This information can potentially be used for screening and preventive interventions as well as for new targets for anti-virulence antibiotic-sparing therapies.

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

Pathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries. IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

Dysbiosis in Children With Neurological Impairment and Long-Term Enteral Nutrition.

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.

Biotics in pediatrics: a short overview.

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.

Correction: Morroni et al. Antimicrobial Activity of Aztreonam in Combination with Old and New ß-Lactamase Inhibitors against MBL and ESBL Co-Producing Gram-Negative Clinical Isolates: Possible Options for the Treatment of Complicated Infections. Antibiotics 2021, 10, 1341.

Results have been implemented with additional data, which have been commented in the following paragraphs [...].

Antimicrobial Susceptibility, Virulence, and Genomic Features of a Hypervirulent Serotype K2, ST65 Klebsiella pneumoniae Causing Meningitis in Italy.

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.

Wolbachia in Aedes koreicus: Rare Detections and Possible Implications.

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.