EVOLUTION OF WOLBACHIA SYMBIOSIS IN ARTHOPODS AND NEMATODES: INSIGTHS FROM PHYLOGENETICS AND COMPARATIVE GENOMICS

Wolbachia is a bacterium observed in relationship with a wide array of arthropod and nematode species. This is an obligate intracellular symbiont, maternally transferred through the host oocytes. In arthropods Wolbachia is able to manipulate reproduction, using multiple strategies to increase the fitness of infected females. In nematodes the bacterium has a fundamental, and not completely understood, role in larvae development. Wolbachia infects ~50% of all the arthropod species worldwide, and in some of them it can be considered the most important sex determination factor. In contrast, Wolbachia presence is much more limited in nematodes, being present in a limited number of filarial species. The taxonomic status within the Wolbachia genus is highly debated, with the current classification dividing all strains in 14 'supergroups'. During my Ph.D. I studied the evolution of the symbiotic relationship between Wolbachia and its arthropod and nematode hosts, using genomic approaches. Indeed, during the evolution of the Wolbachia-host relationship, genetic signs have been left in the Wolbachia genomes. I worked to identify these genomic signs and to evaluate them within an evolutionary frame, in order to obtain a better understanding of how the Wolbachia-host symbiosis evolved. The work here presented can be organized in three major sections: i) the sequencing and analysis of the genome of the filarial nematode Dirofilaria immitis and of its symbiotic Wolbachia strain, wDi; ii) the sequencing of the genome of Wolbachia endosymbiont of Litomosoides sigmodontis, and the phylogenomic reconstruction of the Wolbachia supergroups A-D; iii) a comparison of the genomes of 26 Wolbachia strains spanning the A to F supergroups. Here a schematic summary of the results is reported: 1. Dirofilaria immitis and the Wolbachia symbiont wDi show metabolic complementarity for fundamental pathways 2. The metabolic pathway for the synthesis of wDi membrane proteins is one evolving the fastest in the genome of the bacterium 3. Nematode Wolbachia belonging to supergroups C and D are monophyletic, indicating that a single transition to mutualism likely occurred during the evolution of Wolbachia 4. Wolbachia strains of the C supergroup show genomic features that are unique in the genus, such as a much higher level of synteny compared to the rest of Wolbachia supergroups, and a newly generated pattern of GC skew curves, typically observed in free-living bacteria genomes 5. Wolbachia supergroups show conserved genomic features, which suggest genomic isolation among them

Three cases of mcr-1-positive colistin-resistant Escherichia coli bloodstream infections in Italy, August 2016 to January 2017

We describe three cases of bloodstream infection caused by colistin-resistant Escherichia coli in patients in a tertiary hospital in Italy, between August 2016 and January 2017. Whole genome sequencing detected the mcr-1 gene in three isolated strains belonging to different sequence types (STs). This occurrence of three cases with mcr-1-positive E. coli belonging to different STs in six months suggests a widespread problem in settings where high multidrug resistance is endemic such as in Italy

EasyPrimer: user-friendly tool for pan-PCR/HRM primers design. Development of an HRM protocol on wzi gene for fast Klebsiella pneumoniae typing

In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the consensus sequence. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer pairs on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer pairs on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer pairs. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer pairs required for bacterial typing allowing to perform cost saving, large-scale surveillance programs

Intra-instar larval cannibalism in Anopheles gambiae (s.s.) and Anopheles stephensi (Diptera: Culicidae)

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

Multiple Klebsiella pneumoniae KPC Clones Contribute to an Extended Hospital Outbreak

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

In vivo acquisition and risk of inter-species spread of blaKPC-3-plasmid from Klebsiella pneumoniae to Serratia marcescens in the lower respiratory tract

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

SeqDeχ : a sequence deconvolution tool for genome separation of endosymbionts from mixed sequencing samples

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

Antimicrobial activity of aztreonam in combination with old and new β-lactamase inhibitors against mbl and esbl co-producing gram-negative clinical isolates: Possible options for the treatment of complicated infections

none14noMetallo-β-lactamases (MBLs) are among the most challenging bacterial enzymes to over-come. Aztreonam (ATM) is the only β-lactam not hydrolyzed by MBLs but is often inactivated by co-produced extended-spectrum β-lactamases (ESBL). We assessed the activity of the combination of ATM with old and new β-lactamases inhibitors (BLIs) against MBL and ESBL co-producing Gram-negative clinical isolates. Six Enterobacterales and three non-fermenting bacilli co-producing MBL and ESBL determinants were selected as difficult-to-treat pathogens. ESBLs and MBLs genes were characterized by PCR and sequencing. The activity of ATM in combination with seven different BLIs (clavulanate, sulbactam, tazobactam, vaborbactam, avibactam, relebactam, zidebactam) was assessed by microdilution assay and time–kill curve. ATM plus avibactam was the most effective combination, able to restore ATM susceptibility in four out of nine tested isolates, reaching in some cases a 128-fold reduction of the MIC of ATM. In addition, relebactam and zidebactam showed to be effective, but with lesser reduction of the MIC of ATM. E. meningoseptica and C. indologenes were not inhibited by any ATM–BLI combination. ATM–BLI combinations demonstrated to be promising against MBL and ESBL co-producers, hence providing multiple options for treatment of related infections. However, no effective combination was found for some non-fermentative bacilli, suggesting the presence of additional resistance mechanisms that complicate the choice of an active therapy.openMorroni G.; Bressan R.; Fioriti S.; D'Achille G.; Mingoia M.; Cirioni O.; Di Bella S.; Piazza A.; Comandatore F.; Mauri C.; Migliavacca R.; Luzzaro F.; Principe L.; Lagatolla C.Morroni, G.; Bressan, R.; Fioriti, S.; D'Achille, G.; Mingoia, M.; Cirioni, O.; Di Bella, S.; Piazza, A.; Comandatore, F.; Mauri, C.; Migliavacca, R.; Luzzaro, F.; Principe, L.; Lagatolla, C

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