EVOLUTION, COMPARATIVE GENOMICS AND GENOMIC EPIDEMIOLOGY OF BACTERIA OF PUBLIC HEALTH IMPORTANCE

La presente tesi \ue8 incentrata sull'epidemiologia genomica delle infezioni batteriche ospedaliere. L'ambiente ospedaliero \ue8 peculiare, in quanto al suo interno si concentrano un elevato numero di agenti batterici, pazienti con un sistema immunitario debole e un uso massiccio di sostanze antimicrobiche. Questa combinazione favorisce lo sviluppo e la selezione di ceppi resistenti agli antibiotici e la diffusione di infezioni opportunistiche: in generale il prosperare dei patogeni nosocomiali. Alcune tecniche all'avanguardia per lo studio di questo tipo di infezioni sono basate sull\u2019uso della genomica e di approcci evoluzionistici: esse permettono di conoscere le caratteristiche genomiche dei ceppi batterici e di ricostruire la loro storia evolutiva. Grazie alla possibilit\ue0 di sequenziare il DNA ad un prezzo sempre pi\uf9 economico, i progetti di ricerca sono supportati da un numero sempre crescente di genomi e i dati genomici depositati nelle banche dati sono in crescita esponenziale: questo rende possibile eseguire una variet\ue0 sempre maggiore di analisi. Il primo lavoro qui riportato descrive l'evoluzione del Clonal Complex 258 (CC258) di Klebsiella pneumoniae. Le mutazioni puntiformi (single nucleotide polymorphism, SNP) hanno permesso di ricostruire la filogenesi globale di tutta la specie e di collocare il CC258 nel suo contesto evolutivo. Successivamente, \ue8 stato possibile rilevare la presenza di una ricombinazione di 1,3 Mb nei genomi del clade in analisi. Un\u2019analisi del molecular clock ha poi consentito di datare sia questo che gli altri eventi di ricombinazione scoperti in lavori precedenti. Questi risultati sono stati usati per completare il quadro della storia evolutiva del CC258, caratterizzata da frequenti eventi di macro-ricombinazione. Un\u2019evoluzione rapida e caratterizzata da scambi di elevate quantit\ue0 di informazioni genomiche \ue8 una caratteristica comune ad altri patogeni nosocomiali che sviluppano fenotipi da "superbatteri". Sebbene frequente, il modello di evoluzione per macro-ricombinazioni non \ue8 comune a tutti i batteri responsabili di infezioni nosocomiali. Un\u2019eccezione \ue8 il ceppo SMAL di Acinetobacter baumannii, presentato in un altro sottoprogetto di questa tesi. In questo lavoro sono stati analizzati i genomi del sequence type (ST) 78 di A. baumannii. La filogenesi e la genomica comparativa hanno rivelato la presenza di due differenti cladi all'interno del ST che presentano differenti "stili" evolutivi. Un gruppo (contenente i genomi SMAL) \ue8 caratterizzato da una minore variabilit\ue0 del contenuto genico e dalla presenza di un numero pi\uf9 elevato di copie di insertion sequence (IS). Una IS interrompe il gene comEC/rec2 in tutti i genomi SMAL. Questo gene codifica per una proteina coinvolta nell\u2019acquisizione del DNA esogeno, quindi la sua inattivazione limita lo scambio di geni. Questo suggerisce una spiegazione per la bassa plasticit\ue0 genomica. In un altro lavoro presentato in questa tesi, l'epidemiologia genomica \ue8 stata applicata per ricostruire la diffusione di un focolaio epidemico di K. pneumoniae in un\u2019unit\ue0 di terapia intensiva ospedaliera. In un primo momento, \ue8 stato utilizzato un approccio filogenetico per separare gli isolati appartenenti all'epidemia da quelli sporadici. Poi le date di isolamento e gli SNP genomici hanno permesso di costruire una rete genomica che modellasse la propagazione delle infezioni nel reparto. La ricostruzione ha indicato una diffusione radiale del patogeno dal paziente zero a tutti gli altri infetti, rivelando cos\uec un errore sistematico nelle procedure di biosicurezza dell'ospedale. Questa applicazione quasi forense dell'epidemiologia genomica \ue8 stata utilizzata anche in altri due lavori qui presentati, entrambi riguardanti la ricostruzione di infezioni alimentari. In uno degli articoli, incentrato su Salmonella enterica, l\u2019analisi filogenetica \ue8 stata eseguita solamente con gli SNP sinonimi al fine di filtrare le mutazioni patoadattative. Nell'altro lavoro sono stati utilizzati dati epidemiologici, tipizzazione molecolare e filogenesi basata sugli SNP per studiare l'infezione di nove isolati di Listeria monocytogenes, che si ritenevano essere parte dello stesso focolaio e alla fine sono risultati genomicamente non correlati. Infine, viene qui presentato anche un articolo di review riguardante l'epidemiologia genomica. L'articolo \ue8 focalizzato sulle ultime pubblicazioni ad alto impatto che analizzano l'evoluzione genomica degli agenti patogeni batterici e le dinamiche di propagazione delle epidemie in brevi periodi di tempo. L'articolo descrive, infine, le ultime ricostruzioni epidemiologiche a livello storico, che sono possibili grazie alle moderne tecnologie di isolamento e sequenza del DNA.The present thesis is focused on genomic epidemiology of bacterial hospital infections. The hospital environment is unique, as it concentrates a high number of bacterial agents, frequent antibiotic use, and patients with weak immune systems. This combination favours the development and selection of antibiotic resistant strains and the spread of opportunistic infections: in general the thriving of nosocomial pathogens. Genomics and evolutionary approaches have emerged as the cutting edge tools for studying this kind of infections, allowing to study the genomic features of bacterial strains and their evolution. Thanks to the possibility to sequence DNA at a constantly cheaper price, research projects are supported by a growing number of genomes and a considerable amount of genomic data is available in the databases, expanding the amount of possible investigations that can be performed. The first work presented here describes the evolution of the Clonal Complex 258 (CC258) of Klebsiella pneumoniae. Single nucleotide polymorphisms (SNPs) allowed to reconstruct the global phylogeny of the entire species and to collocate the CC258 in its evolutionary context. Furthermore, it was possible to detect the presence of a 1.3 Mb recombination in the genomes of the clade in analysis. A molecular clock approach allowed to date this and other previously discovered recombination events. These findings were used to complete the picture of the evolutionary history of CC258, which is characterized by frequent macro-recombination events. A quick evolutive strategy characterized by exchange of high amount of information is a common feature to other nosocomial pathogens, which develop \u201csuperbug\u201d phenotypes. Although common, the macro-recombination evolution model is not shared by all nosocomial infection bacteria. One exception is the SMAL strain of Acinetobacter baumannii, presented in another subproject of this thesis. In this work, the genomes of Sequence Type (ST) 78 of A. baumannii were analyzed. Phylogeny and comparative genomics revealed the presence of two different clades within the ST, presenting different evolutive \u201clifestyles\u201d. One group (containing the SMAL genomes) was characterized by a lower gene content variability and by the presence of a higher copy number of insertion sequences (ISs). One IS interrupts the comEC/rec2 gene in all the SMAL genomes. This gene codes for a protein involved in the exogenous DNA importation, thus its inactivation limits the gene exchange, suggesting an explanation for the low genomic plasticity. In another work presented in this document, genomic epidemiology was applied to reconstruct the spreading routes of a K. pneumoniae epidemic event in an hospital intensive care unit. At first, a phylogenetic approach was used to separate the isolates that belonged to the outbreak from the sporadic ones. Then the isolation dates and genomic SNPs allowed to build a genomic network, which modelled the chain of infection events in the ward. The reconstruction suggested a star-like diffusion of the pathogen from patient zero to the other infected ones, thus revealing a systematic error in the biosafety procedures of the hospital. This almost-forensic application of genomic epidemiology was also used in two other works presented, both of them concerning the reconstruction of food-borne infections. In one of the works, focused on Salmonella enterica, only synonymous SNPs were used as input to a phylogenetic based investigation, in order to filter out pathoadaptative mutations. In the other article, epidemiological data, molecular typing and SNP-based phylogeny were used to investigate the infection of nine Listeria monocytogenes isolates, which were believed to be part of the same outbreak and in the end proved to be genomically unrelated. Lastly, a review paper on genomic epidemiology is also presented. The article is focused on the latest high impact publications analyzing the genome evolution of bacterial pathogens as well as the propagation dynamics of epidemic outbreaks in very short periods of time. The article also describes the latest historical epidemiological studies, which are possible thanks to modern DNA isolation and sequencing technologies

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

Effects of global changes on the climatic niche of the tick Ixodes ricinus inferred by species distribution modelling

Background: Global climate change can seriously impact on the epidemiological dynamics of vector-borne diseases. In this study we investigated how future climatic changes could affect the climatic niche of Ixodes ricinus (Acari, Ixodida), among the most important vectors of pathogens of medical and veterinary concern in Europe. Methods. Species Distribution Modelling (SDM) was used to reconstruct the climatic niche of I. ricinus, and to project it into the future conditions for 2050 and 2080, under two scenarios: a continuous human demographic growth and a severe increase of gas emissions (scenario A2), and a scenario that proposes lower human demographic growth than A2, and a more sustainable gas emissions (scenario B2). Models were reconstructed using the algorithm of "maximum entropy", as implemented in the software Maxent 3.3.3e; 4,544 occurrence points and 15 bioclimatic variables were used. Results: In both scenarios an increase of climatic niche of about two times greater than the current area was predicted as well as a higher climatic suitability under the scenario B2 than A2. Such an increase occurred both in a latitudinal and longitudinal way, including northern Eurasian regions (e.g. Sweden and Russia), that were previously unsuitable for the species. Conclusions: Our models are congruent with the predictions of range expansion already observed in I. ricinus at a regional scale and provide a qualitative and quantitative assessment of the future climatically suitable areas for I. ricinus at a continental scale. Although the use of SDM at a higher resolution should be integrated by a more refined analysis of further abiotic and biotic data, the results presented here suggest that under future climatic scenarios most of the current distribution area of I. ricinus could remain suitable and significantly increase at a continental geographic scale. Therefore disease outbreaks of pathogens transmitted by this tick species could emerge in previous non-endemic geographic areas. Further studies will implement and refine present data toward a better understanding of the risk represented by I. ricinus to human health

Differential single nucleotide polymorphism-based analysis of an outbreak caused by Salmonella enterica serovar Manhattan reveals epidemiological details missed by standard pulsed-field gel electrophoresis

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n = 15) and food, feed, animal, and environmental sources (n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB-BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system

Draft Genome Sequence of Clostridium tyrobutyricum Strain DIVETGP, Isolated from Cow's Milk for Grana Padano Production

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999\ua0bp, with a G+C content of 30.8%

Genomic epidemiology of Klebsiella pneumoniae in Italy and novel insights into the origin and global evolution of its resistance to carbapenem antibiotics

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an 3c1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy

A Network Perspective for Community Assembly

Species interactions are responsible for many key mechanisms that govern the dynamics of ecological communities. Variation in the way interactions are organized among species results in different network structures, which translates into a community's ability to resist collapse and change. To better understand the factors involved in dictating ongoing dynamics in a community at a given time, we must unravel how interactions affect the assembly process. Here, we build a novel, integrative conceptual model for understanding how ecological communities assemble that combines ecological networks and island biogeography theory, as well as the principles of niche theory. Through our conceptual model, we show how the rate of species turnover and gene flow within communities will influence the structure of ecological networks. We conduct a preliminary test of our predictions using plant-herbivore networks from differently-aged sites in the Hawaiian archipelago. Our approach will allow future modeling and empirical studies to develop a better understanding of the role of the assembly process in shaping patterns of biodiversity