Sara Tagliazucchi

Genomic characterization of Nontuberculous Mycobacteria

Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Characterization of 17 strains belonging to the Mycobacterium simiae complex and description of Mycobacterium paraense sp. nov.

Fourteen mycobacterial strains isolated from pulmonary samples of independent patients in the state of Pará (Brazil), and three strains isolated in Italy, were characterized using a polyphasic approach. Thorough genetic investigation, including whole-genome sequencing, demonstrated that the strains belong to the M. simiae complex, being most closely related to Mycobacterium interjectum. For 14 of the strains, evidence emerged supporting their inclusion in a previously unreported species of the genus Mycobacterium, for which the name Mycobacterium paraense sp. nov. is proposed (type strain, IEC26(T) = DSM 46749(T) = CCUG 66121(T)). The novel species is characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies, and a HPLC mycolic acid profile different from other known mycobacteria. In different genetic regions, high sequence microheterogeneity was detected.

The new phylogeny of the genus Mycobacterium: The old and the news

BACKGROUND Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus. RESULTS Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous. CONCLUSIONS Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences.

Prevalence of Usutu and West Nile virus antibodies in human sera, Modena, Italy, 2012: FAGGIONI et al.

A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North‐eastern Italy.

Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation

Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. We retrospectively analysed a consecutive series of 486 human immunodeficiency virus‐negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61·5%) and 187 (38·5%) cases, respectively. Of note, seven of the 111 (6·3%) patients with benign lymphadenopathy without well‐defined aetiology, showed chronic/recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reactive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)‐6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV‐6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well‐known aetiology and without recurrences, positivity for HHV‐6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter‐follicular regions. Immunohistochemistry for HHV‐6A and HHV‐6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV‐6B reactivation and chronic/recurrent benign lymphadenopathy.

Widespread circulation of echovirus 6 causing aseptic meningitis in paediatric patients in the area of Modena, Italy, in 2011

Introduction : Between May and November 2011, enterovirus RNA was detected in the cerebrospinal fluids (CSFs) of 72 children with signs of aseptic meningitis admitted to paediatric departments of different Hospitals of the prefecture of Modena, Emilia Romagna region, Italy. Enterovirus RNA was detected in 34 CSFs by commercial reverse transcriptase-polymerase chain reaction (RT-PCR). Twenty-one samples, resulted human enterovirus B by species-specific RT-nested PCR, were submitted to sequencing of the 3’ terminus of the VP1 gene. Materials and Methods: Upon sequencing and interrogation of the National Center for Biotechnology Information database, all 21 viruses were characterized as echovirus 6 (E6), and posses a 100% nucleotide identity each other. Results : This study reports the molecular detection and typing of E6 isolated from clinical specimens from paediatric patients with aseptic meningitis in the wide area of Modena, Italy, in 2011.

RIPETUTI CLUSTERS DA S. MARCESCENS IN UN REPARTO DI TERAPIA INTENSIVA NEONATALE: ASPETTI MICROBIOLOGICI E CLINICO-EPIDEMIOLOGICI

Introduzione. Serratia marcescens è stata segnalata negli ultimi anni come causa di epidemie, talora difficili da eradicare, in reparti di terapia intensiva neonatale. Scopo della ricerca è la valutazione degli aspetti microbiologici e clinicoepidemiologici di ripetuti clusters di infezioni da S. marcescens, occorsi nel reparto di Neonatologia del Policlinico di Modena nell’arco di 3 anni. Metodi. È stato condotto uno studio molecolare con RFLPPCR su ceppi di S.marcescens isolati da campioni clinici di 55 neonati. Sono stati valutati i fattori di rischio e le misure di controllo intraprese. Risultati. 38 bambini erano colonizzati e 17 affetti da infezioni di diversa gravità; di questi 6, prematuri di basso peso, hanno sviluppato setticemia ed uno è deceduto. La tipizzazione molecolare dei ceppi ha identificato 7 diversi genotipi (A-G) dei quali due prevalenti: A, 12 casi, e B, 27 casi. Complessivamente sono stati riscontrati 4 clusters di diversa entità: I, gennaio aprile 2003, 9 casi, genotipo A; II, luglio 2003, 3 casi, genotipo B; III, gennaio giugno 2004, 20 casi, genotipi C (8), B (5), D (2), E (3); IV, giugno 2005 febbraio 2006, 24 casi, genotipi A (3), B (18), C (1), G (1). Ripetuti campioni prelevati dall’ambiente e dal personale sono risultati costantemente negativi. I principali fattori di rischio identificati sono stati: basso peso alla nascita, prematurità, ventilazione forzata e catetere venoso centrale. Conclusioni. Genotipi eterogenei sono stati riscontrati nei ceppi studiati. I cloni A e B identificati nei primi 2 clusters, sono stati nuovamente repertati dopo 3 anni nell’ultimo cluster, dimostrando una persistenza nell’ ambiente. Non è stata identificata una fonte comune. Il sovraffollamento del reparto in rapporto al calo del personale è stato collegato ai clusters di infezioni. La trasmissione crociata tramite le mani è stata probabilmente una importante via di diffusione. Screening microbiologici ravvicinati, cohorting degli infetti e implementazione delle misure igieniche sono risultati aspetti fondamentali nel contenimento dell’epidemia. 141