Multicenter epidemiological investigation and genetic characterization of respiratory syncytial virus and metapneumovirus infections in the pre-pandemic 2018-2019 season in northern and central Italy.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause a high burden of disease, particularly in children and the elderly. With the aim to add knowledge on RSV and HMPV infections in Italy, a prospective, multicenter study was conducted by eight centers of the Working Group on Respiratory Virus Infections (GLIViRe), from December 2018-April 2019. Weekly distribution and patients' demographic and clinical data were compared in 1300 RSV and 222 HMPV-positive cases. Phylogenetic analysis of the G-glycoprotein coding region was performed to characterize circulating strains. RSV positivity ranged from 6.4% in outpatients of all ages to 31.7% in hospitalized children; HMPV positivity was 4-1.2% with no age-association. RSV season peaked in February and ended in mid-April: HMPV circulation was higher when RSV decreased in early spring. RSV was more frequent in infants, whereas HMPV infected comparatively more elderly adults; despite, their clinical course was similar. RSV-B cases were two-thirds of the total and had similar clinical severity compared to RSV-A. Phylogenetic analysis showed the circulation of RSV-A ON1 variants and the predominance of RSV-B genotype BA10. HMPV genotype A2c was the prevalent one and presented insertions of different lengths in G. This first multicenter Italian report on seasonality, age-specific distribution, and clinical presentation of RSV and HMPV demonstrated their substantial disease burden in young patients but also in the elderly. These data may provide the basis for a national respiratory virus surveillance network.

SARS-CoV-2 variants of concern surveillance including Omicron using RT-PCR-based genotyping offers comparable performance to whole genome sequencing.

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.

Air and waterborne microbiome of a pharmaceutical plant provide insights on spatiotemporal variations and community resilience after disturbance.

BACKGROUND: The presence of microrganisms in pharmaceutical production plant environments is typically monitored by cultural methods, however these cannot detect the unculturable fraction of the microbial community. To get more accurate information on the composition of these indoor microbial communities, both water and air microbiome from a pharmaceutical production plant were profiled by 16S amplicon sequencing. RESULTS: In the water system, we found taxa which typically characterize surface freshwater, groundwater and oligotrophic environments. The airborne microbiome resulted dominated by taxa usually found in outdoor air in combination with human-associated taxa. The alpha- and beta- diversity values showed that the heat-based sanitization process of the water plant affects the composition of the water microbiome by transiently increasing both diversity and evenness. Taxonomic compositional shifts were also detected in response to sanitization, consisting in an increase of Firmicutes and α-Proteobacteria. On the other hand, seasonality seems to be the main driver of bacterial community composition in air of this work environment. CONCLUSIONS: This approach resulted useful to describe the taxonomy of these indoor microbiomes and could be further applied to other built environments, in which the knowledge of the microbiome composition is of relevance. In addition, this study could assist in the design of new guidelines to improve microbiological quality control in indoor work environments.

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.

Evolution of Stenotrophomonas maltophilia in Cystic Fibrosis Lung over Chronic Infection: A Genomic and Phenotypic Population Study.

Stenotrophomonas maltophilia has been recognized as an emerging multi-drug resistant opportunistic pathogen in cystic fibrosis (CF) patients. We report a comparative genomic and phenotypic analysis of 91 S. maltophilia strains from 10 CF patients over a 12-year period. Draft genome analyses included in silico Multi-Locus Sequence Typing (MLST), Single-Nucleotide Polymorphisms (SNPs), and pangenome characterization. Growth rate, biofilm formation, motility, mutation frequency, in vivo virulence, and in vitro antibiotic susceptibility were determined and compared with population structure over time. The population consisted of 20 different sequence types (STs), 11 of which are new ones. Pangenome and SNPs data showed that this population is composed of three major phylogenetic lineages. All patients were colonized by multiple STs, although most of them were found in a single patient and showed persistence over years. Only few phenotypes showed some correlation with population phylogenetic structure. Our results show that S. maltophilia adaptation to CF lung is associated with consistent genotypic and phenotypic heterogeneity. Stenotrophomonas maltophilia infecting multiple hosts likely experiences different selection pressures depending on the host environment. The poor genotype-phenotype correlation suggests the existence of complex regulatory mechanisms that need to be explored in order to better design therapeutic strategies.

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.

Lack of evidence for an association between seminoma and human papillomavirus infection using GP5+/GP6+ consensus primers.

Testicular germ cell tumors account for about 1% of all cancers. The incidence of these tumors is increasing and they represent the most common solid malignancies of young men aged 15-40 years with seminoma being one of the most common histotype. Pathogenesis of testicular germ cell tumors remains unknown and, although cryptorchidism is considered the main risk factor, there is evidence of an association with environmental and genetic risk factors. Human papillomaviruses (HPV) are a family of DNA viruses and represent a major risk factor for cervical cancer. In addition, they have been associated with other human non-malignant and malignant diseases, including breast and head and neck cancer. HPV sequences have been detected throughout the male lower genitourinary tract as well as in seminal fluid and an increased testicular tumorigenesis has been reported in HPV transgenic mice. Aim of this study was to evaluate the potential involvement of HPV in human testicular tumorigenesis. Real-time PCR employing GP5+/GP6+ consensus HPV primers was used to examine the presence of HPV sequences in a subset of human seminoma (n = 61) and normal testicles (n = 23). None of the specimens tested displayed the presence of HPV DNA. These findings do not support an association between HPV and human seminoma and warrant further studies to assess definitively the role of these viruses in human testicular tumorigenesis.