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

Abstract 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

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

RICERCA DI MARCATORI PER LA DIAGNOSI DI INFEZIONE FUNGINA INVASIVAE (IFI) MEDIANTE APPROCCIO DIAGNOSTICO COMBINATO

Introduzione L'incidenza di IFI ed in particolare delle candidiasi nei pazienti ricoverati in terapia intensiva è in costante aumento ed è associata ad un’elevata mortalità. Evidenze recenti sottolineano l’importanza di indagini multidirezionali e multi-parametriche ai fini di una diagnosi più rapida e precoce possibile. Scopo del presente studio è valutare l’efficacia di un approccio combinato che prevede la ricerca dell’antigene beta-glucano e di anticorpi specifici verso antigeni selezionati di Candida. Metodi 18 pazienti (Reparto di Terapia Intensiva, AOU-Policlinico Modena) sono stati arruolati e suddivisi in due gruppi: gruppo IFI (pazienti con aspergillosi o candidiasi invasiva proven/probable o con diagnosi clinica di pneumocistosi polmonare) e gruppo non-IFI (soggetti ospedalizzati con diagnosi diversa da IFI). In totale, sono stati saggiati 42 sieri con il saggio panfungino (1-3)-β-D-glucano (BG, Fungitell ®) e mediante un saggio sierologico “home-made” basato su microarray proteico per la determinazione quantitativa della risposta anticorpale nei confronti di 11 antigeni di Candida albicans. Risultati Il saggio BG ha mostrato sensibilità, specificità, valore predittivo positivo e negativo di 100%, 85,7%, 88,9 % e 100% rispettivamente; l’analisi delle curve ROC ha restituito una AUC di 0.857 (95% CI: 0.577-1). Il saggio sierologico ha rilevato nei pazienti con emocoltura positiva, una risposta anticorpale nei confronti di 3 antigeni (Bgl2, Pgk1 e Grp2), recentemente descritti come marker diagnostici di candidasi invasiva (Ardizzoni et al., 2013 – submitted). Nessuna risposta anticorpale significativa è invece emersa dall’analisi del siero di pazienti con probabile/possibile IFI e di pazienti non-IFI. Conclusioni La combinazione dei risultati del BG assay e del saggio sierologico in microarray, condotti parallelamente all’emocoltura, può fornire indicazioni diagnostiche e prognostiche aggiuntive in pazienti con provata o sospetta candidiasi invasiva. Riteniamo che questo approccio diagnostico combinato possa rivelarsi particolarmente utile soprattutto nei pazienti con emocoltura negativa

Detection of Pneumocystis jirovecii and Aspergillus spp. DNa in bronchoalveolar lavage fluids by commercial real-time PCr assays: comparison with conventional diagnostic tests

The present study employed two commercial real-time PCR kits, MycAssay™ Pneumocystis (PJ-PCR) and MycAssay™ Aspergillus (ASP-PCR), for the search of fungal DNA on 44 bronchoalveolar lavage (BAL) fluids from patients at risk of invasive fungal disease. Operationally, on the basis of clinical diagnosis and according to the European Organization for Research and Treatment Cancer/Mycoses Study Group (EORTC/MSG) criteria, patients were clustered in 3 groups: a P. jirovecii pneumonia (PCP) group, an invasive aspergillosis (IA) group and a control (CTRL) group, consisting of 8, 10 and 24 patients, respectively. The results were compared to those obtained with conventional diagnostic assays, including BAL culture, galactomannan-ELISA (GM) and immunofluorescence (IF). The PJ-PCR assay returned a sensitivity and specificity of 100% and 94.4%, respectively. The ASP-PCR assay showed a sensitivity and specificity of 80% and 97.1%. When compared to the culture assay, the ASP-PCR showed enhanced sensitivity, and a good level of agreement (kappa = 0.63) was observed between ASP-PCR and GM assays. Overall, our data emphasize the diagnostic usefulness of the two commercial real-time PCR assays, especially in high-risk patients where timing is critical and a low fungal burden may hamper correct and prompt diagnosis by conventional tests

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