Exploring nasopharyngeal microbiota profile in children affected by SARS-CoV-2 infection.

The relationship between COVID-19 and nasopharyngeal (NP) microbiota has been investigated mainly in the adult population. We explored the NP profile of children affected by COVID-19, compared to healthy controls (CTRLs). NP swabs of children with COVID-19, collected between March and September 2020, were investigated at the admission (T0), 72 h to 7 days (T1), and at the discharge (T2) of the patients. NP microbiota was analyzed by 16S rRNA targeted-metagenomics. Data from sequencing were investigated by QIIME 2.0 and PICRUSt 2. Multiple machine learning (ML) models were exploited to classify patients compared to CTRLs. The NP microbiota of COVID-19 patients (N = 71) was characterized by reduction of α-diversity compared to CTRLs (N = 59). The NP microbiota of COVID-19 cohort appeared significantly enriched in Streptococcus, Haemophilus, Staphylococcus, Veillonella, Enterococcus, Neisseria, Moraxella, Enterobacteriaceae, Gemella, Bacillus, and reduced in Faecalibacterium, Akkermansia, Blautia, Bifidobacterium, Ruminococcus, and Bacteroides, compared to CTRLs (FDR < 0.001). Exploiting ML models, Enterococcus, Pseudomonas, Streptococcus, Capnocytopagha, Tepidiphilus, Porphyromonas, Staphylococcus, and Veillonella resulted as NP microbiota biomarkers, in COVID-19 patients. No statistically significant differences were found comparing the NP microbiota profile of COVID-19 patients during the time-points or grouping patients on the basis of high, medium, and low viral load (VL). This evidence provides specific pathobiont signatures of the NP microbiota in pediatric COVID-19 patients, and the reduction of anaerobic protective commensals. Our data suggest that the NP microbiota may have a specific disease-related signature since infection onset without changes during disease progression, regardless of the SARS-CoV-2 VL. IMPORTANCE: Since the beginning of pandemic, we know that children are less susceptible to severe COVID-19 disease. A potential role of the nasopharyngeal (NP) microbiota has been hypothesized but to date, most of the studies have been focused on adults. We studied the NP microbiota modifications in children affected by SARS-CoV-2 infection showing a specific NP microbiome profile, mainly composed by pathobionts and almost missing protective anaerobic commensals. Moreover, in our study, specific microbial signatures appear since the first days of infection independently from SARS-CoV-2 viral load.

Nasopharyngeal microbiota in hospitalized children with Bordetella pertussis and Rhinovirus infection.

Despite great advances in describing Bordetella pertussis infection, the role of the host microbiota in pertussis pathogenesis remains unexplored. Indeed, the microbiota plays important role in defending against bacterial and viral respiratory infections. We investigated the nasopharyngeal microbiota in infants infected by B. pertussis (Bp), Rhinovirus (Rv) and simultaneously by both infectious agents (Bp + Rv). We demonstrated a specific nasopharyngeal microbiome profiles for Bp group, compared to Rv and Bp + Rv groups, and a reduction of microbial richness during coinfection compared to the single infections. The comparison amongst the three groups showed the increase of Alcaligenaceae and Achromobacter in Bp and Moraxellaceae and Moraxella in Rv group. Furthermore, correlation analysis between patients' features and nasopharyngeal microbiota profile highlighted a link between delivery and feeding modality, antibiotic administration and B. pertussis infection. A model classification demonstrated a microbiota fingerprinting specific of Bp and Rv infections. In conclusion, external factors since the first moments of life contribute to the alteration of nasopharyngeal microbiota, indeed increasing the susceptibility of the host to the pathogens' infections. When the infection is triggered, the presence of infectious agents modifies the microbiota favoring the overgrowth of commensal bacteria that turn in pathobionts, hence contributing to the disease severity.

Choice of next-generation sequencing pipelines.

The next-generation sequencing (NGS) technologies are revolutionary tools which have made possible achieving remarkable advances in genetics since the beginning of the twenty-first century. Thanks to the possibility to produce large amount of sequence data, these tools are going to completely substitute other high-throughput technologies. Moreover, the large applications of NGS protocols are increasing the genetic decoding of biological systems through studies of genome anatomy and gene mapping, coupled to the transcriptome pictures. The application of NGS pipelines such as (1) de-novo genomic sequencing by mate-paired and whole-genome shotgun strategies; (2) specific gene sequencing on large bacterial communities; and (3) RNA-seq methods including whole transcriptome sequencing and Serial Analysis of Gene Expression (Sage-analysis) are fundamental in the genome-wide fields like metagenomics. Recently, the availability of these advanced protocols has allowed to overcome the usual sequencing technical issues related to the mapping specificity over standard shotgun library sequencing, the detection of large structural genomes variations and bridging sequencing gaps, as well as more precise gene annotation. In this chapter we will discuss how to manage a successful NGS pipeline from the planning of sequencing projects through the choice of the platforms up to the data analysis management.

Proteomics boosts translational and clinical microbiology.

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient. Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Microbial tracking of multidrug-resistant Klebsiella pneumoniae isolates in a pediatric hospital setting.

We investigated the clonal relatedness of seven multi-drug-resistant (MDR) Klebsiella pneumoniae isolates, as well as three susceptible K. pneumoniae isolates collected during hospital outbreaks and outbreak-related microbiological surveillance, respectively. The relatedness among K. pneumoniae isolates was assessed by pulsed field gel electrophoresis (PFGE) and automated repetitive-sequence-based PCR (rep-PCR) genotyping and the results were compared to a proteomic phenotyping performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All typing methods agreed on the generation of three different clusters of K. pneumoniae isogenetic/related MDR strains. After strengthening hospital infection control measures, no other spreading events involving MDR-K. pneumoniae were reported until the end of the observation period. This preliminary investigation suggests that, in a hierarchical approach to bacterial typing, MALDI-TOF MS proteome profiling might offer a fast and valuable preliminary screening tool able to support microbiologists during nosocomial outbreak surveys.

Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples.

Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium spp. is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is age-dependent. The method herein reported represents a step forward routine diagnosis and improves epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages.

Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG).

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Evaluation by real-time PCR of the expression of S. flexneri virulence-associated genes ospB and phoN2 under different genetical backgrounds.

Under conditions of activated type III secretion Shigella flexneri up-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospB and phoN2 genes. ospB and phoN2 are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time PCR to measure transcription of ospB and phoN2 of wild-type S. flexneri strain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospB and phoN2 genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a mxiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2 operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. flexneri.

Molecular characterization of virulence determinants of Stenotrophomonas maltophilia strains isolated from patients affected by cystic fibrosis.

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study 13 S. maltophilia strains (11 isolated from the airways of independent CF patients, and two non-CF respiratory reference strains) have been characterized for the expression of several virulence-associated factors. In particular, the ability to form biofilm on abiotic surfaces has been determined and correlated with different features, such as motility, adherence and the ability to invade A549 respiratory epithelial cells. Moreover, the presence of a flagellum-associated gene as well as that of the StmPr1 gene, which encodes an extracellular protease, have been determined by Southern blot hybridization. Our data indicate that the different degree of biofilm formation exhibited by the 11 CF isolates does not correlate with motility, ability to adhere to and invade A549 cells, or with the presence of flagella. On the other hand, among the CF isolates the StmPr1 gene was found only in two strains, both able to establish chronic lung infections in CF patients. Moreover, only four of the strains analyzed show a temperature-independent antibiotic-resistance profile, suggesting either a different origin of these strains or an intervening adaptation to host tissues.

Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread.

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.