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Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC-MS(2)-spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2(ko) mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2(ko) mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia., Biological Significance: The manuscript deals with a "frontier" topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly "instable", and the relative balance between mucosal and fecal bacteria starts its dynamics of "fight" to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a "very" low complexity, was herein selected as model to design a LC-MS(2)-based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics., (© 2013. Published by Elsevier B.V. All rights reserved.)

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., (© 2013. Published by Elsevier B.V. All rights reserved.)

Background: Chlamydia pneumoniae has been repeatedly associated with atherosclerotic cardiovascular diseases. We investigated the pattern of distribution of C. pneumoniae among patients with carotid atherosclerotic disease evaluating chlamydial load in carotid plaque, peripheral blood mononuclear cells (PBMC) and lymph node from same patient., Methods and Results: Thirty carotid plaques, 30 PBMC and 30 lymph nodes were examined by real-time PCR assay. C. pneumoniae DNA was detected, in carotid plaques, PBMC and lymph nodes in 11 patients; in carotid plaques and PBMC in five patients; in PBMC and lymph nodes in four patients; in lymph nodes in two patients; and in PBMC only in one patient. C. pneumoniae DNA in PBMC significantly coincided with the presence of the respective DNA in carotid plaque (p=0.0001) and lymph node (p=0.02). A higher chlamydial load was detected in PBMC than in lymph nodes and carotid plaques. More than 90% of patients with carotid plaques, PBMC and lymph nodes positive to C. pneumoniae were symptomatic, smokers, hypertensives, dyslipidemics and showed carotid plaques with rupture on the surface, hemorrhage and thrombosis., Conclusion: The measurement of chlamydial load in PBMC may be helpful in the future to assess the state of C. pneumoniae infection and the risk of developing sequelae.

Bacteria of the Burkholderia cepacia complex consist of a number of closely related genomic species (genomovars) potentially pathogenic for cystic fibrosis (CF) patients, collectively referred to as the B. cepacia complex. The genomovar status and epidemiological relatedness of B. cepacia complex strains recovered from CF patients, attending a CF Center at the University Hospital "Policlinico Umberto I" of Rome, were investigated using 16S rRNA PCR-RFLP, recA PCR-RFLP, genomovar-specific PCR, and RAPD. Forty-seven isolates identified as B. cepacia by commercial systems were repeatedly recovered from 19 CF patients. The taxonomy approach used in this study showed that 17 of the 19 patients were colonized by B. cepacia complex strains. Genomovar III (11 strains) was the most prevalent genomovar. Two strains of genomovar I, one B. stabilis (genomovar IV), one B. multivorans (genomovar II), and 4 strains of B. anthina (genomovar VIII) were also identified. This is the first report of multiple patient colonization by B. anthina in a CF center. The epidemiological and genetic relatedness as well as the presence of molecular markers associated with virulence and transmissibility of the B. cepacia complex strains were determined and probable patient-to-patient spread was observed.

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.