In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion,the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing

Valutazione qualitativa e quantitativa di due nuove formulazioni probiotiche e analisi del loro comportamento in vitro in condizioni che mimano quelle del tratto gastrointestinale

Secondo l’Organizzazione Mondiale della Sanità (OMS), i probiotici sono microrganismi vivi che, se somministrati in adeguate quantità, sono in grado di esercitare effetti benefici sulla salute dell’ospite. Un microrganismo probiotico deve essere sicuro, geneticamente stabile, non presentare antibiotico-resistenze trasmissibili ed essere in grado di sopravvivere durante il transito nello stomaco e di moltiplicarsi una volta raggiunto l’intestino, determinandone una temporanea colonizzazione ed esprimendo, in questo distretto, le sue proprietà benefiche. Le principali formulazioni probiotiche sono costituite da singole specie o miscele di diverse specie microbiche appartenenti ai generi Lactobacillus, Streptococcus, Bifidobacterium, Bacillus e Saccharomyces. Il lavoro svolto durante in presente internato di Tesi si è posto come principale obiettivo l’analisi qualitativa e quantitativa di due nuove formulazioni probiotiche (A monospecie e B multispecie) di imminente commercializzazione nel Regno Unito. La prima parte del progetto di ricerca è stata dedicata alla determinazione dell’effettivo contenuto microbico di entrambe le formulazioni probiotiche, impiegando sia metodiche di microbiologia convenzionale, per la conta dei microrganismi e l’isolamento delle diverse specie, sia tecniche molecolari per l’identificazione di specie, tra cui la spettrometria di massa MALDI-TOF MS e il sequenziamento dell’rDNA 16s e 18s. I risultati ottenuti hanno evidenziato la validità dei diversi approcci metodologici utilizzati per l’identificazione della specie di lievito Saccharomyces boulardii costituente la formulazione A e di due specie di lattobacilli (Lactobacillus rhamnosus e Lactobacillus helveticus) presenti nella formulazione B. Ulteriore obiettivo del progetto di Tesi è stato quello di ottenere informazioni sulla tolleranza gastrica e sulla resistenza ai sali biliari dei microrganismi presenti nelle formulazioni probiotiche, per meglio comprenderne l’effettivo valore probiotico. Sono stati condotti studi in vitro con due succhi gastrici artificiali, ASTM e USP, preparati entrambi a pH 1.5 e pH 3.0 e con il succo intestinale artificiale contenente pancreatina e sali biliari a pH 8.0. I risultati ottenuti hanno mostrato una forte tolleranza all’acidità gastrica e al succo intestinale artificiale per entrambi i prodotti. Sebbene con i limiti di uno studio in vitro, i risultati ottenuti suggeriscono che entrambe le formulazioni testate siano dei prodotti che rispecchiano quanto dichiarato sulle rispettive confezioni, sia in termini quantitativi che per il contenuto delle specie microbiche. Inoltre, l’elevata sopravvivenza in ambiente gastrico e la tolleranza all’ambiente intestinale simulati anche per tempi prolungati costituiscono i requisiti fondamentali affinché queste formulazioni possano esercitare la loro attività probiotica. The World Health Organization (WHO) defines probiotics as living microorganisms that, if administered in adequate amounts, are capable of exerting beneficial effects on the host. A probiotic microorganism must be safe, genetically stable, not present transmissible antibiotic resistances, be able to survive to the harsh gastric environment and survive, multiply and temporarily colonize the intestine, where it can exert its beneficial properties. The main probiotic formulations are constituted by single species or mixtures of different microbial species mostly belonging to the Lactobacillus, Streptococcus, Bifidobacterium, Bacillus and Saccharomyces genera. The main objective of this Thesis was to perform a qualitative and quantitative analysis of two new probiotic formulations (A, monospecies and B, multispecies) of imminent commercialization on the United Kingdom market. The first part of the research project was dedicated to the analysis of the actual microbial content of both probiotic formulations, using both conventional microbiology methods, for the count of microorganisms and the isolation of different species, and molecular techniques for species identification, including MALDI-TOF MS mass spectrometry and metagenomics analysis by 16s and 18s rDNA sequencing. The results obtained highlighted the validity of the different methodological approaches used for the identification of the yeast species Saccharomyces boulardii constituting formulation A and of two species of lactobacilli (Lactobacillus rhamnosus and Lactobacillus helveticus) present in formulation B. A further objective of the Thesis was to obtain information on the gastric tolerance and resistance to bile salts of the microorganisms constituting the probiotic formulations, to better understand their effective probiotic value. In vitro studies were conducted with two artificial gastric juices, ASTM and USP, both prepared at pH 1.5 and pH 3.0 and with artificial intestinal juice containing pancreatin and bile salts at pH 8.0. The results obtained showed a strong tolerance to gastric acidity and artificial intestinal juice for both products. Although with the limitations of an in vitro study, the overall results indicate that both formulations contain the number and the species of microorganisms that are declared on the respective labels, thus being safe and free of contaminants. In addition, the high survival in the gastric environment and the tolerance to the intestinal environment, even for a long time, constitute the fundamental requirements for these formulations to behave as probiotics

In vitro modelling of Chlamydia trachomatis infection in the Etiopathogenesis of male infertility and reactive arthritis

Chlamydia trachomatis is an obligate, intracellular bacterium responsible for a range of diseases of public health importance, since C. trachomatis infection is often asymptomatic and, hence, untreated, leading to chronic complications, including prostatitis, infertility, and reactive arthritis. The ample spectrum of diseases caused by C. trachomatis infection is reflected in its ability to infect and multiply within a wide range of different cell types. Cervical epithelial cells, to date, have been the most studied cellular infection model, highlighting the peculiar features of the host-cell inflammatory and immune responses to the infection. Herein, we provide the up-to-date evidence on the interaction between C. trachomatis and human prostate epithelial, Sertoli and synovial cells

Oxidative Stress and Inflammation in SARS-CoV-2- and Chlamydia pneumoniae-Associated Cardiovascular Diseases

Throughout the years, a growing number of studies have provided evidence that oxidative stress and inflammation may be involved in the pathogenesis of infectious agent-related cardiovascular diseases. Amongst the numerous respiratory pathogens, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus responsible for the global ongoing pandemic, and Chlamydia pneumoniae, a widely known intracellular obligate bacteria, seem to have an essential role in promoting reactive oxygen species and cytokine production. The present review highlights the common oxidative and inflammatory molecular pathways underlying the cardiovascular diseases associated with SARS-CoV-2 or C. pneumoniae infections. The main therapeutic and preventive approaches using natural antioxidant compounds will be also discussed

Potential IFNγ Modulation of Inflammasome Pathway in Chlamydia trachomatis Infected Synovial Cells

Following a Chlamydia trachomatis infection, the host immune response is characterized by its recognition via Toll-like and Nod-like Receptors, and the subsequent activation of interferon (IFN)-γ-mediated signaling pathways. Recently, the inflammasome-mediated host cell response has emerged to play a role in the physiopathology of C. trachomatis infection. Here we investigated, for the first time, the interaction of IFN-γ and inflammasome in an in vitro model of C. trachomatis-infected primary human synovial cells. Chlamydial replication as well as the expression of caspase-1, IL-1β, as well as IL-18 and IL-6, were assayed. Our results demonstrated the inhibitory activity of IFN-γ by interfering with the inflammasome network through the downregulation of caspase-1 mRNA expression. In addition, the ability of C. trachomatis to hinder the inflammasome pathway favoring its intracellular survival within synovial cells, was observed. Overall, our data suggest a potential mechanism of immune evasion by C. trachomatis in synovial cells, that may be contested by IFN-γ

A 12-month follow-up of the immune response to SARS-CoV-2 primary vaccination: evidence from a real-world study

A real-world population-based longitudinal study, aimed at determining the magnitude and duration of immunity induced by different types of vaccines against COVID-19, started in 2021 by enrolling a cohort of 2,497 individuals at time of their first vaccination. The study cohort included both healthy adults aged ≤65 years and elderly subjects aged >65 years with two or more co-morbidities. Here, patterns of anti-SARS-CoV-2 humoral and cell-mediated specific immune response, assessed on 1,182 remaining subjects, at 6 (T6) and 12 months (T12) after the first vaccine dose, are described. At T12 median anti-Spike IgG antibody levels were increased compared to T6. The determinants of increased anti-Spike IgG were the receipt of a third vaccine dose between T6 and T12 and being positive for anti-Nucleocapside IgG at T12, a marker of recent infection, while age had no significant effect. The capacity of T12 sera to neutralize in vitro the ancestral B strain and the Omicron BA.5 variant was assessed in a subgroup of vaccinated subjects. A correlation between anti-S IgG levels and sera neutralizing capacity was identified and higher neutralizing capacity was evident in healthy adults compared to frail elderly subjects and in those who were positive for anti-Nucleocapside IgG at T12. Remarkably, one third of T12 sera from anti-Nucleocapside IgG negative older individuals were unable to neutralize the BA.5 variant strain. Finally, the evaluation of T-cell mediated immunity showed that most analysed subjects, independently from age and comorbidity, displayed Spike-specific responses with a high degree of polyfunctionality, especially in the CD8 compartment. In conclusion, vaccinated subjects had high levels of circulating antibodies against SARS-CoV-2 Spike protein 12 months after the primary vaccination, which increased as compared to T6. The enhancing effect could be attributable to the administration of a third vaccine dose but also to the occurrence of breakthrough infection. Older individuals, especially those who were anti-Nucleocapside IgG negative, displayed an impaired capacity to neutralize the BA.5 variant strain. Spike specific T-cell responses, able to sustain immunity and maintain the ability to fight the infection, were present in most of older and younger subjects assayed at T12