The Use of Experimental Evolution to Study the Response of Pseudomonas aeruginosa to Single or Double Antibiotic Treatment

The widespread use of antibiotics promotes the evolution and dissemination of drug resistance and tolerance. Both mechanisms promote survival during antibiotic exposure and their role and development can be studied in vitro with different assays to document the gradual adaptation through the selective enrichment of resistant or tolerant mutant variants. Here, we describe the use of experimental evolution in combination with time-resolved genome analysis as a powerful tool to study the interaction of antibiotic tolerance and resistance in the human pathogen Pseudomonas aeruginosa . This method guides the identification of components involved in alleviating antibiotic stress and helps to unravel specific molecular pathways leading to drug tolerance or resistance. We discuss the influence of single or double drug treatment regimens and environmental aspects on the evolution of antibiotic resilience mechanisms

Chromosome Organization and Replisome Dynamics in Mycobacterium smegmatis

Subcellular organization of the bacterial nucleoid and spatiotemporal dynamics of DNA replication and segregation have been studied intensively, but the functional link between these processes remains poorly understood. Here we use quantitative time-lapse fluorescence microscopy for single-cell analysis of chromosome organization and DNA replisome dynamics in Mycobacterium smegmatis. We report that DNA replication takes place near midcell, where, following assembly of the replisome on the replication origin, the left and right replication forks colocalize throughout the replication cycle. From its initial position near the cell pole, a fluorescently tagged chromosomal locus (attB, 245 degrees from the origin) moves rapidly to the replisome complex just before it is replicated. The newly duplicated attB loci then segregate to mirror-symmetric positions relative to midcell. Genetic ablation of ParB, a component of the ParABS chromosome segregation system, causes marked defects in chromosome organization, condensation, and segregation. ParB deficiency also results in mislocalization of the DNA replication machinery and SMC (structural maintenance of chromosome) protein. These observations suggest that ParB and SMC play important and overlapping roles in chromosome organization and replisome dynamics in mycobacteria. IMPORTANCE We studied the spatiotemporal organization of the chromosome and DNA replication machinery in Mycobacterium smegmatis, a fast-growing relative of the human pathogen Mycobacterium tuberculosis. We show that genetic ablation of the DNA-binding proteins ParB and SMC disturbs the organization of the chromosome and causes a severe defect in subcellular localization and movement of the DNA replication complexes. These observations suggest that ParB and SMC provide a functional link between chromosome organization and DNA replication dynamics. This work also reveals important differences in the biological roles of the ParABS and SMC systems in mycobacteria versus better-characterized model organisms, such as Escherichia coli and Bacillus subtilis

Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

During the bacterial cell cycle, chromosome replication and cell division must be coordinated with overall cell growth in order to maintain the correct ploidy and cell size. The spatial and temporal coordination of these processes in mycobacteria is not understood. Here we use microfluidics and time-lapse fluorescence microscopy to measure the dynamics of cell growth, division and chromosome replication in single cells of Mycobacterium smegmatis. We find that single-cell growth is size-dependent (large cells grow faster than small cells), which implicates a size-control mechanism in cell-size homoeostasis. Asymmetric division of mother cells gives rise to unequally sized sibling cells that grow at different velocities but show no differential sensitivity to antibiotics. Individual cells are restricted to one round of chromosome replication per cell division cycle, although replication usually initiates in the mother cell before cytokinesis and terminates in the daughter cells after cytokinesis. These studies reveal important differences between cell cycle organization in mycobacteria compared with better-studied model organisms

Long-Acting GLP-1 Receptor Agonist Exenatide Influence on the Autonomic Cardiac Sympatho-Vagal Balance

Long-acting glucagon-like peptide 1 receptor agonists are increasingly used to treat type 2 diabetes. An increase of heart rate (HR) has been observed with their use. To elucidate the role of the cardiac sympatho-vagal balance as a possible mediator of the reported increase in HR, we performed power spectral analysis of HR variability (HRV) in patients receiving exenatide extended-release (ER). Twenty-eight ambulatory patients with type 2 diabetes underwent evaluation at initiation of exenatide-ER and thereafter at 3 and at 6 months. To obtain spectral analyses of HRV, a computerized acquisition of 10 minutes of RR electrocardiogram intervals (mean values of ~700 RR intervals) were recorded both in lying and in standing positions. All patients showed a substantial increase of HR both in lying and in standing positions. Systolic blood pressure, body weight, and glycated hemoglobin A1c significantly decreased both at 3 and 6 months compared with basal levels. The low-frequency/high-frequency ratio varied from 3.05 \ub1 0.4 to 1.64 \ub1 0.2 (P < 0.001) after 3 months and to 1.57 \ub1 0.3 (P < 0.001) after 6 months in a lying position and from 4.56 \ub1 0.8 to 2.24 \ub1 0.3 (P < 0.001) after 3 months and to 2.38 \ub1 0.4 (P < 0.001) after 6 months in a standing position compared with basal values, respectively. HR variations, induced by exenatide-ER treatment, do not appear to be related to sympathetic autonomic tone. Of note, we observed a relative increase of vagal influence on the heart

COMVC-19: A Program to protect healthcare workers' mental health during the COVID-19 Pandemic. What we have learned

OBJECTIVE: In 2020, the COVID-19 pandemic brought a work and stress overload to healthcare workers, increasing their vulnerability to mental health impairments. In response, the authors created the COMVC-19 program. The program offered preventive actions and mental health treatment for the 22,000 workers of The Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP). This paper aims to describe its implementation and share what we have learned from this experience. METHODS: Workers were able to easily access the program through a 24/7 hotline. Additionally, a mobile phone app that screened for signs and symptoms of emotional distress and offered psychoeducation and/or referral to treatment was made available. Data from both these sources as well as any subsequent psychiatric evaluations were collected. RESULTS: The first 20 weeks of our project revealed that most participants were female, and part of the nursing staff working directly with COVID-19 patients. The most frequently reported symptoms were: anxiety, depression and sleep disturbances. The most common diagnoses were Adjustment, Anxiety, and Mood disorders. CONCLUSIONS: Implementing a mental health program in a multimodal intervention was feasible in a major quaternary public hospital. Our data also suggests that preventive actions should primarily be aimed at anxiety and depression symptoms, with a particular focus on the nursing staff. &nbsp

Abstracts of 51st EASD Annual Meeting

Background and aims: Presence and frequency of beta cell (BC) dysfunction(BCD) and insulin resistance (IR) in patients with newly diagnosedtype 2 diabetes mellitus (NDT2D) are imperfectly known, becauseprevious studies used small cohorts and/or only surrogate indexes of BCfunction and IR.We sought to assess BC function and IR with state-of-artmethods in the VNDS.Materials and methods: In 712 GADA-negative, drug naïve, consecutiveItalian NDT2D patients we assessed: 1. standard parameters; 2. insulinsensitivity (IS) by the euglycaemic insulin clamp); 3. BC functionby state-of-art modeling of prolonged (5 hours) OGTT-derived glucose/C-peptide curves. Thresholds for BCD and IR were the 25th percentilesof BC function and IS assessed with the same methods of the VNDS inItalian subjects with normal glucose regulation of the GENFIEV (n=340)and GISIR (n=386) studies, respectively.Results: In the VNDS, 89.8% [95% C.I.: 87.6 - 92.0%] and87.8% [85.4 - 90.2] patients had BCD and IR, respectively. Patientswith only one defect were 19.7% [16.8 - 22.6]. IsolatedBCD and isolated IR were present in 10.9% [8.6 - 13.2] and8.9% [6.8 - 11.0] patients, respectively. Coexistence of BCDand IR was observed in 78.9% [75.9 - 81.9] of the patients.1.4% [0.5 - 2.3] of the patients had no detectable alterations inBC function and IS. Patients (19.7%) with only one metabolicdefect had lower BMI, fasting glucose, HbA1c, triglycerides andBC function, and higher HDL-cholesterol and IS than patientswith both BCD and IR (p<0.01 or less after Bonferroni’scorrection).Conclusion: In conclusion, in NDT2DM patients: 1. at least 75.9% haveboth BCD and IR; 2. At least 87.6% and 85.4% have BCD and IR,respectively; 3. At least 16.8% have only one defect and a significantlydifferent (milder) metabolic phenotype compared to patients with bothdefects. These findings may be relevant to therapeutic strategies centeredon the metabolic phenotype of the patient.Clinical Trial Registration Number: NCT00879801; NCT01526720Supported by: University of Veron

Adaptive Response of Group B Streptococcus to High Glucose Conditions: New Insights on the CovRS Regulation Network

Although the contribution of carbohydrate catabolism to bacterial colonization and infection is well recognized, the transcriptional changes during these processes are still unknown. In this study, we have performed comparative global gene expression analysis of GBS in sugar-free versus high glucose milieu. The analysis revealed a differential expression of genes involved in metabolism, transport and host-pathogen interaction. Many of them appeared to be among the genes previously reported to be controlled by the CovRS two-component system. Indeed, the transcription profile of a Delta covRS strain grown in high-glucose conditions was profoundly affected. In particular, of the total genes described to be regulated by glucose, similar to 27% were under CovRS control with a functional role in protein synthesis, transport, energy metabolism and regulation. Among the CovRS dependent genes, we found bibA, a recently characterized adhesin involved in bacterial serum resistance and here reported to be down-regulated by glucose. ChIP analysis revealed that in the presence of glucose, CovR binds bibA promoter in vivo, suggesting that CovR may act as a negative regulator or a repressor. We also demonstrated that, as for other target promoters, chemical phosphorylation of CovR in aspartic acid increases its affinity for the bibA promoter region. The data reported in this study contribute to the understanding of the molecular mechanisms modulating the adaptation of GBS to glucose

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections

Updated Italian Tetrapod Ichnology Reference List

We provide a list of contribution by Italian scientists to tetrapod ichnology with papers on both material from Italy and abroad. Foreign author’s contributions on tetrapod ichnology based on material from Italy are also considered. The list updates the previous one published by D’Orazi Porchetti et al. (2008) and, as a result, includes works from 1869 up to now. Following the previous reference list, papers of non-Italian researchers on foreign material are reported when the material was found on Italian territory at the time of publication

Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections

The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed inpatient microevolution of Pseudomonas aeruginosa , an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multi-drug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections