Multicenter Evaluation of the Novel ETEST Fosfomycin for Antimicrobial Susceptibility Testing of Enterobacterales, Enterococcus faecalis, and Staphylococcus Species.

Fosfomycin is a phosphonic acid derivative active against a wide spectrum of Gram-positive and Gram-negative pathogens. It is used for the treatment of uncomplicated urinary tract infections (uUTI) or severe infections by oral or intravenous (i.v.) administration. In order to improve its performance and robustness, the fosfomycin strip, an antibiotic gradient diffusion strip, was redeveloped and evaluated in the multicenter study summarized in this paper. ETEST fosfomycin (ETEST FO) clinical performance was evaluated by three study sites on 152 Enterococcus faecalis, 100 Staphylococcus spp. and 330 Enterobacterales in comparison with the CLSI and EUCAST agar dilution reference method. Referring to FDA performance criteria, the ETEST FO achieved 91.0% of essential (EA) and 99.0% of categorical agreement (CA) for Escherichia coli. In addition, 98.0% EA and 93.4% CA were achieved for E. faecalis, with no very major errors (VME) or major errors (ME). According to EUCAST breakpoints for intravenous fosfomycin use, Enterobacterales and Staphylococcus spp. also met ISO acceptance criteria for EA and CA (EA 91.5%, 94.0%, respectively, and CA 98.0% for both). A VME rate of 8.8% was observed for Enterobacterales but the MICs were within EA. A trend to predict lower MICs for Citrobacter spp., E. coli and Salmonella enterica and to predict higher MICs for Klebsiella pneumoniae MICs was observed, while ETEST FO should not be used for Enterobacter cloacae, because of low EA and a high VME rate. The study results support the efficiency of the novel ETEST FO, making it an easy-to-handle tool as a substitute to the classical agar dilution method.

Digital antimicrobial susceptibility testing using the MilliDrop technology.

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.

TET2 gene expression and 5-hydroxymethylcytosine level in multiple sclerosis peripheral blood cells.

Aberrant DNA methylation can lead to genome destabilization and to deregulated gene expression. Recently, 5-hydroxymethylcytosine (5hmC), derived from oxidation of 5-methylcytosine (5mC) by the Ten-Eleven Translocation (TET) enzymes, has been detected. 5hmC is now considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the DNA methylation/demethylation machinery in MS. We assessed the expression of enzymes involved in DNA methylation/demethylation in peripheral blood mononuclear cells (PBMCs) from 40 subjects with MS and 40 matched healthy controls. We performed also, DNA methylation analysis of specific promoters and analysis of global levels of 5mC and 5hmC. We show that TET2 and DNMT1 expression is significantly down-regulated in MS PBMCs and it is associated with aberrant methylation of their promoters. Furthermore, 5hmC is decreased in MS PBMCs, probably as a result of the diminished TET2 level.

Metformin improves putative longevity effectors in peripheral mononuclear cells from subjects with prediabetes. A randomized controlled trial.

BACKGROUND AND AIMS: Prediabetes increases cardiovascular risk and is associated with excess mortality. In preclinical models, metformin has been shown to exert anti-ageing effects. In this study, we sought to assess whether metformin modulates putative effector longevity programs in prediabetic subjects. METHODS AND RESULTS: In a randomized, single-blind, placebo-controlled trial, 38 prediabetic subjects received metformin (1500 mg/day) or placebo for 2 months. At baseline and after treatment, we collected anthropometric and metabolic parameters. Gene and protein levels of SIRT1, mTOR, p53, p66Shc, SIRT1 activity, AMPK activation, telomere length, and SIRT1 promoter chromatin accessibility were determined in peripheral blood mononuclear cells (PBMCs). Plasma N-glycans, non-invasive surrogate markers of ageing, were also analysed. Compared to baseline, metformin significantly improved metabolic parameters and insulin sensitivity, increased SIRT1 gene/protein expression and SIRT1 promoter chromatin accessibility, elevated mTOR gene expression with concomitant reduction in p70S6K phosphorylation in subjects' PBMCs, and modified the plasma N-glycan profile. Compared to placebo, metformin increased SIRT1 protein expression and reduced p70S6K phosphorylation (a proxy of mTOR activity). Plasma N-glycans were also favourably modified by metformin compared to placebo. CONCLUSION: In individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. ClinicalTrials. gov identifier: NCT01765946 - January 2013.

Alpine Skiing With total knee ArthroPlasty (ASWAP): impact on molecular and architectural features of musculo-skeletal ageing.

This study investigated features of skeletal muscle ageing in elderly individuals having previously undergone unilateral total knee arthroplasty (TKA) and whether markers of sarcopenia could be mitigated by a 12-week alpine skiing intervention. Novel biomarkers agrin, indicative of neuromuscular junction (NMJ) degeneration, tumor suppressor protein p53, associated with muscle atrophy, and a new ultrasound-based muscle architecture biomarker were used to characterize sarcopenia. Participant details and study design are presented by Kösters et al. (2015). The results of this study show that NMJ degeneration is widespread among active septuagenarians previously subjected to TKA: all participants showed elevated agrin levels upon recruitment. At least 50% of individuals were identified as sarcopenic based on their muscle architecture, supporting the hypothesis that NMJ alterations precede sarcopenia. Notably, sarcopenia was strongly associated with the expression of p53, which seems to confirm its validity as a biomarker of muscle atrophy. Training did not significantly modify any of these biomarkers. In view of the lack of accretion of muscle mass in response to the alpine skiing intervention, we hypothesize that local muscle inflammation and oxidative stress may have blunted the anabolic response to training and promoted muscle breakdown in this elderly post-TKA population.

16(th) IHIW: immunogenetics of aging.

Ageing is a process characterised by progressive loss of function in multiple different organ systems, such as the nervous, endocrine and immune systems. Current data showing that ageing processes may be associated with alterations in the immune system suggest that some of the genetic determinants of senescence might be polymorphic genes that regulate immune responses. The 'Immunogenetics of Aging' programme was a component introduced in the 14th International HLA and Immunogenetics Workshop (IHIWS) and developed further within the 15th and 16th. The aim of this component was to determine the contribution of immune genes to successful ageing and an increased capacity to reach the extreme limits of lifespan. Within the 16th IHIWS, new populations were included, and the number of samples analysed was increased. Analysis was focused on innate immunity genes (KIR and MBL2) and their correlation with CMV serostatus. Collaborative studies suggested that both activating and inhibitory KIR and functionally relevant MBL2 haplotypes are important factors for control of CMV infection in the elderly and therefore for chronic low-grade inflammation. Results showed that these genes might be predictive biomarkers in ageing and longevity. Prevalence of MBL2 haplotypes determining absence of the protein (LYPB, LYQC and HYPD) was observed in elderly people with a higher CMV antibody titre. The high CMV titre was also associated with a decreased frequency of the activatory KIR2DS5 and A1B10 haplotypes in elderly. Due to the role of KIR and low or deficient MBL haplotypes in viral infections, these genetic markers could be considered as indicators of a need for CMV prophylaxis at younger age and therefore increased probability of longer lifespan.

Immunology of Aging: the Birth of Inflammaging.

The inflammaging concept was introduced in 2000 by Prof. Franceschi. This was an evolutionary or rather a revolutionary conceptualization of the immune changes in response to a lifelong stress. This conceptualization permitted to consider the lifelong proinflammatory process as an adaptation which could eventually lead to either beneficial or detrimental consequences. This dichotomy is influenced by both the genetics and the environment. Depending on which way prevails in an individual, the outcome may be healthy longevity or pathological aging burdened with aging-related diseases. The concept of inflammaging has also revealed the complex, systemic nature of aging. Thus, this conceptualization opens the way to consider age-related processes in their complexity, meaning that not only the process but also all counter-processes should be considered. It has also opened the way to add new concepts to the original one, leading to better understanding of the nature of inflammaging and of aging itself. Finally, it showed the way towards potential multimodal interventions involving a holistic approach to optimize the aging process towards a healthy longevity.

Use of chloroprocaine in orthopedic day surgery: a brief report in a cohort of patients undergoing knee arthroscopy.

OBJECTIVE: Spinal anesthesia with local anesthetics is a viable alternative to general anesthesia in orthopedic surgery, and it is currently considered the standard of care for knee arthroscopy. The use of chloroprocaine may offer several potential advantages over other local anesthetics, including, above all, its rapid onset and short duration of action. The aim of the present retrospective study is to evaluate the post-surgical outcomes of patients who underwent knee arthroscopy using spinal anesthesia with chloroprocaine in an outpatient orthopedic setting. PATIENTS AND METHODS: Data from patients who underwent elective knee arthroscopy between January 2022 and December 2022 were collected for the present study. Spinal anesthesia with chloroprocaine 10 mg/mL was administered in the designated subarachnoid space (L3-L4 in the majority of patients). A dosage of 40 mg was used to obtain a satisfactory sensory and motor block. RESULTS: A total number of 302 patients met the inclusion criteria. No complications were reported during surgery in the present series of patients. None of the patients required bladder catheterization. In 84% of cases, the PADSS (Post-Anesthetic Discharge Scoring System) score at discharge was 10, whereas in 16% of cases, the PADSS score was 9. The mean time from anesthesia induction to first urination was 75±9.4 minutes, while the mean time from the anesthesia induction to the discharge from the hospital was 152±18.5 minutes. CONCLUSIONS: Spinal chloroprocaine for knee arthroscopy demonstrated a short motor block duration, resulting in a fast time to discharge. These limited data show that chloroprocaine may be safely and effectively applied in outpatient knee arthroscopy procedures. However, more studies, possibly with a randomized design, are required to confirm these findings.

A new cognitive clock matching phenotypic and epigenetic ages.

Cognitive abilities decline with age, constituting a major manifestation of aging. The quantitative biomarkers of this process, as well as the correspondence to different biological clocks, remain largely an open problem. In this paper we employ the following cognitive tests: 1. differentiation of shades (campimetry); 2. evaluation of the arithmetic correctness and 3. detection of reversed letters and identify the most significant age-related cognitive indices. Based on their subsets we construct a machine learning-based Cognitive Clock that predicts chronological age with a mean absolute error of 8.62 years. Remarkably, epigenetic and phenotypic ages are predicted by Cognitive Clock with an even better accuracy. We also demonstrate the presence of correlations between cognitive, phenotypic and epigenetic age accelerations that suggests a deep connection between cognitive performance and aging status of an individual.

Living in endemic area for infectious diseases accelerates epigenetic age.

Inflammaging is a low-grade inflammatory state generated by the aging process that can contribute to frailty and age-related diseases in the elderly. However, it can have distinct effects in the elderly living in endemic areas for infectious diseases. An increased inflammatory response may confer protection against infectious agents in these areas, although this advantage can cause accelerating epigenetic aging. In this study, we evaluated the inflammatory profile and the epigenetic age of infected and noninfected individuals from an endemic area in Brazil. The profile of cytokines, chemokines and growth factors analyzed in the sera of the two groups of individuals showed similarities, although infected individuals had a higher concentration of these mediators. A significant increase in IL-1ra, CXCL8, CCL2, CCL3 and CCL4 production was associated with leprosy infection. Notably, elderly individuals displayed distinct immune responses associated with their infection status when compared to adults suggesting an adaptive remodelling of their immune responses. Epigenetic analysis also showed that there was no difference in epigenetic age between the two groups of individuals. However, individuals from the endemic area had a significant accelerated aging when compared to individuals from São Paulo, a non-endemic area in Brazil. Moreover, the latter cohort was also epigenetically aged in relation to an Italian cohort. Our data shows that living in endemic areas for chronic infectious diseases results in remodelling of inflammaging and acceleration of epigenetic aging in individuals regardless of their infectious status. It also highlights that geographical, genetic and environmental factors influence aging and immunosenescence in their pace and profile.

'Immunogenetics of Aging': report on the activities of the 15th International HLA and Immunogenetics Working Group and 15th International HLA and Immunogenetics Workshop.

'Immunogenetics of Aging' is a component that was first included in the 14th International HLA and Immunogenetics Workshop (IHIWS) and developed further within the 15th Workshop. The aim of this component was to assess the impact of human leukocyte antigen (HLA) genes, cytokine genes, and some innate immunity genes such as killer-cell immunoglobulin-like receptors (KIRs) and mannose-binding lectin 2 (MBL2) in successful aging and their contribution to the better understanding of immune dysfunction in old age. Within the 15th IHIWS new populations were included in the analysis. Additional cytokine gene polymorphisms were assessed and innate immunity genes were analyzed for possible relevance in longevity. The results showed that longevity might be associated with anti-inflammatory cytokine gene profiles, decreased frequency of interleukin-10 (IL-10) and transforming growth factor-B1 haplotypes associated with a low level of gene expression, and increased frequency of haplotypes determining a high level of expression. Extended tumor necrosis factor-A and IL-12B genotypes were also likely relevant to longevity. Data also showed that innate immunity genes are associated with susceptibility to infections in the elderly and showed that these genes might be an important genetic marker in aging. Decreased frequencies of KIR2DS5 and A1B10 haplotypes, and an increased proportion of MBL2-deficient haplotypes were found in the group with higher cytomegalovirus-specific IgG antibody levels. Together, these studies emphasize the relevance of genes regulating immune functions in maintaining human longevity and stress the importance of further clarifying their impact on successful aging.

Leukocyte telomere length is associated with complications of type 2 diabetes mellitus.

OBJECTIVE: The key goal of diabetes management is to prevent complications. While the patho-physiological mechanisms responsible for diabetes complications have been extensively studied, at present it is impossible to predict which patient with diabetes could develop complications. In recent years, the role of leukocyte telomere length in the pathogenesis of cardiovascular disease and Type 2 diabetes has been investigated. However, studies aiming to investigate the role of telomeres in the development and progression of Type 2 diabetes, as well as diabetic complications, are still lacking. As a consequence, this study aimed to verify whether leukocyte telomere length is associated with the presence and the number of diabetic complications in a sample of patients with Type 2 diabetes. METHODS: This is a cross-sectional study. Nine hundred and one subjects were enrolled, including 501 patients with Type 2 diabetes, of whom 284 had at least one complication and 217 were without complications, and 400 control subjects. Leukocyte telomere length was measured by quantitative real-time PCR. RESULTS: Patients with diabetes complications had significantly shorter leukocyte telomere length than both patients without diabetes complications and healthy control subjects. Moreover, among patients with diabetes complications, leukocyte telomere length became significantly and gradually shorter with the increasing number of diabetes complications. The magnitude of the effect of the decrease of the abundance of telomeric template vs. a single-copy gene length (T/S ratio) on complications is described by the estimated odds ratio OR=5.44 (95%CI 3.52-8.42). CONCLUSIONS: The results of the study support the hypothesis that telomere attrition may be a marker associated with the presence and the number of diabetic complications.

Gut microbiota as a candidate for lifespan extension: an ecological/evolutionary perspective targeted on living organisms as metaorganisms.

An emerging central concept in evolutionary biology suggests that symbiosis is a universal characteristic of living organisms that can help in understanding complex traits and phenotypes. During evolution, an integrative circuitry fundamental for survival has been established between commensal gut microbiota and host. On the basis of recent knowledge in worms, flies, and humans, an important role of the gut microbiota in aging and longevity is emerging. The complex bacterial community that populates the gut and that represents an evolutionary adapted ecosystem correlated with nutrition appears to limit the accumulation of pathobionts and infections in all taxa, being able of affecting the efficiency of the host immune system and exerting systemic metabolic effects. There is an urgent need to disentangle the underpinning molecular mechanisms, which could shed light on the basic mechanisms of aging in an ecological perspective. Thus, it appears possible to extend healthy aging and lifespan by targeting the host as a metaorganism by manipulating the complex symbiotic ecosystem of gut microbiota, as well as other possible ecosystems of the body.

Disentangling age-dependent DNA methylation: deterministic, stochastic, and nonlinear.

DNA methylation variability arises due to concurrent genetic and environmental influences. Each of them is a mixture of regular and noisy sources, whose relative contribution has not been satisfactorily understood yet. We conduct a systematic assessment of the age-dependent methylation by the signal-to-noise ratio and identify a wealth of "deterministic" CpG probes (about 90%), whose methylation variability likely originates due to genetic and general environmental factors. The remaining 10% of "stochastic" CpG probes are arguably governed by the biological noise or incidental environmental factors. Investigating the mathematical functional relationship between methylation levels and variability, we find that in about 90% of the age-associated differentially methylated positions, the variability changes as the square of the methylation level, whereas in the most of the remaining cases the dependence is linear. Furthermore, we demonstrate that the methylation level itself in more than 15% cases varies nonlinearly with age (according to the power law), in contrast to the previously assumed linear changes. Our findings present ample evidence of the ubiquity of strong DNA methylation regulation, resulting in the individual age-dependent and nonlinear methylation trajectories, whose divergence explains the cross-sectional variability. It may also serve a basis for constructing novel nonlinear epigenetic clocks.

Sex-Specific Age-Related Changes in Methylation of Certain Genes.

The aim of the study was to conduct a functional analysis of sex-specific age-related changes in DNA methylation. MATERIALS AND METHODS: The study used a GSE87571 methylation dataset obtained from the blood DNA of 729 individuals aged 14 to 94 using the Illumina Infinium HumanMethylation450K BeadChip (USA). Gene ontology analysis was performed for 3 groups of genes (females, males, and duplicates) using the PANTHER database. The DAVID platform was used to perform KEGG metabolic pathway analysis. RESULTS: The studies revealed unique for males and females changes in methylation of CpG sites, associated with certain metabolic processes. It was demonstrated that most of the CpG sites, for which methylation changes with age were revealed in both sexes, are associated with the genes responsible for the development and functioning of the nervous system. In males, unique age-related methylation changes affect CpG sites associated with changes in the immune system and lipid metabolism. In females, most CpGs are associated with changes involved in transcription and translation processes. Analysis of biological functions by KEGG revealed that a unique process associated with age-related changes in methylation of the glutamatergic system is typical for males. In females, unique biological processes with age-related changes include genes responsible for the development of diabetes and genes associated with cAMP signaling cascades (KEGG:04024). CONCLUSION: Our studies reveal fundamental features of sex-dependent changes in methylation of CpG sites with variance increasing, which may indicate differences in age-related changes.

Changes in the ceca microbiota of broilers vaccinated for coccidiosis or supplemented with salinomycin.

The objective of this study was to characterize differences in the cecal microbiota of chickens vaccinated for coccidiosis or receiving salinomycin in the diet. In this study, 140 male 1-day-old broiler chickens were divided in 2 groups: vaccine group (live vaccine) vaccinated at the first day and salinomycin group (125 ppm/kg since the first day until 35 d of age). Each treatment was composed for 7 replicates of 10 birds per pen. At 28 d, the cecal content of one bird per replicate was collected for microbiota analysis. The genetic sequencing was conducted by the Miseq Illumina platform. Vaccine group showed lower body weight, weight gain, and poorer feed conversion in the total period (P < 0.05). Bacterial 16S rRNA genes were classified as 3 major phyla (Bacteroidetes, Firmicutes, and Proteobacteria), accounting for more than 98% of the total bacterial community. The microbiota complexity in the cecal was estimated based on the α-diversity indices. The vaccine did not reduce species richness and diversity (P > 0.05). The richness distribution in the salinomycin group was larger and more uniform than the vaccinated birds. Salinomycin group was related to the enrichment of Bacteroidetes, whereas Firmicutes and Proteobacteria phyla were in greater proportions in the vaccine group. The last phylum includes a wide variety of pathogenic bacteria. The vaccine did not decrease the species richness but decreased the percentage of Bacteroidetes, a phylum composed by genera that produce short-chain fatty acids improving intestinal health. Vaccine group also had higher Proteobacteria phylum, which may help explain its poorer performance.

Self-rated health in individuals with and without disease is associated with multiple biomarkers representing multiple biological domains.

Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.

Maturation of the immkune response in vitro. Focal fluctuation and changes in affinity of anti-beta-D-galactosidase activating antibody.

We have cultivated lymph node microfragments from beta-D-galactosidase (Escherichia coli) primed rabbits and have measured their secondary response directed towards the whole molecule (precipitating antibodies) and to a single determinant (activating antibodies) of the antigen. By decreasing the size of the fragments to 10(5) cells, we began to observe heterogeneity among identical cultures in terms of positivity of response, antibody specificity, and titers. The affinity of "early" activating antibodies was inversely proportional to the dose of challenge. While no maturation was seen in low and excessive challenge, in all cultures receiving intermediate doses the association constant was raised several orders of magnitude within periods of 20 days. The relevance of these data to the mechanism of affinity selection of antigen-sensitive cells is discussed.

Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.