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Background: Bacillus cereus is a widespread environmental Gram-positive bacterium which is especially common in soil and dust. It produces two types of toxins that cause vomiting and diarrhea. At present, foodborne outbreaks due to Bacillus cereus group bacteria (especially Bacillus cereus sensu stricto) are rising, representing a serious problem in the agri-food supply chain. Methods: In this work, we analyzed 118 strains belonging to the Bacillus cereus group, isolated from several food sources, for which in vitro and in silico antibiotic resistance assessments were performed. Results: Many strains showed intermediate susceptibility to clindamycin, erythromycin, and tetracycline, suggesting an evolving acquisition of resistance against these antibiotics. Moreover, one strain showed intermediate resistance to meropenem, an antibiotic currently used to treat infections caused by Bacillus cereus . In addition to the phenotypic antimicrobial resistance profile, all strains were screened for the presence/absence of antimicrobial genes via whole-genome sequencing. There was inconsistency between the in vitro and in silico analyses, such as in the case of vancomycin, for which different isolates harbored resistance genes but, phenotypically, the same strains were sensitive. Conclusions: This would suggest that antibiotic resistance is a complex phenomenon due to a variety of genetic, epigenetic, and biochemical mechanisms.

Fourier transform infrared spectroscopy (FTIRS) is a diagnostic technique historically used in the microbiological field for the characterization of bacterial strains in relation to the specific composition of their lipid, protein, and polysaccharide components. For each bacterial strain, it is possible to obtain a unique absorption spectrum that represents the fingerprint obtained based on the components of the outer cell membrane. In this study, FTIRS was applied for the first time as an experimental diagnostic tool for the discrimination of two pathogenic species belonging to the Bacillus cereus group, Bacillus anthracis and Bacillus cereus sensu stricto; these are two closely related species that are not so easy to differentiate using classical microbiological methods, representing an innovative technology in the field of animal health.

Introduction: Brucellosis is a widespread zoonosis of great economic importance for livestock farming in many areas of the world. It is a highly infectious disease which is diagnosed using conventional serological and microbiological methods. The aim of this study was to assess the efficiency of a specific real-time PCR in combination with broth cultivation in detecting Brucella spp. in organs of infected cattle, in order to compare the sensitivity of the two approaches and the time needed in them until a correct diagnosis is made., Material and Methods: We examined 67 organs collected from 10 cattle slaughtered following a brucellosis outbreak which occurred in February 2016 in southern Italy. The research was carried out by enrichment broth cultivations in combination with a real-time PCR every week for six weeks., Results: Brucella strains were isolated by cultivation from 44 enrichment broths of organs. All isolates were later identified as Brucella abortus by real-time PCR. Using this method in combination with cultivation made it possible to identify the same percentage of infected animals faster than by cultivation alone. Moreover, the same diagnostic results were obtained, on average two weeks before they would have been using only cultivation. In almost all cases, Brucella was detected by real-time PCR after the first week of cultivation in pre-enrichment Brucella broth, while the bacterial growth was evident usually after 2 or 3 weeks., Conclusion: Real-time PCR has allowed results to be obtained faster than in the classical microbiological method, reducing the response times to identify positive animals by half., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2022 V. Manzulli et al. published by Sciendo.)

The Delta variant raised concern regarding its ability to evade SARS-CoV-2 vaccines. We evaluated a serum neutralizing response of 172 Italian healthcare workers, three months after complete Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer) vaccination, testing their sera against viral isolates of Alpha, Gamma and Delta variants, including 36 subjects with a previous SARS-CoV-2 infection. We assessed whether IgG anti-spike TRIM levels and serum neutralizing activity by seroneutralization assay were associated. Concerning Gamma variant, a two-fold reduction in neutralizing titres compared to the Alpha variant was observed, while a four-fold reduction of Delta virus compared to Alpha was found. A gender difference was observed in neutralizing titres only for the Gamma variant. The serum samples of 36 previously infected SARS-CoV-2 individuals neutralized Alpha, Gamma and Delta variants, demonstrating respectively a nearly three-fold and a five-fold reduction in neutralizing titres compared to Alpha variant. IgG anti-spike TRIM levels were positively correlated with serum neutralizing titres against the three variants. The Comirnaty vaccine provides sustained neutralizing antibody activity towards the Alpha variant, but it is less effective against Gamma and even less against Delta variants.

Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) technology is currently increasingly used in diagnostic laboratories as a cost effective, rapid and reliable routine technique for the identification and typing of microorganisms. In this study, we used MALDI-TOF MS to analyze a collection of 160 strains belonging to the Bacillus cereus group (57 B. anthracis , 49 B. cereus , 1 B. mycoides , 18 B. wiedmannii , 27 B. thuringiensis , 7 B. toyonensis and 1 B. weihenstephanensis ) and to detect specific biomarkers which would allow an unequivocal identification. The Main Spectra Profiles (MSPs) were added to an in-house reference library, expanding the current commercial library which does not include B. toyonensis and B. wiedmannii mass spectra. The obtained mass spectra were statistically compared by Principal Component Analysis (PCA) that revealed seven different clusters. Moreover, for the identification purpose, were generated dedicate algorithms for a rapid and automatic detection of characteristic ion peaks after the mass spectra acquisition. The presence of specific biomarkers can be used to differentiate strains within the B. cereus group and to make a reliable identification of Bacillus anthracis , etiologic agent of anthrax, which is the most pathogenic and feared bacterium of the group. This could offer a critical time advantage for the diagnosis and for the clinical management of human anthrax even in case of bioterror attacks.

In 2011, in Bangladesh, 11 anthrax outbreaks occurred in six districts of the country. Different types of samples were collected from May to September in the six districts where anthrax had occurred in order to detect and type Bacillus anthracis (B. anthracis) strains. Anthrax was detected in 46.6% of the samples analysed, in particular in soils, but also in bone samples, water, animal feed, and rumen ingesta of dead animals. Canonical single nucleotide polymorphisms (CanSNPs) analysis showed that all the isolates belonged to the major lineage A, sublineage A.Br.001/002 of China and Southeast Asia while the multi-locus variable number of tandem repeats (VNTRs) analysis (MLVA) with 15 VNTRs demonstrated the presence of five genotypes, of which two resulted to be new genotypes. The single nucleotide repeats (SNRs) analysis showed 13 SNR types; nevertheless, due to its higher discriminatory power, the presence of two isolates with different SNR-type polymorphisms was detected within two MLVA genotypes. This study assumes that soil is not the only reason for the spread of the disease in Bangladesh; contaminated feed and water can also play an important role in the epidemiology of anthrax. Possible explanations for these epidemiological relationships are discussed.

The coronavirus disease 2019 (Covid-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and presents a global health emergency that needs urgent intervention. Viruses constantly change through mutation, and new variants of a virus are expected to occur over time. In the United Kingdom (UK), a new variant called B.1.1.7 has emerged with an unusually large number of mutations. The aim of this study is to evaluate the level of protection of sera from 12 patients infected and later healed in Apulia Region (Italy) with Covid-19 between March and November 2020, when the English variant was not circulating in this territory yet, against the new VOC 202012/01 variant by seroneutralization assay. The sera of patients had already been tested before, using a virus belonging to the lineage B.1 and showed an antibody neutralizing titer ranging between 1:160 and 1:320. All the 12 sera donors confirmed the same titers of neutralizing antibodies obtained with a strain belonging to the lineage B.1.1.7 (VOC 202012/01). These data indicate that antibodies produced in subjects infected with variants of Sars-CoV-2 strain before the appearance of the English one, seem to have a neutralizing power also against this variant.

Background: The highly variable manifestation of the COVID-19 disease, from completely asymptomatic to fatal, is both a clinical and a public health issue. The criteria for discharge of hospitalized patients have been based so far on the negative result of Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) tests, but the persistence of viral fragments may exceed that of the integral virus by weeks. The aim of our study was to verify the clearance of the virus at viral culture in patients hospitalized for COVID-19 that have clinically recovered but are still positive on nasopharyngeal swab., Methods: The study was conducted in hospitalized patients with positive RT-PCR on nasopharyngeal swab. Patients included were from asymptomatic to severe cases and performed nasopharyngeal control swabbing on day 14 for asymptomatic patient or at least three days after remission of symptoms. RT-PCR positive specimens were sent to a biosafety level 3 laboratory for viral culture., Results: We performed a combined analysis of RT-PCR and a highly sensitive in vitro culture from 84 samples of hospitalized patients. The average age was 46 ± 20.29, and 40.5% of the subjects had radiologically confirmed pneumonia, with average PaO 2 of 72.35 ± 12.12and P/F ratio of 315 ± 83.15. Ct values for the N gene were lower in the first swab than in the control one ( p < 0.001). The samples from 83 patients were negative at viral culture, and RT-PCR on the respective supernatants always confirmed the absence of viral growth., Conclusions: Our preliminary results demonstrate that patients clinically recovered for at least three days show the viral clearance at viral culture, and presumably they continued to not be contagious.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis., Competing Interests: The authors have declared that no competing interests exist.

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.

A unique population of Foxp3(+)CD4(+) regulatory T (Treg) cells, with a distinct transcriptome and antigen-receptor repertoire, resides in visceral adipose tissue (VAT) of lean individuals. These cells regulate local inflammation and both local and systemic metabolic indices. Here we focus on expansion of the VAT Treg compartment in aging lean mice-assessing these cells' phenotypic conversion from conventional CD4(+) T cells, influx from lymphoid organs, and local population dynamics. Our findings establish that the VAT Treg compartment is seeded from thymocytes generated during the first weeks of life and expands beyond 10 weeks of age due to indolent proliferation, of certain clones in particular, coupled with enhanced survival. Accumulation of VAT Tregs depends on the antigen(s) presented by MHC class-II molecules and soluble mediators, notably interleukin(IL)-33. Addressing such factors therapeutically promises novel approaches for harnessing Tregs to stem the growing epidemic of obesity and consequent metabolic abnormalities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A unique population of Foxp3(+)CD4(+) regulatory T (Treg) cells resides in visceral adipose tissue (VAT) of lean mice, especially in the epididymal fat depot. VAT Tregs are unusual in their very high representation within the CD4(+) T-cell compartment, their transcriptome, and their repertoire of antigen-specific T-cell receptors. They are important regulators of local and systemic inflammation and metabolism. The overall goal of this study was to learn how the VAT Treg transcriptome adapts to different stimuli; in particular, its response to aging in lean mice, to metabolic perturbations associated with obesity, and to certain signaling events routed through PPARγ, the "master-regulator" of adipocyte differentiation. We show that the VAT Treg signature is imposed early in life, well before age-dependent expansion of the adipose-tissue Treg population. VAT Tregs in obese mice lose the signature typical of lean individuals but gain an additional set of over- and underrepresented transcripts. This obese mouse VAT Treg signature depends on phosphorylation of the serine residue at position 273 of PPARγ, in striking parallel to a pathway recently elucidated in adipocytes. These findings are important to consider in designing drugs to target type 2 diabetes and other features of the "metabolic syndrome."

Foxp3(+)  CD4(+) regulatory T (Treg) cells, recognized to be one of the most important defences of the human body against an inappropriate immune response, have recently gained attention from those outside immunology thanks to the compelling evidence for their capability to exert non-canonical immune functions in a variety of tissues in health and disease. The recent discovery of the differences between tissue-resident Treg cells and those derived from lymphoid organs is affecting the mindset of many investigators now questioning the broad applicability of observations originally based on peripheral blood/lymphoid organ cells. So far, the best characterized 'Treg flavour' comes from studies focused on their role in suppressing adipose tissue inflammation and obesity-driven insulin resistance. Adipose tissue derived Treg cells are distinct from their counterparts in lymphoid organs based on their transcriptional profile, T-cell receptor repertoire, and cytokine and chemokine receptor expression pattern. These cells are abundant in visceral adipose tissue of lean mice but their number is greatly reduced in insulin-resistant animal models of obesity. Interestingly, peroxisome-proliferator-activated receptor γ expression by visceral adipose tissue Treg cells is crucial for their accumulation, phenotype and function in the fat and surprisingly necessary for complete restoration of insulin sensitivity in obese mice by the anti-diabetic drug Pioglitazone. This review surveys recent findings relating to the unique phenotype and function of adipose tissue-resident Treg cells, speculates on the nature of their dynamics in lean and obese mouse models, and analyses their potential therapeutic application in the treatment of type 2 diabetes., (© 2014 John Wiley & Sons Ltd.)

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T (Treg) cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the 'master regulator' of adipocyte differentiation, as a crucial molecular orchestrator of VAT Treg cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT Treg cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of Treg cells with unique functions can be precisely targeted to therapeutic ends.

Maintenance of lymphoid homeostasis in a number of immunological and inflammatory contexts is served by a variety of regulatory T (Treg) cell subtypes and depends on interaction of the transcription factor FoxP3 with specific transcriptional cofactors. We report that a commonly used insertional mutant of FoxP3 (GFP-Foxp3) modified its molecular interactions, blocking HIF-1α but increasing IRF4 interactions. The transcriptional profile of these Treg cells was subtly altered, with an overrepresentation of IRF4-dependent transcripts. In keeping with IRF4-dependent function of Treg cells to preferentially suppress T cell help to B cells and Th2 and Th17 cell-type differentiation, GFP-FoxP3 mice showed a divergent susceptibility to autoimmune disease: protection against antibody-mediated arthritis in the K/BxN model, but greater susceptibility to diabetes on the NOD background. Thus, specific subfunctions of Treg cells and the immune diseases they regulate can be influenced by FoxP3's molecular interactions, which result in divergent immunoregulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Foxp3+CD4+ regulatory T (T(reg)) cells are a key population in controlling the immune response. Recently, their roles have been expanded to broader, non-immune, contexts, in particular the metabolic consequences downstream of obesity-induced inflammation, e.g. type-2 diabetes and cardiovascular disease. This review highlights the major innate and adaptive immune cell subsets contributing to adipose-tissue inflammation, the key role played by fat-resident T(regs), and the potential of T(reg)-based therapies for treatment of the metabolic syndrome., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Obesity is accompanied by chronic, low-grade inflammation of adipose tissue, which promotes insulin resistance and type-2 diabetes. These findings raise the question of how fat inflammation can escape the powerful armamentarium of cells and molecules normally responsible for guarding against a runaway immune response. CD4(+) Foxp3(+) T regulatory (T(reg)) cells with a unique phenotype were highly enriched in the abdominal fat of normal mice, but their numbers were strikingly and specifically reduced at this site in insulin-resistant models of obesity. Loss-of-function and gain-of-function experiments revealed that these T(reg) cells influenced the inflammatory state of adipose tissue and, thus, insulin resistance. Cytokines differentially synthesized by fat-resident regulatory and conventional T cells directly affected the synthesis of inflammatory mediators and glucose uptake by cultured adipocytes. These observations suggest that harnessing the anti-inflammatory properties of T(reg) cells to inhibit elements of the metabolic syndrome may have therapeutic potential.

Several diseases are characterized by chronic inflammation, a condition frequently associated with angiogenesis and fibrogenesis that account for the development of granulation tissue. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a crucial modulator of intracellular prosurvival signaling pathways and is implicated in the pathogenesis of inflammatory process. In this study, we have investigated the role of NF-kappaB in the angiogenic and fibrogenic response induced by lambda-carrageenin in a rat model of chronic inflammation at 1, 3, and 5 days. The subcutaneous implant of lambda-carrageenin-soaked sponges in rat induced a time-related increase of granulation tissue formation accompanied by intense neovascularization. These lambda-carrageenin-induced changes were significantly reduced by coinjection of wild-type oligodeoxynucleotide (WT ODN) decoy to NF-kappaB. Molecular, morphological, and ultrastructural analysis performed on whole granulation tissue demonstrated: (1) inhibition of NF-kappaB/DNA binding activity; (2) downregulation of cyclooxygenase-2, matrix metalloproteinase-9, tumor necrosis factor-alpha, and vascular endothelial growth factor; (3) upregulation of thrombospondin (TSP)-1 at 1 day and TSP-2 at 5 days; and (4) increase in Bax to Bcl-2 ratio. Our findings show that the blockade of NF-kappaB activation by WT ODN decoy prevents the development of granulation tissue induced by lambda-carrageenin-soaked sponge implant upregulating Bax as well as TSP-1 and TSP-2 expression.