Phenotypic and genotypic antimicrobial resistance correlation and plasmid characterization in Salmonella spp. isolates from Italy reveal high heterogeneity among serovars

IntroductionThe spread of antimicrobial resistance among zoonotic pathogens such as Salmonella is a serious health threat, and mobile genetic elements (MGEs) carrying antimicrobial resistance genes favor this phenomenon. In this work, phenotypic antimicrobial resistance to commonly used antimicrobials was studied, and the antimicrobial resistance genes (ARGs) and plasmid replicons associated with the resistances were determined.MethodsEighty-eight Italian Salmonella enterica strains (n = 88), from human, animal and food sources, isolated between 2009 and 2019, were selected to represent serovars with different frequency of isolation in human cases of salmonellosis. The presence of plasmid replicons was also investigated.Results and discussionResistances to sulphonamides (23.9%), ciprofloxacin (27.3%), ampicillin (29.5%), and tetracycline (32.9%) were the most found phenotypes. ARGs identified in the genomes correlated with the phenotypical results, with blaTEM-1B, sul1, sul2, tetA and tetB genes being frequently identified. Point mutations in gyrA and parC genes were also detected, in addition to many different aminoglycoside-modifying genes, which, however, did not cause phenotypic resistance to aminoglycosides. Many genomes presented plasmid replicons, however, only a limited number of ARGs were predicted to be located on the contigs carrying these replicons. As an expectation of this, multiple ARGs were identified on contigs with IncQ1 plasmid replicon in strains belonging to the monophasic variant of Salmonella Typhimurium. In general, high variability in ARGs and plasmid replicons content was observed among isolates, highlighting a high level of heterogeneity in Salmonella enterica. Irrespective of the serovar., many of the ARGs, especially those associated with critically and highly important antimicrobials for human medicine were located together with plasmid replicons, thus favoring their successful dissemination

Whole-genome characterisation of TEM-1 and CMY-2 β-lactamase-producing Salmonella Kentucky ST198 in Lebanese broiler chain

Objectives : Salmonella enterica subsp. enterica serovar Kentucky has been associated with the worldwide ciprofloxacin-resistant (CIPR) Salmonella Kentucky sequence type 198 (ST198) epidemic clone, mostly recovered from poultry farms and products. The aim of this study was to examine whether this expanding clone exists in the Lebanese broiler chain. Methods : Eight CIPR and extended-spectrum cephalosporin-resistant Salmonella Kentucky isolates previously recovered from Lebanese broilers were genetically characterised by whole-genome sequencing. Results : Seven of the eight isolates belonged to ST198 and were phylogenetically closely related. They all harboured mutations in the chromosomal quinolone resistance genesgyrA and parC with double and single substitutions, respectively. The blaTEM-1B and blaCMY-2 genes were both detected in six isolates. Insertion sequence ISEcp1 was located upstream of blaCMY-2, harboured by IncI1 plasmids in four strains. An IS10 transposition coupled to homologous recombination at transposition sites mediated CMY-2 plasmid integration into the chromosome of one strain. Resistance genes to aminoglycosides [aadA7 and aac(3)-Id], tetracyclines [tet(A)] and sulfonamides (sul1) were detected in five strains, among which four were positive for the presence of Salmonella genomic island 1 (SGI1) variant SGI1-K. All studied isolates harboured a variety of Salmonella pathogenicity islands (SPIs) as well as common regulatory and virulence genes. Conclusion : Here we report for the first time in Lebanon the detection and dissemination of the emerging highly drug-resistantSalmonella Kentucky ST198. Our findings shed new light on this clone as a potential public-health threat

Non-random distribution of deleterious mutations in the DNA and protein-binding domains of IRF6 are associated with Van Der Woude syndrome

Background: The development of the face occurs during the early days of intrauterine life by the formation of facial processes from the first Pharyngeal arch. Derangement in these well-organized fusion events results in Orofacial clefts (OFC). Van der Woude syndrome (VWS) is one of the most common causes of syndromic cleft lip and/or palate accounting for 2% of all cases. Mutations in the IRF6 gene account for 70% of cases with the majority of these mutations located in the DNA-binding (exon 3, 4) or protein-binding domains (exon 7-9). The current study was designed to update the list of IRF6 variants reported for VWS by compiling all the published mutations from 2013 to date as well as including the previously unreported VWS cases from Africa and Puerto Rico.Methods: We used PubMed with the search terms; "Van der Woude syndrome," "Popliteal pterygium syndrome," "IRF6," and "Orofacial cleft" to identify eligible studies. We compiled the CADD score for all the mutations to determine the percentage of deleterious variants.Results: Twenty-one new mutations were identified from nine papers. The majority of these mutations were in exon 4. Mutations in exon 3 and 4 had CADD scores between 20 and 30 and mutations in exon 7-9 had CADD scores between 30 and 40. The presence of higher CADD scores in the protein-binding domain (exon 7-9) further confirms the crucial role played by this domain in the function of IRF6. In the new cases, we identified five IRF6 mutations, three novel missense mutations (p.Phe36Tyr, p.Lys109Thr, and p.Gln438Leu), and two previously reported nonsense mutations (p.Ser424*and p.Arg250*).Conclusion: Mutations in the protein and DNA-binding domains of IRF6 ranked among the top 0.1% and 1% most deleterious genetic mutations, respectively. Overall, these findings expand the range of VWS mutations and are important for diagnostic and counseling purposes.</p

On Assessing Trustworthy AI in Healthcare. Machine Learning as a Supportive Tool to Recognize Cardiac Arrest in Emergency Calls

Artificial Intelligence (AI) has the potential to greatly improve the delivery of healthcare and other services that advance population health and wellbeing. However, the use of AI in healthcare also brings potential risks that may cause unintended harm. To guide future developments in AI, the High-Level Expert Group on AI set up by the European Commission (EC), recently published ethics guidelines for what it terms “trustworthy” AI. These guidelines are aimed at a variety of stakeholders, especially guiding practitioners toward more ethical and more robust applications of AI. In line with efforts of the EC, AI ethics scholarship focuses increasingly on converting abstract principles into actionable recommendations. However, the interpretation, relevance, and implementation of trustworthy AI depend on the domain and the context in which the AI system is used. The main contribution of this paper is to demonstrate how to use the general AI HLEG trustworthy AI guidelines in practice in the healthcare domain. To this end, we present a best practice of assessing the use of machine learning as a supportive tool to recognize cardiac arrest in emergency calls. The AI system under assessment is currently in use in the city of Copenhagen in Denmark. The assessment is accomplished by an independent team composed of philosophers, policy makers, social scientists, technical, legal, and medical experts. By leveraging an interdisciplinary team, we aim to expose the complex trade-offs and the necessity for such thorough human review when tackling socio-technical applications of AI in healthcare. For the assessment, we use a process to assess trustworthy AI, called 1Z-Inspection® to identify specific challenges and potential ethical trade-offs when we consider AI in practice.</jats:p

Production of cytokines in response to macrophage stimulation with different strains of Group B Streptococcus

Streptokoki skupine B (GBS) so vodilni vzrok za bolezni in smrt novorojenckov po vsem svetu. Prav tako imajo pomemben vpliv na nosecnice, starejše in imunsko oslabljene osebe. Prehod iz asimptomatske kolonizacije v invazivno okužbo še ni povsem razumljen. V naši raziskavi smo s pomocjo pretocnega citometra analizirali tvorbo citokinov po stimulaciji makrofagov, da bi raziskali vpliv razlicnih sevov GBS na potek in resnost bolezni. Ugotovili smo razlike v tvorbi vnetnih in regulatornih citokinov ter kemokinov 3 in 24 ur po stimulaciji makrofagov ter razlike v tvorbi citokinov med posameznimi serotipi GBS. Opazili smo, da na profil citokinov vpliva razlicna citotoksicnost posameznih sevov. Na podlagi meritev IL-1ß in IL-18 predvidevamo, da doloceni sevi GBS povzrocajo piroptozo v stimuliranih makrofagih. Z analizo tvorbe IL-12 smo ugotovili tudi, da prihaja do razlik v aktivaciji sekundarnega imunskega odziva med sevi GBS. Pri hipervirulentnem sekvencnem tipu ST-17 pa smo ugotovili nižjo produkcijo vnetnih citokinov na zacetku okužbe ter povišano tvorbo regulatornega citokina IL-10. To povezujemo z njegovo sposobnostjo izmikanja imunskemu sistemu in povzrocanjem invazivnih okužb.Group B Streptococcus (GBS) is one of the most common causes of neonatal illness and death worldwide. Pregnant women, the elderly, and immunocompromised individuals are also severely affected by GBS. The transition from asymptomatic colonization to invasive disease is not yet fully understood. In our study, we used flow cytometry to analyze the production of cytokines after stimulation of macrophages to investigate the effects of different GBS strains on the severity of disease progression. We found differences in the production of inflammatory cytokines, regulatory cytokines and chemokines 3 and 24 hours after macrophage stimulation, as well as differences in cytokine production between different GBS serotypes. We observed the influence of different cytotoxic GBS strains on the profile of cytokine production. Based on the measurements of IL-1ß and IL-18, we hypothesize that certain GBS strains induce pyroptosis in stimulated macrophages. By analyzing IL-12 production, we found differences in activation of the secondary immune response between GBS strains. In the case of the hypervirulent sequence type ST-17, we observed lower production of inflammatory cytokines and increased production of the regulatory cytokine IL-10 at the onset of infection, which we related to its ability to evade the immune system and cause invasive infections

Effect of pH and Salinity on the Ability of Salmonella Serotypes to Form Biofilm

Salmonella is a major cause of food-borne infections in Europe, and the majority of human infections are caused by only a few serotypes, among them are Salmonella enterica subsp. enterica serotype Enteritidis (hereafter Salmonella Enteritidis), Salmonella Typhimurium, and the monophasic variant of S. Typhimurium. The reason for this is not fully understood, but could include virulence factors as well as increased ability to transfer via the external environment. Formation of biofilm is considered an adaptation strategy used by bacteria to overcome environmental stresses. In order to assess the capability of different Salmonella serotypes to produce biofilm and establish whether this is affected by pH and salinity, 88 Salmonella isolates collected from animal, food, and human sources and belonging to 15 serotypes, including those most frequently responsible for human infections, were tested. Strains were grown in tryptic soy broth (TSB), TSB with 4% NaCl pH 4.5, TSB with 10% NaCl pH 4.5, TSB with 4% NaCl pH 7, or TSB with 10% NaCl pH 7, and biofilm production was assessed after 24 h at 37°C using crystal violet staining. A linear mixed effect model was applied to compare results from the different experimental conditions. Among the tested serotypes, S. Dublin showed the greatest ability to form biofilm even at pH 4.5, which inhibited biofilm production in the other tested serotypes. Salmonella Senftenberg and the monophasic variant of S. Typhimurium showed the highest biofilm production in TSB with 10% NaCl pH 7. In general, pH had a high influence on the ability to form biofilm, and most of the tested strains were not able to produce biofilm at pH 4.5. In contrast, salinity only had a limited influence on biofilm production. In general, serotypes causing the highest number of human infections showed a limited ability to produce biofilm in the tested conditions, indicating that biofilm formation is not a crucial factor in the success of these clones