p66Shc signaling and autophagy impact on C2C12 myoblast differentiation during senescence.

During aging, muscle regenerative capacities decline, which is concomitant with the loss of satellite cells that enter in a state of irreversible senescence. However, what mechanisms are involved in myogenic senescence and differentiation are largely unknown. Here, we showed that early-passage or "young" C2C12 myoblasts activated the redox-sensitive p66Shc signaling pathway, exhibited a strong antioxidant protection and a bioenergetic profile relying predominantly on OXPHOS, responses that decrease progressively during differentiation. Furthermore, autophagy was increased in myotubes. Otherwise, late-passage or "senescent" myoblasts led to a highly metabolic profile, relying on both OXPHOS and glycolysis, that may be influenced by the loss of SQSTM1/p62 which tightly regulates the metabolic shift from aerobic glycolysis to OXPHOS. Furthermore, during differentiation of late-passage C2C12 cells, both p66Shc signaling and autophagy were impaired and this coincides with reduced myogenic capacity. Our findings recognized that the lack of p66Shc compromises the proliferation and the onset of the differentiation of C2C12 myoblasts. Moreover, the Atg7 silencing favored myoblasts growth, whereas interfered in the viability of differentiated myotubes. Then, our work demonstrates that the p66Shc signaling pathway, which highly influences cellular metabolic status and oxidative environment, is critical for the myogenic commitment and differentiation of C2C12 cells. Our findings also support that autophagy is essential for the metabolic switch observed during the differentiation of C2C12 myoblasts, confirming how its regulation determines cell fate. The regulatory roles of p66Shc and autophagy mechanisms on myogenesis require future attention as possible tools that could predict and measure the aging-related state of frailty and disability.

Putting everything in its place: using the INSDC compliant Pathogen Data Object Model to better structure genomic data submitted for public health applications.

Fast, efficient public health actions require well-organized and coordinated systems that can supply timely and accurate knowledge. Public databases of pathogen genomic data, such as the International Nucleotide Sequence Database Collaboration (INSDC), have become essential tools for efficient public health decisions. However, these international resources began primarily for academic purposes, rather than for surveillance or interventions. Now, queries need to access not only the whole genomes of multiple pathogens but also make connections using robust contextual metadata to identify issues of public health relevance. Databases that over time developed a patchwork of submission formats and requirements need to be consistently organized and coordinated internationally to allow effective searches.To help resolve these issues, we propose a common pathogen data structure called the Pathogen Data Object Model (DOM) that will formalize the minimum pieces of sequence data and contextual data necessary for general public health uses, while recognizing that submitters will likely withhold a wide range of non-public contextual data. Further, we propose contributors use the Pathogen DOM for all pathogen submissions (bacterial, viral, fungal, and parasites), which will simplify data submissions and provide a consistent and transparent data structure for downstream data analyses. We also highlight how improved submission tools can support the Pathogen DOM, offering users additional easy-to-use methods to ensure this structure is followed.

Flebite associada a cateter venoso periférico e a administração de medicamentos: Análise retrospetiva de incidentes / Peripheral intravenous catheter-associated phlebitis and drug administration: Retrospective incident analysis / Flebitis asociada al catéter venoso periférico y a la administración de fármacos: Análisis retrospectivo de incidentes

Resumo Enquadramento: A flebite é uma complicação associada à utilização de cateter venoso periférico, classificada como evento adverso. Objetivos: Analisar os incidentes de flebite associada ao cateter venoso periférico e aos medicamentos administrados em doentes adultos internados e as suas consequências. Metodologia: Estudo quantitativo, retrospetivo, descritivo e transversal. Técnica de amostragem não probabilística por conveniência sendo selecionados 96 doentes adultos internados em 2019, na área da medicina de um Centro Hospitalar em Lisboa. O grau de flebite foi avaliado pela escala Visual Infusion Phlebitis Score Português Portugal (VIP PT-PT) traduzida e adaptada para Português Europeu. O dano foi categorizado de acordo com a classificação internacional sobre segurança do doente (CISD). Resultados: Dos incidentes de flebite documentados, 78% classificados com score 2 pela escala VIP PT-PT. Pela CISD, 87,5% resultaram em dano ligeiro e 12,5% em moderado. Os antibióticos foram o grupo terapêutico mais frequente. Conclusão: A flebite tem impacto na segurança do doente. Reforça-se a importância dos cuidados de enfermagem com enfoque na vigilância e deteção precoce de flebite.

Abstract Background: Phlebitis is a complication associated with peripheral intravenous catheters and an adverse event. Objective: To analyze peripheral intravenous catheter-associated phlebitis incidents associated and drug administration to adult inpatients and their consequences. Methodology: Quantitative, retrospective, descriptive, and cross-sectional study. Non-probabilistic convenience sampling technique was used, with 96 adult patients hospitalized in 2019 selected in the area of medicine at a Hospital Center in Lisbon. The type of phlebitis incident was characterized using the Visual Infusion Phlebitis scale adapted and translated to European Portuguese. Patient harm was categorized according to the International Classification for Patient Safety (ICPS). Results: Of the documented phlebitis incidents, 78% obtained a score 2 on the VIP PT-PT scale. According to the ICPS, 87.5% resulted in mild harm and 12.5% in moderate harm. Antibiotics were the most frequent therapeutic group. Conclusion: Phlebitis has an impact on patient safety. The importance of nursing care is reinforced, with a focus on surveillance and early detection of phlebitis to prevent complications.

Resumen Marco contextual: La flebitis es una complicación asociada al uso de catéteres venosos periféricos clasificada como acontecimiento adverso. Objetivos: Analizar los incidentes de flebitis asociada al empleo de catéteres venosos periféricos y a la administración de fármacos en pacientes adultos hospitalizados y sus consecuencias. Metodología: Estudio cuantitativo, retrospectivo, descriptivo y transversal. Técnica de muestreo no probabilístico por conveniencia, se seleccionaron 96 pacientes adultos ingresados en 2019 en el área médica de un centro hospitalario de Lisboa. El grado de flebitis se evaluó mediante la escala Visual Infusion Phlebitis Score Portugués Portugal (VIP PT-PT), traducida y adaptada al portugués europeo. El daño se categorizó según la Clasificación Internacional sobre Seguridad del Paciente (CISD). Resultados: De los incidentes de flebitis documentados, el 78% se clasificó con una puntuación de 2 según la escala VIP PT-PT. Por la CISD, el 87,5% resultó en daño leve y el 12,5% en moderado. Los antibióticos fueron el grupo terapéutico más frecuente. Conclusión: La flebitis repercute en la seguridad del paciente. Se refuerza la importancia de los cuidados de enfermería centrados en la vigilancia y la detección precoz de la flebitis.

Accelerating bioinformatics implementation in public health.

We have adopted an open bioinformatics ecosystem to address the challenges of bioinformatics implementation in public health laboratories (PHLs). Bioinformatics implementation for public health requires practitioners to undertake standardized bioinformatic analyses and generate reproducible, validated and auditable results. It is essential that data storage and analysis are scalable, portable and secure, and that implementation of bioinformatics fits within the operational constraints of the laboratory. We address these requirements using Terra, a web-based data analysis platform with a graphical user interface connecting users to bioinformatics analyses without the use of code. We have developed bioinformatics workflows for use with Terra that specifically meet the needs of public health practitioners. These Theiagen workflows perform genome assembly, quality control, and characterization, as well as construction of phylogeny for insights into genomic epidemiology. Additonally, these workflows use open-source containerized software and the WDL workflow language to ensure standardization and interoperability with other bioinformatics solutions, whilst being adaptable by the user. They are all open source and publicly available in Dockstore with the version-controlled code available in public GitHub repositories. They have been written to generate outputs in standardized file formats to allow for further downstream analysis and visualization with separate genomic epidemiology software. Testament to this solution meeting the requirements for bioinformatic implementation in public health, Theiagen workflows have collectively been used for over 5 million sample analyses in the last 2 years by over 90 public health laboratories in at least 40 different countries. Continued adoption of technological innovations and development of further workflows will ensure that this ecosystem continues to benefit PHLs.

Pediatric Thyroidectomy: Experience From a Portuguese Hospital.

Background and objective Pediatric thyroid disease requiring surgery is rare. Thyroid nodules are a frequent indication for surgery and are mostly benign. However, up to 25% of cases can be malignant. In this study, we aimed to describe our center's experience with regard to pediatric thyroid surgery. Methods This was a retrospective transverse study involving pediatric patients who underwent thyroid surgery at a tertiary hospital between January 2010 and December 2021. Results A total of 14 patients underwent 15 surgeries. The main reason for referral to pediatric endocrinology was thyroid nodules (n=10). Thirteen fine needle aspirations (FNAs) were performed, with follicular tumor (n=6) being the most common finding. The median age of patients at surgery was 15.9 years [interquartile range (IQR): 14.0-16.8]. The most common surgical indications were the presence of a follicular tumor on FNA (n=5) and thyroid nodule size causing symptoms (n=5). There was one case of prophylactic thyroidectomy due to the identification of a multiple endocrine neoplasia type 2A (MEN2A) mutation. The most frequently described histopathology results were follicular adenoma (n=6) and colloid nodular goiter (n=6). Three postoperative complications were observed in three different patients: bilateral lesion of the recurrent laryngeal nerve, cervical hematoma, and transient hypoparathyroidism with hypocalcemia. Conclusion In our study, the most frequent surgical indication was a follicular tumor. A good correlation was found between FNA cytology and final histopathology results, which is in accordance with previous studies. This reinforces the importance of FNA in diagnosis and surgical planning. The rate of complications in our study is comparable to that in larger single-center series in the literature.

Type 1 diabetes mellitus - Population characterization and metabolic control outcomes in a Portuguese patient sample.

AIMS: To characterize a cohort of T1D patients and to compare diabetes control between patients using different regimen of insulin therapy and glucose monitoring. METHODS: Were included all T1D patients followed at the Pediatric Endocrinology Unit, between April 1st and June 30th, 2021. Several clinical and demographic variables were analyzed. RESULTS: Our sample included 208 patients, 56.7 % males, mean age of 12.7 ± 4.6 years. The median HbA1c was 7.3 %. Most patients, 78.8% were treated with continuous subcutaneous insulin infusion (CSII) and 81.3 % used continuous glucose monitoring (CGM). CSII had a lower HbAc compared with multiple daily injections (MDI) users (7.1vs 8.1 %, p < 0.01). In the CSII group, those who used CGM had a lower HbAc (7.1 vs 7.5 %,p = 0.02). Analyzing the data of the ambulatory glucose report, the CSII users had a lower glucose management indicator, (7.2 % vs 7.6 %, p < 0.01), more time in range (58.0 % vs 52.4 %;p < 0.01) and less time above range > 250 mg/dL (12.4 % vs 20.5 %;p < 0.01) than MDI users. CONCLUSIONS: The median HbA1c was 7.3% very close to the recommended target. In Portugal, pediatric patients can access a CSII provided by the national health service and a CGM system due to an elevated reimbursement of their cost. This healthy policy allows us to achieve better goals without the risk of hypoglycemia.

LMAS: evaluating metagenomic short de novo assembly methods through defined communities.

BACKGROUND: The de novo assembly of raw sequence data is key in metagenomic analysis. It allows recovering draft genomes from a pool of mixed raw reads, yielding longer sequences that offer contextual information and provide a more complete picture of the microbial community. FINDINGS: To better compare de novo assemblers for metagenomic analysis, LMAS (Last Metagenomic Assembler Standing) was developed as a flexible platform allowing users to evaluate assembler performance given known standard communities. Overall, in our test datasets, k-mer De Bruijn graph assemblers outperformed the alternative approaches but came with a greater computational cost. Furthermore, assemblers branded as metagenomic specific did not consistently outperform other genomic assemblers in metagenomic samples. Some assemblers still in use, such as ABySS, MetaHipmer2, minia, and VelvetOptimiser, perform relatively poorly and should be used with caution when assembling complex samples. Meaningful strain resolution at the single-nucleotide polymorphism level was not achieved, even by the best assemblers tested. CONCLUSIONS: The choice of a de novo assembler depends on the computational resources available, the replicon of interest, and the major goals of the analysis. No single assembler appeared an ideal choice for short-read metagenomic prokaryote replicon assembly, each showing specific strengths. The choice of metagenomic assembler should be guided by user requirements and characteristics of the sample of interest, and LMAS provides an interactive evaluation platform for this purpose. LMAS is open source, and the workflow and its documentation are available at https://github.com/B-UMMI/LMAS and https://lmas.readthedocs.io/, respectively.

Future-proofing and maximizing the utility of metadata: The PHA4GE SARS-CoV-2 contextual data specification package.

BACKGROUND: The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and share best practices, improve the availability of critical bioinformatics tools and resources, and advocate for greater openness, interoperability, accessibility, and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a need for a fit-for-purpose, open-source SARS-CoV-2 contextual data standard. RESULTS: As such, we have developed a SARS-CoV-2 contextual data specification package based on harmonizable, publicly available community standards. The specification can be implemented via a collection template, as well as an array of protocols and tools to support both the harmonization and submission of sequence data and contextual information to public biorepositories. CONCLUSIONS: Well-structured, rich contextual data add value, promote reuse, and enable aggregation and integration of disparate datasets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19. The package is now supported by the NCBI's BioSample database.

Histological Inflammation in the Endoscopically Uninflamed Mucosa is Associated With Worse Outcomes in Limited Ulcerative Colitis.

BACKGROUND: The Montreal classification categorizes patients with ulcerative colitis (UC) based on their macroscopic disease extent. Independent of endoscopic extent, biopsies through all colonic segments should be retrieved during index colonoscopy. However, the prognostic value of histological inflammation at diagnosis in the inflamed and uninflamed regions of the colon has never been assessed. METHODS: This was a multicenter retrospective cohort study of newly diagnosed patients with treatment-naïve proctitis and left-sided UC. Biopsies from at least 2 colonic segments (endoscopically inflamed and uninflamed mucosa) were retrieved and reviewed by 2 pathologists. Histological features in the endoscopically inflamed and uninflamed mucosa were scored using the Nancy score. The primary outcomes were disease complications (proximal disease extension, need for hospitalization or colectomy) and higher therapeutic requirements (need for steroids or for therapy escalation). RESULTS: Overall, 93 treatment-naïve patients were included, with a median follow-up of 44 months (range, 2-329). The prevalence of any histological inflammation above the endoscopic margin was 71%. Proximal disease extension was more frequent in patients with histological inflammation in the endoscopically uninflamed mucosa at diagnosis (21.5% vs 3.4%, P = 0.04). Histological involvement above the endoscopic margin was the only predictor associated with an earlier need for therapy escalation (adjusted hazard ratio, 3.69; 95% confidence interval, 1.05-13.0); P = 0.04) and disease complications (adjusted hazard ratio, 4.79; 95% confidence interval, 1.10-20.9; P = 0.04). CONCLUSIONS: The presence of histological inflammation in the endoscopically uninflamed mucosa at the time of diagnosis was associated with worse outcomes in limited UC.

Oxidative Stress in Amyotrophic Lateral Sclerosis: Pathophysiology and Opportunities for Pharmacological Intervention.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or Charcot disease, is a fatal neurodegenerative disease that affects motor neurons (MNs) and leads to death within 2-5 years of diagnosis, without any effective therapy available. Although the pathological mechanisms leading to ALS are still unknown, a wealth of evidence indicates that an excessive reactive oxygen species (ROS) production associated with an inefficient antioxidant defense represents an important pathological feature in ALS. Substantial evidence indicates that oxidative stress (OS) is implicated in the loss of MNs and in mitochondrial dysfunction, contributing decisively to neurodegeneration in ALS. Although the modulation of OS represents a promising approach to protect MNs from degeneration, the fact that several antioxidants with beneficial effects in animal models failed to show any therapeutic benefit in patients raises several questions that should be analyzed. Using specific queries for literature search on PubMed, we review here the role of OS-related mechanisms in ALS, including the involvement of altered mitochondrial function with repercussions in neurodegeneration. We also describe antioxidant compounds that have been mostly tested in preclinical and clinical trials of ALS, also describing their respective mechanisms of action. While the description of OS mechanism in the different mutations identified in ALS has as principal objective to clarify the contribution of OS in ALS, the description of positive and negative outcomes for each antioxidant is aimed at paving the way for novel opportunities for intervention. In conclusion, although antioxidant strategies represent a very promising approach to slow the progression of the disease, it is of utmost need to invest on the characterization of OS profiles representative of each subtype of patient, in order to develop personalized therapies, allowing to understand the characteristics of antioxidants that have beneficial effects on different subtypes of patients.

Capacitação do familiar cuidador do idoso dependente / Training the family carer of the dependent elderly

Com o aumento do índice de envelhecimento e, por sua vez, do grau de dependência dos idosos, tem surgido cada vez mais, a preocupação com os cuidados às pessoas desta faixa etária. Na maioria dos casos, estes cuidados são prestados pela família. O familiar cuidador é assim o principal pilar da prestação de cuidados ao idoso dependente, sendo necessário conhecer as suas necessidades/dificuldades. Ancorado na Metodologia do Planeamento em Saúde foi desenvolvido o projeto "Capacitação do Familiar Cuidador do Idoso Dependente". A partir da caraterização dos familiares cuidadores e dos seus discursos sobre as suas práticas com o familiar dependente, necessidades e apoios, constatou-se como principais dificuldades a aceitação, adaptação à doença e forma como lidam com a doença do familiar. As necessidades expressas foram de apoio formal, nomeadamente, existência de estruturas de centro de dia. Verificaram-se também, necessidades de formação ao nível da higiene, dos cuidados à pele e da gestão do tempo. No sentido de dar resposta às necessidades/dificuldades encontradas, emergiu o Manual do Cuidador "Ao Cuidar de MIM, Cuido do OUTRO", instrumento de suporte facilitador da capacitação do familiar cuidador. A avaliação do projeto, pela perspetiva da equipa multiprofissional da UCC, atesta a sua relevância na capacitação do cuidador, no aumento da literacia do cuidador e nas estratégias para o cuidador cuidar de si próprio.

With the increase in the aging rate and, in turn, the degree of dependence of the elderly, there has been an increasing concern with care for people of this age group. In most cases, this care is provided by the family. The family caregiver is thus the main pillar of care for the dependent elderly, and it is necessary to know their needs / difficulties. Anchored in the Health Planning Methodology, the project "Training the Family Caregiver for the Dependent Elderly" was developed. From the characterization of the family caregivers and their speeches about their practices with the dependent family member, needs and support, it was found that the main difficulties were acceptance, adaptation to the disease and the way they deal with the family member's disease. The expressed needs were for formal support, namely, the existence of day care facilities. There were also training needs in terms of hygiene, skin care and time management. In order to respond to the needs / difficulties encountered, the Caregiver Manual "When Caring for ME, I take care of the OTHER" emerged, a support instrument that facilitates the training of the family caregiver. The evaluation of the project, from the perspective of the UCC multiprofessional team, attests to its relevance in the training of the caregiver, in increasing the literacy of the caregiver and in the strategies for the caregiver to take care of himself.

DEN-IM: dengue virus genotyping from amplicon and shotgun metagenomic sequencing.

Dengue virus (DENV) represents a public health threat and economic burden in affected countries. The availability of genomic data is key to understanding viral evolution and dynamics, supporting improved control strategies. Currently, the use of high-throughput sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR-amplified viral sequences (amplicon sequencing), is potentially the most informative approach to monitor viral dissemination and genetic diversity by providing, in a single methodological step, identification and characterization of the whole viral genome at the nucleotide level. Despite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper the reproducibility and comparison of results. Here we present DEN-IM, a one-stop, user-friendly, containerized and reproducible workflow for the analysis of DENV short-read sequencing data from both amplicon and shotgun metagenomics approaches. It is able to infer the DENV coding sequence (CDS), identify the serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement for extensive bioinformatics expertise. Using DEN-IM, we successfully analysed two types of DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples from patients with variable serotypes and genotypes, including an in vitro spiked sample containing the four known serotypes. The second consisted of 106 paired-end and 76 single-end amplicon sequences of DENV 3 genotype III and DENV 1 genotype I, respectively, where DEN-IM allowed detection of the intra-genotype diversity. The DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM).

Insecure striving as an exacerbator of the toxic effect of shame feelings on disordered eating.

BACKGROUND: Empirical evidence has previously shown the impact of shame and insecure striving on the emergence of eating psychopathology. However, interactions between these variables and the development of pathological eating remain unclear. OBJECTIVE: The present study aimed at exploring the moderator effect of insecure striving on the relationship between shame and disordered eating, while controlling for BMI effects. METHODS: Participants of this study were 458 women from the general population, with a mean age of 30.74 (SD = 12.15) and a mean BMI of 23.14 kg/m2 (SD = 3.99), who completed an online investigation protocol. Out of these participants, 7.0% were underweight, 67.9% had a normal weight, 18.8% were overweight and 6.3% were obese. RESULTS: Results showed that the study variables were positive and significantly intercorrelated. A multiple regression analysis revealed that insecure striving and shame explained 20.5% of disordered eating. The exacerbating role of insecure striving on the relationship between shame and disordered eating was further confirmed through path analysis. The model explained 38% of disordered eating and the plotted graph revealed that, for any level of shame, women who manifested a greater need to strive to avoid inferiority, expressed higher levels of disordered eating, in comparison to those who felt a lower pressure to prove others that they are attractive through the achievement of excellence goals. CONCLUSIONS: These results support the notion that eating psychopathology may be regarded as a striving to avoid inferiority strategy, establishing an important avenue for disordered eating-related investigation and clinical practice. LEVEL OF EVIDENCE: Level V, cross-sectional descriptive study.

A social rank approach to disordered eating: Exploring relationships between shame, fears of compassion, striving, and body shame.

OBJECTIVES: The impact of a social rank mentality on eating-related difficulties has been previously recognized, although mediating processes involved remain to be clarified. The current study aimed to explore the roles of insecure striving and body image shame on the relationships between external shame and fears of receiving compassion from others with eating difficulties, while controlling for BMI effects. METHODS: A total of 335 women from the general population participated in this study, aged between 18 and 62 years, who completed an investigation protocol, with self-report measures. RESULTS: Path analysis results showed that, when controlling for BMI, external shame and fears of compassion from others presented an indirect effect on eating difficulties, mediated by insecure striving and body image shame. The model explained 63% of the variance of eating-related difficulties. CONCLUSIONS: Findings suggest that, in Western societies, women who highly feel that others negatively evaluate the self and present higher levels of fears of receiving compassion from others may endorse maladaptive strategies (as insecure striving) which in turn may lead to experiencing greater levels of body image shame and eating-related difficulties. PRACTITIONER POINTS: This study suggests that therapists may observe that individuals who are vulnerable to developing body image and eating-related difficulties present high levels of shame and fears of compassion from others, as well as a need to compete to avoid inferiority. Therapeutic targets for people with eating-related difficulties may focus on shame, insecure striving, and fears of receiving compassionate and affiliative emotions. Addressing fears of receiving compassion and reducing shame may decrease the need to compete to avoid undesired inferiority and facilitate adaptive eating attitudes and behaviours.

Distinct Phenotypic and Genomic Signatures Underlie Contrasting Pathogenic Potential of Staphylococcus epidermidis Clonal Lineages.

Background: Staphylococcus epidermidis is a common skin commensal that has emerged as a pathogen in hospitals, mainly related to medical devices-associated infections. Noteworthy, infection rates by S. epidermidis have the tendency to rise steeply in next decades together with medical devices use and immunocompromized population growth. Staphylococcus epidermidis population structure includes two major clonal lineages (A/C and B) that present contrasting pathogenic potentials. To address this distinction and explore the basis of increased pathogenicity of A/C lineage, we performed a detailed comparative analysis using phylogenetic and integrated pangenome-wide-association study (panGWAS) approaches and compared the lineages's phenotypes in in vitro conditions mimicking carriage and infection. Results: Each S. epidermidis lineage had distinct phenotypic signatures in skin and infection conditions and differed in genomic content. Combination of phenotypic and genotypic data revealed that both lineages were well adapted to skin environmental cues. However, they appear to occupy different skin niches, perform distinct biological functions in the skin and use different mechanisms to complete the same function: lineage B strains showed evidence of specialization to survival in microaerobic and lipid rich environment, characteristic of hair follicle and sebaceous glands; lineage A/C strains showed evidence for adaption to diverse osmotic and pH conditions, potentially allowing them to occupy a broader and more superficial skin niche. In infection conditions, A/C strains had an advantage, having the potential to bind blood-associated host matrix proteins, form biofilms at blood pH, resist antibiotics and macrophage acidity and to produce proteases. These features were observed to be rare in the lineage B strains. PanGWAS analysis produced a catalog of putative S. epidermidis virulence factors and identified an epidemiological molecular marker for the more pathogenic lineage. Conclusion: The prevalence of A/C lineage in infection is probably related to a higher metabolic and genomic versatility that allows rapid adaptation during transition from a commensal to a pathogenic lifestyle. The putative virulence and phenotypic factors associated to A/C lineage constitute a reliable framework for future studies on S. epidermidis pathogenesis and the finding of an epidemiological marker for the more pathogenic lineage is an asset for the management of S. epidermidis infections.

Author Correction: Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Streptococcus pneumoniae Serotype 3 in Mexico (1994 to 2017): Decrease of the Unusual Clonal Complex 4909 Lineage following PCV13 Introduction.

Streptococcus pneumoniae expressing serotype 3 has a high virulence and a high case fatality ratio. Most studies of serotype 3 pneumococci have focused on a single lineage, the widespread sequence type 180 (ST180). To evaluate the serotype 3 lineages causing infections in Mexico, we characterized 196 isolates recovered from 1994 to 2017. The isolates were mostly susceptible to all antimicrobials tested. A single meningitis isolate was resistant to penicillin, and the resistance to erythromycin was 5.2%. The isolates represented the widely disseminated clonal complex 180 (CC180; n = 140), the unusual CC4909 (n = 42), CC260 (n = 11), and a few singletons (n = 3). CC260 was less frequent among pneumococcal invasive disease isolates than CC180 and CC4909 (P = 0.015). There was a decrease of CC4909 (P < 0.001) following PCV13 introduction (2012 to 2017). The CC4909 isolates were represented mostly by ST1119 (n = 40), seemingly having a restricted geographic origin, with isolates in the PubMLST database having been recovered only in Mexico, the United States, and Germany. A genomic analysis of publicly available genomes showed that ST1119 isolates have less than 32% similarity with ST180 isolates, indicating that these lineages are more separated than revealed by traditional multilocus sequence typing. Considering the suggestions of a lower efficacy of the 13-valent pneumococcal conjugate vaccine against serotype 3, the different dynamics of the two major serotype 3 lineages in Mexico following the introduction of PCV13 should be closely monitored.

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.