Factors Contributing to the Evolution of mecA-Mediated β-lactam Resistance in Staphylococci: Update and New Insights From Whole Genome Sequencing (WGS)

The understanding of the mechanisms of antibiotic resistance development are fundamental to alert and preview beforehand, the large scale dissemination of resistance to antibiotics, enabling the design of strategies to prevent its spread. The mecA-mediated methicillin resistance conferring resistance to broad-spectrum β-lactams is globally spread in staphylococci including hospitals, farms and community environments, turning ineffective the most widely used and efficient class of antibiotics to treat staphylococcal infections. The use of whole genome sequencing (WGS) technologies at a bacterial population level has provided a considerable progress in the identification of key steps that led to mecA-mediated β-lactam resistance development and dissemination. Data obtained from multiple studies indicated that mecA developed from a harmless core gene (mecA1) encoding the penicillin-binding protein D (PbpD) from staphylococcal species of animal origin (S. sciuri group) due to extensive β-lactams use in human created environments. Emergence of the resistance determinant involved distortion of PbpD active site, increase in mecA1 expression, addition of regulators (mecR1, mecI) and integration into a mobile genetic element (SCCmec). SCCmec was then transferred into species of coagulase-negative staphylococci (CoNS) that are able to colonize both animals and humans and subsequently transferred to S. aureus of human origin. Adaptation of S. aureus to the exogenously acquired SCCmec involved, deletion and mutation of genes implicated in general metabolism (auxiliary genes) and general stress response and the adjustment of metabolic networks, what was accompanied by an increase in β-lactams minimal inhibitory concentration and the transition from a heterogeneous to homogeneous resistance profile. Nowadays, methicillin-resistant S. aureus (MRSA) carrying SCCmec constitutes one of the most important worldwide pandemics. The stages of development of mecA-mediated β-lactam resistance described here may serve as a model for previewing and preventing the emergence of resistance to other classes of antibiotics

A compreensão de textos literários : estratégias de intervenção na sala de aula

Esta dissertação relaciona-se com a didática da leitura, dada a importância capital que assume na vida dos indivíduos na sociedade atual. Ler é compreender e requer do leitor um papel ativo na construção do significado do texto. Os programas da disciplina de Português refletem estas preocupações dada a ênfase no domínio da leitura e no desenvolvimento de estratégias de leitura no Ensino Básico. Com este estudo pretende-se: 1) caraterizar a compreensão na leitura dos alunos em textos literários, ao iniciarem o 5.º ano de escolaridade; 2) melhorar a compreensão na leitura dos alunos do 5.º ano de escolaridade, em textos literários, através do ensino explícito de estratégias de leitura. Numa primeira fase, avaliamos o nível de desempenho dos alunos em tarefas de compreensão na leitura, através da aplicação de uma prova de leitura aos alunos que iniciam o 5.º ano de escolaridade. Numa segunda fase, implementamos uma intervenção didática em duas turmas do 5.º ano, destinada ao ensino explícito de estratégias de leitura visando a melhoria dos níveis de compreensão. Na terceira fase, avaliamos a modificação de comportamentos dos alunos relativamente ao desempenho em atividades de compreensão na leitura. Utilizamos a metodologia de investigação-ação. Na avaliação quantitativa dos efeitos da intervenção, consideramos os resultados dos alunos nas variáveis: compreensão literal, inferencial, crítica e reorganização. No tratamento estatístico, recorremos a testes não paramétricos. Os resultados mostram que após a intervenção se verificam ganhos em todos os domínios, ainda que no literal não sejam estatisticamente significativos. Estes resultados permitem-nos concluir que o ensino sistemático de estratégias de leitura desenvolve a compreensão nos domínios analisados e constitui uma alternativa metodológica para a educação literária.This Master Thesis deals with issues related to teaching to read due to the fact that reading is a very important competence in modern societies. Reading means to understand and it requires the reader an active role in the construction of meaning. The Portuguese National Curriculum, especially in Programas de Português do Ensino Básico reflects these concerns and it emphasizes the importance of reading competences as well as the development of reading strategies in Basic Education. The aims of this research are: 1) characterize the reading comprehension abilities of students in literary texts, when they start 5 th grade; 2) improve their reading comprehension abilities through explicit teaching of reading strategies. On a first stage, it was diagnosed the level of our students‟ performance in reading comprehension tasks by applying a test to all the students who started 5th grade. On a second stage, we implemented a pedagogical-didactic intervention in two classes of 5th grade students based on the explicit teaching of reading strategies aiming to improve their level of reading skills. On the third and last stage, we evaluated the performance of students in reading comprehension tasks in order to assess the efficiency of our intervention. We chose action-research project where reflection is formed as a key point for change in building environments aimed at promoting literacy. In the quantitative analysis of the intervention effects, four variables were considered: literal comprehension, inferential comprehension, critical and reorganization. In the statistic analysis Wilcoxon tests were used. This research project shows encouraging results with substantial gains in all variables except in literal comprehension because the results are not statistically significant. These results suggest that a pedagogical practice which values a structured and systematic didactics on behalf of teaching reading strategies stimulates and develops the competences we have studied and it constitutes an alternative methodology for literary education

Molecular Epidemiology of Methicillin-Resistant Staphylococcus hominis (MRSHo): Low Clonality and Reservoirs of SCCmec Structural Elements

BACKGROUND: Methicillin resistant Staphylococcus hominis (MRSHo) are important human pathogens in immunocompromised patients. However, little is known regarding its population structure and staphylococcal chromosomal cassette mec (SCCmec) content. METHODOLOGY/PRINCIPAL FINDINGS: To assess the population structure and the SCCmec content of S. hominis, 34 MRSHo and 11 methicillin-susceptible S. hominis (MSSHo) from neutropenic patients collected over a 3-year period were studied. The genetic backgrounds of S. hominis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and SCCmec types were determined by PCR. Cassette chromosome recombinases (ccr) were characterized by PCR and ccrB sequencing. The 34 S. hominis isolates were classified into as many as 28 types and 32 subtypes (SID = 99.82%); clonal dissemination was occasionally observed. The main SCCmec structures identified were SCCmec type VI (4B) (20%), SCCmec VIII (4A) (15%), and a new SCCmec composed of mec complex A in association with ccrAB1 (38%); 27% of the isolates harbored non-typeable SCCmec. Overall, a high prevalence of mec complex A (73.5%), ccrAB1 (50%) and ccrAB4 (44%) were found. Importantly, ccrB1 and ccrB4 from both MRSHo and MSSHo showed a high nucleotide sequence homology with those found in S. aureus SCCmec I, VI and VIII respectively (>95%). CONCLUSIONS/SIGNIFICANCE: The S. hominis population showed a limited clonality and a low genetic diversity in the allotypes of ccr and classes of mec complex. Moreover, our data suggest that S. hominis might have been a privileged source of mec complex A, ccrB1 and ccrB4, for the assembly of primordial SCCmec types

A aplicação da pedagogia Waldorf em alunos com síndrome de DAMP

O presente estudo na área de educação especial tem como objecto um aluno que em termos clínicos enquadra os défices apresentados no síndrome de DAMP e que, no ano de 2010-2011, frequentou em simultâneo o ensino regular e a pedagogia Waldorf. O aluno manifestou, ao longo do seu percurso escolar, dificuldades de adaptação e de aprendizagem em todas as áreas logo no primeiro ano de escolaridade. No decorrer do 1º Ciclo integrou a Educação Especial e beneficiou de algumas das medidas educativas previstas no Decreto-Lei 3/ 2008. Ao transitar para o 5º Ano, além das medidas que já usufruía, foi-lhe adicionada a frequência de actividades ocupacionais em parceria com a Casa Santa Isabel, visando o desenvolvimento de estratégias de educação específicas da pedagogia Waldorf. Decorrido o ano lectivo, apurámos uma relativa unanimidade no que diz respeito às dificuldades apresentadas inicialmente pelo aluno e às suas melhorias, principalmente ao nível da motricidade fina, integração pessoal/social e atenção/concentração. Os resultados mostram que as actividades oferecidas pela pedagogia Waldorf foram benéficas para o aluno, como forma de superação ainda que ligeira de algumas das dificuldades que apresentava, embora o trabalho tenha acabado por ser inconclusivo, atendendo à integração tardia do aluno nesta pedagogia e ao pouco tempo em que nela esteve inserido.This study regarding special education has as subject matter a student who, in clinical terms, exhibits all characteristics of DAMP syndrome and who throughout the school year of 2010-2011 attended a regular school system as well as the Waldorf education. Throughout his educational path and during his first year the student experienced adjustment and learning difficulties in all subjects. During the first cycle the student was included in a special education program and benefited from all the educational measures established by the decree- law 3/2008. During fifth grade, along with the measures already set, he was also included in hands-on learning activities in a partnership with Casa Santa Isabel, aiming to develop specific educational standards set by Waldorf education. At the end of the school year we were able to reach an unanimous evaluation of the difficulties initially presented by the student as well as of his improvements as to motor skills, personal/social integration and attention/ concentration. The results show that all the activities offered by the Waldorf Education were beneficial for the student and helped to improve, although scarcely, some of his difficulties. The study ended inconclusive due to the late integration of the student in this sort of education, as well as due to the short period of attendance in this activity

Uncovering Beta-Lactam Susceptibility Patterns in Clinical Isolates of Mycobacterium tuberculosis through Whole-Genome Sequencing

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9431576/The increasing threat of drug resistance and a stagnated pipeline of novel therapeutics endanger the eradication of tuberculosis. Beta-lactams constitute promising additions to the current therapeutic arsenal and two carbapenems are included in group C of medicines recommended by the WHO for use in longer multidrug-resistant tuberculosis regimens. However, the determinants underlining diverse Mycobacterium tuberculosis phenotypes to beta-lactams remain largely undefined. To decipher these, we present a proof-of-concept study based on a large-scale beta-lactam susceptibility screening for 172 M. tuberculosis clinical isolates from Portugal, including 72 antimycobacterial drug-resistant strains. MICs were determined for multiple beta-lactams and strains were subjected to whole-genome sequencing to identify core-genome single-nucleotide variant-based profiles. Global and cell wall-targeted approaches were then followed to detect putative drivers of beta-lactam response. We found that drug-resistant strains were more susceptible to beta-lactams, but significant differences were not observed between distinct drug-resistance profiles. Sublineage 4.3.4.2 strains were significantly more susceptible to beta-lactams, while the contrary was observed for Beijing and 4.1.2.1 sublineages. While mutations in beta-lactamase or cell wall biosynthesis genes were uncommon, a rise in beta-lactam MICs was detected in parallel with the accumulation of mutations in peptidoglycan cross-linking or cell division genes. Finally, we exposed that putative beta-lactam resistance markers occurred in genes for which relevant roles in cell wall processes have been ascribed, such as rpfC or pknA. Genetic studies to validate the relevance of the identified mutations for beta-lactam susceptibility and further improvement of the phenotype-genotype associations are needed in the future. IMPORTANCE Associations between differential M. tuberculosis beta-lactam phenotypes and preexisting antimycobacterial drug resistance, strain sublineage, or specific mutational patterns were established. Importantly, we reveal that highly drug-resistant isolates of sublineage 4.3.4.2 have an increased susceptibility to beta-lactams compared with other strains. Thus, directing beta-lactams to treat infections by specific M. tuberculosis strains and refraining its use from others emerges as a potentially important strategy to avoid resistance development. Individual mutations in blaC or genes encoding canonical beta-lactam targets, such as peptidoglycan transpeptidases, are infrequent and do not greatly impact the MICs of potent carbapenem plus clavulanic acid combinations. An improved understanding of the global effect of cumulative mutations in relevant gene sets for peptidoglycan and cell division processes on beta-lactam susceptibility is also provided.This work was supported by Fundação para a Ciência e Tecnologia (PTDC/BIA-MIC/31233/2017 to M.J.C, SFRH/BD/136853/2018 to F.O. and 2021.05446.BD to C.S.) and by the European Society of Clinical Microbiology and Infectious Diseases (Research Grant 2018 to M.J.C.).info:eu-repo/semantics/publishedVersio

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus

This work was financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. This work was also supported by FCT through grants PTDC/BIA-MIC/31645/2017 (awarded to R.G.S.) and PTDC/CVT-CVT/29510/2017 (awarded to M.M.); Projects LISBOA-01-0145-FEDER007660 (Microbiologia Molecular, Estrutural e Celular) and UID/Multi/04378/2019 funded by FEDER funds through COMPETE2020—Programa Operacional de Competitividade e Internacionalização (POCI); and by ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI—“Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa2020” and by national funds through FCT.In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to β-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosomepublishersversionpublishe

WGS-based dual strategy for the identification of key targets to enhance beta-lactam activity in mycobacterium tuberculosis

info:eu-repo/semantics/publishedVersio

Farnesol antimicrobial role as biofilm cell detachment inducer in S. epidermidis biofilms

Objetives: Farnesol is a naturally-occurring sesquiterpene that was originally isolated from essential oils found in many plants has been described to have antimicrobial potential against several bacteria, including S. epidermidis. However, farnesol mechanism of action is not yet fully understood and some contradictory findings have been reported. We recently described that while farnesol was not efficient at killing biofilm bacteria, a strong reduction on biofilm biomass was detected, and we hypothesize that farnesol could be inducing biofilm detachment. Here, we address this hypothesis. Methods: To test our hypothesis we used 36 representative clinical strains of S. epidermidis from different parts of the world and characterized them in terms of genetic variability, biofilm formation and on the effect of farnesol on biofilm physiology and gene expression. Results: Farnesol had no bactericidal effect on stationary phase populations equal or above 108 CFU/mL. In exponential phase planktonic bacteria, farnesol showed a bacteriostatic effect after cell density reached 108 CFU/mL. In any of the growth phases studied, farnesol was effective in killing above 90% of bacteria in 4 h when cell density was 107 CFU/mL or below. Confocal microscopy and flow citometry analysis confirmed that in biofilms bacteria were not killed by farnesol but nevertheless cell wall integrity was affected. Gene expression studies revealed differential responses to farnesol, depending on the bacterial strain tested. Farnesol cell detachement from biofilms was also straindependent. Conclusions: We found that while farnesol cannot kill high density bacterial communities, such as biofilms, it was nevertheless able to induce biofilm detachment in 50% of the strains that formed biofilm

Distinct phenotypic and genomic signatures underlie contrasting pathogenic potential of Staphylococcus epidermidis clonal lineages

Copyright © 2019 Espadinha, Sobral, Mendes, Méric, Sheppard, Carriço, de Lencastre and Miragaia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.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.DE and CM were supported by Ph.D. grants PD/BD/52206/2013 and SFRH/BD/129483/2017, respectively, from the Fundação para a Ciência e Tecnologia (FCT). This work was partially supported by project PTDC/FIS-NAN/0117/2014, project PTDC/CVT-CVT/29510/2017, project PTDC/BIA-MIC/31645/2017, and project EXPOSE - SAICT-POL/23222/2016 from FCT; Projects LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) and UID/Multi/04378/2019) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI); by ONEIDA project (LISBOA-01-0145-FEDER- 016417) co-funded by FEEI - “Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa2020” and by national funds through FCT; Operacional Competitividade e Internacionalização, Programa Operacional Regional de Lisboa (FEDER) and Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

Genetic Diversity of Arginine Catabolic Mobile Element in Staphylococcus epidermidis

BACKGROUND:The methicillin-resistant Staphylococcus aureus clone USA300 contains a novel mobile genetic element, arginine catabolic mobile element (ACME), that contributes to its enhanced capacity to grow and survive within the host. Although ACME appears to have been transferred into USA300 from S. epidermidis, the genetic diversity of ACME in the latter species remains poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS:To assess the prevalence and genetic diversity of ACME, 127 geographically diverse S. epidermidis isolates representing 86 different multilocus sequence types (STs) were characterized. ACME was found in 51% (65/127) of S. epidermidis isolates. The vast majority (57/65) of ACME-containing isolates belonged to the predominant S. epidermidis clonal complex CC2. ACME was often found in association with different allotypes of staphylococcal chromosome cassette mec (SCCmec) which also encodes the recombinase function that facilities mobilization ACME from the S. epidermidis chromosome. Restriction fragment length polymorphism, PCR scanning and DNA sequencing allowed for identification of 39 distinct ACME genetic variants that differ from one another in gene content, thereby revealing a hitherto uncharacterized genetic diversity within ACME. All but one ACME variants were represented by a single S. epidermidis isolate; the singular variant, termed ACME-I.02, was found in 27 isolates, all of which belonged to the CC2 lineage. An evolutionary model constructed based on the eBURST algorithm revealed that ACME-I.02 was acquired at least on 15 different occasions by strains belonging to the CC2 lineage. CONCLUSIONS/SIGNIFICANCE:ACME-I.02 in diverse S. epidermidis isolates were nearly identical in sequence to the prototypical ACME found in USA300 MRSA clone, providing further evidence for the interspecies transfer of ACME from S. epidermidis into USA300