Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae.

Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways essential under fluoroquinolone stress and potential novel targets to revitalize use of this antibiotic class, we perform genome-wide CRISPRi-seq screens and generate antibiotic-gene essentiality signatures. Expectedly, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, including recA, recJ, recF, recO, rexAB, and ruvAB. Surprisingly, specific downregulation of the gene encoding the histidine kinase LiaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that liaS deletion causes LiaR hyperphosphorylation and upregulation of the LiaR regulon. We use RNA-seq to refine the LiaR regulon, highlighting the role of heat-shock response and pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin, restoring sensitivity in fluoroquinolone-resistant strains in vitro. Furthermore, bacitracin/levofloxacin combination therapy is effective in vivo and improved treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. These findings offer a starting point for identification and validation of potent combination therapies to treat antibiotic-resistant pneumococcal infections

Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid

The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus. Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT. The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureus sgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly

Multidrug Intrinsic Resistance Factors inStaphylococcus aureusIdentified by Profiling Fitness within High-Diversity Transposon Libraries

Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The MIC of an antibiotic against S. aureus, as well as other microbes, is determined by the affinity of the antibiotic for its target in addition to a complex interplay of many other cellular factors. Identifying nontarget factors impacting resistance to multiple antibiotics could inform the design of new compounds and lead to more-effective antimicrobial strategies. We examined large collections of transposon insertion mutants in S. aureus using transposon sequencing (Tn-Seq) to detect transposon mutants with reduced fitness in the presence of six clinically important antibiotics-ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin, and vancomycin. This approach allowed us to assess the relative fitness of many mutants simultaneously within these libraries. We identified pathways/genes previously known to be involved in resistance to individual antibiotics, including graRS and vraFG (graRS/vraFG), mprF, and fmtA, validating the approach, and found several to be important across multiple classes of antibiotics. We also identified two new, previously uncharacterized genes, SAOUHSC_01025 and SAOUHSC_01050, encoding polytopic membrane proteins, as important in limiting the effectiveness of multiple antibiotics. Machine learning identified similarities in the fitness profiles of graXRS/vraFG, SAOUHSC_01025, and SAOUHSC_01050 mutants upon antibiotic treatment, connecting these genes of unknown function to modulation of crucial cell envelope properties. Therapeutic strategies that combine a known antibiotic with a compound that targets these or other intrinsic resistance factors may be of value for enhancing the activity of existing antibiotics for treating otherwise-resistant S. aureus strains

Exploración de características genéticas asociadas al desarrollo del fenotipo de resistencia intermedia heterogénea a vancomicina (hVISA), en aislamientos de Staphylococcus aureus resistente a meticilina (MRSA) causantes de bacteriemia

Vancomycin is the main therapeutic option to treat severe methicillin-resistant Staphylococcus aureus (MRSA) infections; therefore, the emergence of isolates with decreased susceptibility to this antibiotic is a concern. Isolates with heterogeneous intermediate resistance to vancomycin (hVISA) are a clinical and epidemiological challenge, as they are associated with therapeutic and long hospital stays, it is not detectable by standard methodologies, and the genetic mechanism of resistance is not completely understood. However, numerous genes and mutations have been related with this phenotype. For this reason, the aim of this study was to identify genetic characteristics associated with the development of the hVISA phenotype, in MRSA isolates causing bacteremia in 9 Latin American countries. From 538 MRSA isolates, 30 exhibited a positive result for the hVISA phenotype by E-test based methods, however, from these, only 3 were confirmed by population analysis profile/area under the curve (PAP/AUC). Whole genome sequencing was performed to the 30 isolates and amino acid substitutions were identified in 46 proteins associated with intermediate resistance phenotypes (VISA/hVISA) in previous studies. In total, 98 substitutions were found, most related to cell wall biogenesis, DNA/RNA processing, membrane biosynthesis and regulatory systems, from which the most frequent were in VraT (E156G), WalK (L14I), and Atl (Y38H). In addition, changes were also explored in subpopulations of hVISA isolates that grew at concentrations ≥3μg/mL of vancomycin and maintained the hVISA phenotype (no VISA subpopulations). Consistently, no amino acid substitutions were detected other than those found in the isolates from which they were obtained, except for the Y121D change in transport protein PotD. Likewise, a low number of SNPs was detected in the subpopulations when compared with the genomes of the hVISA isolates from which they belonged. In this study, the genetic characterization of hVISA isolates causing bacteremia in patients in South America was performed; previously reported and unreported changes associated with alterations in functions of regulatory systems, cell wall biogenesis, DNA/RNA processing, and membrane biosynthesis were detected, potentially explaining the phenotypic characteristics of hVISA. On another hand, the analysis of hVISA subpopulations showed that the changes detected were consistent with the genomes of the hVISA isolates, suggesting that the mechanisms for the development and regulation of heterogeneous resistance are additional to those reported in the VISA phenotype.Vancomicina es la principal opción terapéutica para tratar infecciones severas por Staphylococcus aureus resistente a meticilina (MRSA); por lo tanto, la emergencia de aislamientos con susceptibilidad disminuida a este antibiótico es preocupante. Los aislamientos con resistencia intermedia heterogénea a vancomicina (hVISA) son un reto clínico y epidemiológico, pues están asociados con fallas terapéuticas y largas estancias hospitalarias, no es detectable por metodologías estándar, y el mecanismo genético de resistencia no está completamente elucidado. No obstante, numerosos genes y mutaciones han sido relacionados con este fenotipo. Por esta razón, el objetivo de este estudio fue identificar características genéticas asociadas al fenotipo hVISA, en aislamientos de MRSA causantes de bacteriemia en 9 países latinoamericanos. A partir de 538 aislamientos MRSA, 30 presentaron un resultado positivo para el fenotipo hVISA por métodos basados en E-test, sin embargo, de estos, solo 3 fueron confirmados mediante perfil de análisis poblacional/área bajo la curva (PAP/AUC). A los 30 aislamientos se les realizó secuenciación de genoma completo, con el fin de identificar sustituciones de aminoácidos en 46 proteínas que han sido asociadas a los fenotipos de resistencia intermedia (VISA/hVISA) en estudios previos. En total se encontraron 98 sustituciones, la mayoría relacionadas a biogénesis de pared celular, procesamiento de DNA/RNA, biosíntesis de membrana y sistemas reguladores, de las cuales, las mas comunes se presentaron en VraT (E156G), WalK (L14I) y Atl (Y38H). Adicionalmente, se exploraron cambios en las subpoblaciones de los aislamientos hVISA que crecieron a concentraciones ≥3μg/mL de vancomicina y mantuvieron el fenotipo hVISA (no hubo selección de colonias VISA). De forma consistente, no se detectaron sustituciones de aminoácidos diferentes a las de los aislamientos de donde se obtuvieron, excepto por el cambio Y121D en la proteína de transporte PotD. Así mismo, se detectó un número bajo de SNPs en las subpoblaciones cuando se compararon con los genomas de los aislamientos hVISA de donde provenían. En este estudio se realizó la caracterización genética de aislamientos hVISA causantes de bacteriemia en pacientes de Sur América; se detectaron cambios previamente reportados y cambios no reportados asociados a alteraciones en funciones de sistemas reguladores, biogénesis de pared celular, procesamiento de DNA/RNA, y biosíntesis de membrana, que potencialmente explican las características fenotípicas de hVISA. Por otro lado, el análisis de subpoblaciones de hVISA demostró que los cambios detectados fueron consistentes con los genomas de los aislamientos hVISA, lo cual sugiere que los mecanismos para el desarrollo y regulación de la resistencia heterogénea son adicionales a aquellos reportados en el fenotipo VISA.Magíster en Ciencias - Microbiología. Línea de Investigación: Resistencia antimicrobiana en enterococos y estafilococosMaestrí

Modelling the anti-methicillin-resistant staphylococcus aureus (MRSA) activity of cannabinoids: a QSAR and Docking study

Twenty-four cannabinoids active against MRSA SA1199B and XU212 were optimized at WB97XD/6-31G(d,p), and several molecular descriptors were obtained. Using a multiple linear regression method, several mathematical models with statistical significance were obtained. The robustness of the models was validated, employing the leave-one-out cross-validation and Y-scrambling methods. The entire data set was docked against penicillin-binding protein, iso-tyrosyl tRNA synthetase, and DNA gyrase. The most active cannabinoids had high affinity to penicillin-binding protein (PBP), whereas the least active compounds had low affinities for all of the targets. Among the cannabinoid compounds, Cannabinoid 2 was highlighted due to its suitable combination of both antimicrobial activity and higher scoring values against the selected target; therefore, its docking performance was compared to that of oxacillin, a commercial PBP inhibitor. The 2D figures reveal that both compounds hit the protein in the active site with a similar type of molecular interaction, where the hydroxyl groups in the aromatic ring of cannabinoids play a pivotal role in the biological activity. These results provide some evidence that the anti-Staphylococcus aureus activity of these cannabinoids may be related to the inhibition of the PBP protein; besides, the robustness of the models along with the docking and Quantitative Structure–Activity Relationship (QSAR) results allow the proposal of three new compounds; the predicted activity combined with the scoring values against PBP should encourage future synthesis and experimental testing

Resistência total à vancomicina em Staphylococcus aureus e Staphylococcus aureus tipo-VanA : uma revisão de 2016

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2017Este estudo pretende rever a literatura existente relativamente a VRSA (segundo a classificação do CLSI) e Staphylococcus aureus tipo-VanA em todo o Mundo. Embora ambos os termos tenham sobreposição significativa, existem diferenças importantes. Os artigos relevantes foram pesquisados nos motores de busca Google Académico e PubMed, tendo sido excluídos os artigos originais não indexados na Web of Science e os estudos negativos para o isolamento de estirpes VRSA. Após a descrição do primeiro caso, em 2002 nos EUA, seguiram-se vários outros casos, principalmente em países asiáticos, mas a maioria do conhecimento sobre estas estirpes provém da caracterização minuciosa das 16 estirpes americanas confirmadas. Apesar de tudo VRSA parece ser ainda um organismo extremamente raro e o potencial para vir a tornar-se um problema comum é desconhecido. Estas estirpes surgem quando há transferência do operão vanA de uma estirpe VRE para uma estirpe receptora de Staphylococcus aureus, que passa então a produzir precursores de peptidoglicano modificados aos quais a vancomicina se liga com uma afinidade muito baixa. A sua emergência parece exigir condições cuja conjugação é extremamente rara e, mesmo quando estas estirpes surgem e causam infecção, geralmente tem havido várias opções para tratar tais infecções com sucesso. O diagnóstico laboratorial destas estirpes é relativamente simples, mas pode ser dificultado pela utilização de métodos considerados inválidos.The present study is aimed at reviewing the existing literature on VRSA (after the CLSI classification) and VanA-type Staphylococcus aureus around the World. Even though both terms show significant overlap, there are important differences. The relevant papers were retrieved from the Google Scholar and PubMed search engines; those not indexed on Web of Science, as well as those reporting none VRSA strain isolated, have been excluded. Since the first case report, which happened in 2002 in the USA, several other cases have been published, most of them in Asian countries, but most of the current knowledge on these strains is derived from the careful description of the 16 confirmed American isolates. Nevertheless, VRSA still seems to be an extremely rare organism, and the potential for it to become a common problem is unknown. These strains emerge when a vanA operon from a VRE strain is transferred to a receptor Staphylococcus aureus strain, which begins producing modified peptidoglycan precursors with a dramatically lower binding affinity to vancomycin. Such an emergence seems to demand a certain set of conditions, the combination of which is extremely rare. Even when these strains emerge and cause infection, in general there have been several options left to treat such infections with success. Laboratory diagnosis is relatively straightforward, but may be hindered by the use of non-validated methods

The Chemistry of Nitrogenous Food Stuffs

This 18 page thesis contains analyses of various nitrogenous foods

The unusual mode of action of the polyketide glycoside antibiotic cervimycin C

Cervimycins A–D are bis-glycosylated polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated Staphylococcus aureus (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concen trations. Indeed, artificial downregulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell