Nonoptimal DNA topoisomerases allow maintenance of supercoiling levels and improve fitness of Streptococcus pneumoniae

Fluoroquinolones, which target gyrase and topoisomerase IV, are used for treating Streptococcus pneumoniae infections. Fluoroquinolone resistance in this bacterium can arise via point mutation or interspecific recombination with genetically related streptococci. Our previous study on the fitness cost of resistance mutations and recombinant topoisomerases identified GyrAE85K as a high-cost change. However, this cost was compensated for by the presence of a recombinant topoisomerase IV (parC and parE recombinant genes) in strain T14. In this study, we purified wild-type and mutant topoisomerases and compared their enzymatic activities. In strain T14, both gyrase carrying GyrAE85K and recombinant topoisomerase IV showed lower activities (from 2.0- to 3.7-fold) than the wild-type enzymes. These variations of in vitro activity corresponded to changes of in vivo supercoiling levels that were analyzed by two-dimensional electrophoresis of an internal plasmid. Strains carrying GyrAE85K and nonrecombinant topoisomerases had lower (11.1% to 14.3%) supercoiling density (σ) values than the wild type. Those carrying GyrAE85K and recombinant topoisomerases showed either partial or total supercoiling level restoration, with σ values being 7.9% (recombinant ParC) and 1.6% (recombinant ParC and recombinant ParE) lower than those for the wild type. These data suggested that changes acquired by interspecific recombination might be selected because they reduce the fitness cost associated with fluoroquinolone resistance mutations. An increase in the incidence of fluoroquinolone resistance, even in the absence of further antibiotic exposure, is envisaged.This study was supported by grants BIO2008-02154 from Plan Nacional de I+D+I of the Ministerio de Ciencia e Innovación and COMBACT-S-BIO-0260/2006 from the Comunidad de Madrid. Ciber Enfermedades Respiratorias is an initiative from the Instituto de Salud Carlos III. A. G. de la Campa is an Investigador Científico from the CSI

Fluoroquinolone resistance in penicillin-resistant streptococcus pneumoniae clones, Spain

Among 2,882 Streptococcus pneumoniae sent to the Spanish Reference Laboratory during 2002, 75 (2.6%) were ciprofloxacin-resistant. Resistance was associated with older patients (3.9% in adults and 7.2% in patients > or =65 years of age), with isolation from noninvasive sites (4.3% vs. 1.0%), and with penicillin and macrolide resistance. Among 14 low-level resistant (MIC 4-8 microg/mL) strains, 1 had a fluoroquinolone efflux phenotype, and 13 showed single ParC changes. The 61 high-level ciprofloxacin-resistant (MIC > or =16 microg/mL) strains showed either two or three changes at ParC, ParE, and GyrA. Resistance was acquired either by point mutation (70 strains) or by recombination with viridans streptococci (4 strains) at the topoisomerase II genes. Although 36 pulsed-field gel electrophoresis patterns were observed, 5 international multiresistant clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5 and Sweden15A-25) accounted for 35 (46.7%) of the ciprofloxacin-resistant strains. Continuous surveillance is needed to prevent the dissemination of these clones

Changes in Fluoroquinolone-Resistant Streptococcus pneumonia after 7-Valent Conjugate Vaccination, Spain

Four new genotypes appeared in 2006 after childhood vaccination was begun

Human IgMhiCD300a+ B cells are circulating marginal zone memory B cells that respond to pneumococcal polysaccharides and their frequency is decreased in people living with HIV

CD300a is differentially expressed among B cell subsets, although its expression on IgM+ B cells is not well known. We have identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). Results showed that IgMhiCD300a+ B cells were CD10-CD27+CD25+IgDloCD21hiCD23-CD38loCD1chi, suggesting that they are circulating marginal zone IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a- counterpart. Moreover, the shorter CDR3 AA length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire, indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a TLR9 agonist than IgM+CD300a- cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated to the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.This work was supported by the following grants to F.B.: Agencia Estatal de Investigación (PID2019-109583RB-I00/AEI/10.13039/501100011033) and Gilead Fellowship Program (GLD15/00303). A grant to B.d.A.: Agencia Estatal de Investigación-ISCIII (PI22CIII/00030). J.V. and I.T. are recipients of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2018_2_0242 and PRE_2021_2_0215). A.O and I.T. are recipients of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB18/002 and FJGB19/002). L.A. is an Ikerbasque Research Fellow, Ikerbasque, Basque Foundation for Science. F.B. is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science. We want to particularly acknowledge the patients in this study for their participation, to the HIV BioBank and the collaborating centres for the generous gifts of the clinical samples used in this study. The HIV BioBank is supported by Instituto de Salud Carlos III (PT20/00138) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN (CB22/01/00041). CoRIS cohort is supported by CIBER—Consorcio Centro de Investigación Biomédica en Red—(CB21/13/00091), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. This study would not have been possible without the collaboration of all patients, medical and nursing staff and data managers who have taken part in the Project. CoRIS cohort is supported by CIBER—Consorcio Centro de Investigación Biomédica en Red—(CB21/13/00091), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU

Fluoroquinolone Resistance in Penicillin-resistant Streptococcus pneumoniae Clones, Spain

Of 75 clones isolated, 1 had ciprofloxacin efflux, and 74 had mutations at the DNA topoisomerase gene

Human IgMhiCD300a+ B Cells Are Circulating Marginal Zone Memory B Cells That Respond to Pneumococcal Polysaccharides and Their Frequency Is Decreased in People Living with HIV

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).CD300a is differentially expressed among B cell subsets, although its expression in immunoglobulin (Ig)M+ B cells is not well known. We identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). The results showed that IgMhiCD300a+ B cells were CD10−CD27+CD25+IgDloCD21hiCD23−CD38loCD1chi, suggesting that they are circulating marginal zone (MZ) IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a− counterpart. Moreover, the shorter complementarity-determining region 3 (CDR3) amino acid (AA) length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a toll-like receptor 9 (TLR9) agonist than IgM+CD300a− cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated with the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.This work was supported by the following grants to F.B.: Agencia Estatal de Investigación (PID2019-109583RB-I00/AEI/10.13039/501100011033) and Gilead Fellowship Program (GLD15/00303). A grant to B.d.A.: Agencia Estatal de Investigación-ISCIII (PI22CIII/00030). J.V. and I.T. are recipients of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2018_2_0242 and PRE_2021_2_0215). A.O and I.T. are recipients of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB18/002 and FJGB19/002). L.A. is an Ikerbasque Research Fellow, Ikerbasque, Basque Foundation for Science. F.B. is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science. We want to particularly acknowledge the patients in this study for their participation, to the HIV BioBank and the collaborating centres for the generous gifts of the clinical samples used in this study. The HIV BioBank is supported by Instituto de Salud Carlos III (PT20/00138) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN (CB22/01/00041). CoRIS cohort is supported by CIBER—Consorcio Centro de Investigación Biomédica en Red—(CB21/13/00091), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. This study would not have been possible without the collaboration of all patients, medical and nursing staff and data managers who have taken part in the Project. CoRIS cohort is supported by CIBER—Consorcio Centro de Investigación Biomédica en Red—(CB21/13/00091), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU.Peer reviewe

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Impact of the Nutritional Supplementation with DHA on Cystic Fibrosis Microbiota

Curs 2017-2018Cystic fibrosis (CF) is a genetic disease characterized by the formation of thick secretions in the gut and airways, among other anatomical locations, which leads to an altered microbiota. Understanding the significance of bacterial species that colonize and persist in CF patients requires a detailed examination of bacterial community structures. In addition, deficiency in fatty acids, such as docosahexaenoic acid (DHA), is a common co-morbidity in CF patients who are malnourished. Thus, the restoration of DHA levels may have influence in the bacterial composition of CF microbiota. The aims of this study were, first, to analyze the gut and airway microbiota of CF patients using next generation sequencing (NGS) tools, secondly, to determine the DHA supplementation impact in the microbiota of both ecosystems, and finally, to associate particular bacterial profiles to different clinical variables as nutritional status and lung function. Forty-eight CF patients were randomized into two groups and receiving either DHA supplemented diet or placebo for 6 months. Fecal or sputum samples were collected before and after finishing the DHA or placebo supplementation (two samples per each patient). We performed V3-V4 16S rDNA NGS analysis to characterize the gut and lung microbiota at basal status in these patients. The results showed that gut and airways CF microbiota was aberrant, with significant inter-individual variability in their community structure. The core CF-gut microbiota consisted of Lachnospiraceae family and Streptococcus and Blautia genera. Airways in children were dominated by Streptococcus and Haemophilus whereas in adults Streptococcus was the only genera over 10% abundance. Lachnospiraceae abundance in CF-gut and children airways was linked to the effect of DHA supplementation in the bacterial composition. Only considering the top-28 most abundant genera in adult CF-airways, we were able to detect an association between strict anaerobes, such as Prevotella, Veillonella and Lachnospiraceae, and CF patients with lower lung function.Director/a: Rosa del Campo Moren

Impact of the Nutritional Supplementation with DHA on Cystic Fibrosis Microbiota

Cystic fibrosis (CF) is a genetic disease characterized by the formation of thick secretions in the gut and airways, among other anatomical locations, which leads to an altered microbiota. Understanding the significance of bacterial species that colonize and persist in CF patients requires a detailed examination of bacterial community structures. In addition, deficiency in fatty acids, such as docosahexaenoic acid (DHA), is a common co-morbidity in CF patients who are malnourished. Thus, the restoration of DHA levels may have influence in the bacterial composition of CF microbiota. The aims of this study were, first, to analyze the gut and airway microbiota of CF patients using next generation sequencing (NGS) tools, secondly, to determine the DHA supplementation impact in the microbiota of both ecosystems, and finally, to associate particular bacterial profiles to different clinical variables as nutritional status and lung function. Forty-eight CF patients were randomized into two groups and receiving either DHA supplemented diet or placebo for 6 months. Fecal or sputum samples were collected before and after finishing the DHA or placebo supplementation (two samples per each patient). We performed V3-V4 16S rDNA NGS analysis to characterize the gut and lung microbiota at basal status in these patients. The results showed that gut and airways CF microbiota was aberrant, with significant inter-individual variability in their community structure. The core CF-gut microbiota consisted of Lachnospiraceae family and Streptococcus and Blautia genera. Airways in children were dominated by Streptococcus and Haemophilus whereas in adults Streptococcus was the only genera over 10% abundance. Lachnospiraceae abundance in CF-gut and children airways was linked to the effect of DHA supplementation in the bacterial composition. Only considering the top-28 most abundant genera in adult CF-airways, we were able to detect an association between strict anaerobes, such as Prevotella, Veillonella and Lachnospiraceae, and CF patients with lower lung function

Fitness of Streptococcus pneumoniae Fluoroquinolone-Resistant Strains with Topoisomerase IV Recombinant Genes▿

The low prevalence of ciprofloxacin-resistant (Cpr) Streptococcus pneumoniae isolates carrying recombinant topoisomerase IV genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal parE-parC intergenic regions. A study of the transcription of these genes and of the fitness cost for 24 isogenic Cpr strains was performed. Six first-level transformants were obtained either with PCR products containing the parC quinolone resistance-determining regions (QRDRs) of S. pneumoniae Cpr mutants with point mutations or with a PCR product that includes parE-QRDR-ant-parC-QRDR from a Cpr Streptococcus mitis isolate. The latter yielded two strains, T6 and T11, carrying parC-QRDR and parE-QRDR-ant-parC-QRDR, respectively. These first-level transformants were used as recipients in further transformations with the gyrA-QRDR PCR products to obtain 18 second-level transformants. In addition, strain Tr7 (which contains the GyrA E85K change) was used. Reverse transcription-PCR experiments showed that parE and parC were cotranscribed in R6, T6, and T11; and a single promoter located upstream of parE was identified in R6 by primer extension. The fitness of the transformants was estimated by pairwise competition with R6 in both one-cycle and two-cycle experiments. In the one-cycle experiments, most strains carrying the GyrA E85K change showed a fitness cost; the exception was recombinant T14. In the two-cycle experiments, a fitness cost was observed in most first-level transformants carrying the ParC changes S79F, S79Y, and D83Y and the GyrA E85K change; the exceptions were recombinants T6 and T11. The results suggest that there is no impediment due to a fitness cost for the spread of recombinant Cpr S. pneumoniae isolates, since some recombinants (T6, T11, and T14) exhibited an ability to compensate for the cost