s pneumoniae d39 rpsl k56t  (ATCC)

Journal: Apmis

Article Title: Critical Steps in Shotgun Metagenomics‐Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing

doi: 10.1111/apm.13511

Figure Lengend Snippet: Droplet digital PCR on spike‐in bacterial samples. Mean (of duplicates) droplet digital PCR (ddPCR) gene copies/μL of (a) nuc , (b) lytA and (c) uidA genes, specific for Staphylococcus aureus , Streptococcus pneumoniae and Escherichia coli , respectively, in spiked samples at two different concentrations between 10 3 –10 5 CFU/mL, is shown in green. The ddPCR copies/μL of the human gene, RPP30 , spiked in each sample is shown in black. The RPP30 gene was not detected in S. aureus ‐spiked WB samples. The result from ddPCR gives copies/μL from the extracted DNA.

Article Snippet: The S. aureus , E. coli and S. pneumoniae were cultured overnight on blood agar plates (Columbia Blood Agar Base (Acumedia Neogen Corporation, Lansing, MI, USA) and 6% horse blood) at 36.5°C.

Techniques: Digital PCR

Journal: Apmis

Article Title: Critical Steps in Shotgun Metagenomics‐Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing

doi: 10.1111/apm.13511

Figure Lengend Snippet: Proportion of classified reads from shotgun metagenomics sequencing from the bacterial spiked samples. Whole blood (WB) samples spiked with two concentrations and corresponding retrieved plasma (P) samples were centrifuged at 100 g or 180 g , with (a) S. aureus (10 4 and 10 5 CFU/mL), (b) E. coli (10 3 and 10 4 CFU/mL) and (c) S. pneumoniae (10 4 and 10 5 CFU/mL). The pooled DNA of duplicates for each spiked sample was sequenced. The number of reads classified for each bacterium is written above each bar.

Article Snippet: The S. aureus , E. coli and S. pneumoniae were cultured overnight on blood agar plates (Columbia Blood Agar Base (Acumedia Neogen Corporation, Lansing, MI, USA) and 6% horse blood) at 36.5°C.

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: DNA transformation in S. pneumoniae depends on environment and genetic background. ( A ) The transformation efficiency of S. pneumoniae D39 grown as biofilm or planktonic cells was monitored in C + Y and CDM media following exposure to a D39-FolA I100L lysate or D39 WT lysate. The acquisition of the mutation leading to the FolA I100L substitution confers TMP resistance to the transformants. For the C + Y medium, we compared transformation efficiency in the presence (+) and absence (−) of exogenous CSP-1. ( B – E ) The natural transformation efficiency of S. pneumoniae D39, TIGR4, ATCC 49619 and CCRI-14647 grown as biofilms was monitored in media C + Y, BHI, CDM or Columbia following exposure to a D39-RpsL K56T lysate. Transformation efficiency is reported as the number of antibiotic-resistant transformants over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared using the Kruskal–Wallis test followed by Dunn’s test. Significance is indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For biofilm natural transformation, S. pneumoniae D39, TIGR4, CCRI-14647 or ATCC-49647 biofilms were grown in CDM, C + Y, BHI or Columbia medium for 24 h at 35°C under a 5% CO 2 atmosphere, after which the culture supernatant was carefully removed and changed for a 1:25 mixture of either S. pneumoniae D39-RpsL K56T or D39-FolA I100L lysates diluted in fresh medium.

Techniques: Transformation Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Selection for increased transformation efficacy in S. pneumoniae D39 using Mut-Seq. ( A ) Overview of the Mut-Seq screen. S. pneumoniae D39 mutagenized with EMS and grown as biofilm in C + Y medium in 24-well plates were exposed to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant, which confers resistance to TMP. Transformants were selected on TMP-containing agar plates and grown again as biofilms in C + Y medium in 24-well plates. No TMP-resistant transformants were obtained from nonmutagenized control cells processed in parallel. To ascertain that the TMP-resistant phenotype results from increased transformation efficiency and not directly from mutagenesis, 110 randomly picked TMP-resistant transformants were subjected to a second round of transformation using a cell lysate derived from S. pneumoniae D39 coding for the RpsL K56T variant, which confers resistance to STR. This led to 39 transformants resistant to both TMP and STR, whose genomes were sequenced. Of these, 19 had acquired the expected FolA I100L at the first round of transformation and were as such considered to have genuine increased natural transformation capacities. ( B ) The increased natural transformation efficiency of the 19 S. pneumoniae D39 FolA I100L -positive transformants derived from the Mut-Seq screen was further assessed as biofilm in C + Y following exposure to a D39-RpsL K56T lysate. Transformation efficiency is reported as the number of STR-resistant transformants over the number of viable cells. Error bars represent the standard error calculated from biological replicates ( n = 6). Transformation efficiencies were compared with the one of D39 wild type (WT) using the Kruskal–Wallis test followed by Dunn’s test. ** P < 0.01. ( C ) A selection of six mutations detected by Mut-Seq in at least two independent mutants (see Table ) along with two mutations not expected to increase natural transformation were introduced in the genome of S. pneumoniae D39. The natural transformation efficiency of the transformants was monitored with cells grown as biofilms in C + Y following exposure to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant. Transformation efficiency is reported as the number of TMP-resistant cells over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared with the one of D39 using the Kruskal–Wallis test followed by Dunn’s test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For biofilm natural transformation, S. pneumoniae D39, TIGR4, CCRI-14647 or ATCC-49647 biofilms were grown in CDM, C + Y, BHI or Columbia medium for 24 h at 35°C under a 5% CO 2 atmosphere, after which the culture supernatant was carefully removed and changed for a 1:25 mixture of either S. pneumoniae D39-RpsL K56T or D39-FolA I100L lysates diluted in fresh medium.

Techniques: Selection, Transformation Assay, Derivative Assay, Variant Assay, Control, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Mut-Seq derived mutations in genes detected in at least two S. pneumoniae D39 transformants

Article Snippet: For biofilm natural transformation, S. pneumoniae D39, TIGR4, CCRI-14647 or ATCC-49647 biofilms were grown in CDM, C + Y, BHI or Columbia medium for 24 h at 35°C under a 5% CO 2 atmosphere, after which the culture supernatant was carefully removed and changed for a 1:25 mixture of either S. pneumoniae D39-RpsL K56T or D39-FolA I100L lysates diluted in fresh medium.

Techniques: Derivative Assay

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Mutations in diverse genes intersecting with Mut-Seq and GWAS screens increase natural transformation in S. pneumoniae . Mutations detected in PurD ( A ), PurH ( B ), Cps2O ( C ) and ScrR ( D ) by Mut-Seq (blue) or in the two GWAS screens (tan, GWAS-1; beige, GWAS-2) were introduced in S. pneumoniae D39. The natural transformation efficiency of the transformants was monitored with planktonic cells grown in C + Y media following exposure to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant. Transformation efficiency is reported as the number of TMP-resistant cells over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared with those of D39 using the Kruskal–Wallis test followed by Dunn’s test. Significance is indicated above the whiskers as follows: * P < 0.05; ** P < 0.01; *** P < 0.001. For the GWAS screens, mutations positively associated with MDR are aligned under the “+” sign whereas those either not associated with MDR (GWAS-1) or positively associated with antibiotic susceptibility (GWAS-2) are aligned under the “−” sign. The significance levels of the association with MDR (GWAS-1, + and −; GWAS-2, +) or susceptibility (GWAS-2, −) prior to and after correction for population structure are indicated within parentheses (uncorrected/corrected) below the mutations on the x -axis as follows: * P = 10 −06 –10 −10 ; ** P = 10 −10 –10 −20 ; *** P = 10 −20 –10 −40 . See Table for the complete GWAS-1 and GWAS-2 data. NA on the x -axis indicates that no mutation was found for the screen in the gene.

Article Snippet: For biofilm natural transformation, S. pneumoniae D39, TIGR4, CCRI-14647 or ATCC-49647 biofilms were grown in CDM, C + Y, BHI or Columbia medium for 24 h at 35°C under a 5% CO 2 atmosphere, after which the culture supernatant was carefully removed and changed for a 1:25 mixture of either S. pneumoniae D39-RpsL K56T or D39-FolA I100L lysates diluted in fresh medium.

Techniques: Transformation Assay, Derivative Assay, Variant Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: DNA transformation in S. pneumoniae depends on environment and genetic background. ( A ) The transformation efficiency of S. pneumoniae D39 grown as biofilm or planktonic cells was monitored in C + Y and CDM media following exposure to a D39-FolA I100L lysate or D39 WT lysate. The acquisition of the mutation leading to the FolA I100L substitution confers TMP resistance to the transformants. For the C + Y medium, we compared transformation efficiency in the presence (+) and absence (−) of exogenous CSP-1. ( B – E ) The natural transformation efficiency of S. pneumoniae D39, TIGR4, ATCC 49619 and CCRI-14647 grown as biofilms was monitored in media C + Y, BHI, CDM or Columbia following exposure to a D39-RpsL K56T lysate. Transformation efficiency is reported as the number of antibiotic-resistant transformants over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared using the Kruskal–Wallis test followed by Dunn’s test. Significance is indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To extend this observation, we tested the ability of four strains (D39, TIGR4, ATCC 49619 and CCRI-14647) to transform a RpsL K56T mutation when exposed to a lysate of S. pneumoniae D39 coding for this S12 ribosomal protein variant, which confers resistance to STR, in four media in the absence of exogenous CSP.

Techniques: Transformation Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Selection for increased transformation efficacy in S. pneumoniae D39 using Mut-Seq. ( A ) Overview of the Mut-Seq screen. S. pneumoniae D39 mutagenized with EMS and grown as biofilm in C + Y medium in 24-well plates were exposed to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant, which confers resistance to TMP. Transformants were selected on TMP-containing agar plates and grown again as biofilms in C + Y medium in 24-well plates. No TMP-resistant transformants were obtained from nonmutagenized control cells processed in parallel. To ascertain that the TMP-resistant phenotype results from increased transformation efficiency and not directly from mutagenesis, 110 randomly picked TMP-resistant transformants were subjected to a second round of transformation using a cell lysate derived from S. pneumoniae D39 coding for the RpsL K56T variant, which confers resistance to STR. This led to 39 transformants resistant to both TMP and STR, whose genomes were sequenced. Of these, 19 had acquired the expected FolA I100L at the first round of transformation and were as such considered to have genuine increased natural transformation capacities. ( B ) The increased natural transformation efficiency of the 19 S. pneumoniae D39 FolA I100L -positive transformants derived from the Mut-Seq screen was further assessed as biofilm in C + Y following exposure to a D39-RpsL K56T lysate. Transformation efficiency is reported as the number of STR-resistant transformants over the number of viable cells. Error bars represent the standard error calculated from biological replicates ( n = 6). Transformation efficiencies were compared with the one of D39 wild type (WT) using the Kruskal–Wallis test followed by Dunn’s test. ** P < 0.01. ( C ) A selection of six mutations detected by Mut-Seq in at least two independent mutants (see Table ) along with two mutations not expected to increase natural transformation were introduced in the genome of S. pneumoniae D39. The natural transformation efficiency of the transformants was monitored with cells grown as biofilms in C + Y following exposure to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant. Transformation efficiency is reported as the number of TMP-resistant cells over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared with the one of D39 using the Kruskal–Wallis test followed by Dunn’s test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To extend this observation, we tested the ability of four strains (D39, TIGR4, ATCC 49619 and CCRI-14647) to transform a RpsL K56T mutation when exposed to a lysate of S. pneumoniae D39 coding for this S12 ribosomal protein variant, which confers resistance to STR, in four media in the absence of exogenous CSP.

Techniques: Selection, Transformation Assay, Derivative Assay, Variant Assay, Control, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Mut-Seq derived mutations in genes detected in at least two S. pneumoniae D39 transformants

Article Snippet: To extend this observation, we tested the ability of four strains (D39, TIGR4, ATCC 49619 and CCRI-14647) to transform a RpsL K56T mutation when exposed to a lysate of S. pneumoniae D39 coding for this S12 ribosomal protein variant, which confers resistance to STR, in four media in the absence of exogenous CSP.

Techniques: Derivative Assay

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Mutations in diverse genes intersecting with Mut-Seq and GWAS screens increase natural transformation in S. pneumoniae . Mutations detected in PurD ( A ), PurH ( B ), Cps2O ( C ) and ScrR ( D ) by Mut-Seq (blue) or in the two GWAS screens (tan, GWAS-1; beige, GWAS-2) were introduced in S. pneumoniae D39. The natural transformation efficiency of the transformants was monitored with planktonic cells grown in C + Y media following exposure to a cell lysate derived from S. pneumoniae D39 coding for the FolA I100L variant. Transformation efficiency is reported as the number of TMP-resistant cells over the number of viable cells. Each point represents a biological replicate. Transformation efficiencies were compared with those of D39 using the Kruskal–Wallis test followed by Dunn’s test. Significance is indicated above the whiskers as follows: * P < 0.05; ** P < 0.01; *** P < 0.001. For the GWAS screens, mutations positively associated with MDR are aligned under the “+” sign whereas those either not associated with MDR (GWAS-1) or positively associated with antibiotic susceptibility (GWAS-2) are aligned under the “−” sign. The significance levels of the association with MDR (GWAS-1, + and −; GWAS-2, +) or susceptibility (GWAS-2, −) prior to and after correction for population structure are indicated within parentheses (uncorrected/corrected) below the mutations on the x -axis as follows: * P = 10 −06 –10 −10 ; ** P = 10 −10 –10 −20 ; *** P = 10 −20 –10 −40 . See Table for the complete GWAS-1 and GWAS-2 data. NA on the x -axis indicates that no mutation was found for the screen in the gene.

Article Snippet: To extend this observation, we tested the ability of four strains (D39, TIGR4, ATCC 49619 and CCRI-14647) to transform a RpsL K56T mutation when exposed to a lysate of S. pneumoniae D39 coding for this S12 ribosomal protein variant, which confers resistance to STR, in four media in the absence of exogenous CSP.

Techniques: Transformation Assay, Derivative Assay, Variant Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Point mutations in functionally diverse genes are associated with increased natural DNA transformation in multidrug resistant Streptococcus pneumoniae

doi: 10.1093/nar/gkae1140

Figure Lengend Snippet: Phylogenetic distribution among MDR strains of GWAS mutations in genes also detected by Mut-Seq. Phylogeny was inferred from a recombination-pruned core SNP alignment of the 216 S. pneumoniae genomes used for our pilot GWAS-1 ( A ) and 850 S. pneumoniae genomes used for our GWAS-2 ( B ). Coloring at the tip of the branches refers to the antibiotic susceptibility status of the strains (red, AMR; green, susceptible). The heatmap on the right indicates the presence (blue) or absence (white) in the genomes of the mutations leading to the amino acid substitutions listed on top. Interactive versions of the figures are available at the following links: https://microreact.org/project/1uP9V6z4HzTheCNWDrM93M-spnmdr216august2024 ; https://microreact.org/project/oFajQo1ffGbFEVysRU1t3b-spnmdr850august2024 .

Article Snippet: To extend this observation, we tested the ability of four strains (D39, TIGR4, ATCC 49619 and CCRI-14647) to transform a RpsL K56T mutation when exposed to a lysate of S. pneumoniae D39 coding for this S12 ribosomal protein variant, which confers resistance to STR, in four media in the absence of exogenous CSP.

Techniques:

Journal: bioRxiv

Article Title: Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth

doi: 10.1101/2025.01.10.632320

Figure Lengend Snippet:

Article Snippet: In order to expose S. pneumoniae to commensal bacteria, 1 CFU of each pneumococcal isolate was inoculated into four culture conditions, resulting in the following pneumococcal broth cultures: (i) S. pneumoniae cultured alone in 2 ml of BBL Todd-Hewitt broth (Becton Dickinson) as negative controls (S); (ii) S. pneumoniae co-cultured with D. pigrum (SD); (iii) S. pneumoniae co-cultured with C. pseudodiphtheriticum (SC); and (iv) S. pneumoniae co-cultured with both (SDC).

Techniques: Bacteria

Journal: bioRxiv

Article Title: Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth

doi: 10.1101/2025.01.10.632320

Figure Lengend Snippet:

Article Snippet: In order to expose S. pneumoniae to commensal bacteria, 1 CFU of each pneumococcal isolate was inoculated into four culture conditions, resulting in the following pneumococcal broth cultures: (i) S. pneumoniae cultured alone in 2 ml of BBL Todd-Hewitt broth (Becton Dickinson) as negative controls (S); (ii) S. pneumoniae co-cultured with D. pigrum (SD); (iii) S. pneumoniae co-cultured with C. pseudodiphtheriticum (SC); and (iv) S. pneumoniae co-cultured with both (SDC).

Techniques: Bacteria