type strain rd kw20  (ATCC)

Journal: bioRxiv

Article Title: Evolution of β-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen Haemophilus influenzae

doi: 10.1101/2025.05.02.651845

Figure Lengend Snippet: Increasing number of mutations and changes of beta-lactam MICs in six evolution experiments. Number of mutations (black) and minimum inhibitory concentration fold-increase compared to the parental strain Rd KW20 for ampicillin (Amp, red), cefotaxime (Ctx, green), and ceftriaxone (Cro, blue) in selected clones over 20 passages (two or five clones per passage). Bacterial population A - without antibiotic exposure and exposed to increasing concentrations of B - ampicillin, C - cefotaxime, D - ceftriaxone replicate 1, E - ceftriaxone replicate 2, F - ceftriaxone replicate 3. Shaded areas represent the 95% confidence interval.

Article Snippet: Briefly, short reads were mapped to the reference genome H. influenzae Rd KW20 (ATCC 51907) (BioSample SAMN39831887) using the Burrows-Wheeler Aligner (BWA) ( ) and allele calling was done with the Genome Analysis Toolkit (GATK) ( ).

Techniques: Concentration Assay, Clone Assay

Journal: bioRxiv

Article Title: Evolution of β-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen Haemophilus influenzae

doi: 10.1101/2025.05.02.651845

Figure Lengend Snippet: Maximum likelihood phylogeny of H. influenzae Rd KW20 clones evolved in the presence of ampicillin (A, 53 clones), cefotaxime (B, 56 clones), and ceftriaxone (C, 57 clones). Amino acid substitutions in the penicillin binding protein 3 (PBP3) and presence of mutations in specified genes as well as minimum inhibitory concentration (MIC) changes over time are color coded. Strains that exceeded the clinical breakpoint for ampicillin/cefotaxime/ceftriaxone according to EUCAST breakpoints are marked with a white star at the MIC heatmap. The specified genes are those in which at least 20 clones from the whole set of selected clones exhibited mutations but did not mutate under antibiotic-free conditions.

Article Snippet: Briefly, short reads were mapped to the reference genome H. influenzae Rd KW20 (ATCC 51907) (BioSample SAMN39831887) using the Burrows-Wheeler Aligner (BWA) ( ) and allele calling was done with the Genome Analysis Toolkit (GATK) ( ).

Techniques: Clone Assay, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Evolution of β-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen Haemophilus influenzae

doi: 10.1101/2025.05.02.651845

Figure Lengend Snippet: Crystal structure of transpeptidase domain (residues 254-610) of the penicillin binding protein 3 (PBP3) of H. influenzae (Bellini et al. [2019]) with superimposed ampicillin (A, colored in cyan) and cefotaxime (B, colored in magenta) molecules. In vitro selected amino acid substitutions in PBP3 Thr332Ile, Thr443Ala, Ala530Ser, Asp551Tyr, and Ala561Glu are colored in red. Known positions for amino acid substitutions defining established PBP3 resistance groups are colored in green.

Article Snippet: Briefly, short reads were mapped to the reference genome H. influenzae Rd KW20 (ATCC 51907) (BioSample SAMN39831887) using the Burrows-Wheeler Aligner (BWA) ( ) and allele calling was done with the Genome Analysis Toolkit (GATK) ( ).

Techniques: Binding Assay, In Vitro

Journal: bioRxiv

Article Title: Evolution of β-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen Haemophilus influenzae

doi: 10.1101/2025.05.02.651845

Figure Lengend Snippet: Bacterial fitness of selected clones derived from evolving populations exposed to ampicillin, cefotaxime, and ceftriaxone. Bacterial fitness measured as relative growth difference between the ancestral strain ( H. influenzae Rd KW20) and the selected clone. A relative fitness of 1 indicates no fitness cost, whereas a value greater or less than 1 indicates increased or decreased fitness, respectively. The black bars represent standard deviation. *padj<0.05; **padj<0.01; ***padj<0.001. The heatmap indicates MIC values of ampicillin (Amp), cefotaxime (Ctx) and ceftriaxone (Cro) in mg/L of the respective strain (already presented in ). Stars indicate clinical resistance above the respective EUCAST breakpoints. Hatched panels indicate heteroresistance. Clones that derived from the same evolution experiment are grouped together on the y-axis.

Article Snippet: Briefly, short reads were mapped to the reference genome H. influenzae Rd KW20 (ATCC 51907) (BioSample SAMN39831887) using the Burrows-Wheeler Aligner (BWA) ( ) and allele calling was done with the Genome Analysis Toolkit (GATK) ( ).

Techniques: Clone Assay, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: Evolution of β-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen Haemophilus influenzae

doi: 10.1101/2025.05.02.651845

Figure Lengend Snippet: Collateral effects of increased MICs against individual beta-lactam antibiotics. Heat map of log2-transformed relative increase in MIC of ampicillin, cefotaxime, ceftriaxone (underlying MIC values already presented in ), meropenem (beta-lactams), amikacin, kanamycin, gentamicin (aminoglycosides), levofloxacin, moxifloxacin (fluoroquinolones), clarithromycin (macrolide), vancomycin (glycopeptide), rifampicin (ansamycin), colistin (polymyxin) and tetracycline (tetracycline) in mutant clones relative to the MIC of the parental strain H. influenzae Rd KW20. Selected clones were evolved in the multi-step evolution experiment. Stars indicate clinical resistance above the respective EUCAST breakpoints (not available for aminoglycosides, clarithromycin, vancomycin, and colistin). Hatched panels indicate heteroresistance. Clones that derived from the same evolution experiment are grouped together on the y-axis.

Article Snippet: Briefly, short reads were mapped to the reference genome H. influenzae Rd KW20 (ATCC 51907) (BioSample SAMN39831887) using the Burrows-Wheeler Aligner (BWA) ( ) and allele calling was done with the Genome Analysis Toolkit (GATK) ( ).

Techniques: Transformation Assay, Glycoproteomics, Mutagenesis, Clone Assay, Derivative Assay

Journal: bioRxiv

Article Title: Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

doi: 10.1101/2024.07.09.602704

Figure Lengend Snippet: Serial dilutions of H. influenzae ΔHindII/III transformants harboring either no plasmid, pHflu1, pHflu2, pHflu3, or pHflu8 were plated on sBHI supplemented with antibiotics as indicated. Images were taken after one and two days of growth at 37 °C.

Article Snippet: Single colonies of H. influenzae Rd KW20 WT and ΔHindII/III were used to inoculate 3 mL of Difco sBHI in sterile 15-mL conical centrifuge tubes and incubated overnight at 37°C with shaking at 225 rpm.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

doi: 10.1101/2024.07.09.602704

Figure Lengend Snippet: ( A ) Red fluorescence provided by pHflu4 is visible in ambient light, while green provided by pHflu6 and pHflu7 is better observed under UV light. H. influenzae ΔHindII/III transformants were serially diluted and spot-plated on sBHI supplemented with 5 µg/ml chloramphenicol and grown for 2 days at 37 °C. The image below was taken under UV light. ( B ) H. influenzae ΔHindII/III carrying pHflu1, pHflu4, pHflu6, and pHflu7 as observed by confocal fluorescence microscopy. Red channel excitation/emission wavelength: 594/613 nm; red channel: 488/543 nm

Article Snippet: Single colonies of H. influenzae Rd KW20 WT and ΔHindII/III were used to inoculate 3 mL of Difco sBHI in sterile 15-mL conical centrifuge tubes and incubated overnight at 37°C with shaking at 225 rpm.

Techniques: Fluorescence, Microscopy