atcc 25922 transconjugants  (ATCC)

Journal: Infection and Immunity

Article Title: Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

doi: 10.1128/IAI.00756-19

Figure Lengend Snippet: Screening of β-lactamase-producing K. pneumoniae in the cell culture supernatant of HT-29 cells reveals altered susceptibility to CTX. (A to L) K. pneumoniae strains with the indicated resistance mechanisms (Table 1) were screened for susceptibility to 16 mg/liter CTX (in the resistant range) in RPMI medium plus 5% LB or in the cell supernatant (Sup) plus 5% LB by measuring growth curves in a Bioscreen C device and by viable CFU counts. (M to T) VIM-1-producing K. pneumoniae (AO15200) was screened for susceptibility to ciprofloxacin, azithromycin, fosfomycin, imipenem, amoxicillin, doxycycline, cefotaxime, and ceftriaxone in RPMI plus 5% LB or in the supernatant plus 5% LB. T0 represents initial inoculum. Data are presented as the means of results from three or more independent experiments. CFU counts are presented, with statistical analysis of log-transformed CFU per milliliter with an independent t test. Statistical significance, presented as P values of <0.05, are indicated (****, P < 0.0001).

Article Snippet: The transfer was confirmed by colony PCR and gel electrophoresis , showing that the VIM enzyme was successfully transferred to the recipient strains. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l -cysteine were measured with a Bioscreen growth curve, and the OD 600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922 VIM-1 and MG1655 VIM-1 was measured with a Bioscreen assay in the presence of l -cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655.

Techniques: Cell Culture, Transformation Assay

Journal: Infection and Immunity

Article Title: Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

doi: 10.1128/IAI.00756-19

Figure Lengend Snippet: The altered susceptibility of VIM-1 metallo-β-lactamase to CTX is a result of zinc chelation in the supernatant. (A) VIM-1-producing K. pneumoniae strain (AO15200) cultured in RPMI medium plus 5% LB, with the addition of EDTA (0.1 mM) and CTX (16 mg/liter). (B) AO15200 was cultured in RPMI medium plus 5% LB or in the supernatant (Sup) plus 5% LB, with supplementation with the divalent metals FeSO4, ZnSO4, MgSO4, and CaCl2 (0.5 mM). T0 represents initial inoculum. (C) Sytox green cell permeability measured with EDTA and compared to that of heat-killed bacteria. Graphs represent means of data from three or more independent experiments, with statistical significance presented as P values of <0.05, as indicated (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).

Article Snippet: The transfer was confirmed by colony PCR and gel electrophoresis , showing that the VIM enzyme was successfully transferred to the recipient strains. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l -cysteine were measured with a Bioscreen growth curve, and the OD 600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922 VIM-1 and MG1655 VIM-1 was measured with a Bioscreen assay in the presence of l -cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655.

Techniques: Cell Culture, Permeability

Journal: Infection and Immunity

Article Title: Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

doi: 10.1128/IAI.00756-19

Figure Lengend Snippet: The altered susceptibility of VIM-1-producing K. pneumoniae results from the reduction of l-cystine to l-cysteine in the cell culture supernatant of HT-29 cells. (A) The supernatant was subjected to a size exclusion filter where the low-molecular-weight fraction (<3 kDa) was loaded onto an Oasis column, enriching for peptides and proteins. K. pneumoniae (AO15200) was cultured in the different fractions in the presence of 16 mg/liter CTX. (B) The K. pneumoniae strain was cultured in the presence of l-cystine and l-cysteine (0.5 mM) with and without 16 mg/liter CTX, after which viability was determined by a colony count assay. T0 represents initial inoculum. (C) The Oasis column flowthrough fractions of supernatants from HT-29 cells cultured in either DMEM or DMEM/− were loaded onto a reverse-phase column (RPC Resource column) and fractionated with an isocratic flow of 0.1% TFA. Fractions were collected for 15 min (flow rate, 1 ml/min). The thiol content was measured in the fractions with a GSH standard, and fractions where l-cysteine was loaded onto the column constituted a positive control. (D) Colony count assay of the K. pneumoniae strain (AO15200) cultured in the presence of fractions 6 and 7 with or without 16 mg/liter CTX. (E) Colony count assay of the K. pneumoniae strain cultured with glutathione (oxidized [ox] and reduced [red]) or l-cysteine in the presence or absence of 16 mg/liter CTX. (F) The supernatant collected from HT-29 cells was analyzed for reducing properties with BODIPY FL-Cys conversion of l-cystine to l-cysteine. Samples include cells with fresh RPMI medium, cells with RPMI medium after 24 h, and the supernatant alone. Values are given relative to the value for the RPMI control. (G) Measurement of l-cystine-to-l-cysteine conversion with BODIPY FL-Cys in the HT-29 cell supernatant in the presence of the indicated concentrations of the thioredoxin antagonist PX-12. Means were compared to the value for the supernatant with 0 μM PX-12. Graphs represent means of data from three independent experiments, with statistical significance presented as P values of <0.05, as indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Statistical significance was measured on the means of log-transformed CFU per milliliter with an independent t test, and for relative fluorescence, one-way ANOVA was performed with Tukey’s multiple-comparison test.

Article Snippet: The transfer was confirmed by colony PCR and gel electrophoresis , showing that the VIM enzyme was successfully transferred to the recipient strains. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l -cysteine were measured with a Bioscreen growth curve, and the OD 600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922 VIM-1 and MG1655 VIM-1 was measured with a Bioscreen assay in the presence of l -cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655.

Techniques: Cell Culture, Molecular Weight, Positive Control, Transformation Assay, Fluorescence

Journal: Infection and Immunity

Article Title: Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

doi: 10.1128/IAI.00756-19

Figure Lengend Snippet: MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l-cysteine were measured with a Bioscreen growth curve, and the OD600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922VIM-1 and MG1655VIM-1 was measured with a Bioscreen assay in the presence of l-cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655. Graphs represent means of data from three independent experiments.

Article Snippet: The transfer was confirmed by colony PCR and gel electrophoresis , showing that the VIM enzyme was successfully transferred to the recipient strains. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l -cysteine were measured with a Bioscreen growth curve, and the OD 600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922 VIM-1 and MG1655 VIM-1 was measured with a Bioscreen assay in the presence of l -cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655.

Techniques: Expressing, Nucleic Acid Electrophoresis, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

doi: 10.1128/IAI.00756-19

Figure Lengend Snippet: Zinc-chelating thiols in urine resensitize VIM-1-producing K. pneumoniae to CTX. (A) Colony count assay to determine the susceptibility of the K. pneumoniae strain (AO15200) to CTX in both urine and the HT-29 supernatant. (B) The urine samples were subjected to the same size exclusion, peptide/protein enrichment (Oasis column), and reverse-phase fractionation as the HT-29 cell culture supernatant. The fractions obtained after the reverse-phase step were used in a colony count assay with the K. pneumoniae strain (AO15200) in RPMI medium plus 5% LB in the presence or absence of CTX. (C) The thiol content in the fractions was measured with a fluorometric thiol assay and compared to a GSH standard. (D) Urine samples were diluted from 100% to 5% in PBS and tested for inhibition of growth of K. pneumoniae (AO15200) with CTX. The dilution before resensitization was lost (evaluated for each donor sample) was used in panel E (green circle for a representative sample). (E) Pure or diluted urine was used to culture the K. pneumoniae strain in the presence of CTX and with or without the addition of ZnSO4 (10 μM). T0 represents initial inoculum. Statistical significance was measured on the means of data from three or more independent experiments of log-transformed CFU per milliliter with an independent t test. P values are indicated (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The transfer was confirmed by colony PCR and gel electrophoresis , showing that the VIM enzyme was successfully transferred to the recipient strains. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 4 caption a7 MBL expression in a neutral background confirms the thiol-mediated effect on VIM and NDM. (A and B) The MICs of CTX for AO15200 and an NDM-1-expressing E. coli DH5α strain with and without l -cysteine were measured with a Bioscreen growth curve, and the OD 600 after 10 h was evaluated. (C) The growth of the VIM-1 transconjugants ATCC 25922 VIM-1 and MG1655 VIM-1 was measured with a Bioscreen assay in the presence of l -cysteine and CTX. (D) PCR and gel electrophoresis confirm plasmid transfer from AO15200 to ATCC 25922 and MG1655.

Techniques: Protein Enrichment, Fractionation, Cell Culture, Inhibition, Transformation Assay