Journal: Journal of Antimicrobial Chemotherapy

Article Title: Affinity of cefotiam for the alternative penicillin binding protein PBP3 SAL used by Salmonella inside host eukaryotic cells

doi: 10.1093/jac/dkac422

Figure Lengend Snippet: Identification of cefotiam in a screening designed to identify compounds that inhibit cell division by targeting PBP3 SAL . (a) Higher final OD 600 values detected in S. Typhimurium wild-type and ΔPBP3 isogenic strains incubated in the presence of 0.001 mg/mL (2.3 µM) aztreonam in LB medium pH 4.6. Microscope images were taken at 6 h after the onset of growth. Bar: 25 µm. (b) Effect on growth of S. Typhimurium wild-type, ΔPBP3 and ΔPBP3 SAL isogenic strains incubated in LB medium pH 4.6 with the indicated concentrations of cefotiam hydrochloride. For these assays, the antibiotic was taken from the aliquot supplied at 10 mM in the Prestwick Chemical Library. (c) Effect of cefotiam on cell division visualized through the microscope in the indicated S. Typhimurium strains and antibiotic concentrations using LB medium pH 4.6. Note the differential effect when used at 0.00032 mg/mL (0.51 µM) causing blockage of cell division only in the strains producing PBP3 SAL . The cefotiam used in these assays was acquired commercially with a purity of ≥98%. Bar: 25 µm. (d) Phenotype of the indicated isogenic S. Typhimurium single or double mutants lacking PBPs in response to commercially acquired cefotiam. Note the lack of growth at 0.0008 mg/mL (1.28 µM) cefotiam of the strains producing PBP3 SAL as the only enzyme promoting cell division (ΔPBP3 genetic background). Assays were performed for a minimum of two independent biological replicates. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC .

Article Snippet: Cefotiam hydrochloride (ref. 66309-69-1, Molekula GmbH, Munich, Germany) was added to 0.02 mg membrane extracts prepared in 50 mM sodium phosphate buffer pH 4.6 to reach final concentrations of the antibiotic in the 0.00005–0.005 mg/mL range.

Techniques: Incubation, Microscopy

Journal: Journal of Antimicrobial Chemotherapy

Article Title: Affinity of cefotiam for the alternative penicillin binding protein PBP3 SAL used by Salmonella inside host eukaryotic cells

doi: 10.1093/jac/dkac422

Figure Lengend Snippet: Cefotiam binds to PBP3 SAL with higher affinity than to PBP3. (a) Representative BOCILLIN-FL competition binding assays performed in membrane extracts obtained from ΔPBP3 and ΔPBP3 SAL isogenic mutants grown in LB medium pH 4.6. Binding conditions were also pH 4.6. Asterisks point to protein used in the densitometry analyses to adjust values for protein content. (b) Determination of the IC 50 of cefotiam for competing BOCILLIN-FL binding to PBP1A/1B, PBP3 and PBP3 SAL . For PBP1A/1B, data are shown separately corresponding to the data of ΔPBP3 and ΔPBP3 SAL , respectively. Data are from three independent experiments and were analysed by Student’s t -test. *, P ≤ 0.05; **, P ≤ 0.005. ns, not significant. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC .

Article Snippet: Cefotiam hydrochloride (ref. 66309-69-1, Molekula GmbH, Munich, Germany) was added to 0.02 mg membrane extracts prepared in 50 mM sodium phosphate buffer pH 4.6 to reach final concentrations of the antibiotic in the 0.00005–0.005 mg/mL range.

Techniques: Binding Assay, Membrane

Journal: Journal of Antimicrobial Chemotherapy

Article Title: Affinity of cefotiam for the alternative penicillin binding protein PBP3 SAL used by Salmonella inside host eukaryotic cells

doi: 10.1093/jac/dkac422

Figure Lengend Snippet: Cefotiam shows stronger viability inhibition in intracellular S. Typhimurium compared with other cephalosporins like cefuroxime. (a) Viability of the series of S. Typhimurium isogenic single and double mutants defective in PBPs inside NRK-49F rat fibroblasts at distinct post-infection times (2 hpi, 24 hpi) determined by the drop assay. Shown are serial dilutions of the overnight culture used for infection (inoculum) and the extracts obtained from the infected NRK-49F fibroblast culture. Cefotiam (0.001 mg/mL, 1.6 µM) was added to the tissue culture medium at 2 hpi. (b) Ratios of viable intracellular bacteria at 24 hpi versus 2 hpi determined for the indicated strains in the absence/presence of cefotiam. The values are shown relative to the samples not treated with the antibiotic and are the mean and SD of a total of the four independent biological replicates. Experimental mean of the 24 hpi/2 hpi ratios corresponding to four independent assays for samples without antibiotic were: 0.675 (wild type), 0.675 (ΔPBP3) and 0.738 (ΔPBP3 SAL ). (c) Structures of the cephalosporins cefotiam and cefuroxime, which show high affinity for PBP3 SAL and PBP3, respectively. (d) Drop assay depicting the viability of S. Typhimurium wild-type, ΔPBP3 and ΔPBP3 SAL strains inside NRK-49F fibroblasts at 2 hpi and 24 hpi. Shown are serial dilutions corresponding to inoculum (extracellular bacteria) and extracts containing intracellular bacteria. Cefuroxime (0.02 mg/mL, 47 µM) was added to the tissue culture medium at 2 hpi. (e) Ratios of viable intracellular bacteria at 24 hpi versus 2 hpi obtained for the indicated strains in the absence/presence of cefuroxime. The values are shown relative to the samples with no β-lactam added and are the mean and SD of four independent biological replicates. Data were analysed by Student’s t -test. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.001. n.s., not significant. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC .

Article Snippet: Cefotiam hydrochloride (ref. 66309-69-1, Molekula GmbH, Munich, Germany) was added to 0.02 mg membrane extracts prepared in 50 mM sodium phosphate buffer pH 4.6 to reach final concentrations of the antibiotic in the 0.00005–0.005 mg/mL range.

Techniques: Inhibition, Infection, Bacteria