Other members of the Shewanella genus,

Other members of the Shewanella genus, 1516647 S. algae and S. xiamenensis, has been shown to be capable of hydrolyzing carbapenem and imipenem [33,36]. Heterogeneous expression of any of these blactamases in E. coli elevated the corresponding MICs for amoxicillin, ticarcillin, and piperacillin to at least 256 mg/ml, indicating that they were functional [25,33,36]. However, it has been assumed that BlaA has a dispensable role in the resistance of S. oneidensis to b-lactam antibiotics because of its extremely low basal expression and weak induction by imipenem and cefoxitin at subinhibitory concentrations (0.5? mg/ml) [25]. In this study, however, we have identified a previously undescribed phenomenon that certain b-lactams at modest concentrations delay growth and induce cell lysis. We have shown that resistance of S. oneidensis to b-lactams is due to BlaA. In its absence, typical b-lactams are as potent as ciprofloxacin, the most effective antibiotic against Shewanella tested to date. We then presented evidence that the growth inhibition and cell lysis by subMIC ampicillin is largely due to low expression of blaA, resulting in a slow removal of the antibiotic. This observation is consistent with OXA-type b-lactamases from P. aeruginosa which are not inducibleExpression of blaA in S. oneidensisFigure 3. Cell lysis caused by ampicillin at 2.5 mg/ml. Cultures of late-exponential phase cells (,0.6 of OD600) were diluted 1:100 with LB broth, and incubated at 30uC in a shaker at 200 rpm. (A) Growth of S. oneidensis in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/ ml) levels. (B) Microscopic images of cells at various times in the presence of ampicillin at 2.5 mg/ml. Arrows point to knobs and branches characterstic of treated cells. (C) Growth of cultures varying in initial cell density in the presence of ampicillin at 2.5 mg/ml. (D) Amounts of ampicillin remaining at the 4EGI-1 indicated times in cultures supplemented initially with ampicillin at 50 mg/ml or 2.5 mg/ml. In all panels, experiments were performed at least in triplicate and the error bars PD168393 cost represent standard deviation (SD). doi:10.1371/journal.pone.0060460.gby imipenem and cefoxitin at subinhibitory concentrations (0.2?1 mg/ml), suggesting that this group of b-lactamases may be regulated by similar mechanisms [25,38]. Prompt and substantial production of b-lactamases is a metabolically costly endeavor for growing bacteria, but necessaryfor survival when high concentrations of b-lactam antibiotics are encountered. It is therefore not surprising that cells increase production of BlaA extensively when 50 mg/ml ampicillin was added. However, Shewanella are mainly found in marine and freshwater environments, where the concentrations of antibioticsFigure 4. Impact of the loss of blaA on growth. (A) Growth of the nblaA strain in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/ml). Hc and Mc represent the nblaA strain complemented in trans. (B) Susceptibility assay of the nblaA strain to ampicillin. nblaAc represents the nblaA strain complemented in trans. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (A). doi:10.1371/journal.pone.0060460.gExpression of blaA in S. oneidensisTable 2. MICs (mg/ml) of b-lactams for S. oneidensis wild type and derivative strains.MIC (mg/ml)a Ampicillin Penicillin Carbenicillin DblaAc 64 128 .128 DdacAc 16 32WT 16 32DblaA ,1 ,1 ,DSO0541 16 32DSO0914 16 32Damp.Other members of the Shewanella genus, 1516647 S. algae and S. xiamenensis, has been shown to be capable of hydrolyzing carbapenem and imipenem [33,36]. Heterogeneous expression of any of these blactamases in E. coli elevated the corresponding MICs for amoxicillin, ticarcillin, and piperacillin to at least 256 mg/ml, indicating that they were functional [25,33,36]. However, it has been assumed that BlaA has a dispensable role in the resistance of S. oneidensis to b-lactam antibiotics because of its extremely low basal expression and weak induction by imipenem and cefoxitin at subinhibitory concentrations (0.5? mg/ml) [25]. In this study, however, we have identified a previously undescribed phenomenon that certain b-lactams at modest concentrations delay growth and induce cell lysis. We have shown that resistance of S. oneidensis to b-lactams is due to BlaA. In its absence, typical b-lactams are as potent as ciprofloxacin, the most effective antibiotic against Shewanella tested to date. We then presented evidence that the growth inhibition and cell lysis by subMIC ampicillin is largely due to low expression of blaA, resulting in a slow removal of the antibiotic. This observation is consistent with OXA-type b-lactamases from P. aeruginosa which are not inducibleExpression of blaA in S. oneidensisFigure 3. Cell lysis caused by ampicillin at 2.5 mg/ml. Cultures of late-exponential phase cells (,0.6 of OD600) were diluted 1:100 with LB broth, and incubated at 30uC in a shaker at 200 rpm. (A) Growth of S. oneidensis in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/ ml) levels. (B) Microscopic images of cells at various times in the presence of ampicillin at 2.5 mg/ml. Arrows point to knobs and branches characterstic of treated cells. (C) Growth of cultures varying in initial cell density in the presence of ampicillin at 2.5 mg/ml. (D) Amounts of ampicillin remaining at the indicated times in cultures supplemented initially with ampicillin at 50 mg/ml or 2.5 mg/ml. In all panels, experiments were performed at least in triplicate and the error bars represent standard deviation (SD). doi:10.1371/journal.pone.0060460.gby imipenem and cefoxitin at subinhibitory concentrations (0.2?1 mg/ml), suggesting that this group of b-lactamases may be regulated by similar mechanisms [25,38]. Prompt and substantial production of b-lactamases is a metabolically costly endeavor for growing bacteria, but necessaryfor survival when high concentrations of b-lactam antibiotics are encountered. It is therefore not surprising that cells increase production of BlaA extensively when 50 mg/ml ampicillin was added. However, Shewanella are mainly found in marine and freshwater environments, where the concentrations of antibioticsFigure 4. Impact of the loss of blaA on growth. (A) Growth of the nblaA strain in the presence of ampicillin at H (50 mg/ml), M (2.5 mg/ml) or L (0.125 mg/ml). Hc and Mc represent the nblaA strain complemented in trans. (B) Susceptibility assay of the nblaA strain to ampicillin. nblaAc represents the nblaA strain complemented in trans. Experiments were performed at least in triplicate and the error bars represent standard deviation (SD) as in (A). doi:10.1371/journal.pone.0060460.gExpression of blaA in S. oneidensisTable 2. MICs (mg/ml) of b-lactams for S. oneidensis wild type and derivative strains.MIC (mg/ml)a Ampicillin Penicillin Carbenicillin DblaAc 64 128 .128 DdacAc 16 32WT 16 32DblaA ,1 ,1 ,DSO0541 16 32DSO0914 16 32Damp.