Antibiotic susceptibility by the Kirby-Bauer disk diffusion test.^a

a<p>Growth inhibition halo diameters were measured after 20 (PAO1) or 40 h (PAO1 Δ<i>tolB araC-</i>P_BAD<i>tolB</i>) of growth at 37°C on MH agar plates, containing or not arabinose at the indicated concentration. Values are the average ±SD of at least three independent assays. Asterisks indicate statistically significant differences compared to wild type (one-way ANOVA; ^*<i>P</i><0.05; ^**<i>P</i><0.01; ^***<i>P</i><0.001).</p>b<p>Abbreviations: Gm, gentamycin; Sm; streptomycin, Tc, tetracyclin, Ap, ampicillin; Ipm, imipenem; Cip, ciprofloxacin, Caz, ceftazidime, Ct, colistin, PmB, polymyxin B.</p

Scheme of the strategy used to generate the <i>P. aeruginosa</i> PAO1 <i>tolB</i> conditional mutant.

<p>An exogenous copy of the <i>tolB</i> coding sequence under the control of an arabinose-dependent promoter was inserted into the <i>attB</i> neutral site of the <i>P. aeruginosa</i> chromosome, by using the integration-proficient plasmid mini-CTX1-<i>araC</i>P_BAD<i>tolB</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103784#pone-0103784-t001" target="_blank">Table 1</a>). After Flp-mediated removal of the mini-CTX1 backbone (not shown), the resulting strain (PAO1 <i>araC</i>P_BAD<i>tolB</i>) is a merodiploid for <i>tolB</i>. In-frame deletion of the endogenous copy of <i>tolB</i> was obtained using the suicide plasmid pDM4Δ<i>tolB</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103784#pone-0103784-t001" target="_blank">Table 1</a>). Sucrose selection was carried out in the presence of arabinose to select removal of the pDM4 backbone, followed by PCR screening to identify clones carrying the <i>tolB</i> in-frame deletion. One of these clones was selected and used for the following analyses. This conditional mutant was named PAO1 Δ<i>tolB araC</i>P_BAD<i>tolB</i>.</p

TolB-deficient cells show defects in outer membrane stability and cell division.

<p>SEM and TEM analysis (left and right panels, respectively) of PAO1 wild-type cells (A,B), TolB-deficient mutant cells (C,D) and TolB-proficient mutant cells (E,F), grown as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103784#pone-0103784-g003" target="_blank">Fig. 3A</a>. Bars: 3 µm (left panels) or 1 µm (right panels). The inset in panel D shows an enlargement of the boxed area. Abbreviations: IM, inner membrane; OM, outer membrane.</p

TolB is crucial for <i>P. aeruginosa</i> resistance and persistence.

<p>(A) Growth of PAO1 (circles) and PAO1 <i>tolB</i> conditional mutant (diamonds) at 37°C in MH broth at 200 rpm in flasks after two successive subcultures in the presence (filled symbols) or in the absence (open symbols) of 0.2% arabinose. The graph is representative of several assays giving similar results. (B) Lytic effect of SDS (0–5%), measured as decrease in cell suspension turbidity (OD₆₀₀), on PAO1 wild-type cells (WT, filled circles), TolB-deficient mutant cells (<i>tolB</i>, open diamonds) and TolB-proficient mutant cells (<i>tolB</i> TolB⁺, filled diamonds). (C) Resistance of WT, <i>tolB</i> and <i>tolB</i> TolB⁺ to the bactericidal activity of 50% human serum or (D) to the bactericidal antibiotic ofloxacin (0.5 mg/L), expressed as percent survival compared to untreated cells. Results in panels B–D are the mean (± SD) of four independent experiments. (E) Persistence of WT, <i>tolB</i> and <i>tolB</i> TolB⁺ cells in <i>G. mellonella</i> larvae at 2 h post-infection. Sixteen larvae per group were infected in three independent assays. ***, P<0.001 (one-way ANOVA).</p

TolB is essential for <i>P. aeruginosa</i> growth <i>in vitro</i>.

<p>(A) Growth curves of the wild-type strain PAO1 (filled circles) and the PAO1 <i>tolB</i> conditional mutant in the presence (filled diamonds) or in the absence (open diamonds) of 0.2% arabinose in MH broth at 37°C in microtiter plates at 200 rpm. Results are the mean (± SD) of three independent experiments performed in triplicate. (B) Growth of PAO1 and the PAO1 <i>tolB</i> conditional mutant on MH agar plates with or without 0.2% arabinose (ARA) at 16 h. (C) Growth of the PAO1 <i>tolB</i> conditional mutant as described in legend to panel A in the presence of increasing concentrations of arabinose (0–0.2%), measured as OD₆₀₀ (left panel) or CFU/ml (right panel). The graphs are representative of at least two independent experiments giving similar results. (D) Growth curves of <i>P. aeruginosa</i> PA14 or (E) the clinical strain TR1 (filled circles) and their corresponding <i>tolB</i> conditional mutants in the presence (filled squares) or in the absence (open squares) of 0.2% arabinose in MH broth at 37°C in microtiter plates at 200 rpm. Results are the mean (± SD) of two independent experiments performed in triplicate.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

TolB depletion increases <i>P. aeruginosa</i> sensitivity to polymyxins in a killing assay.

<p>Survival of PAO1 wild-type cells (filled circles) and TolB-deficient mutant cells (open diamonds), obtained as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103784#pone-0103784-g003" target="_blank">Figure 3A</a>, after 1-h treatment with 4, 1 or 0.25 mg/L of colistin (left panel), or 2, 0.5 or 0.125 mg/L of polymyxin B (right panel). Values are expressed as percent survival compared to untreated cells, and the results represent the mean (± SD) of three independent experiments. **, P<0.01 (one-way ANOVA).</p

Image_3_Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy.PDF

<p>Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD₅₀ and LD₉₀) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.</p

Table_1_Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy.DOCX

<p>Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD₅₀ and LD₉₀) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.</p

Table_2_Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy.xlsx

<p>Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD₅₀ and LD₉₀) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.</p