A comparison of 2-year Kaplan–Meier survival curves of PLA patients with and without septic ocular or CNS complications (p = 0.502, by log-rank test).

<p>A comparison of 2-year Kaplan–Meier survival curves of PLA patients with and without septic ocular or CNS complications (p = 0.502, by log-rank test).</p

A comparison of 2-year Kaplan–Meier survival curves of PLA patients aged <65 years and a Charlson comorbidity index ≤1 (i.e. between 0 and 1) with and without septic ocular or CNS complications (p = 0.001, by log-rank test).

<p>A comparison of 2-year Kaplan–Meier survival curves of PLA patients aged <65 years and a Charlson comorbidity index ≤1 (i.e. between 0 and 1) with and without septic ocular or CNS complications (p = 0.001, by log-rank test).</p

The frequency of PLA cases with septic ocular or central nervous system complications.

<p><b>NOTE.</b> PLA, pyogenic liver abscess.</p

Clinical characteristics of PLA patients with and without septic ocular or central nervous system complications.

<p><b>NOTE.</b> PLA, pyogenic liver abscess.</p

The physiological functions evaluation between wild-type KJ and its derived mutants.

<p>The data represent means of three repetitions. Error bars indicate the standard deviation for three triplicate samples. *, <i>p</i>≤0.05 significance calculated by a Student’s <i>t</i>-test. (A) Motility ability. Five microliter bacterial cell suspension was inoculated at the swimming agar (1% tryptone, 0.5% NaCl and 0.15% agar), and the diameter (mm) of swimming zone was measured after 48 h incubation at 37°C. (B) H₂O₂ susceptibility test. Sterile filter paper with 20 μl of 10% H₂O₂ was placed onto MH agar, which was uniformly spread with bacterial cell suspension. The diameter of a zone of growth inhibition was measured after 24 h incubation at 37°C. (C) Secreted protease activity assay. Forty microliter bacterial cell suspension was dipped on LB agar containing 1% skin milk. The proteolytic activity of bacteria was assessed by measuring the transparent zones around the bacteria after incubation at 37°C for 72 h.</p

Transcriptomic analysis of genes differentially expressed in the <i>smeRySy</i> mutant compared to the wild-type <i>S</i>. <i>maltophilia</i> KJ.

<p>Transcriptomic analysis of genes differentially expressed in the <i>smeRySy</i> mutant compared to the wild-type <i>S</i>. <i>maltophilia</i> KJ.</p

A Linkage between SmeIJK Efflux Pump, Cell Envelope Integrity, and σ^E-Mediated Envelope Stress Response in <i>Stenotrophomonas maltophilia</i>

<div><p>Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of <i>Stenotrophomonas maltophilia</i>, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σ^E-mediated envelope stress response (ESR) of <i>S. maltophilia</i> was assessed. SmeIJK was involved in the intrinsic resistance of <i>S. maltophilia</i> KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the <i>smeIJK</i> deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σ^E-mediated ESR. Moreover, the expression of <i>smeIJK</i> was further induced by sub-lethal concentrations of MDAs or surfactants in an σ^E-dependent manner. These data collectively suggested an alternative physiological role of <i>smeIJK</i> in cell envelope integrity maintenance and σ^E-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting <i>S. maltophilia</i> from the envelope stress, <i>smeIJK</i> is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σ^E-mediated ESR in <i>S. maltophilia</i>.</p></div

Inactivation of SmeSyRy Two-Component Regulatory System Inversely Regulates the Expression of SmeYZ and SmeDEF Efflux Pumps in <i>Stenotrophomonas maltophilia</i>

<div><p>SmeYZ efflux pump is a critical pump responsible for aminoglycosides resistance, virulence-related characteristics (oxidative stress susceptibility, motility, and secreted protease activity), and virulence in <i>Stenotrophomonas maltophilia</i>. However, the regulatory circuit involved in SmeYZ expression is little known. A two-component regulatory system (TCS), <i>smeRySy</i>, transcribed divergently from the <i>smeYZ</i> operon is the first candidate to be considered. To assess the role of SmeRySy in <i>smeYZ</i> expression, the <i>smeRySy</i> isogenic deletion mutant, KJΔRSy, was constructed by gene replacement strategy. Inactivation of <i>smeSyRy</i> correlated with a higher susceptibility to aminoglycosides concomitant with an increased resistance to chloramphenicol, ciprofloxacin, tetracycline, and macrolides. To elucidate the underlying mechanism responsible for the antimicrobials susceptibility profiles, the SmeRySy regulon was firstly revealed by transcriptome analysis and further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and promoter transcription fusion constructs assay. The results demonstrate that inactivation of <i>smeRySy</i> decreased the expression of SmeYZ pump and increased the expression of SmeDEF pump, which underlies the <i>ΔsmeSyRy</i>-mediated antimicrobials susceptibility profile. To elucidate the cognate relationship between SmeSy and SmeRy, a single mutant, KJΔRy, was constructed and the complementation assay of KJΔRSy with <i>smeRy</i> were performed. The results support that SmeSy-SmeRy TCS is responsible for the regulation of <i>smeYZ</i> operon; whereas SmeSy may be cognate with another unidentified response regulator for the regulation of <i>smeDEF</i> operon. The impact of inverse expression of SmeYZ and SmeDEF pumps on physiological functions was evaluated by mutants construction, H₂O₂ susceptibility test, swimming, and secreted protease activity assay. The increased expression of SmeDEF pump in KJΔRSy may compensate, to some extents, the SmeYZ downexpression-mediated compromise with respect to its role in secreted protease activity.</p></div

The expression of <i>smeIJK</i> operon in different stresses and culture media.

<p>Plasmid containing a transcriptional fusion of the upstream region of <i>smeI</i> to the <i>xylE</i> gene (pSmeI_xylE) was transferred into the wild-type KJ. The C23O activities of the logarithmic-phase cultures of KJ(pSmeI_xylE) were determined. Each bar represents the mean of three independent experiments. Each bar represents the mean of three independent experiments. *, <i>p</i>≤0.01 significance calculated by a Student's <i>t</i>-test. (A) The expression of <i>smeIJK</i> operon in different stresses. The concentrations of the stressors added were: Triton X-100, 100 µg/ml; benzalkonium chloride (BC), 10 µg/ml; cetyltributylammonium bromide (CTAB), 10 µg/ml; gentamicin (Gm), 1 µg/ml; amikacin (Ami), 1 µg/ml; and leucomycin (Leu), 0.5 µg/ml. (B) The expression of <i>smeIJK</i> operon in different culture media, including the LB, the LB without NaCl, the LB with casein hydrolysate, and the MH.</p

Antimicrobial susceptibilities of <i>S. maltophilia</i> KJ and its derived deletion mutants.

<p>Antimicrobial susceptibilities of <i>S. maltophilia</i> KJ and its derived deletion mutants.</p