DataSheet_1_A comprehensive description of the TolC effect on the antimicrobial susceptibility profile in Enterobacter bugandensis.pdf

BackgroundEnterobacter bugandensis is an emerging human pathogen in which multidrug resistant strains have been continuously isolated from various environments. Thus, this organism possesses the potential to pose challenges in human healthcare. However, the mechanisms, especially the efflux pumps, responsible for the multidrug resistance in E. bugandensis remain to be well elucidated.MethodsThe Enterobacter strain CMCC(B) 45301 was specifically identified using whole genome sequencing. The specific CMCC(B) 45301 homologues of the TolC dependent efflux-pump genes characterized in Escherichia coli were identified. The tolC deletion mutant in CMCC(B) 45301 was constructed and subjected to susceptibility tests using 26 different antimicrobial agents, along with the wild type strain. The synergistic effects combining the Bacillus crude extract (BCE) and several other TolC-affected compounds against CMCC(B) 45301 were assayed.ResultsWe reclassified the Enterobacter CMCC(B) 45301 strain from species cloacae to bugandensis, on the basis of its whole genome sequence. We found that the CMCC(B) 45301 TolC, AcrAB, AcrD, AcrEF, MdtABC, EmrAB, and MacAB exhibit high similarity with their respective homologues in E. coli and Enterobacter cloacae. Our results for the susceptibility tests revealed that lacking tolC causes 4- to 256-fold decrease in the minimal inhibitory concentrations of piperacillin, gentamicin, kanamycin, tetracycline, norfloxacin, ciprofloxacin, chloramphenicol, and erythromycin against CMCC(B) 45301. In addition, the inhibition zones formed by cefuroxime, cefoperazone, amikacin, streptomycin, minocycline, doxycycline, levofloxacin, florfenicol, trimethoprim-sulfamethoxazole, azithromycin, lincomycin, and clindamycin for the tolC mutant were larger or more obvious than that for the parent. Our data suggested the important role played by TolC in CMCC(B) 45301 susceptibility to common antibiotic families covering ß-lactam, aminoglycoside, tetracycline, fluoroquinolone, phenicol, folate pathway antagonist, macrolide, and lincosamide. Deletion for tolC also increased the susceptibility of CMCC(B) 45301 to berberine hydrochloride and BCE, two natural product-based agents. Finally, we found that erythromycin, norfloxacin, and ciprofloxacin can potentiate the antibacterial activity of BCE against CMCC(B) 45301.DiscussionThe present study elaborated the comprehensive TolC effect on the antimicrobial susceptibility profile in E. bugandensis, which might contribute to the development of more therapeutic options against this nosocomial pathogen.</p

DataSheet_2_A comprehensive description of the TolC effect on the antimicrobial susceptibility profile in Enterobacter bugandensis.pdf

BackgroundEnterobacter bugandensis is an emerging human pathogen in which multidrug resistant strains have been continuously isolated from various environments. Thus, this organism possesses the potential to pose challenges in human healthcare. However, the mechanisms, especially the efflux pumps, responsible for the multidrug resistance in E. bugandensis remain to be well elucidated.MethodsThe Enterobacter strain CMCC(B) 45301 was specifically identified using whole genome sequencing. The specific CMCC(B) 45301 homologues of the TolC dependent efflux-pump genes characterized in Escherichia coli were identified. The tolC deletion mutant in CMCC(B) 45301 was constructed and subjected to susceptibility tests using 26 different antimicrobial agents, along with the wild type strain. The synergistic effects combining the Bacillus crude extract (BCE) and several other TolC-affected compounds against CMCC(B) 45301 were assayed.ResultsWe reclassified the Enterobacter CMCC(B) 45301 strain from species cloacae to bugandensis, on the basis of its whole genome sequence. We found that the CMCC(B) 45301 TolC, AcrAB, AcrD, AcrEF, MdtABC, EmrAB, and MacAB exhibit high similarity with their respective homologues in E. coli and Enterobacter cloacae. Our results for the susceptibility tests revealed that lacking tolC causes 4- to 256-fold decrease in the minimal inhibitory concentrations of piperacillin, gentamicin, kanamycin, tetracycline, norfloxacin, ciprofloxacin, chloramphenicol, and erythromycin against CMCC(B) 45301. In addition, the inhibition zones formed by cefuroxime, cefoperazone, amikacin, streptomycin, minocycline, doxycycline, levofloxacin, florfenicol, trimethoprim-sulfamethoxazole, azithromycin, lincomycin, and clindamycin for the tolC mutant were larger or more obvious than that for the parent. Our data suggested the important role played by TolC in CMCC(B) 45301 susceptibility to common antibiotic families covering ß-lactam, aminoglycoside, tetracycline, fluoroquinolone, phenicol, folate pathway antagonist, macrolide, and lincosamide. Deletion for tolC also increased the susceptibility of CMCC(B) 45301 to berberine hydrochloride and BCE, two natural product-based agents. Finally, we found that erythromycin, norfloxacin, and ciprofloxacin can potentiate the antibacterial activity of BCE against CMCC(B) 45301.DiscussionThe present study elaborated the comprehensive TolC effect on the antimicrobial susceptibility profile in E. bugandensis, which might contribute to the development of more therapeutic options against this nosocomial pathogen.</p

The relationship between the distribution of bacteria and archaea (7 phyla and classes) and 5 environmental factors in four sites of SPG analyzed by CCA.

<p>The relationship between the distribution of bacteria and archaea (7 phyla and classes) and 5 environmental factors in four sites of SPG analyzed by CCA.</p

The diversity indices and richness estimators of the bacterial and archaeal 16S rRNA gene clone libraries. OTUs of the 16S rRNA gene sequences were determined at 3% for bacteria and 2% for archaea sequence distance cut-off using the DOTUR program.

<p>The coverage (<i>C),</i> Shannon-Weiner (<i>H), Simpson (D)</i> and <i>evenness (J) indices,</i> and <i>S_ACE</i> and <i>S_chao1</i> richness estimators were calculated using the OTU data.</p

Decreased clearance of IgAN patient IgA by CD89-expressing Kupffer cells.

<p>(A) IgA, purified from a normal donor and from a patient with IgAN, was stained with Cy3-anti-human IgA Ab, and subsequently incubated with Kupffer cells from CD89 transgenic mice at a concentration of 1 μg/mL and a temperature of 37°C. After 1-h-incubation, intracellular vesicles containing IgA were diminished when IgAN patient-derived IgA endocytosis was assessed and compared with normal human IgA. (B) sCD89-IgA complexes in cell supernatant of (A) were detected by ELISA using A3 and anti-mouse IgA mAb, using 1 μg/mL human IgA binding to 5 μg/mL recCD89 as an internal control. The sCD89-IgA complexes were detected in the Kupffer cell supernatant after 1h of incubation with either control IgA or patient IgA. (C) Decreased clearance of 10 μg IgAN patient-derived IgA in C57BL/6-Tg mice when compared to normal human IgA. A total of 10 μg of each of the proteins was injected into the tail vein of C57BL/6-Tg (n = 5 per group). At the indicated times after injection, sera were obtained and IgA levels were determined. IgA from IgAN patients was cleared less quickly in Tg mice compared with normal IgA. (D) Induction of mesangial IgA/sCD89 deposits by injecting patient IgA into 6-wk-old C57BL/6-Tg mice. Double staining by anti-human IgA and anti-CD89 in kidney 48 h after constant injection of patient IgA into C57BL/6-Tg mice(n = 4) is shown. Bar = 10μm.</p

Phylogenetic tree of the 45 representative OTUs from bacterial 16S rRNA gene clone libraries (could represented all OTUs) and 32 cultivated bacteria from the four stations, constructed using neighbor-joining method of Phylip 3.66 based on the blast results of RDP classifer and EzTaxon server 2.1.

<p>The * represents the bacterial strains which were potential novel bacterial species. The phylogenetic neighbours were from the database of type strains with validly published prokaryotic names in the EzTaxon server (<a href="http://www.eztaxon.org/" target="_blank">http://www.eztaxon.org/</a>).</p

CD89 transgenic mice do not develop IgAN.

<p>(A) From the left to the right: histology of kidney sections after periodic acid-Schiff (PAS), IgA and CD89 staining of kidneys from CD89 Tg mice (10 mice per group) at different ages. No mesangial IgA deposits or periglomerular CD89 cells were seen in kidneys from CD89 Tg mice; Bar: 25μm. (B and C) The IgA and CD89 IOD were quantified in 20 randomly chosen fields for each mouse at 400 magnification. n = 4 mice <i>per</i> group; (D and E) Hematuria (×1000 red cells/mL; D) and urine protein (μg/mL) were measured in the urine of 12 to 32-wk-old WT, CD89 Tg mice (7–14 mice per group). No difference was detected between WT and CD89 Tg mice. (F) BUN levels in CD89 Tg and WT mice.</p

CD89 expression on the surface of monocytes/macrophages of CD89 transgenic mice.

(A) Flow cytometric analysis of CD89 surface expression on mouse blood cells. Cells of non-transgenic and transgenic mice were stained with anti-CD89-FITC. Cells were stained with Gr-1-PE, CD45/B220-PE or Ly6C-PE to identify granulocytes, lymphocytes and monocytes/macrophages, respectively. Experiments shown are representative of at least three independent experiments, yielding essentially identical results. (B, C) CD89 Expression of bone marrow monocytes (B) and peritoneal macrophages (C) in Tg mice and WT mice, detected by anti-CD89-FITC mAb.</p

CD89 expression in the tissues of transgenic mice.

(A)Western blot analysis of CD89 transgenic mice: Protein levels of the CD89 were measured in lysates of livers, lungs and spleens from CD89 transgenic mice and littermate WT mice. (B) Double immunofluorescent staining for CD68 (red) and CD89 (green) in frozen sections of liver (Kupffer cells), lung (dust cells) and spleen macrophages from CD89 Tg mice versus liver from WT mice; Cell nuclei were stained with DAPI (blue). Bar:10μm.</p

Critical Role of Kupffer Cell CD89 Expression in Experimental IgA Nephropathy

<div><p>Although IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide, its etiology remains only partly understood. It is clear that the pathogenesis of IgAN involves the formation of macromolecular IgA1 complexes and increased levels of serum IgA1 and IgA1-immune complexes(IC), due to defective IgA1 clearance. Previous studies suggest that the blood and tissue myeloid cell-expressed IgA Fc receptor (FcαR/CD89) mediates IgA-IC clearance and its dysfunction, via decreased activity or excessive levels of soluble FcαR/sCD89 induces IgAN. Such a mechanism requires robust stimulation of IgAN levels via forced expression of CD89. In the absence of unequivocal evidence supporting such a mechanism to date, we attempted to test the extent of CD89-evoked IgAN by generating a transgenic mouse strain expressing human CD89 under the control of murine CD14 promotor. No deposition of IgA-CD89 complexes or glomerulonephritis was detected, however. Further studies showed that elimination of murine IgA was mediated by Kupffer cells. In patients, however, CD89/IgA complexes were detected, and injection of patient IgA induced IgAN-like features in CD89 Tg mice. In transgenic mice, IgAN pathogenesis involves impaired clearance of abnormal IgA via CD89, primarily by the Kupffer cells. Conditional IgAN progression in CD89 transgenic mice thus reveals important aspects of IgAN pathogenesis.</p></div