[Show abstract][Hide abstract] ABSTRACT: The biological characteristics and molecular epidemiology of Pseudomonas aeruginosa associated with mink hemorrhagic pneumonia from Shandong province of eastern China were determined in this study. From 2010 to 2011, 30 mink P. aeruginosa isolates were identified from lung, fecal and feed samples of clinical cases and subjected to serotyping, antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) using SpeI. The P. aeruginosa isolates belonged to four serotypes—21 of type G, four of type I, three of type M, one of type B, and one non-typable strain. The strains were divided into four large groups as determined by PFGE. Isolates from the group 2 were highly homologous and were obtained from the same region as an epidemic. All of the isolates were sensitive to piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, amikacin, gentamicin and tobramycin and resistant to ampicillin, cefuroxime and cefuroxime axetil. A high frequency of resistance was found to ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, nitrofurantoin, and trimethoprim/sulfamethoxazole (96.7%). Resistance to ticarcillin/clavulanic acid, ciprofloxacin and levofloxacin was less common (13.3%). There was no relationship between antibiotic resistance and serotype distribution of the isolates. The epidemic serotype of P. aeruginosa from the mink hemorrhagic pneumonia in Shandong province was type G, which was a clone of commonly found in this province. These findings reveal the genetic similarities and antimicrobial susceptibility profiles of P. aeruginosa from clinical cases of mink hemorrhagic pneumonia and will facilitate the prevention and control of the disease in Shandong province of China.
[Show abstract][Hide abstract] ABSTRACT: Although enrofloxacin (ENR) is widely used for therapy of bacterial infections in the veterinary clinical, bacterial resistance to ENR is becoming an increasing worldwide problem. The primary global response of Escherichia coli to ENR exposure before resistance is largely unknown on the proteomic level. The purpose of this study was to understand the physiological response of E. coli to a subinhibitory concentration of ENR using proteomic methods. Differentially expressed proteins of the whole-cell extracts were visualized by two-dimensional gel electrophoresis, and the selected proteins were purified and identified by MALDI-TOF/mass spectrometry analysis. The result showed that the number of proteins (mean±standard deviation) detected in the ENR-treated strains was significantly (p<0.05) reduced from 1115±25 to 732±19. In total, 42 differentially expressed proteins with more than twofold difference were identified, including 13 upregulated proteins (p<0.05) and 17 downregulated proteins (p<0.05), as well as the specific proteins expressed in the group with or without ENR-treated cells. The results show that the differentially expressed proteins identified in E. coli exposed to ENR included proteins involved with a classic resistance mechanism, such as bacterial cell membrane permeability mediated by OmpX and OmpW, and other adaptive changes that appear to represent the physiological basis and background of resistance to ENR.
[Show abstract][Hide abstract] ABSTRACT: We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the multidrug-resistance gene cfr. A pair of outer primers and a pair of inner primers and one loop primer were specially designed for recognizing seven distinct sequences on the target cfr gene. The amplification reaction was performed within only 35 min under isothermal conditions at 63 °C in a regular water bath with visual measurement. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of 1 pg per tube of chicken Staphylococcus sciuri genomic DNA. The detection rate of cfr gene for 50 Staphylococcus clinical strains by LAMP assays was 16% and appeared 100% consistence with the result by PCR method. The LAMP method reported here demonstrated a potential and valuable means for detection of the multidrug-resistance gene cfr: easy, rapid, visual, specific, accurate and sensitive, especially useful for on-the-spot investigation.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms behind reduced bacterial sensitivity to drugs and the nature of this phenomenon were explored. Escherichia coli were treated with a sub-inhibitory dose or a repeatedly doubled dose of enrofloxacin (EN) in vitro. After 10 serial passages,
the minimal inhibitory concentration (MIC) of EN to the bacteria was assessed by changes in expression of genes involved in
the SOS response, drug efflux pumps, porins and EN targets, and the mutation of the quinolone resistance determining region
(QRDR). The results showed that the MIC of EN to E. coli was higher after a single passage when treated with either concentration of EN compared with that of the controls (0.5 μg/ml).
From this point onwards, the MIC of E. coli treated with the sub-inhibitory concentration tended to be stable, whilst it increased continuously to 64 μg/ml when treated
with the doubling dose. Regardless of the EN concentration that bacteria were exposed to, their efflux pumps were started,
the expression of the SOS regulatory genes recA and umuC and EN target gene parC were significantly up-regulated, and the expression of the porins gene ompW was significantly down-regulated. No mutations were detected in the QRDR of E. coli treated with the sub-inhibitory concentration of EN, but mutations at three sites occurred after treatment with the repeatedly
doubling dose. The current results suggest that the reduction in sensitivity of bacteria to drugs is positively, but non-linearly,
correlated with the concentration of drugs used. The E. coli studied initiated mechanisms to adapt to the enrofloxacin stress which included an SOS response, target gene mutations and
efflux pump activity and so on. These adaptive mechanisms are interactive, but the inherited and non-inherited mechanisms
play different roles in the adaptation of bacteria to drugs.
Annals of Microbiology 03/2012; 62(1). DOI:10.1007/s13213-011-0260-3 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.
[Show abstract][Hide abstract] ABSTRACT: The effect on gene expression and cell morphology of Escherichia coli strain ATCC 25922 of exposure to the antibiotic enrofloxacin was studied. The growth rate of the cells was compromised, and
the cells took on a filamentous shape. Microarray analysis showed that the expression of 519 genes was altered by the treatment,
of which 239 were up- and 280 down-regulated. The transcription of 34 genes was increased at least three fold, and that of
32 genes decreased at least three fold. The functions of these differentially expressed genes included the SOS response, cell
division, the stress response, biosynthesis, metabolism, transport and transcription regulation. The overall response was
consistent with the notion that exposure to enrofloxacin initiated the SOS response and inhibited cell division. Although
there was extensive physiological tolerance to the stress, there was no evidence for any mutation to resistance.
KeywordsEnrofloxacin-Gene expression profiles microarray-
Annals of Microbiology 12/2010; 60(4):653-660. DOI:10.1007/s13213-010-0105-5 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel reductive compound with molecular weight of about 1000Da, named Pc reducer, was purified from the liquid culture of a white-rot basidiomycete Phanerochaetechrysosporium. It was likely to have an alkene-ester structure according to Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectra. Pc reducer reduced the hydroxyl radical HO(*) and the stable nitroxide radical under certain conditions. It inhibited the repolymerization of the products from the oxidation of phenolic lignin-model compounds by reducing certain intermediate radicals. The activity of manganese peroxidases was promoted by Pc reducer at certain concentrations. Pc reducer could also weaken the repolymerization of fragments from the oxidation of Na-lignosulfonate by lignin peroxidases and manganese peroxidases. It has potential ability to improve the ligninolytic efficiency of peroxidases in P. chrysosporium.
[Show abstract][Hide abstract] ABSTRACT: The lignin peroxidase (LiP) catalyzed oxidation of pyrogallol red (PR) in the absence and presence of veratryl alcohol (3,4-dimethoxybenzyl
alcohol, VA) was carried out in bis (2-ethylhexyl) sulfosuccinate sodium (AOT)/ polyoxyethylene lauryl ether (Brij30) reversed
micelles to elucidate the role of VA. Results indicated that VA could accelerate the LiP catalyzed oxidation of PR, especially
at low H2O2 concentrations. Unlike in bulk aqueous medium, the protection of LiP by VA in the present medium was relatively unsubstantial,
even at high H2O2 concentrations. Analysis of data from a series of experiments showed that the enhancement of the PR oxidation caused by VA
was mainly due to the indirect oxidation of PR by VA+∙ from the LiP catalyzed oxidation of VA. It was also found that at the same protector concentration (40 μM), VA (the physiological substrate of LiP) was less effective than PR (a phenolic compound) in protecting LiP from the H2O2 derived inactivation. This novel phenomenon deserves further study.
Central European Journal of Chemistry 08/2007; 5(3):672-687. DOI:10.2478/s11532-007-0032-x · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The H(2)O(2) supply strategy was one of crucial factors for high efficient degradation of pollutants with lignin peroxidase (LiP). In this paper, an attempt was made to couple a H(2)O(2) producing enzymatic reaction to the LiP catalyzed oxidation of dyes. H(2)O(2) needed was generated by glucose oxidase (GOD) and its substrate glucose. The generation rate of H(2)O(2) could be easily controlled by adjusting the pH of the degradation system and the amount of GOD added. Due to the controlled release of H(2)O(2), a sustainable constant activity of LiP was observed. The inhibition of LiP by high level H(2)O(2) supplied externally by a single addition at the beginning of the experiments could be avoided. Degradation of three dyes (xylene cyanol, fuchsine and rhodamine B) with LiP coupled with GOD indicated that the present H(2)O(2) supply strategy was very effective for improvement of the efficiency of the decolourization of dyes.
Journal of Biotechnology 07/2006; 123(4):483-90. DOI:10.1016/j.jbiotec.2005.12.034 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new low-molecular-weight peptide with phenol oxidase activity, named Pc factor, was isolated and purified from liquid culture of a white-rot basidiomycete Phanerochaete chrysosporium. Its molecular weight was about 600 Da estimated by gel-filtration. Three amino acids Glu, Gly and Val were detected in hydrolysate. Absorption peaks corresponding to amino acids and peptide were observed by UV and IR spectra analysis. And the signal of Ca of amino acid was also detected by 13C-NMR method. Pc factor had high thermostability and remained active in weakly alkalescent pH range. It could chelate Fe3+ and reduce it to Fe2+, but no hydroxyl radical HO* could be detected during the reaction process. It could oxidize phenolic lignin-model compounds such as 2,6-dimethoxyphenol (2,6-DMP), 2,2'-azinobis (3-ethylbenzathiazoline-6-sulfinic acid) (ABTS) and syringaldazine in the absence of Mn2+ and H2O2. These characteristics differed greatly from those of manganese peroxidases. The oxidative catalysis of Pc factor can be enhanced by certain metal ions such as Cu2+ and Mn2+ etc., and O2 molecule was necessary for this reaction. In summary, Pc factor may function as an electron carrier in this novel oxidation-reduction system.
Science in China Series C Life Sciences 07/2006; 49(3):243-50. DOI:10.1007/s11427-006-0243-y · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.
Biochemical and Biophysical Research Communications 12/2003; 311(2):491-4. DOI:10.1016/j.bbrc.2003.10.029 · 2.30 Impact Factor