Jing Qi

A Loop-Mediated Isothermal Amplification Method for Rapid Detection of NDM-1 Gene

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

The identification, typing, and antimicrobial susceptibility of Pseudomonas aeruginosa isolated from mink with hemorrhagic pneumonia

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.

A loop-mediated isothermal amplification method for rapid detection of the multidrug-resistance gene cfr

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.

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

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.

Identification of Wohlfahrtiimonas chitiniclastica isolated from an infected cow with hoof fetlow, China.

Wohlfahrtiimonas chitiniclastica is a rare but an emerging zoonotic pathogen. Here, we report the isolation and identification of W. chitiniclastica strain DZ2015 from hoof pus of an infected cow with hoof fetlow in Shandong, China by 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing and mouse infection experiments showed that the strain of W. chitiniclastica had broad susceptibility and was pathogenic to mice. This is the first report of the W. chitiniclastica isolated from an infected domestic animal in China.

The prevalence of colistin resistance in escherichia coli and klebsiella pneumoniae isolated from food animals in China: coexistence of mcr-1 and bla NDM with low fitness cost

Increasing colistin resistance is a global concern because colistin is used as a last resort for the treatment of carbapenem-resistant Enterobacteriaceae infections. The plasmid-mediated colistin resistance gene, mcr-1 was found in distinct bacterial species isolated from humans, animals, and the environment. In this study, farms in four different agricultural provinces in China were investigated to determine the occurrence of the antimicrobial resistance and related genes. A total of 373 Escherichia coli and 54 Klebsiella pneumoniae were isolated from 510 non-duplicated samples. Of the E. coli and K. pneumoniae isolates, 72.7% and 66.7%, respectively, were susceptible to colistin. Isolates resistant to colistin comprised 46.6% of the samples isolated from Shandong, and 17.8% and 16.4% of the samples from Jilin and Henan, respectively. Twenty-six carbapenem-resistant E. coli isolates were resistant to colistin, in which both mcr-1 and blaNDM were present. Specifically, the co-existence was found in isolates from animals and sewage. Most of the resistance genes were located on plasmids and were 40-244 kilobases. Growth curves of transconjugants carrying mcr-1, blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-9 showed a low fitness cost compared with the recipient. In conclusion, mcr-1 was widespread in E. coli and K. pneumoniae isolated from farms in China. Co-existence of mcr-1 and blaNDM-9 was identified in different sequence types of E. coli with low fitness cost from various origins, indicating an urgent need to take measures for decreasing dissemination.

Global Protein Expression Profile Response of Escherichia coli ATCC 25922 Exposed to Enrofloxacin

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.