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      Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China

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          Abstract

          Pseudomonas aeruginosa is a major opportunistic pathogen in hospital-acquired infections and exhibits increasing antibiotic resistance. A rapid and sensitive molecular method for its detection in clinical samples is needed to guide therapeutic treatment and to control P. aeruginosa outbreaks. In this study, we established a polymerase spiral reaction (PSR) method for rapid detection of P. aeruginosa by targeting the toxA gene, which regulates exotoxin A synthesis. Real-time turbidity monitoring and a chromogenic visualization using hydroxynaphthol blue were used to assess the reaction. All 17 non- P. aeruginosa strains tested negative, indicating the high specificity of the PSR primers. The detection limit was 2.3 pg/μl within 60 min at isothermal temperature (65°C), 10-fold more sensitive than conventional PCR. Then, the PSR assay was applied to a clinical surveillance of P. aeruginosa in three top hospitals in Beijing, China. Of the 130 sputum samples collected from ICU patients with suspected multi-resistant infections, 37 P. aeruginosa isolates were identified from the positive samples. All clinical strains belonged to 10 different P. aeruginosa multilocus sequence typing groups and exhibited high resistance to carbapenems, cephalosporins, and aminoglycosides. Interestingly, of the 33 imipenem-resistant isolates, 30 (90.9%) had lost the outer membrane porin oprD gene. Moreover, isolate SY-95, containing multiple antibiotic resistance genes, possessed the ability to hydrolyze all antibiotics used in clinic and was susceptible only to polymyxin B. Our study showed the high level of antibiotic resistance and co-occurrence of resistance genes in the clinical strains, indicating a rapid and continuing evolution of P. aeruginosa. In conclusion, we developed a P. aeruginosa PSR assay, which could be a useful tool for clinical screening, especially in case of poor resources, or for point-of-care testing.

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          Most cited references25

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          Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.

          Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
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            Urinary tract infections caused by Pseudomonas aeruginosa: a minireview.

            Urinary tract infections (UTIs) are a serious health problem affecting millions of people each year. Infections of the urinary tract are the second most common type of infection in the body. Catheterization of the urinary tract is the most common factor, which predisposes the host to these infections. Catheter-associated UTI (CAUTI) is responsible for 40% of nosocomial infections, making it the most common cause of nosocomial infection. CAUTI accounts for more than 1 million cases in hospitals and nursing homes annually and often involve uropathogens other than Escherichia coli. While the epidemiology and pathogenic mechanisms of uropathogenic Escherichia coli have been extensively studied, little is known about the pathogenesis of UTIs caused by other organisms like Pseudomonas aeruginosa. Scanty available information regarding pathogenesis of UTIs caused by P. aeruginosa is an important bottleneck in developing effective preventive approaches. The aim of this review is to summarize some of the advances made in the field of P. aeruginosa induced UTIs and draws attention of the workers that more basic research at the level of pathogenesis is needed so that novel strategies can be designed.
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              Strand displacement amplification--an isothermal, in vitro DNA amplification technique.

              Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                06 October 2015
                2015
                : 6
                : 1100
                Affiliations
                [1] 1Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China
                [2] 2Department of Digestive System, The Second Affiliated Hospital of Dalian Medical University Dalian, China
                Author notes

                Edited by: Tzi Bun Ng, The Chinese University of Hong Kong, Hong Kong

                Reviewed by: M. Habibur Rahman, Rajshahi University, Bangladesh; Elizabeth Fozo, University of Tennessee, USA

                *Correspondence: Liuyu Huang and Wei Liu, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai District, Beijing 100071, China, huangliuyuly@ 123456163.com ; liuwei5269@ 123456qq.com

                These authors have contributed equally to this work.

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2015.01100
                4594016
                9f0c46aa-6fb8-4442-9a39-5debc2f88a3c
                Copyright © 2015 Dong, Zou, Liu, Yang, Huang, Liu, He, Liu and Huang.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 29 July 2015
                : 23 September 2015
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 30, Pages: 7, Words: 0
                Funding
                Funded by: Mega-projects of Science and Technology Research of China
                Award ID: 2013ZX10004-203
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                p. aeruginosa,psr,toxa,rapid diagnosis,isothermal
                Microbiology & Virology
                p. aeruginosa, psr, toxa, rapid diagnosis, isothermal

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