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      Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China

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          Abstract

          Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

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          Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

          Y. Mori, K Nagamine, N Tomita … (2001)
          The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity. Copyright 2001 Academic Press.
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            Carbapenemases: molecular diversity and clinical consequences.

            Laurent Poirel, Johann D D Pitout, Patrice Nordmann (2007)
            Carbapenemases are beta-lactamases that hydrolyze most beta-lactams including carbapenems. Carbapenemases are classified in four molecular classes; those belonging to class A are the chromosomally-encoded and clavulanic acid-inhibited IMI, NMC-A and SME, identified in Enterobacter cloacae and Serratia marcescens; the plasmid-encoded KPC enzymes identified in Enterobacteriaceae (and rarely in Pseudomonas aeruginosa); and the GES-type enzymes identified in Enterobacteriaceae and P. aeruginosa. The class B enzymes are the most clinically-significant carbapenemases; they are metallo-beta-lactamases, mostly of the IMP and the VIM series. They have been reported worldwide and their genes are plasmid- and integron-located, hydrolyzing all beta-lactams with the exception of aztreonam. One single plasmid-mediated AmpC beta-lactamase, CMY-10, identified in an Enterobacter aerogenes isolate, has been shown to be a cephaslosporinase with some carbapenemase properties. Finally, the class D carbapenemases are being increasingly reported, mostly in Acinetobacter baumannii, and they compromise the efficacy of imipenem and meropenem significantly.
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              An Increasing Prominent Disease of Klebsiella pneumoniae Liver Abscess: Etiology, Diagnosis, and Treatment

              Yun Liu, Ji-Yao Wang, Wei Jiang (2013)
              Background. During the past two decades, Klebsiella pneumoniae (K. pneumoniae) had surpassed Escherichia coli (E. coli) as the predominant isolate from patients with pyogenic liver abscess (PLA) in Asian countries, the United States, and Europe, and it tended to spread globally. Since the clinical symptom is atypical, the accurate and effective diagnosis and treatment of K. pneumoniae liver abscesses (KLAs) are very necessary. Methods. Here, we have comprehensively clarified the epidemiology and pathogenesis of KLA, put emphases on the clinical presentations especially the characteristic radiographic findings of KLA, and thoroughly elucidated the most effective antibiotic strategy of KLA. Results. K1 serotype is strongly associated with KLA especially in diabetic patients. Computed tomography (CT) and ultrasound (US) were two main diagnostic methods of KLA in the past. Most of KLAs have solitary, septal lobular abscesses in the right lobe of liver, and they are mainly monomicrobial. Broad-spectrum antibiotics combined with the US-guided percutaneous drainage of liver abscesses can increase their survival rates, but surgical intervention still has its irreplaceable position. Conclusion. The imaging features contribute to the early diagnosis, and the percutaneous intervention combined with an aminoglycoside plus either an extended-spectrum betalactam or a second- or third-generation cephalosporin is a timely and effective treatment of KLA.
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                Author and article information

                Contributors
                Journal
                Journal ID (nlm-ta): Front Microbiol
                Journal ID (iso-abbrev): Front Microbiol
                Journal ID (publisher-id): Front. Microbiol.
                Title: Frontiers in Microbiology
                Publisher: Frontiers Media S.A.
                ISSN (Electronic): 1664-302X
                Publication date (Electronic): 22 May 2015
                Publication date Collection: 2015
                Volume: 6
                Electronic Location Identifier: 519
                Affiliations
                [1] 1Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China
                [2] 2Department of Laboratory Medicine, The General Hospital of Chinese People's Armed Police Forces Beijing, China
                [3] 3State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology Beijing, China
                Author notes

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

                Reviewed by: Dmitri Debabov, NovaBay Pharmaceuticals, USA; Yuji Morita, Aichi Gakuin University, Japan

                *Correspondence: Dongsheng Zhou, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, No.20 Dongda Street, Fengtai District, Beijing 100071, China dongshengzhou1977@ 123456gmail.com
                Liuyu Huang and Jing Yuan, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, No.20 Dongda Street, Fengtai District, Beijing 100071, China huangliuyuly@ 123456163.com ; yuanjing6216@ 123456163.com

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

                †These authors have contributed equally to this work.

                Article
                DOI: 10.3389/fmicb.2015.00519
                PMC ID: 4440914
                PubMed ID: 26052327
                SO-VID: 7dcd556f-bc99-40bb-a3bb-77684e815780
                Copyright © Copyright © 2015 Dong, Liu, Li, Wang, Li, Zou, Yang, Huang, Zhou, Huang and Yuan.
                License:

                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
                Date received : 18 March 2015
                Date accepted : 09 May 2015
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 25, Pages: 6, Words: 4443
                Categories
                Subject: Microbiology
                Subject: Original Research

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: klebsiella pneumoniae,loop-mediated isothermal amplification,lamp,rapid detection
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: klebsiella pneumoniae, loop-mediated isothermal amplification, lamp, rapid detection

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