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      Rapid simultaneous detection of bla oxa-23, Ade-B, int-1, and ISCR-1 in multidrug-resistant Acinetobacter baumannii using single-tube multiplex PCR and high resolution melting assay

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          Abstract

          Objective: The aim of this study was to develop a multiplex PCR system for the rapid and simultaneous detection of bla oxa-23, Ade-B, int-1, and ISCR-1 genes in multidrug-resistant Acinetobacter baumannii (MDRAB) using high resolution melting (HRM) assay.

          Methods: Four pairs of primers were designed, and PCR amplification products were sequenced and compared with NCBI GeneBank sequences to ensure primer specificity. Multiplex PCR was performed using a dedicated HRM reagent, and melting curves and temperatures were able to distinguish the four genes. This method was subsequently used to detect these genes in 79 MDRAB isolates from the Third Affiliated Hospital of Southern Medical University in southern China.

          Results: Using the HRM assay, 73 out of 79 isolates were found to carry both bla oxa-23 and Ade-B, one isolate carried int-1, two isolates carried both int-1 and ISCR-1, and three isolates carried Ade-B, int-1, and ISCR-1. No isolates carried all four genes.

          Conclusion: Compared with traditional resistance gene detection methods–PCR and agarose gel electrophoresis-based resistance gene detection methods–the multiplex PCR and HRM assay method was simple, rapid, highly efficient, and cost-effective. Our results showed that bla oxa-23 and Ade-B were the main resistance genotypes in MDRAB.

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          Acinetobacter: an old friend, but a new enemy.

          K.J. Towner (2009)
          Acinetobacter emerged as a significant nosocomial pathogen during the late 1970s, probably as a consequence, at least in part, of increasing use of broad-spectrum antibiotics in hospitals. Most clinically significant isolates belong to the species Acinetobacter baumannii or its close relatives, with many infections concentrated in intensive care, burns or high dependency units treating severely ill or debilitated patients. Large outbreaks can occur in such units, involving the infection or colonisation of numerous patients by specific epidemic strains of A. baumannii. Recently, a particular problem has concerned cross-infection of injured military patients repatriated from combat regions of the world (e.g. Iraq and Afghanistan). Carbapenems have previously been the treatment of choice for infected patients, but increasing reports worldwide now describe A. baumannii isolates resistant to all conventional antimicrobial regimens. Data to support therapeutic use of the limited number of new antimicrobial agents (e.g. tigecycline) with in-vitro activity against these pathogens are still very limited. Detailed advice concerning prevention and control of outbreaks caused by multidrug-resistant strains of acinetobacter is available from the UK Health Protection Agency. In addition to antibiotic prescribing policies and audit, these measures focus on reinforcing standard infection control procedures and precautions, with particular attention to thorough cleaning of patient areas to take account of the long-term survival of acinetobacter after drying and inadequate disinfection. Despite these measures, the problem continues to escalate, with many hospitals worldwide now reporting outbreaks caused by multidrug-resistant strains of acinetobacter.
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            Laboratory diagnosis, clinical management and infection control of the infections caused by extensively drug-resistant Gram-negative bacilli: a Chinese consensus statement.

            J. Hu, B. Hu, L He … (2016)
            Extensively drug-resistant (XDR) Gram-negative bacilli (GNB) are defined as bacterial isolates susceptible to two or fewer antimicrobial categories. XDR-GNB mainly occur in Enterobacteriaceae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The prevalence of XDR-GNB is on the rise in China and in other countries, and it poses a major public health threat as a result of the lack of adequate therapeutic options. A group of Chinese clinical experts, microbiologists and pharmacologists came together to discuss and draft a consensus on the laboratory diagnosis, clinical management and infection control of XDR-GNB infections. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created according to documents from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). Multiple risk factors of XDR-GNB infections are analyzed, with long-term exposure to extended-spectrum antimicrobials being the most important one. Combination therapeutic regimens are summarized for treatment of XDR-GNB infections caused by different bacteria based on limited clinical studies and/or laboratory data. Most frequently used antimicrobials used for the combination therapies include aminoglycosides, carbapenems, colistin, fosfomycin and tigecycline. Strict infection control measures including hand hygiene, contact isolation, active screening, environmental surface disinfections, decolonization and restrictive antibiotic stewardship are recommended to curb the XDR-GNB spread.
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              Distribution of different efflux pump genes in clinical isolates of multidrug-resistant Acinetobacter baumannii and their correlation with antimicrobial resistance.

              Ming-Feng Lin, Yun-You Lin, Chi-Chao Tu … (2015)
              Efflux pumps are one of the major mechanisms of antimicrobial resistance in Acinetobacter baumannii. This study aimed to understand the distribution of different types of pump genes in clinical isolates of multidrug-resistant A. baumannii (MDRAB) and to reveal the relationship between their presence and expression with antimicrobial resistance.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Infect Drug Resist
                Journal ID (iso-abbrev): Infect Drug Resist
                Journal ID (publisher-id): IDR
                Journal ID (pmc): idr
                Title: Infection and Drug Resistance
                Publisher: Dove
                ISSN (Electronic): 1178-6973
                Publication date (Electronic): 07 June 2019
                Publication date Collection: 2019
                Volume: 12
                Pages: 1573-1581
                Affiliations
                [1 ]Department of Clinical Laboratory, The Third Affiliated Hospital of Southern Medical University , Guangzhou 510630, People’s Republic of China
                [2 ]Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University , Guangzhou 510515, People’s Republic of China
                Author notes
                Correspondence: Fu XiongDepartment of Medical Genetics, School of Basic Medical Sciences, Southern Medical University , No. 1023-1063, South of Shatai Road, Baiyun District, Guangzhou510515, People’s Republic of ChinaTel +86 20 6164 8510Email xiongfu@ 123456smu.edu.cn
                Youming ChenDepartment of Clinical Laboratory, The Third Affiliated Hospital of Southern Medical University , No. 183, West of Zhongshan Avenue, Tianhe District, Guangzhou510630, People’s Republic of ChinaTel +86 20 6278 4789Email cym38432@ 123456163.com
                [*]

                These authors contributed equally to this work

                Article
                Publisher ID: 207225
                DOI: 10.2147/IDR.S207225
                PMC ID: 6565808
                PubMed ID: 31289445
                SO-VID: b8ed64a3-6463-4159-9091-2dac0808a381
                Copyright © © 2019 Sun et al.
                License:

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                History
                Date received : 01 March 2019
                Date accepted : 13 May 2019
                Page count
                Figures: 7, Tables: 2, References: 28, Pages: 9
                Categories
                Subject: Original Research

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: blaoxa-23,ade-b,int-1,iscr-1,multiplex pcr,high resolution melting
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: blaoxa-23, ade-b, int-1, iscr-1, multiplex pcr, high resolution melting

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